Fe 2

The growth profile of strain A7 was checked to tolerate an increasing concentration of aluminum (AlCl₃) (0–4 mM) and other metal compounds using the Single Plate Serial Dilution Spotting (SP-SDS) method on modified M9 agar plates [80 (link)]. The liquid culture was in the M9 broth supplemented with 0.1% proteases peptone. Similarly, the effect of Fe (III) from 0–5 mM, Fe (II) from 0–3 mM, NaCl (0–8%) (Sigma Aldrich, St. Louis, MO, USA) [81 (link)], pH (1–14) and temperature (4–50 °C) was checked in three biological replicates [82 (link),83 (link)]. Freshly grown bacterial cells (0.1 OD₆₀₀) were serially diluted from 10⁻¹ to 10⁻⁶, and 3 µL from each dilution was inoculated into the demarcated areas of SP-SDP and in a 10⁻¹ dilution in a 50 mL broth. The Petri dishes were incubated for 18–48 h, and the flask was held in an orbital shaker at 160 rpm at 30 ± 2 °C for 48 h. The optical density of the cultures was taken at 600 nm (PerkinElmer UV/VIS spectrometer Lamda 25, USA). The relative growth (RG) was calculated using the formula [84 (link)]. Relative Growth (%) = A_t/A_c ∗ 100, where A_c = optical density of the average of three biological replicates of controls, and A_t = optical density of average three biological replicates of the treatments. Bacterial 20% and 50% growth inhibitory concentration (IC) IC₂₀ and IC₅₀ values were calculated using the dose–response data.

All chemicals including lead(II) nitrate, cadmium(II) sulphate, mercury(II) nitrate, atomic absorption spectrometric standard of metals (Ni(II), Zn(II), Cu(II), As(III), Hg(I), Se(II), and Fe(II)), and nitric acid (HNO₃, ultraclean) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) unless noted otherwise. Stock solutions were prepared in ACS-purity water immediately prior to use.

SPIO were prepared as described previously [15 (link)]. In brief, 0.1M Fe(III) (Sigma) and 0.2M Fe(II) (Sigma) aqueous solutions were prepared by dissolving FeCl₃ and FeCl₂, respectively. For production of Fe₃O₄ nanoparticles, 4 mL of the Fe(III) and 1 mL of the Fe(II) solutions were mixed at room temperature, and the pH was adjusted to 11 using 5M NaOH. After exposure to a magnet, the precipitates were washed with deionized water after which 3 g of organic acid was added to achieve complete coating of the particle surface. The precipitates were redispersed in deionized water after excess adherents were removed by centrifugation. Before labeling, 50 μg/mL of SPIO was coated by mixing 0.75 μg/mL poly-L-lysine (Sigma) into the culture medium at room temperature for 1 h to facilitate their endocytosis. MSCs were then seeded in a 6-well plate at a density of 4 × 10⁴/ well and cultured for 24 h. The MSCs were incubated in SPIO-containing medium for 24 h and thoroughly washed with PBS.