Hfx. mediterranei Biomass Elemental Analysis

Once the cultures reached the stationary phase, they were centrifuged at 720 rpm for 20 min at a temperature of 4 °C to try to preserve the integrity of the samples. After centrifugation, the pellet was washed twice with salt water at 15%, which consisted of resuspending the pellet in salt water at 15%, centrifuging under the same conditions as previously described, and removing the supernatant. After, samples were dried in an oven until the complete elimination of moisture to obtain the dry mass. Then, they were digested by the addition of 1.5 g 69% HNO₃ in a sand bath at a temperature of approximately 70 °C for 6 h for complete digestion of the samples. If lower temperatures than indicated are used, the digestion is not completely carried out, thus affecting the accuracy and precision of the results. Next, digests were made up to 20 g for a final HNO₃ concentration of 5% w/w. Finally, aliquots of the latter solution were diluted 1:10 and 1:100. Nitric acid concentration was kept constant for these solutions at 5% w/w.
According to Hfx. mediterranei biomass composition, 1:100 and undigested samples were, respectively, employed for the analysis of major (Ca, K, Mg and Na) and trace elements (Fe, Mn, and Zn) by ICP-OES. To improve accuracy and minimize matrix effects, prior to ICP-OES determinations, 1:100 and undiluted samples digests were spiked with Sc as the internal standard (final concentration 0.5 mg L). Determination of ultra-trace elements by ICP-MS was carried out using 1:10 and undiluted samples. The former solution was used for measuring Al, Ba, Cu, Ni and Sr, while the latter allowed Co determinations. Both sample solutions were spiked with Ru as the internal standard (final concentration of 10 µg L⁻¹) [51 (link),52 (link)].