Physiological, biochemical, and molecular characterization of Bacillus isolates from soils using selective enrichment strategies.

Soil ecosystems represent an important reservoir of microbial diversity with high potential for biotechnological exploitation. Among soil microorganisms, Bacillus species are particularly valued due to their metabolic versatility, stress tolerance, and capacity to produce industrially relevant enzymes and bioactive compounds. This study investigates the impact of controlled soil pretreatments on microbial community dynamics and the subsequent isolation of stress-tolerant Bacillus strains from four climatically distinct regions of Tunisia (Nabeul, Siliana, Medenine, and Tozeur). To selectively enrich spore-forming bacteria, soil samples were subjected to controlled thermal treatments (heat at 40 °C, 60 °C, and 80 °C for 3 h) and calcium carbonate amendments (at concentrations of 5%, 10%, and 15% (w/w)) prior to microbial isolation. Pretreatment of soil samples significantly (p < 0.05) affected microbial counts, revealing differential responses among the studied sites. A total of 40 bacterial isolates were obtained, and 15 representative ones underwent detailed biochemical, physiological, and molecular characterization. 16S rRNA sequencing revealed diverse Bacillus taxa, including B. velezensis, B. subtilis, B. amyloliquefaciens, Cytobacillus horneckiae, together with one unresolved isolate designated Bacillus sp. 107. Physiological profiling revealed that the isolates were mesophilic, moderately halotolerant, and capable of growth under neutral to alkaline conditions. This integrative approach highlights the novelty of combining soil conditioning and microbial isolation strategies. It expands the understanding of soil-derived Bacillus diversity and generates a collection of physiologically robust isolates that may serve as candidates for future functional screening and application-oriented studies.