Suprapure nitric acid

The suprapure nitric acid, nitric acid, acetic acid, percholoric acid, hydrocholoric acid, sodium hydroxide, oxalic acid, pure lead of 1000 ppm, mercury and potassium chloride were purchased from Merck Chemical Company (pro-analysis grade) (Darmstadt, Germany). All chemicals and reagents were analytical grade and used without further purification. All experiments were carried out at room temperature. We also used repeated dilutions with deionized double distilled water to prepare low concentration solutions.
Blood serum samples preparation 10 mL of blood sample was taken for the subjects. 2 mL of the sample together with 2 mL of suprapure concentrated nitric acid were transferred into a cleaned test tube, and then 2 mL of HClO 4 was added. The resulting solution was heated to nearly boiling point and the appearance of yellow color. Then 2 mL of nitric acid was added to the solution and heated upto a boiling point for ten minutes, and then on the residual ash, 2 mL of HCL was added and evaporated until nearly dryness. The acidity of the solution was neutralized with 2 M NaOH (pH = 4.6). The final sample (2 mL) was transferred into a 10 mL volumetric flask and diluted with 8 mL deionized double distilled water. To protect of the lead, 5 ml of sodium oxalate (1 mM) buffer was added to the final sample (totally 15 mL) which was ready for injection into the voltammetry cell [21] .

The capacity of B2-DHA strain to decrease the chromium contents in the liquid medium was determined by the inductively coupled plasma atomic emission spectroscopy (ICP-AES). [27] The strain B2-DHA was grown in 50 mL LB medium supplemented with 100 µg/mL of chromium and incubated at 37°C with shaking at 180 rpm for five days. The B2-DHA cells grown similarly but without potassium chromate were used as controls. Five sets of experiments were performed with three replicates along with controls. The cell free medium was collected by centrifugation (10000 rpm for 10 min) using a Sorvall rotor (Sorvall Super T21, USA). The cell free broth was filtered through 0.2 μm filter and acidified to pH 2.0 with 30% suprapure nitric acid (Merck, Germany). The acidified cell free broth was analyzed for total chromium content by using ICP-AES. [27]

The bioaccumulation of chromium was determined in the B2-DHA strain grown in 50 mL LB medium supplemented with 100 µg/mL of chromium and incubated at 37°C for five days.
After incubation, samples were collected and centrifuged at 10000 rpm for 10 min at Sorvall rotor (Sorvall Super T21, USA). The pellet was washed with 0.9 % saline twice and air-dried until a constant dry weight was achieved. The entire contents of the flask were harvested and the dry weight of the cells was recorded. Cells were digested with 30% suprapure nitric acid (Merck, Germany) according to ratio of 7.5 mL nitric acid per g dry biomass using microwave digestion. The samples were brought to a constant volume prior to determination of chromium contents. Measurement of chromium was also carried out similarly in the control experiments using media containing chromium but not exposed to B2-DHA and in media devoid of chromium but treated with B2-DHA. All analyses were carried out after filtration of the cell digest through 0.2 μm filter. The chromium present in the dried pellets was determined by the inductively coupled plasma mass spectroscopy (ICP -MS). [38]