Transport, motility, biofilm forming potential and survival of Bacillus subtilis exposed to cold temperature and freeze–thaw

• First report of the transport behavior of Bacillus subtilis after exposure to freeze–thaw.
• Observed greater retention of B. subtilis on sand after freeze–thaw.
• Increased survival of B. subtilis at low temperature in higher ionic strength.
• Lower bacterial motility and biofilm formation observed after freeze–thaw.
• Observed weaker bonds between the bacteria and quartz after freeze–thaw.

In cold climate regions, microorganisms in upper layers of soil are subject to low temperatures and repeated freeze–thaw (FT) conditions during the winter. We studied the effects of cold temperature and FT cycles on the viability and survival strategies (namely motility and biofilm formation) of the common soil bacterium and model pathogen Bacillus subtilis. We also examined the effect of FT on the transport behavior of B. subtilis at two solution ionic strengths (IS: 10 and 100 mM) in quartz sand packed columns. Finally, to study the mechanical properties of the bacteria-surface bond, a quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor changes in bond stiffness when B. subtilis attached to a quartz substrate (model sand surface) under different environmental conditions. We observed that increasing the number of FT cycles decreased bacterial viability and that B. subtilis survived for longer time periods in higher IS solution. FT treatment decreased bacterial swimming motility and the transcription of flagellin encoding genes. Although FT exposure had no significant effect on the bacterial growth rate, it substantially decreased B. subtilis biofilm formation and correspondingly decreased the transcription of matrix production genes in higher IS solution. As demonstrated with QCM-D, the bond stiffness between B. subtilis and the quartz surface decreased after FT. Moreover, column transport studies showed higher bacterial retention onto sand grains after exposure to FT. This investigation demonstrates how temperature variations around the freezing point in upper layers of soil can influence key bacterial properties and behavior, including survival and subsequent transport.

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