2.2 Preparation of synthetic biofilm and growth ofPseudomonas aeruginosa biofilm
Synthetic biofilms were prepared from alginate (Acros Organics, USA) with internal release of calcium ions using D-glucono-δ-lactone (GDL) and calcium carbonate. As a slowly hydrolyzing lactone, GDL can control the pH and slow the release of Ca2+ ions, which crosslink the alginate gel internally (Pawar & Edgar, 2012). This results in more homogeneous mechanical properties. The method was modified from Draget et al. (1991). Bacterial cells were grown from using sludge from the local South Bend Wastewater Treatment Plant, South Bend, Indiana, USA (See SI). The mixture of alginate/Ca2+/bacteria was naturally dried at room temperature for 24 h to achieve full gelation. After full gelation, the alginate sample was tested immediately in the rheometer or with deformation experiments. The detailed information of synthetic biofilm preparation is provided in the SI.
P. aeruginosa PAO1 (ATCC 15692) labeled with green fluorescent protein (GFP) was used for the biofilm experiments. The growth medium (see SI) contained 100 mg/L of acetate as an electron donor and 15 µg/mL of gentamicin sulfate (Sigma-Aldrich, USA) to maintain axenic conditions. The P. aeruginosa biofilm was grown in a membrane-aerated biofilm reactor (MABR), following Aybar et al. (2019). The biofilm was collected from the membrane after two weeks of growth, at which time it was approximately 800 µm thick. Approximately 2 g (wet) of biofilm was collected. The sample was mixed and homogenized by gently stirring with spatula, and tested immediately with rheometry and deformation experiments.