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.