## 3 Results and Discussion
### Effects of pH and Temperature on the Accumulation of Hg by BL21-7  
To characterize the effects of pH on the ability of the engineered bacteria to remove Hg from the environment, BL21-7 was cultured in LB liquid medium overnight at 37 \({}^{\circ}\)C. One microliter of this culture was transferred to 100 mL of nutrition-poor PB medium (pH = 5, 6, 7, 8 or 9) containing 20 umol/L HgCl\({}_{2}\) and was cultured continually. The OD\({}_{600}\) values of the cultures were measured every two h. After the cultures entered stationary phase, the Hg contents in the bacteria and medium were measured. The results showed that all of the growth curves of the cultures showed an S shape. No significant differences were found among the growth curves of cultures at pH = 5, 7 and 8 (Figure 7). For example, after BL21-7 was cultured for 30 h, the OD\({}_{600}\) values of the cultures (pH = 5, 7 and 8) were 1.21 \(\pm\) 0.03, 1.22 \(\pm\) 0.03 and 1.2 \(\pm\) 0.03, respectively. Among the five PB media (pH = 5, 6, 7, 8 and 9), the OD\({}_{600}\) values of BL21-7 cultured in PB at pH = 6 were the highest in stationary phase. The engineered bacteria could also grow in PB at pH = 9. However, they grew very slowly (Figure 7). The final OD\({}_{600}\) of the culture at pH = 9 was also the lowest among the five treatments. After BL21-7 was cultured for 40 h, the Hg contents in the supernatant of the PB media (pH = 5, 6, 7, 8 and 9) containing 20 umol/L HgCl\({}_{2}\) were 6.1 \(\pm\) 0.10 umol/L, 5.2 \(\pm\) 0.11 umol/L, 5.9 \(\pm\) 0.12 umol/L, 7.6 \(\pm\) 0.12 umol/L and 12.5 \(\pm\) 0.11 umol/L, respectively. The Hg content in the supernatant of the PB at pH = 6 containing 20 umol/L HgCl\({}_{2}\) was the lowest among the five treatments. The engineered bacterial strain accumulated the most Hg in the liquid medium at a pH of 6 and it worked well in the media with pH values from 5 to 8. To characterize the effects of temperature on the engineered strain's growth and accumulation of Hg, BL21-7 was cultured in LB liquid medium overnight at 37 \({}^{\circ}\)C. One microliter of this culture was transferred to 100 mL of nutrition-poor PB medium containing 20 umol/L HgCl\({}_{2}\) and was cultured at 25 \({}^{\circ}\)C, 30 \({}^{\circ}\)C and 37 \({}^{\circ}\)C. The OD\({}_{600}\) values and Hg content were measured as described above. The results showed that although BL21-7 grew more slowly at 25 \({}^{\circ}\)C than at 30 \({}^{\circ}\)C or 37 \({}^{\circ}\)C, the OD\({}_{600}\) values of these three treatments were similar at stationary phase (Figure 8). For example, although \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Metals** & \begin{tabular}{c} **HgCl\({}_{2}\) Removed** \\ **(umol/L)** \\ \end{tabular} & \
\begin{tabular}{c} **MMC Removed** \\ **(umol/L)** \\ \end{tabular} &
\begin{tabular}{c} **PMC Removed** \\ **(umol/L)** \\ \end{tabular} \\ \hline None & 8.80 \(\pm\) 0.16 & 5.47 \(\pm\) 0.28 & 7.80 \(\pm\) 0.17 \\ Cd\({}^{2+}\) & 8.66 \(\pm\) 0.15 & 5.53 \(\pm\) 0.23 & 7.76 \(\pm\) 0.15 \\ Pb\({}^{2+}\) & 8.85 \(\pm\) 0.09 & 5.41 \(\pm\) 0.25 & 7.73 \(\pm\) 0.18 \\ Cu\({}^{2+}\) & 8.95 \(\pm\) 0.12 & 5.44 \(\pm\) 0.26 & 7.83 \(\pm\) 0.17 \\ Cd\({}^{2+}\) + Pb\({}^{2+}\) + Cu\({}^{2+}\) & 8.98 \(\pm\) 0.19 & 5.59 \(\pm\) 0.27 & 7.85 \(\pm\) 0.19 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Mercury removed from LB medium in which BL21-7 was cultured for 5 h in the presence of other metals. the OD\({}_{600}\) values of the cultures (T = 25 \({}^{\circ}\)C, 30 \({}^{\circ}\)C and 37 \({}^{\circ}\)C) at 20 h were 0.6 \(\pm\) 0.04, 1.1 \(\pm\) 0.04 and 1.2 \(\pm\) 0.03, respectively, the OD\({}_{600}\) values of the same cultures at 34 h were all 1.3 \(\pm\) 0.03. After BL21-7 was cultured for 20 h, the Hg contents in the supernatant of the PB medium (T = 25 \({}^{\circ}\)C, 30 \({}^{\circ}\)C and 37 \({}^{\circ}\)C) containing 20 \(\upmu\)mol/L HgCl\({}_{2}\) were 11.2 \(\pm\) 0.12 \(\upmu\)mol/L, 8.2 \(\pm\) 0.15 \(\upmu\)mol/L and 6.5 \(\pm\) 0.11 \(\upmu\)mol/L, respectively.