# Acidic Neutralization by Indigenous Bacteria Isolated from Abandoned Mine Areas
## 3 Results and Discussion
### Acid Neutralization by the Isolated Bacterial Consortia  
The isolated consortia of indigenous bacteria could survive above pH 4.5 and effectively neutralize the acidity (Figure 3), irrespective of the original soil conditions. At an initial pH of 5, regardless of the type of microbial community, bacterial growth started immediately after inoculation with concomitant acid neutralization. In contrast, the bacterial strains proliferated in the initial pH 4.5 after some acclimation to the environmental stress (i.e., low pH). The growth of microorganisms and the increase in pH were accelerated after 2 h in Y1 and C1, whereas the C2 bacterial consortium reached equivalent levels after 6 h. Hence, the C2 consortium has slower growth because it has a longer acclimation time to acidic conditions than the other consortia. Subsequently, they grew rapidly after adapting to environmental changes, so the acidity was neutralized concomitantly with the proliferation of microorganisms and enzymatic activity (i.e., dehydrogenase activity). Complete neutralization from an initial pH of 4.5 and 5 took 24 h and 8 h for Y1, respectively. In the case of C1, neutralization was completed within 12 h and 6 h at an initial pH of 4.5 and 5, respectively. Similarly, in the case of C2, 12 h and 6 h were also needed to neutralize an initial pH of 4.5 and 5, respectively, although it took a longer acclimation time than C1 soil in the beginning. This suggests that all three indigenous bacterial consortia successfully neutralized the acidic medium to pH 7 or higher within 24 h. In addition, as incubation time was increased, the pH increase rate gradually decreased as the bacterial growth was slowed down, as shown in Figure 3. It did not exceed pH 8.5 while maintaining a neutral pH range. It was also found that pH no longer increased after 14 days of stabilization. This neutralization pattern has been consistently confirmed in our previous studies [28; 46]. Figure 2: Microbial community structures at the genus level of bacterial consortia isolated from (**a**) Y1 soil sample and (**b**) C1 and C2 soil samples. In this regard, in acidic environments, some microorganisms consume excess protons to maintain pH homeostasis for their survival in acidic environments [47]. Specifically, in these acid-resistant systems, microorganisms consume protons from nitrogenous compounds such as urea and amino acids to produce alkaline substances (e.g., NH4\({}^{+}\)) [48]. In this study, the NB medium is composed mainly of peptones and beef extracts that can supply abundant nitrogenous compounds that contribute to ammonia production [49]. Consequently, the isolated bacterial consortia can neutralize acidity by ammonium-associated metabolism. Therefore, acidified soil could be ameliorated using indigenous microorganisms through the introduction of appropriate nitrogenous substances such as nitrogen fertilizer and biochar [34; 35; 50] as an alternative nutrient medium. Figure 3: Acid-neutralizing ability of bacterial consortium of **(a)** Y1, **(b)** C1, and **(c)** C2 in the acidic NB medium for six days under the different pH conditions (i.e., pH 4, 4.5, 5, and control). Each column exhibits the temporal variation in pH, optical density at 600 nm (OD\({}_{600}\)), dehydrogenase activity as of absorbance at 481 nm (A\({}_{481}\)), and NH4\({}^{+}\) production yield, respectively. The yield of NH4\({}^{+}\) production also increased continuously except for the initial pH 4, suggesting that the microbial metabolism may be due to NH4\({}^{+}\) production during acid neutralization. Indeed, a strong positive correlation was established between the hydroxyl ion (OH\({}^{-}\)) concentration and the amount of NH4\({}^{+}\) produced with a correlation coefficient (R) of 0.922 (\(p<\) 0.05, Figure 4), regardless of the types of bacterial consortium. That is, the NH4\({}^{+}\) concentration increased with increasing pH, indicating that NH4\({}^{+}\) generation is closely related to acid neutralization.