Freezone 4.5 liter benchtop

The bacterial strains used in this study are summarized in Table 2. For the preparation of inoculums, several colonies of each bacterial strain were inoculated in 500 ml liquid LB medium (Roth, Karlsruhe, Germany) and grown at 30°C and 150 rpm for 24 h (Co1-6, L13-6-12, P69, and 3Re4-18) and for 48 h (Wb2n-11 and Mc5Re-14). To harvest the cells, bacterial suspensions were centrifuged at 13,500 g for 20 min. Pellets were dissolved in 2 ml sucrose solution (1%), serving as cryoprotectant agent, and frozen to −70°C for 5 h. Tubes containing the frozen bacterial suspension were put into ampules and connected to a freeze-dryer (Labconco FreeZone 4.5 Liter Benchtop, USA) for 12 h under vacuum at <0.1 Pa.

After lyophilizing hydrogels (FreeZone 4.5 Liter Benchtop, Labconco, Kansas City, MO, USA) for 48 h at −50 °C under 0.1 mbar, the morphological characterization of hydrogels was realized. Images were acquired with the Quanta 250 FEG microscope (FEI, Eindhoven, The Netherlands) equipped with a large-field electron detector (LFD) at a 218× magnification, 12.2 working distance, 20 kV, 4.5 spot size, 18 °C, 100 Pa, and without humidity.