Sporulation temperature effect on germination heterogeneity of individual Bacillus thuringiensis spores through single-cell optical analysis

The germination heterogeneity of individual bacterial spores significantly impacts spore viability and has critical implications for sterilization processes, food safety, and infection control. Temperature, as a key environmental factor, influences both sporulation and bacterial growth. This study investigates how sporulation temperature affects the germination behavior of individual Bacillus thuringiensis (Bt) spores at the single-cell level using optical approaches. Multivariate curve resolution-alternating least squares analysis showed that the average levels of Ca 2+ dipicolinic acid (CaDPA), protein, and nucleic acid in Bt spores decreased with increasing sporulation temperature. Spores formed at 22°C showed minimal impact on nonnutrient germination but exhibited slower and less efficient nutrient germination compared to those formed at higher temperatures. However, intensive heat-shock treatment (80°C for 30 min) significantly accelerated nutrient germination in 22°C-spores. In contrast, spores formed at 42°C displayed reduced speeds and efficiencies under both nutrient and nonnutrient germination. Germination parameters revealed that sporulation temperature mainly affected the initiation and duration of rapid CaDPA release and germination rate during nutrient germination, while cortex hydrolysis time remained relatively stable. During CaDPA germination, spores formed at lower temperatures released CaDPA faster. During dodecylamine germination, both initiation time and duration of rapid CaDPA release increased with sporulation temperature. Overall, sporulation temperature modulates key spore components, thereby enhancing germination heterogeneity. Lower temperatures may reduce germinant sensitivity and modify inner membrane characteristics, while higher temperatures may alter the structure of CaDPA release channels and impair nucleic acid synthesis. • Single-cell analysis reveals how sporulation temp. influences germination variation. • Sporulation temperature greatly impacts spore makeup and germination performance. • Spores formed under nonoptimal temperatures show higher germination heterogeneity. • Spore formation conditions are critical for food safety and industrial applications.