Measurement of intracellular copper content.

To measure the copper in the mycelia, microwave digestion generation atomic fluorescence spectrometry was performed as described for mycelia by Krakowska et al. (45). Liquid-cultivated mycelia were separated from the fermentation broth at 7 days, washed in distilled water to remove any nonspecifically bound copper, and oven-dried for 2 days at 60°C until reaching a constant weight before determination of the dry weight. The oven-dried samples were ground to a powder for analysis. The dried samples (200 mg) were digested with 8 ml of HNO₃ and 4 ml of hydrogen peroxide using microwave digestion (Milestone Ethos T). The digested sample was carefully transferred to a quartz crucible, and excess reagents were evaporated at 60°C for 45 min. Digestion of two replicates of each sample was performed. The volume of each sample was adjusted to 10 ml with doubly deionized water, and quantitative analysis of the copper in the resulting biomass was performed using the atomic absorption spectrophotometry (AAS) method by using inductively coupled plasma-optical emission spectroscopy (ICP-OES; PerkinElmer Optima 2100DV). The parameters used for the flame AAS method were as follows: type of flame/fuel, acetylene/air; wavelength, 324.8 nm; width of slit, 0.7 nm; linear range, 4.0 mg/liter; measurement time; 2 × 2 s; and curve-fitting method, linear regression. The reagent blank and analytical duplicates were used where appropriate to ensure accuracy and precision in the analysis. A range of concentrations of copper was analyzed using ICP-OES to generate a standard curve for copper.