Effects of sporulation times in liquid or on plates on <i>Bacillus subtilis</i> spore resistance, germination, inner membrane fluidity and permeability, and core contents

This study aimed to determine reasons that longer sporulation times in liquid or on plates have large effects on <i>Bacillus subtilis</i> spore germination and resistance. <i>B. subtilis</i> spores were prepared for 3/30/60 days in liquid or on plates and their germination, resistance, levels of core water and Ca²⁺-dipicolinic acid (CaDPA), and inner membrane (IM) fluidity and permeability were measured. Liquid spores of 3/30/60 days had no differences in wet heat resistance, while plate spores of 30/60 days had higher wet heat resistance than 3-day plate spores. There were minimal increases in 3- to 60-day liquid spores' resistance to UV radiation and chemicals, while plate spores of 30/60 days had increased resistance to chemicals. Germinant receptor (GR) germination with L-valine was identical with 3- to 60-day liquid or plate spores, while plate spores of 30/60 days germinated faster than 3-day spores with the L-asparagine, D-glucose, D-fructose, and KCl mixture. Three-day liquid or plate spores germinated faster than 30- and 60-day spores with the GR-independent germinant, dodecylamine, and 30-day plate spores had lower IM permeability and higher IM rigidity than 3-day plate spores or 30-day liquid spores. However, levels of two spore core components' modulating spore wet heat resistance, water, and CaDPA were identical in spores of 3/30/60 days. Thus, increased IM rigidity and decreased IM permeability appear to be major factors in increased resistance of spores prepared on plates for long periods. However, precisely how these longer times cause increased IM rigidity and lower permeability, but not with spores prepared in liquid, is not clear.IMPORTANCECells of some Bacillota cause food spoilage and human diseases. These organisms' ability to do this is exacerbated by forming hard-to-kill dormant spores because of their resistance and ability to come "back to life" in germination. We examined two <i>Bacillus subtilis</i> sporulation parameters, liquid versus solid media and sporulation time, measuring effects on spore resistance and germination. We found (i) an effector of spore heat resistance, core water content, is not changed by different sporulation media or times; and (ii) spores' IM becomes more rigid and less permeable in spores made on solid media and for longer times. This knowledge may influence how spores are prepared as probiotics or as standards for analyses of autoclave function.