## 2 Materials and Methods
### Apparatus for Continuous Cr(VI) Removal  
A cylindrical packed-bed bioreactor (CPB; length 25 cm, id 16 cm) was constructed from acrylic materials for the immobilized _Ochroductrum_ sp. CUST210-1 biosystem (Figure 1A). Three sampling pores were constructed at the top of the biosystem through which the pH value, dissolved oxygen (DO) content, and Cr(VI) concentration in the wastewater could be measured. Plastic Raschig rings (rosette type, id 2 cm) were used as packing material. The inflow solution, which contained 1/1000 LB supplemented with 300 mg L\({}^{-1}\) Cr(VI) and the CUST210-1 strain (\(3\times 10^{7}\) cfu mL\({}^{-1}\)) stored in the reservoir, was continuously recirculated with the flow directed upward from the inlet at the bottom of the reactor. The liquid retention time (LRT) of the inflow solution was controlled at 12 h. When the outlet Cr(VI) concentration was less than 0.5 mg L\({}^{-1}\), the cell immobilization procedure was regarded as completed.  
Calcium alginate beads are made through external gel formation [32]. Three percent (_w/v_) sodium alginate in 50-mM sodium PB (pH 7.0) was mixed with 5% chromate reductase solution at the ratio of 1:1. The mixture was taken into a syringe and dripped directly into a gently stirred 1.5% CaCl\({}_{2}\) solution from a height of approximately 2 cm; this resulted in the formation of spherical beads of enzyme-entrapped calcium alginate. The beads were left in the CaCl\({}_{2}\) solution for 30 min, recovered by filtration, and thoroughly washed with distilled water thrice. Toxicity evaluation of the Cr(VI) reduction products was conducted using the immobilized chromate reductase beads in batch mode. The beads were packed into a cylindrical column bioreactor (CCB) until the immobilized enzyme biosensor was used for continuous Cr(VI) removal. The CCB (length 32 cm, id 8 cm) was constructed from acrylic materials, with sampling pores installed as in the _Ochroductrum_ sp. CUST210-1 biosystem (Figure 1B).  
Effects of Operating Parameters on Continuous Cr(VI) Removal by Immobilized Ochroductrum sp. CUST210-1 Biosystem  
When the _Ochroductrum_ sp. CUST210-1 biosystem completed immobilization, synthetic wastewater was continuously introduced to the CPB. The effects of different operating parameters, including LRT (1-6 h), pH (7-9), operating temperature (25 \({}^{\circ}\)C-45 \({}^{\circ}\)C), and Cr(VI) concentration (50-400 mg L\({}^{-1}\)), on continuous Cr(VI) removal were assessed, and the synthetic wastewater (1/1000 LB supplemented  
Figure 1: Schematic of (**A**) immobilized _Ochroductrum_ sp. CUST210-1 biosystem and (**B**) immobilized enzyme biosensor: (1) peristaltic pump, (2) flow meter, (3) bioreactor, (4) sampling port, (5) pH electrode, (6) Dissolved oxygen (DO) electrode, and (7) digital pH/DO meter.  
with Cr(VI)) was used in this study. The designed LB concentration simulated organic concentrations in tannery wastewater after treatment by chemical precipitation. Generally, the inflow Cr(VI) concentration, pH, LRT, and operating temperature were set as 200 mg L\({}^{-1}\), 7.0, 4 h, and 35 \({}^{\circ}\)C, respectively. Each operating parameter was continuously operated for at least 6 h, with the results expressed as the average for each parameter.  
Furthermore, the effects of the actual Cr(VI)-containing wastewater, which was collected from the effluent of tannery wastewater after treatment by chemical precipitation (New Taipei City, Taiwan), on Cr(VI) removal were evaluated by continuously feeding the water into the biosystem for 196 days. In the case of this actual wastewater, the LRT, operating temperature, and pH were controlled at 4 h, 35 \({}^{\circ}\)C, and 7.0.