Elan drce 9000

A previous study (Kuffner et al., 2010 (link)) showed that bacterial exudates of Microbacterium spp. cultures mobilize metals in contaminated soils, particularly when collected during the stationary phase. Therefore, bacteria were grown in 50 ml of Landy medium at 27°C and 200 rpm until the stationary phase. Cells were removed by centrifugation (4,700 rpm, 20 min, 4°C) and the supernatants were filtrated through 0.2 μm filters (Millipore). Filtrates were stored at −20°C and pH values were measured prior starting the extraction assay. For each strain two cultures were prepared and from each culture three aliquots were analyzed as follows. Five ml of filtrates were shaken with 1 g of Zn, Cd, and Pb-contaminated soil for 2 h at room temperature. Soil characteristics can be found in Supplementary Table S11. As negative control, 1 g of soil was shaken with 5 ml of sterile Landy medium, which was adjusted to pH values between 6.5 and 3.7 to mimic the medium acidification caused by bacterial growth in some cultures. Soil particles were removed by centrifugation (7,000 rpm, 5 min), the supernatants were filtered through 0.45 μm filters and acidified to 2% HNO₃ (p.a. grade, Sigma-Aldrich, Vienna, Austria). Concentration of Zn, Cd, Pb, Fe, Cu, and Mn were quantified by inductively coupled plasma mass spectrometry (Elan DRCe 9000, PerkinElmer).

Approximately 200 mg of dry, ground plant material was used for acid digestion. The digestion matrix contained 5 ml HNO₃, 1 ml H₂O₂ and one drop 1-Octanol. For biomass < 100 mg half of acid and peroxide was used. The digestions were performed using an open digestion unit (Velp Scientifica). Digests were filled up to a volume of 50 ml/25 ml with HQ-water resulting in ~6.5% HNO₃. If dry weight was lower than 50 mg, digestion was performed with a Multiwave 3000 (64 MG5 rotor) using only 0.5 ml HNO₃ (sub-boiled) and 0.1 ml H₂O₂. After the digestion, micro-digests were acidified with hydrofluoric acid to a final concentration of 0.01% in order to prevent polymerization or precipitation of W.
For measurements of nutrient and W content in nodules, biomass was not always sufficient to perform digestion for individual replicates. If present, nodules of treatments with low biomass (plants supplied with KNO₃ and those treated with W) were pooled. Results only represent estimates and are thus not extensively discussed (Table S2). Micronutrients and W concentrations in the digests were measured with inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer, Elan DRCe 9000, Waltham, MA, USA); macronutrients were analyzed by ICP-OES Optima 3000 XL (Perkin Elmer).