Hydrogen Peroxide Assay

H₂O₂ levels were measured for mineral leachates and mineral suspensions reacted with E. coli growing in LB or MSA media using a method modified from Cohen⁴⁰. Aliquots were sampled over a 24 h period and centrifuged at 13,000 rpm for 2 min. Supernatants (500–20 μl) were transferred into micro centrifuge tubes with the addition of 50 mM EDTA. Buffer solution (1M KH₂PO₄, pH 4.2) was added to a final volume of 1400 μl. Then 50 μl of 5 mM leuco crystal violet was added followed by 50 μl of horseradish peroxidase type II (14.4 units/ml). Absorbance was measured with a spectrophotometer at 590 nm wavelength after 15 min for color development. Hydrogen peroxide standard curves ranging from 0.5–25 μM were prepared in the same manner.