## 3 Results and Discussion
### Determination of MIC and Effects of Mn on Cellular Protein Expression  
The MIC was defined as the lowest metal concentration at which bacterial growth was not observed. The isolated strain was found to be viable in medium with a MnCl\({}_{2}\) concentration up to 800 mM. The MIC was therefore estimated to be 800 mM. The ability to tolerate a high concentration of Mn was established for strains that were isolated from Mn-rich environments. For example, _Marinomonas_ sp. S11-S-4 isolated from sediment collected from the Arctic Ocean was able to grow when the Mn\({}^{2+}\) concentration was up to 100 mM [33], while strains isolated from Sanindipur mines (Odisha, India) showed visible growth at a concentration up to 500 mM of Mn [1; 6]. Interestingly, _Acinetobacter_ sp. MSB 5 isolated from Sanindipur mines was also viable at even a 1000 mM concentration of MnCl\({}_{2}\)[5]. Meanwhile, _Bacillus thuringiensis_ HM7 isolated from Xiangtan Mn ore in China was able to grow at 31.78 mM [34]. The total protein content of the 2 De strain was found to be 29.97 mg mL\({}^{-1}\) for cells cultivated in the presence of 0.5 mM MnO\({}_{2}\), 28.30 mg mL\({}^{-1}\) when bacteria were cultivated in the presence of the soil, and 31.34 mg mL\({}^{-1}\) for control cells. To analyze the effect of a high level of Mn on the protein profiles of the isolated bacterial strain, SDS/PAGE of the whole cell lysate was carried out (Figure 2). Figure 1: Maximum likelihood-based phylogeny derived from 16S rRNA sequence data, representing the relative position of the 2 De strain among other _Pseudomonas_. All positions containing gaps or missing data were deleted. Bootstrap values are represented at the branching points. The bar represents 0.002 substitutions per site, accession number from GenBank for 2 De strain is shown in brackets. When the 2 De strain was cultivated in the absence of Mn, weakly visible bands in the range of 12 to 250 kDa were obtained (Figure 2a), while exposure of the strain both to the soil or MnO\({}_{2}\) influenced most of the protein synthesis, indicated by more intense staining (Figure 2b,c). Mn serves as a cofactor for bacterial enzymes involved in the removal of harmful free radicals produced during metabolism. However, high Mn accumulation exerts toxic effects on cells. Therefore, it is necessary for bacteria to regulate the Mn acquisition system [35]. They adjust to changing environmental conditions due to utilizing numerous transcriptional and biochemical regulators for uptake and export of Mn. These include intracellular accumulation, enhanced activity of efflux pumps, detoxifying enzymes, intracellular, and extracellular sequestration, and Mn exporter protein like MnTE, MntP, MntX, CtpC, and P-type ATPase [2; 4; 18; 19; 20]. These exporters are critical for proper Mn homeostasis and cell function for maintaining sufficient Mn levels. Their metal-binding capability may lead to its preferential association with Mn(II) or Mn(IV) particles [36]. On the other hand, the protein Alx, which has a mechanism of action that is not primarily through Mn export, can increase Mn levels and contribute to cell growth under high exposure to Mn [2]. The presence of Mn might also induce synthesis of oxide-associated proteins including metalloproteinases. This phenomenon was also observed in two bacterial cultures of Mn(II)-oxidizers: _Roseobacter_ sp. AzwK- 3b and _Erythbroacter_ sp. SD- 21 [36]. In turn, [PERSON] and [PERSON] discovered that chitin-associated proteins are involved in the removal of manganese from mine impacted waters through adsorption. These authors showed that the presence of proteins significantly increases the sorption capacity of the crab-shield particles [37]. Considering the increased intensity of protein bands of the 2 De strain after treatment with metallurgical waste or MnO\({}_{2}\), it seems very likely that some of these mechanisms are present in its cells. For _Acinetobacter_ sp.