## 3 Results
### Optimum Growth Temperature of the NW2 Isolate  
The NW2 isolate exhibited optimal growth characteristics at 55 \({}^{\circ}\)C (Figure 5). The lag phase of the growth curve lasted for about 5 h and then reached the exponential phase. The bacterial growth rates at exponential phases were determined by linear regression analysis (R\({}^{2}\) > 0.95). The bacterial growth reached the stationary phase after about 11 h, indicating rapid growth characteristics at its optimum temperature. The NW2 isolate exhibited suboptimal growth at all other temperatures tested (Figure 5). Moreover, the maximum growth rate of the NW2 isolate was exhibited at 55 \({}^{\circ}\)C (Figure 6). These results indicate that the NW2 isolate is a moderate thermophile. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Isolates** & **Accession Number 1** & **Closest Relative** & **Accession Number 3** & **Identity 4** \\ \hline \multirow{3}{*}{NW2} & \multirow{3}{*}{PV489057} & \multicolumn{2}{c}{_Thermaerobacillus_} & \multirow{3}{*}{NR\_115200.1} & \multirow{3}{*}{99.30\%} \\  & & & & & & \\  & & & & & & \\ \cline{1-1}  & & & & & & \\ \cline{1-1}  & & & & & & \\ \hline \multirow{3}{*}{NW3} & \multirow{3}{*}{PV489066} & \multicolumn{2}{c}{_Anoxybacillus gonensis_} & \multirow{3}{*}{NR\_025667.1} & \multirow{3}{*}{96.85\%} \\  & & & & & & \\ \cline{1-1}  & & & & & & \\ \cline{1-1}  & & & & & & \\ \hline \multirow{3}{*}{NW6} & \multirow{3}{*}{PV489038} & \multicolumn{2}{c}{_Bacillus licheniformis_} & \multirow{3}{*}{NR\_074923.1} & \multirow{3}{*}{99.71\%} \\  & & & & & & \\ \cline{1-1}  & & & & & & \\ \end{tabular} \({}^{\dagger}\) NCBI gene bank accession number of thermophilic bacteria isolated in this study. 2 The closest bacterial species with the highest percentage identity match available in the NCBI gene bank. 3 NCBI gene bank accession number of the closest bacterial species. 4 BLSTAR search identity of the closest bacterial species available online: [[https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)]([https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)) Blast.cgi (accessed on 5 May 2025).  
\end{table} 
Table 2: Molecular characterization of thermophilic bacterial isolates by 16S rRNA gene sequencing. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Bacterial Characteristics** & **NW2** & **1 * & **2 * & **3 * & **4 * & **5 * & **6 * & **7 * \\ \hline Gram Stain & + & + & NR & + & + & NR & NR & + \\ \hline Cell shape & Rod & Rod & Rod & Rod & Rod & Rod & Rod \\ \hline \multirow{3}{*}{Culture medium} & Nutrient broth & \multirow{3}{*}{Neutrin broth} & Nutrient broth & Nutrient broth & Nutrient broth & \begin{tabular}{c} Luira- \\ Bertani \\ broth \\ \end{tabular} & T5 & \begin{tabular}{c} Anaerobic \\ basal \\ medium \\ \end{tabular} \\ \hline \multirow{3}{*}{Source of isolation} & Thermal springs & Thermal springs & Thermal springs & Thermal springs & Thermal springs & Thermal springs & \begin{tabular}{c} Thermal \\ springs \\ \end{tabular} & \begin{tabular}{c} Thermal \\ springs \\ \end{tabular} & \begin{tabular}{c} Thermal \\ springs \\ \end{tabular} & \
\begin{tabular}{c} Thermal \\ springs \\ \end{tabular} &
\begin{tabular}{c} Thermal \\ springs \\ \end{tabular} \\ \hline \multirow{3}{*}{Source of isolation} & \multicolumn{2}{c}{* 1—Anoxybacillus mongoliensis MBT001 [23]; 2—Anoxybacillus sp. AH1 [22]; 3—Anoxybacillus sp. KP1 [33]; 4—Anoxybacillus amylolyticus [34]; 5—Anoxybacillus thermarum A4 [35]; 6—Anoxybacillus sp. YIM 342 [36]; 7—Anoxybacillus favithermus DSM 2641 [15].} \\ \end{tabular} \\ \cline{1-1}  & & & & & & & \\ \end{tabular}
\end{table} 
Table 1: _Cont._Figure 4: The phylogenetic analysis of thermophilic bacterial isolates using 16S rRNA gene sequences. The tree was constructed using the neighbor-joining method with MEGA 12 and visualized with modifications using tvBOT [30]. Figure 5: Growth curves of NW2 isolate at different temperatures from 40 \({}^{\circ}\)C to 65 \({}^{\circ}\)C in NB (pH 7.5).