Freezone 2.5 liter benchtop freeze dry system

CM agar plates were covered with sterile cellophane membranes (Bio-Rad) and incubated with 5–10 μl of a spore or cell suspension of the strains PK1.22, NP2.8, NP3.2, NP4.1, N402, BN26.1, BN34.2 or XY1.1, respectively. After 72 h of incubation at 30°C, biomasses were harvested from the plates and freeze-dried (FreeZone 2.5 Liter Benchtop Freeze Dry System, Labconco, USA). The dried biomass was weighted, grinded and resuspended in 50 mM sodium phosphate buffer (pH 7) to a final concentration of 50 μg/ml. After sonication (Bandelin Sonorex TK52 Transistor, Germany) for 10 min at room temperature, cell debris was sedimented at 19100×g for 10 min. Finally, 100 μl of supernatants were transferred to a MTP to measure fluorescence intensity (GloMax^®-Multi+ Detection System, Promega, Germany) at 510–570 nm after excitation at 490 nm. Background signal of phosphate buffer was subtracted from all sample values. Unfortunately, and in contrast to the ΔcreA strains, ΔflbA and ΔbrlA strains showed an auto-fluorescence signal, which made it impossible to analyze the impact of anafp deletion in these strains via fluorescence measurements.

ALG-C was synthesized via EDC-NHS chemistry. Briefly, 100 mg of alginate powder (Sigma-Aldrich, Oakville, ON, USA) was dissolved in 10 mL of distilled water. Then, 143 mg of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), 86 mg of N-hydroxysuccinimide (NHS), and 130 mg of dopamine (Sigma-Aldrich) were added to the alginate solution. The mixture was reacted overnight at room temperature. Then, unreacted EDC, NHS, and dopamine were dialyzed using dialysis membrane (MWCO: 12 K Da, Spectra/Por^®) against distilled water for 3 days. The distilled water was changed three times each day. After lyophilization at −50 °C for 3 days (FreeZone 2.5 Liter Benchtop Freeze Dry System, Labconco), the alginate-catechol conjugate was collected. The degree of substitution was analyzed by ¹H NMR spectroscopy (Avance III 400 FT-NMR, Bruker). Briefly, 5 mg of ALG-C was dissolved in 1 mL of deuterium oxide (D₂O, Sigma-Aldrich). Then, ALG-C solution was loaded in Wilmad^® NMR tube and analyzed. In addition, the synthesis of ALG-C was further characterized by Attenuated Total Reflection-Fourier Transformation Infrared (ATR-FTIR, TENSOR27, Bruker). Briefly, the lyophilized ALG-C was scanned 32 times with a resolution of 8 cm⁻¹ and measured from wavenumbers ranging from 650 to 4000 cm⁻¹. The ALG-C film was also fabricated via the above process using CaCl₂.