Icap rq icp ms

Total metals were measured
following acidification with 2% v/v nitric acid using inductively
coupled plasma mass spectroscopy (iCAP RQ ICP-MS; Thermo Fisher Scientific,
Waltham, WA). Phosphate was measured using a 4500 HACH spectrophotometer
with PhosVer3 ascorbic acid (Method 8048, HACH, Loveland, CO). Total
chlorine and total ammonia were monitored in influent water using
a 4500 HACH spectrophotometer with DPD (Method 8167, HACH) and salicylate
(Method 8155, HACH) and were confirmed to be low (<0.05 mg/L total
chlorine and <0.06 mg/L NH3-N).

The total amounts of Mn²⁺, Fe, and Zn²⁺ were quantified from 5-ml cultures. Cells were grown overnight in LB broth and then inoculated in LBK pH 6.8, and LBK pH 8.4 media supplemented with 1 mM IPTG and appropriate concentration of ampicillin respectively. After growth to the mid-log phase at 37 °C, cells were harvested using centrifugation at 4000g for 10 minutes. Cell pellets were washed with 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) pH 7.5, containing 2 mM EDTA and then washed twice with 10 mM HEPES as described in (Zeinert et al., 2018 (link)). Cell pellets were dried for 1 hour in a centrifuge evaporator. Dried cell pellets were solubilized in 400 μl of 30% (v/v) HNO₃ and incubated at 95 °C for 10 min. Then samples were centrifuged at 20,000g for 5 minutes. Samples were prepared for inductively coupled plasma mass spectrometry (ICP-MS) by diluting 300 μl of supernatant of lysed cells into 2.7 ml of 2.5% (v/v) HNO₃ and run on an iCAP RQ ICP-MS (Thermo Scientific). Metal concentrations are presented as intracellular levels after correction for mean cell volume determined from total protein content (Martin et al., 2015 (link)). The data obtained were presented from three repeats of the experiment.