# Acidic Neutralization by Indigenous Bacteria Isolated from Abandoned Mine Areas
## 2 Experimental Procedure
### Microcosm Acid Neutralization Test  
The acid neutralization potential was evaluated using the indigenous bacterial consortium's isolated from each soil sample. They were isolated by adding 5 g of a soil sample to 50 mL of NaCl solution (0.85% (_w/v_)) and mixed for 15 min at 250 rpm using a funnel shaker (MMV-1000W, EYELA, Tokyo, Japan). Subsequently, 2 mL of the extract was transferred to 100 mL DifcoTM Nutrient Broth (NB, Franklin Lakes, NJ, USA) of pH 7.0, incubated at 30 \({}^{\circ}\)C, and shaken at 160 rpm for 2 days. The bacterial consortium was cultivated in NB medium until the optical density at 600 nm (OD600) reached up to 1.10 \(\pm\) 0.05, and they were then harvested by centrifugation at 14,000\(\times\)_g_ for 5 min followed by washing twice with a sterile 0.85% (_w/v_) NaCl solution. The bacterial cells were then transferred to 500 mL Erlenmeyer flasks containing 300 mL of NB medium, which was acidified to pH 4, 4.5, and 5 by adding a 4 M sulfuric acid (H2 SO4) and 4 M sodium hydroxide (NaOH) solution.  
Figure 1: Sampling sites of (**a**) Y1, (**b**) C1, and (**c**) C2, respectively.  
They were incubated for 6 days at 30 \({}^{\circ}\)C while being agitated continuously at 160 rpm in a shaking incubator (VS-8480 SF, Vision Scientific Co., Ltd., Daejeon, Korea). At the same time, the control was set up in the same manner without adjusting the pH using an acidic or alkaline reagent. The microbial biomass, dehydrogenase activity, pH, and ammonium ion (NH\({}_{4}\)\({}^{+}\)) concentration were monitored daily during the acid neutralization process.  
The OD\({}_{600}\) of the microbial biomass was measured using a UV-Vis spectrometer (UV-3300, Humans, Korea), while the dehydrogenase activity was determined using an INT assay [44]. For the INT assay, 0.5 mL of a bacterial suspension was centrifuged at 10,000 \(\times\)\(g\) for 5 min, after which the supernatant was decanted. A 0.5 mL aliquot of 0.2% (\(\omega\)/\(v\)) INT (iodonitrotetrazolium chloride, TCI, Tokyo, Japan) solution (in 200 mM phosphate buffer, pH 7.6) and 0.1 mL of 1% (\(w\)/\(v\)) sucrose (Daejung Chemicals, Korea) solution were added to the bacterial cell pellet. After incubation at 30 \({}^{\circ}\)C for 30 min, 50 \(\upmu\)L of HCI (35%, Samchun Chemicals, Seoul, Korea) and 1 mL of 1,4-dioxane (Sigma, Saint Louis, MI, USA) were added to block the enzymatic activity and simultaneously extract the INT formazan produced. The dehydrogenase activity was determined by the absorbance at 481 nm.  
The aliquot was centrifuged at 1000\(\times\)\(g\) for 10 min, and the resulting supernatant was used to determine the pH and NH\({}_{4}\)\({}^{+}\) concentration. In this study, pH was measured using a pH meter (BP3001, Trans Instrument, Petro Centre, Singapore), while NH\({}_{4}\)\({}^{+}\) concentration was determined using the Nessler reagent colorimetric method ([PERSON] et al., 2009). For that, the 50-fold diluted supernatant was reacted with the Nessler reagent (HACH, Ames, IA, USA), from which its absorbance was measured comparably at 425 nm against the reagent blank. The observed absorbance was finally converted to the NH\({}_{4}\)\({}^{+}\) concentration (mM) over a calibration curve drawn by NH\({}_{4}\)Cl.