Halophile microorganisms are key ectoine producers with significant industrial applications. Their growth and osmoregulation vary across environmental conditions, necessitating comprehensive investigations of cultivation and osmotic responses. Alkalibacillus haloalkaliphilus was utilized to study ectoine production and secretion, offering insights into bacterial stress responses through analysis of cellular states and membrane characteristics during stress-related processes. A holistic approach began with screening nutrient requirements via microarrays, enhancing understanding of catabolism and identifying metabolic bottlenecks, which led to selecting potential industrial raw materials. The most suitable substrate was then optimized using Design of Experiments (DoE) and Response Surface Methodology (RSM). Given that microbial growth and ectoine production depend on key parameters like salinity, pH, and temperature, the optimization yielded three significant process strategies. Each strategy involved single-cell analysis through a novel staining method to monitor changes in membrane potential. Cells were subjected to thermal and osmotic shocks to release accumulated ectoine. Investigating the bacterial stress response and its impact on membrane properties revealed changes in fluidity and integrity under environmental stress, using fluorescent techniques and single-cell analysis. This established methodology provided deeper insi
