Mineral weathering experiment

We followed the methods of Sun et al. (2010). K and Fe-limited liquid medium (KFM) (containing 1% sucrose, 0.1% (NH₄)₂SO₄, 0.05% Na₂HPO₄, 0.05% MgSO₄, 0.01% NaCl, 0.02% yeast extract, 0.2% biotite powders, pH 7.2) was used to test mineral weathering potentials of the isolates. Unweathered crystal biotite was crushed in a jaw crusher ground and passed through sieves having a 0.149-mm mesh size. The rock powders were ultrasonically cleaned in deionized water to remove fine particles and were leached with 0.1 N HCl to remove exchangeable bases, washed with distilled water until the supernatant became clear. The elemental composition of the rock is as follows: SiO₂ 39.99%, Al₂O₃ 18.98%, K₂O 9.12%, Na₂O 0.28%, Fe₂O₃ 14.75%, CaO 0.07%, and MgO 0.24%. To prepare the inoculum, bacterial strains were initially grown in sterilized improved Gibbson medium (sterilized at 121 °C for 30 min) at 28 °C for 18–20 h in a rotary shaker at 150 rpm, and harvested by centrifugation at 3000 × g for 10 min. Inoculum was washed two times in sterile distilled water, and cell pellets were then resuspended in saline solution (0.85% NaCl) to a final concentration of 10⁸ cells mL⁻¹ before the dissolution experiments were started. Triplicate 250-mL polycarbonate Erlenmeyer culture flasks with vented caps (0.3 µm PTFE membranes) containing 50 mL of sterilized KFM were each inoculated with 2.5 mL of a bacterial suspension (Inoculum). The flasks were incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. Controls with rock but no bacterial cells to monitor the range of abiotic dissolution were treated in the same manner. The weathering of the rock in the presence of bacteria was monitored at 7 days of incubation. Samples for chemical analysis were then filtered through a 5-μm Millipore filter (to retain the rock, but not the bacterial cells); 20 mL of the filtrate from each flask were centrifuged at 10,000 rpm for 10 min to remove cells of suspension. 5 mL of the supernatant were collected for pH determination and another 5 ml of the supernatant were acidified with HNO₃ (final concentration 2% v/v) to avoid precipitation of dissolved chemical species and analyzed for Si, Fe, and K contents by ICP-AES (Atomic Emission Spectrometer).