Isolation and Screening of Bacteria with Biotechnological Potential from Permafrost-Associated Soils of Northern Pakistan

Soil microorganisms from high-altitude and cold environments represent an important source of biotechnologically valuable enzymes and antimicrobial compounds. The present study investigated the bacterial diversity and selected biotechnological potential of soil microorganisms isolated from the northern areas of Pakistan, including soil collected from Babusar Top, an area associated with documented permafrost soils in the Babusar Valley, and Rainbow Bridge. Soil samples were subjected to serial dilution and culture-based isolation, followed by morphological, Gram-staining, biochemical, antimicrobial, and extracellular enzyme screening. Growth from the Babusar Top sample was observed at the 10⁻² dilution, from which four bacterial isolates (BS1, BS2, SB1 and SB2) were obtained, while two additional isolates (RB1 and RB2) were recovered from Rainbow Bridge. All six isolates were Gram-positive rods. Screening for cellulolytic activity showed growth of five isolates (SB2, BS1, BS2, RB1 and RB2; 83.3%) on carboxymethyl cellulose agar under the conditions tested. All six isolates (100%) produced clear zones on milk agar, indicating extracellular protease-mediated casein hydrolysis. Lipase screening showed growth of SB1, BS1, and RB2 (50%) accompanied by grainy precipitate-like appearances around colonies, although no clear hydrolysis zones were observed. All six isolates (100%) were positive for ammonification, demonstrated by a deep yellow color with Nessler's reagent. In antimicrobial screening, SB2 and RB1 (33.3%) produced inhibition against both Escherichia coli and Staphylococcus aureus, demonstrating activity against Gram-negative and Gram-positive test bacteria. These findings demonstrate that bacteria isolated from high-altitude northern Pakistani soils, including the documented permafrost-associated Babusar region, possess diverse enzymatic, metabolic, and antimicrobial properties with potential biotechnological applications.