Each CDFF-grown sample was transferred to a specially designed NMR biofilm reactor to allow the biofilm to continue to grow inside the NMR. The reactor was constructed from Torlon® polyamide-imide plastic (Figure 1). Its single-pass flow system consisted of a medium reservoir bottle, a pulseless dual syringe pump (Pharmacia P-500, Uppsala, Sweden), the NMR biofilm reactor (inside the NMR magnet), and a waste reservoir bottle connected in series with polyetheretherketone (PEEK) plastic tubing. Drip-isolation tubes were placed upstream of the reactor to minimize microbial growth and avoid contamination. Figure 1 shows the NMR biofilm reactor and its location in the NMR probe. The inside of the chamber was 40 mm long, 4 mm wide, and 2 mm tall, giving a total liquid volume of 320 μL. When installed in the magnet, the normal direction to the coverslip surface was perpendicular to the magnet bore and coincident with the Helmholtz radiofrequency detection coil axis.
The influent medium flowed against gravity, and the effluent flow was routed to a waste vessel. O2-saturated minimal medium (SI, Table 1) was pumped through the reactor at a rate of 1 mL/h, which resulted in a laminar flow profile (Reynolds number of 0.1). The hydraulic retention time was 19.2 minutes (dilution rate of 3.13 h-1). A temperature-controlled gas stream delivery unit (FTS Systems, Stone Ridge, NY, USA) maintained a purge of nitrogen gas in the magnet bore and around the sample chamber, keeping the reactor and perfusion lines in the bore at 30 ± 0.2 °C.