Diffusion profiles in L. lactis biofilms under different conditions

Abstract

Despite being an important topic in biofilm research, we still know little about diffusion in biofilms. Emerging biofilms of Lactococcus lactis growing in custom-made flow-cells were monitored and diffusion constants across the height of the biofilms recorded. The biofilms showed different diffusional behavior with regard to flow rate and pH variations, despite growing to similar thickness. At a higher flow rate, the biofilm exhibits slower diffusion compared to the reference cultivation at lower flow rate. By increasing pH, the biofilm exhibited fast growth and little difference in diffusion compared to the reference cultivation. Furthermore, the diffusion inside of the biofilms differed depending on the position in the flow-cell. The present study reveals new insights in how external factors can affect structure and density of biofilms. The method can be reliably used for L. lactis biofilms with a thickness up to 120 μm.

Keywords: FRAP; Lactococcus lactis; biofilm; diffusion; flow‐cell.

FIGURE 1

Flow cell setup. Three evenly‐spaced positions on the 10 × 20 mm sample were chosen: upstream (U), center (C); downstream (D). Dye was applied from the upstream corner of the sample. At each position, five different heights were measured: From the substrate 50 μm (H1), 100 μm (H2), 150 μm (H3), 200 μm (H4), 700 μm (H5, water control)

FIGURE 2

Biofilm after 10 h of cultivation with pH7 (exemplary segment from full biofilm). Height of mushroom structure is 328 μm, height of bulk biofilm is 251 μm. Taken with Thorlabs Ganymede II (Thorlabs GmbH, Lübeck, Germany)

FIGURE 3

D_a, as ratio of D_b/D_w plotted over time and height. From top to bottom according to the positions on the sample (flow cell, left side) – position U (upstream), position C (center), position D (downstream). (1) Reference cultivation at pH 5.5 and 0.25 mL·min⁻¹ flow rate, (2) cultivation at pH 7 and 0.25 mL·min⁻¹; for the sake of completeness the grid is displayed for up to 12 h, (3) cultivation at pH 5.5 and 0.5 mL·min⁻¹ flow rate. Green colour indicates faster diffusion, red colour indicates slower diffusion compared to water

FIGURE 4

Plots for all measurements at position U for reference cultivation (left, 0.25 mL·min⁻¹, pH 5.5) and double flow cultivation (right, 0.5 mL·min⁻¹, pH 5.5)