# Acidic Neutralization by Indigenous Bacteria Isolated from Abandoned Mine Areas
## 3 Results and Discussion
### Acid-Neutralizing Mechanism of Bacterial Strains  
The microbial metabolism for acid neutralization could be predicted based on the biochemical properties of each bacterial strain. The primary metabolism is considered as an NH4\({}^{+}\) production in utilizing nitrogenous compounds under acidic conditions. This microbial NH4\({}^{+}\) production occurs mainly by deamination and decarboxylation through the formation of alkaline products and the consumption of protons [24,48]. It also serves as a major mechanism of bacterial resistance to acidification.  
Several metabolic pathways are involved in NH4\({}^{+}\) production and H\({}^{+}\) consumption, including urease, arginine deaminase, and decarboxylase. First, urea hydrolysis is a mechanism that converts urea to ammonia (NH3) and carbon dioxide (CO2), which can be found in a wide range of microorganisms as one of the most representative ammonia production mechanisms [51,52]. The ammonia produced consumes protons to become ammonium ions (NH4\({}^{+}\)) that neutralize protons. In this study, five bacterial strains (YNA-3, CNA-3, CNA-4, CNB-2, and CNB-5) showed a change in substrate color when expressing urease in [PERSON]'s Urea Agar assay, as shown in Table 3.  
Second, the arginine deaminase (or arginine dihydrolase) system can produce citrulina and ammonia through the breakdown of arginine. The resulting citrulina is converted to ornithine and carbamoyl phosphate by ornithine carbamoyltransferase (OTC), and finally mineralized to NH3 and CO2 by carbamate kinase (CK) [53,54]. The results of the API 20E/20 NE test showed that YNA-3, CNA-1, CNB-2, and CNB-4 strains positively activated arginine dihydrolase (Table 3), which could neutralize acids via the arginine deaminase (ADI) pathway.  
Third, the decarboxylase of some amino acids, such as lysine, ornithine, and histidine, functions at low pH under extracellular conditions and contributes to pH maintenance by importing and consuming protons through decarboxylation [27,55,56]. Decarboxylation has  
Figure 6: Comparison of the acid neutralization properties of the final pH (bar graph), OD600 (dashed line), and normalized concentration of ammonium ion (NH4\({}^{+}\)) (solid line) for single bacterial strains after 48 h of the acid-neutralization test. Control was an acidified medium without bacterial inoculation.  
been reported to produce ammonia and the formation of biogenic amines [49]. As a result of biochemical analysis, the three bacterial strains of YNA-2, CNA-5, and CNB-1 belonging to _Serratia_ species can generally express lysine decarboxylase and ornithine decarboxylase, whereas CNA-2 (_Salmonella_ sp.) can express only lysine decarboxylase (Table 3). In addition, the glutamate decarboxylase (GAD) system is also important for acid resistance to survive in acidic environments [57]. GAD catalyzes the \(\alpha\)-decarboxylation reaction of glutamate to produce \(\gamma\)-aminobutyric acid and carbon dioxide, which ultimately consumes protons [58]. Among the GAD systems, the glutaminase YbaS/GlSA converts glutamine to glutamate and ammonia [59]. It can be expressed in various bacteria [59; 60], including _Escherichia coli_, _Pseudomonas_ spp., and _Serratia_ spp. Therefore, it implied that the selected bacterial strains belonging to the _Pseudomonas_ spp. and _Serratia_ spp. can express glutaminase.  
Overall, the bacterial strains isolated from abandoned mine soils can neutralize the acidity of the medium by generating ammonia from nitrogen compounds through several specific metabolisms, such as urea hydrolysis, arginine deaminase system, and amino acid decarboxylation.