Unimax 2010

The desired amount (1–8 g L⁻¹) of FeOOH, which consists of nanosized akaganeite, goethite, and lepidocrocite [23 (link)] was added to Erlenmeyer flasks with a 50 mL solution of arsenate with an arsenic concentration of 100 mg L⁻¹. The stock solution of arsenate was prepared from Na₂HAsO₄.7H₂O (ACS reagent, ≥98%; Sigma–Aldrich, Taufkirchen, Germany), which was dissolved using redistilled water. A FeOOH-free run was also tested.
The prepared suspensions were placed onto a horizontal shaker (Unimax 2010; Heidolph, Schwabach, Germany), and agitated at 130 rpm for 24 h in the dark at 25 °C. Thereafter, the suspensions were filtered through a 0.45 µm pore-size mixed cellulose esters (MCE) membrane filter. The filtrates were diluted to 100 mL with redistilled water and there the total content of arsenic was determined by flame atomic absorption spectrometry (F-AAS; Perkin Elmer Model 1100 (Waltham, USA), wavelength 193.7 nm, air-acetylene flame, deuterium background correction, LOQ ~ 0.5 mg L⁻¹, the standard expanded uncertainty (k = 2) is 6%) or inductively coupled plasma mass spectrometry (ICP-MS; Thermo Scientific (Waltham, USA) iCap Q in KED (kinetic energy discrimination) mode (He), LOQ ~ 0.005 mg L⁻¹, the standard expanded uncertainty (k = 2) is 4%).

The fine powder of S. obliquus (200 g) was extracted with n-hexane (2 L x 3) for 24 h, centrifuged (Sigma 3-18ks Centrifuge, Germany) at 5000 rpm for 20 min at 25°C to separate cell debris from the supernatant. The supernatants evaporated under a vacuum (Heidolph Unimax 2010, Germany) at 40°C to obtain n-hexane extract (8.77g). All the extraction steps were performed in dim light [3 ].

All strains were screened in microplate (96-well) experiments for their ability to grow (16 and 42 h at 37°C) in a. anaerobiosis (AN; static cultivation in MRS broth in Generbox jars, bioMérieux SA, Marcy-l’Etoile, France, with AnaeroGen bags, Oxoid), b. aerobiosis (AE; in MRS broth, agitation on a rotary shaker at 150 rpm; Unimax 2010, Heidolph Instruments GmbH & Co.KG, Germany) and c. heme-supplemented aerobiosis (AEH; in MRS broth with 2.5 µg/mL hemin, initial pH 6.8, agitation on a rotary shaker at 150 rpm). Microplates (180 µL substrate/well) were inoculated with 20 µL of standardized (OD₄₅₀ = 2.0) overnight anaerobic MRS-pre-cultures. Optical density at 450 nm (OD₄₅₀; Titertek Multiskan Plus 311 BO Microplate Reader) and pH values (Double Pore Slim electrode, Hamilton Company, Reno, Nevada, USA) were measured at 16 h and 42 h on two replicates.
Production of catalase was qualitatively assayed by re-suspending the washed biomass (final OD₆₅₀ = 1.0) derived from 1 mL of AN, AE and AEH cultures (16 h, 37°C) in 100 µL of 3% (v/v) H₂O₂. Bubble formation provided an indication of the presence of catalase activity in cell suspensions [23] .

The pHpzc of the VB samples was determined by batch equilibration technique [45] . 5 g of VB was added to a series of six flasks that contain each 100 mL of 0.01 mol L⁻¹ KNO₃ as a background electrolyte. The initial pH values were adjusted in the pH range of 2–11 using 0.1 mol L⁻¹ of HNO₃ or NaOH. Equilibration was carried out by shaking for 12 hours using Heidolph Unimax 2010 shaker at the speed of 250 rpm at room temperature. Ultimately the dispersions were filtered and the final pH (pH_f) of the solution was determined. The pHpzc was found from a plot pH_f vs. pH_i. This procedure was repeated at various concentrations of 0.05 and 0.1 mol L⁻¹ KNO₃ solutions.

After soaking in water for 10 min, BRG was loaded in the treatment chamber. The operating parameters of PEF-AWE followed by electric field was varied from 2.3 to 5.7 kV/cm, the number of pulses from 318 to 3681 at a constant pulse duration 1 μs and pulse repetition frequency 5 Hz. In all experiments, the initial temperature of the samples were 25.0 ± 1.3 °C. Then, the mixtures were extracted on an electrical shaker (Unimax2010, Heidolph) speed 150 rpm at 24 ± 2 °C for 6 h. The mixtures were filtered through filter paper (Whatman No.1, Merck, Germany) and freeze-dried, and then kept at 4 °C until use for analysis. The brief process diagram of PEF-AWE is shown in Fig. 2.Figure 2

The process diagram of PEF-AWE.

Ferric oxyhydroxides used in this study were prepared by alkaline (40 g NaOH p.a.; Centralchem, Bratislava, Slovakia) precipitation of FeCl₃ (54.06 g FeCl₃.6H₂O p.a.; Centralchem, Bratislava, Slovakia) in 1 L of deionized water under laboratory conditions. After 12 h stirring at 150 rpm (Unimax 2010; Heidolph, Schwabach, Germany), freshly prepared precipitates were filtered, washed with distilled water, and dried at 80 °C overnight. The synthesized ferric precipitate was stored in a sealed container at room temperature before further characterization and experiments.
Manganese oxides used in this study were prepared by alkaline (40 g NaOH) precipitation of MnSO₄ (111.5 g MnSO₄.6H₂O p.a.; Centralchem, Bratislava, Slovakia) in 1 L of deionized water under laboratory conditions. After 5 h heating under reflux, the freshly prepared precipitate was filtered, washed with distilled water, and dried at 80 °C. The precipitate was then oven-heated at 95 °C for one hour prior to use and analysis.
Samples were characterized by X-ray powder diffraction (XRD) analyses elsewhere [22 (link),23 (link)] and were identified as amorphous ferric oxyhydroxides and hausmannite [Mn₃O₄].

An amount of 0.3 g of the sample was extracted with dichloromethane for HPLC ≥99.8% (2.5 mL; Sigma-Aldrich Merck KGaA, Darmstadt, Germany) by a shaker (Unimax 2010, Heidolph, Schwabach, Germany) at laboratory temperature (18 °C ± 0.1 °C) for 4 h and then filtered through a syringe PVDF filter (0.22 µm × 13 mm; Chromservis, Bratislava, Slovakia). Afterward, 150 µL of the sample extracts were stored in 2 mL vials with the microvolume insert (Agilent Technologies Inc., Santa Clara, CA, USA).