NIGHAT MARYAM2025-11-212025-11-212022https://escholar.umt.edu.pk/handle/123456789/11289The main purpose of this study was to the isolation and characterization of thermophilic bacteria from water collected from Khurkondooh hot spring located in northern area of Pakistan. Water samples were serially diluted and plated on nutrient agar to isolate specific colonies using streak plate technique at 50℃. Three bacterial species were isolated which were encoded as Ngt01, Ngt02 and Ngt03. These isolates were found to be thermophilic as their growth was observed from 50 to 70℃. The bacteria were differentiated morphologically on the basis of color and texture on nutrient agar plates. Grams staining and biochemical tests such as indole, motility, citrate utilization, TSI, Methyl Red (MR) and Voges-Proskauer (VP) tests were used that identified the isolates as Bacillus spp.. These thermophilic bacilli were tested for their ability to produce alpha-amylase, lipase, and catalase and further enzyme activity assay was performed at different temperatures. All the enzyme were thermostable and showed good activities at a temperature range of 57ºC to 97℃ but with less incubation time at higher temperature. Enzyme activity increased when temperature was increased with different concentration of metal ions. Maximum enzyme activity observed when isolates Ngt01, Ngt02 and Ngt03 was incubated at 97 ºC with least incubation time. While all three isolates without metal taken 1-hour incubation time to showed enzyme activity. Two of the isolates (i.e. Ngt01 and Ngt03) were further identified as Anoxybacillus spp. through 16S rRNA gene sequencing and performing phylogenetic tree analysis. Hot spring water could be a good source of novel thermophilic species with different thermophilic enzymes of industrial applicationsenThesis