Hpr 1000 6m six position high pressure reactor

AX2 cells were grown for 24 h in M1 ± Fe medium or for 480 h in M2 ± Zn or M3 ± Cu media. A total of 10⁸ cells were washed five times in Soerensen phosphate buffer and pelleted in Eppendorf tubes. Digestion was performed by adding 1 ml of concentrated HNO₃ (70%) to each sample. After complete dissolution, samples were further digested by applying microwave heating (Milestone MicroSYNTH, Microwave labstation equipped with an optical fiber temperature control and HPR-1000/6M six position high-pressure reactor, Bergamo, Italy). After digestion, the volume of each sample was brought to 3 ml with ultrapure water, filtered with 0.45 mm filter and analyzed by ICP-MS, using a Thermo Scientific ELEMENT 2 ICP-MS (Finnigan, Rodano, Italy) (Fiorito et al., 2012 (link)). The same analysis was also performed on 800 μl of M2 or M3 medium. The quantification was obtained through a calibration curve measured by using six Fe/Cu/Zn absorption standard solutions (Sigma-Aldrich) in the range 0.0025–0.3 μg/ml. Samples with metals concentrations higher than the upper limit were diluted opportunely. Sample digestion and metal quantifications were carried out by the facility of the Molecular Imaging Center (Department of Chemistry, University of Torino, Italy).