## 4 Discussion  
Extremophiles are increasingly recognized as valuable sources of industrially relevant enzymes due to their ability to produce extremozymes that remain active under extreme environmental conditions. Many thermally adapted enzymes or thermozymes, such as thermostable amylases, proteases, cellulases, and lipases, used in diverse industrial applications have been extensively derived from thermophilic and other extremophilic microorganisms [6]. Owing to their remarkable resilience and efficiency, extremozymes have enormous economic potential in the biotechnology sector. Among the microbial extremozymes, thermally stable amylases are of significant interest in all starch-related industries [6, 37], while these enzymes account for nearly 25% of the world enzyme market [9]. As a result, thermozymes have received greater attention for their suitability in high-temperature industrial processes, where conventional enzymes often fail to perform optimally [19]. In the present study, three thermophilic bacterial strains were successfully isolated and molecularly characterized from the Nelumwewa geothermal spring in Sri Lanka. These isolates exhibited robust growth at 55 \({}^{\circ}\)C under slightly alkaline conditions (pH 7.5). However, among them, only NW2 isolate exhibited prominent extracellular amylase activity as well as extracellular protease activity, indicating it to be a promising candidate to produce thermostable industrial enzymes. Molecular characterization of NW2 isolate revealed that it shared 99.3% 165 rRNA gene sequence identity with _Thermaerobacillus caldiproteolyticus_ strain SF03, previously classified as _Anoxyobacillus caldiproteolyticus_ or _Geobacillus caldiproteolyticus_[38, 39], a well-documented thermophilic bacterium known for its thermostable enzyme production [40, 41]. Phylogenetic analysis grouped the three isolates into three distinct clusters. The NW2 isolate clustered with _T. caldiproteolyticus_ strains (Cluster A), indicating a close evolutionary relationship, while NW6 and NW3 were grouped into Clusters B and C, respectively, showing phylogenetic affiliation with _Bacillus licheniformis_ and _A. gonensis_. A recent study on bacterial diversity analysis of Mahapelessa geothermal spring in Sri Lanka has identified some bacterial species such as _Pannoinbactor_  
Figure 9: Detection of raw cassava starch hydrolysis by crude extracellular amylase of NW2 isolate under optimum conditions (60 \({}^{\circ}\)C and pH 8.0) using the iodine-starch test. Test tubes 1 to 5 illustrate the hydrolysis of cassava starch at concentrations of 0.5% (Tube 1), 1% (Tube 2), 2% (Tube 3), 3% (Tube 4), and 4% (Tube 5), respectively, demonstrating the enzyme’s capacity to degrade starch substrates. Tube 6 serves as a control, showing the reaction with 1% cassava starch without the addition of crude amylase enzyme. spp., _Gulbenkiania_ spp., _Pigmentipluga_ spp., _Enterobacter_ spp., _Klebsiella_ spp., and _Bacillus_ spp. [25]. However, it did not isolate any _Thermaerobacillus_ spp., _Anoxyobacillus_ spp., or _B. licheniformis_, underscoring the wide bacterial diversity across different geothermal springs in Sri Lanka. Under optimal growth conditions (55 \({}^{\circ}\)C and pH 7.5), NW2 isolate reached the stationary phase after about 10 h in NB medium, indicating its rapid growth characteristics. Some reports have suggested that bacterial amylase production is growth dependent [42; 43]. Moreover, NW2 extracellular amylase exhibited optimal activity at 60 \({}^{\circ}\)C and pH 8.0 which are higher than those of optimal bacterial growth. This is a common trait found in some thermostable enzymes due to their structural rigidity [44], suggesting evolutionary adaptation to the seasonal fluctuation of temperature and pH of geothermal springs. The optimal temperature and pH of the extracellular amylase activity of NW2 isolate are almost consistent with already reported amylase activity of some _Anoxyobacillus_ spp. [22; 23].