# Acidic Neutralization by Indigenous Bacteria Isolated from Abandoned Mine Areas
## 3 Results and Discussion
### Acid Neutralization Properties of the Single Bacterial Strains  
The potential of acid-neutralization of single strains was evaluated by inoculating them individually in an acid medium adjusted to pH 4.5. Of these, 11 bacterial strains increased the pH by an average of 41% within 48 h compared to the initial pH (Figure 6), suggesting that they have acid resistance and neutralization properties. It showed that the YNA-2, CNA-1, CNA-3, and CNB-1 strains had a high neutralizing ability, consistent with the results of previous experiments. On the other hand, CNA-5 and CNB-4, which showed low growth rates in the heterogeneous consortium, had higher growth rates in the single strain culture to accomplish acid neutralization. Conversely, despite both strains being able to neutralize the acidity, they remained vulnerable to nutritional competition for growth over other isolates in the consortium. YNA-3, CNA-2, CNA-4, CNB-2, and CNB-5 strains also have high acid-neutralizing properties under acidic conditions in pH 4.5. In contrast, the growth rates of the three bacterial strains, YNA-1, YNA-4, and CNB-3, were very slow at pH 4.5, which was consistent with the previous results, showing that they are barely found at pH > 5.  
Figure 4: Relationship between the concentrations of hydroxyl ion (OH\({}^{-}\)) and ammonium ion (NH\({}_{4}\)\({}^{+}\)) during acid neutralization using the isolated bacterial consortia.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Isolates** & **Species** & **Identities** & **Gaps** \\ \hline Y1 & YNA-1 & _Bacillus toyonensis_ & 1471/1473 (99\%) & 1/1473 (0\%) \\  & YNA-2 & _Serratia grimesii_ & 1459/1461 (99\%) & 1/1461 (0\%) \\  & YNA-3 & _Pseudomonas protegens_ & 1465/1465 (100\%) & 0/1465 (0\%) \\  & YNA-4 & _Atlanticbacter hermannii_ & 1441/1466 (98\%) & 1/1466 (0\%) \\ \hline C1 & CNA-1 & _Pseudescherichiavulneris_ & 1447/1457 (99\%) & 0/1457 (0\%) \\  & CNA-2 & _Salmonella enterica_ subsp. & 1418/1441 (98\%) & 0/1441 (0\%) \\  & CNA-3 & _Citrobacter youngae_ & 1459/1490 (98\%) & 0/1490 (0\%) \\  & CNA-4 & _Pseudomonas picoglossicida_ & 1470/1474 (99\%) & 4/1474 (0\%) \\  & CNA-5 & _Serratia liquefaciens_ & 1479/1484 (99\%) & 4/1484 (0\%) \\ \hline C2 & CNB-1 & _Serratia grimesii_ & 1453/1455 (99\%) & 0/1455 (0\%) \\  & CNB-2 & _Pseudomonas protegens_ & 1443/1443 (100\%) & 0/1443 (0\%) \\  & CNB-3 & _Aeromonas latavinensis_ & 1451/1461 (99\%) & 2/1461 (0\%) \\  & CNB-4 & _Leclercia alexroxylata_ & 1461/1484 (98\%) & 3/1484 (0\%) \\  & CNB-5 & _Stentrophomonas maltophilia_ & 1475/1493 (99\%) & 2/1493 (0\%) \\ \hline \hline \end{tabular}
\end{table}
Table 2: Identification of acid-neutralizing bacterial strains in the Y1, C1, and C2 consortia by 16S, rRNA gene sequencing analysis.  
Figure 5: Distribution of the bacterial strains (bar type) and variation of population density (marked line graph) of the (**a**) Y1, (**b**) C1, and (**c**) C2 bacterial consortium with an incubation time of acidic neutralization process at an initial pH of 4, 4.5, and 5, respectively.  
Individual bacterial strains of each consortium grew competitively according to the initial conditions and degree of acid neutralization as they had different acclimation times to acidic conditions. In addition, these changes in the microbial community made it possible to identify which types of microorganisms with large contributions to acid neutralization could be taken. Nevertheless, detailed mechanisms of bacterial acid neutralization could not be predicted precisely because of the complex interactions between microorganisms in the consortium. In this study, additional experiments were carried out to determine the neutralization properties of each bacterial strain, which has been discussed in the following sections.