Ice nucleation assays

For ice nucleation assays, ten colonies were taken from three replicates of KBC plates, for a total of 30 colonies from each sampling location. A total of 720 colonies was selected at random (180 from each of the four missions). Each colony was picked with a sterile toothpick and transferred to 140 µL of water. Two droplets of 12 µL of each sample were pipetted onto floating boats made of Parafilm^® M placed on top of a cooling bath (Lauda Alpha RA 12, LAUDA-Brinkmann, LP, Delran, NJ, USA) (Hanlon et al., 2017). Droplets of sterile water were used as negative controls. Samples were loaded onto the floating boats at −5 °C, and the temperature of the bath was then lowered to −12 °C in one degree increments. Freezing temperatures were recorded for all of the frozen droplets. Microbes from droplets freezing at temperatures warmer than −10 °C were considered to be ice nucleation active (designated as ice +), and were identified using portions of 16S rDNA sequences as described by Pietsch, Vinatzer & Schmale III (2017).