2.1 Biofilm cultivation
Biofilms were cultivated in custom-made flow cells composed of sticky-Slides (sticky-Slide I 0.4 Luer, ibidi GmbH, Martinsried, Germany) glued to PVC slides (substrata). Sticky-Slides are made from transparent plastic and serve as the cover of the flow cell forming a flow channel with the size of 50 × 5 × 0.45 mm3(length × width × height, thickness of the sticky-Slide = 1 mm). A number of \(N\) = 10 flow cells were operated for each condition in parallel at volumetric flow rates of = 1 mL/min and= 5 mL/min, respectively (equals mean flow velocities of u = 0.75 cm/s and \(u\) = 3.75 cm/s, respectively).
Flow cells were inoculated with Bacillus subtilis pre-cultures grown at 37°C overnight in Luria Broth (LB) medium. Cells were grown to exponential phase; 10 mL of this pre-culture together with a minimal salts glycerol medium in a mixing ratio of 1:500 were used as inoculation solution. The cultivation medium was adapted from (Wang, Wang, & Hao, 2015) and contained (concentration in mg/L): MnCl2 • 4 H2O (10), L-phenylalanine (5), glycerol (5), MgCl2 • 6 H2O (4), L-tryptophane (3.5) with diverging concentrations of FeCl2 • 4 H2O (≙ 0.25 and 2.5 mg/L Fe2+) in tap water. Salt concentrations of the tap water of Karlsruhe are accessible from the homepage of the Stadtwerke Karlsruhe (https://www.stadtwerke-karlsruhe.de) and contained (mg/L): Ca (112), Na (11), Mg (9.7), Si (5.4), K (1.7), P (< 0.01), Fe (< 0.01) and Mn (< 0.005).
Flow cells were flushed with the inoculum for 15 min. Afterwards flow was stopped for 1 h giving bacteria the possibility to settle. Then biofilm cultivation started in flow-through mode. Biofilm development 25 mm downstream the inlet was monitored daily for ten consecutive days by means of OCT using the EvoBot platform (Faina et al., 2016; Gierl, Stoy, Faína, Horn, & Wagner, 2020). An overview of the conducted experiments is provided in Table 1.