# Acidic Neutralization by Indigenous Bacteria Isolated from Abandoned Mine Areas
## 2 Experimental Procedure  
### Soil Sampling and Characterization  
Acidic soil (Y1) or heavy metal contaminated soil samples (C1 and C2) were collected near an abandoned mine area located in Yong-in Province, Gyeonggi-do (37\({}^{\circ}\)13\({}^{\prime}\)39.34\({}^{\prime\prime}\) N, 127\({}^{\circ}\)07\({}^{\prime}\)48.08\({}^{\prime\prime}\) E, Figure 1a) or Cheonan Province, Chungcheongnam-do, South Korea (36\({}^{\circ}\)54\({}^{\prime}\)33.5\({}^{\prime\prime}\) N, 127\({}^{\circ}\)14\({}^{\prime}\)59.7\({}^{\prime\prime}\) E for C1, Figure 1b; 36\({}^{\circ}\)54\({}^{\prime}\)00.8\({}^{\prime\prime}\) N, 127\({}^{\circ}\)15\({}^{\prime}\)33.8\({}^{\prime\prime}\) E for C2, Figure 1c), respectively. The soil samples were taken from five locations in a sampling point and mixed into a single plastic bag. Subsequently, they were air-dried at room temperature and filtered through a 2 mm standard sieve. Soil samples were characterized according to standard analytical protocols. The pH was measured using a pH meter (BP3001, Trans Instrument, Singapore) after mixing 5 g of soil and 25 mL of distilled water followed by leaving it for an hour. The moisture content was determined by weighing the samples before and after drying at 105 \({}^{\circ}\)C until a constant weight was reached. Organic content was determined using the Tyurin titrimetric method [38; 39]. For that, 0.5 g of soil sample was oxidized with 10 mL of potassium dichromatic solution (K\({}_{2}\)Cr\({}_{2}\)O\({}_{7}\), 0.4 N) by heating on a hot plate. After boiling for 5 min, it was cooled down to room temperature, and 100 mL of distilled water, 5 mL of phosphoric acid (85%), and 5 drops of diphenylamine indicator were added to the digested solution. Excess dichromate was measured by titration with a solution of ammonium ferrous sulfate ((NH\({}_{4}\))\({}_{2}\)Fe(SO\({}_{4}\))\({}_{2}\)-6H\({}_{2}\)O, 0.2 N). It was assumed that the dichromate reduced during the oxidation process was equal to the organic carbon (C) present in the sample. The percentage of organic content was calculated by multiplying the organic carbon by the Van Bemmelen factor of 1.724 [40]. Meanwhile, the ammonium nitrogen (NH4\({}^{+}\)-N) content was measured using the Kjeldahl digestion method [41]. To extract the NH4\({}^{+}\)-N from the soil, 10 g of soil sample was mixed with 50 mL of potassium chloride solution (KCl, 2 M) for 30 min and filtered using a qualitative filter paper (No. 2, WhatmanTM, Maidstone, UK). The filtrate was heated through the Kjeldahl distillation device with the addition of 0.2 g of magnesium oxide (MgO), and the vaporous product was condensed in 10 mL of boric acid solution (H3 BO3, 2% (_w/v_)). After that, NH4\({}^{+}\)-N content was determined by titration by H2 SO4. Particle size distribution analysis was carried out according to the Hydrometer method [42; 43]. For that, an air-dried soil sample was soaked in 100 mL of a dispersing agent, a sodium hexametaphosphate solution ((Na(PO3)6), 5% (_w/v_)) for 18 h. Then, it was vigorously mixed for 5 min and transferred to the measuring cylinder. The final volume was adjusted to 1 L by compensating with distilled water, after which it was homogenized by repeatedly inverting it in a cylinder with a cap. Thereafter, the clay fraction was determined by measuring the specific gravity immediately and after 10 h using a hydrometer (DK-713, measurement range 0.995-1.050, Daekwang Inc., Seoul, Korea). The soil suspension was sieved using a standard sieve with a mesh size of 270 mm, and the remainder was weighed to estimate the sand fraction after drying for 24 h. The silt fraction was finally calculated by excluding the fractions of sand and clay. The concentrations of heavy metals (arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn)) in C1 and C2 soil samples were determined using an inductively coupled plasma-optical emission spectroscopy (ICP-OES, Optima 7300 DV, Perkin Elmer, Waltham, MA, USA) according to the standard methods [41].