# Biosorption of Heavy Metals by the Bacterial Exopolysaccharideaccharide FucoPol
## Materials and Methods  
### FucoPol Production, Purification and Characterization  
FucoPol was obtained by cultivation of the bacterium _Enterobacter_ A47 (DSM 23139) in a 2 L bioreactor (BioStat B-plus, Sartorius, Germany) using mineral Medium E* with glycerol (40 g/L) as carbon source, as described by [PERSON] et al. [31]. FucoPol synthesis is triggered by imposing growth limiting conditions (i.e., oxygen and nitrogen limitation) concomitant with carbon availability during cultivation [31]. Thus, cultivation was performed under a fed-batch mode (feeding solution: Medium E* supplemented with 200 g/L of glycerol), with controlled temperature (30.0 \(\pm\) 0.1 \({}^{\circ}\)C), pH (7.0 \(\pm\) 0.02) and dissolved oxygen concentration (10% of the air saturation). FucoPol was recovered from the cultivation broth after 4 days as previously described [27]. The procedure involved dilution of the broth with deionized water (1:10, _v/v_) to reduce viscosity, centrifugation (13000\(\times\)g, 45 min) for cell removal, thermal treatment (70 \({}^{\circ}\)C, 1 h) of the cell-free supernatant to promote protein denaturation and, finally, centrifugation (13,000\(\times\)g, 45 min) for removal of cell fragments and denatured proteins. For FucoPol purification, the low molecular weight compounds (salts, glycerol, proteins) were removed by diafiltration in a cross-flow module (Sartocon Slide Holder), using a membrane with a surface area of 100 cm\({}^{2}\) and a 100 kDa nominal molecular weight cut-off (Hydrostart ultrafiltration cassette, [PERSON]) [27]. During diafiltration, deionized water was added to the reentate to facilitate the diffusion of low molecular weight solutes across the membrane. Finally, the module was operated in ultrafiltration mode, without water addition, to concentrate the treated supernatant (3:1, _v/v_), and the solution was freeze-dried to obtain the FucoPol (12.4 g). FucoPol was characterized in terms of carbohydrate monomers and acyl groups composition, as well as total protein content and molecular mass distribution. For the determination of the sugar and acyl content, FucoPol (1 g/L, 5 mL) was hydrolyzed with 0.1 mL 99% trifluoroacetic acid (TFA) at 120 \({}^{\circ}\)C for 2 h. The constituent monosaccharides were identified and quantified in the hydrolysate by HPLC using a Carbopac PA10 column (Thermo Scientific(tm) Dionex(tm), Sunnyvale, CA, USA), equipped with an amperometric detector, as described by [PERSON] et al. [31]. The analysis was performed at 30 \({}^{\circ}\)C with sodium hydroxide (NaOH 4 mM) as eluent, at a flow rate of 0.9 mL/min. The acid hydrolysates were also used for the identification and quantification of acyl substituents. The analysis was performed by HPLC with and Aminex HPX-87H 300 \(\times\) 7.8 mm (Biorad, Hercules, CA, USA), coupled to an infrared (IR) detector, using sulphuric acid (H\({}_{2}\)SO\({}_{4}\) 0.01N) as eluent, at a flow rate of 0.6 mL/min and a temperature of 30 \({}^{\circ}\)C.  
The protein content was determined by a modified Lowry method: 5.5 mL FucoPol solutions (4.4 g/L) were mixed with 1 mL 20% NaOH and incubated at 100 \({}^{\circ}\)C, for 5 min. After cooling on ice, 170 \(\mu\)L of CuSO\({}_{4}\).5H\({}_{2}\)O (25% _w/v_) were added and the solution was agitated. Afterwards, the solution was centrifuged (3500\(\times\)_g_, for 5 min) and the optical density was measured at 560 nm. Albumin (Sigma-Aldrich) solutions (0.05-1.0 g/L) were used as protein standards. The average molecular weight (M\({}_{\mathrm{w}}\)) was determined by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), as described by [PERSON] et al. [31]. Briefly, FucoPol solutions (0.2 g/L) were dissolved in 0.1 M Tris-HCl, NaCl (0.2 M), pH 8.09 buffer, which was also the SEC mobile phase.