## 3 Results
### Genome Description and Taxonomic Classification of Geobacillus sp. G4
\begin{table}
\begin{tabular}{c c c} \hline
**Isolated Strain** & **Type Reference Strains** & **ANIb (\%)** & **ANIm (\%)** \\ \hline _G. thermoleovorans_ KCTC3570 & 97.65 & 98.30 \\ _G. kaustophilus_ HTA426 & 97.19 & 97.91 \\ _G. kaustophilus_ NHRC102445 & 97.11 & 97.85 \\ _G. proteinijhilus_ 1017 & 93.15 & 94.06 \\ _Geobacillus_ sp. G4 & _G. thermolevulatus_ BGSC93A1 \\ _G. Jurassicus_ NBRC107829 & 90.57 & 91.52 \\ _Geobacillus vulcanit_ PSS1 & 90.41 & 91.54 \\ _G. stearothermophilus_ ATCC12980 & 88.93 & 90.65 \\ _G. uzenensis_ BGSC92A1 & 85.05 & 87.16 \\ _G. subterraneus_ KCTC3922 & 85.04 & 87.12 \\ _G. icigianus_ G1w11 & 84.64 & 87.28 \\ _G. thermoleutrificans_ DSM465 & 82.64 & 85.04 \\ \hline \end{tabular}
\end{table} 
Table 3: Average nucleotide identity (ANI) calculated for _Geobacillus_ sp. G4 using type strains of each species of the _Geobacillus_ genus, calculated on the JSpeciesWS platform (ANIb and ANIm). Information is available at the following link: ([[https://jspecies.ribohost.com/jspeciesws/](https://jspecies.ribohost.com/jspeciesws/)]([https://jspecies.ribohost.com/jspeciesws/](https://jspecies.ribohost.com/jspeciesws/)), accessed on 11 January 2025). In the dDDH analysis, the _Geobacillus_ sp. G4 strain showed dDDH values above 70% with _Geobacillus kaustoophilus_ MTA426 (79.1%) and _Geobacillus kaustoophilus_ NBRC102445 (78.5%) (Table 4). However, the _Geobacillus thermoleoverans_ KCTC3570 strain exhibited the highest similarity with _Geobacillus_ sp. G4, with a dDDH value of 83.2% and a GC content difference of only 0.31%, shows a high genomic proximity. This suggests that both organisms are closely related, meaning _Geobacillus_ sp. G4 could be a variant of _G. thermoleoverans_. Other species showed dDDH values below 70% and a larger GC content difference. These data indicate that, among the species compared, _Geobacillus thermoleoverans_ is the closest to _Geobacillus_ sp. G4 in terms of genomic similarity. The 165 rRNA gene sequence of the _Geobacillus_ sp. G4 strain showed 99.87% identity with _Geobacillus kaustoophilus_ strain BGSC 90A1 and 99.80% identity with _Geobacillus thermoleoverans_ strain LEH-1 (Supplementary Table S5). The _Geobacillus thermoleoverans_ BGSC 96A1 lineage also had 100% query coverage and 99.61% identity. Other _Geobacillus_ and _Bacillus_ strains showed identity percentages ranging from 99.08% to 99.87%, with query coverages ranging from 92% to 100%. In the phylogenetic tree constructed based on the 165 rRNA gene, the _Geobacillus_ strains are distributed across several clades without statistical support (Figure 3), indicating significant diversification within this genus. However, _Geobacillus_ sp. G4 is positioned within a clade supported by a bootstrap value of 71%, containing the strains _Geobacillus thermoleoverans_ KCTC3570, _Geobacillus kaustoophilus_ HTA426, _Geobacillus kaustoophilus_ NBRC102445, _Geobacillus proteinophilus_ 1017, and _Geobacillus vulcani_ PSS1. In the phylogenomic tree reconstructed using molecular markers extracted with AMPPHORA2, the strain _Geobacillus_ sp. G4 is grouped with _Geobacillus kaustoophilus_ HTA426 and _Geobacillus thermoleoverans_ KCTC3570, forming a clade supported by a bootstrap value of 95% (Supplementary Figure S1). In the phylogenomic tree reconstructed on the TYGS platform (Figure 4), the strain _Geobacillus_ sp. G4 is in the clade with the strain _Geobacillus thermoleoverans_ KCTC3570, supported by a bootstrap of 72%.