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Fig. 1 Schematic representation of physical factors important for physiology of biofilm cells. Role of substrate and metabolite transport in hydrodynamic (a) and static (b) biofilm systems. Laminar flow is indicated by horizontal, pointed arrows. The length indicates the velocity at the specific position in the system. The boundary layer is indicated. Diffusion from the laminar layer into and out of the biofilm are indicated with the vertical, flat-headed arrows. The length of the arrow indicates mass transport by diffusion based on the generated concentration gradients. A In the early stages (left panel) of a forming hydrodynamic biofilm, laminar bulk liquid flow (from left to right) carries substrates across the biofilm. The flow rate next to the biofilm is slower than farther out in the bulk liquid, as indicated by the different arrow lengths. Substrates diffuse from the boundary layer into the biofilm. Substrate consumption by the metabolic activity of the cells is indicated by the tapered substrate label. Note that the more pronounced decrease in concentration is in the layers next to the biofilm and not in the bulk liquid. Conversely, the metabolites generated by the biofilm cells diffuse into the boundary layer and from there into the deeper laminar layers, establishing a gradient with higher concentration in the layers closer to the biofilm. At later stages (rightpanel), substantial biomass has accumulated that catalyzes a more rapid (but at an individual cell level, at a more reduced rate) removal of substrates, thus creating steeper gradients. B In static systems with no laminar flow or mixing, the dominant transport of nutrients into a biofilm occurs by diffusion. Another notable and physiologically consequential difference between the shear flow (A) and the zero flow (B) system is the concentration of substrates and metabolites. While the shear flow system has properties of a chemostat system, i.e., very low but constant steady-state concentrations, metabolites accumulate in the static biofilm systems and affect the physiology of cells similarly as the initial high-substrate concentrations real-world settings, the flow rate of bulk liquid can range from zero (e.g., a fungal biofilm forming at the air-aqueous interface of a standing, half empty coffee cup or static laboratory biofilm systems) to high (e.g., inside of a drinking water pipe or a hydrodynamic laboratory flow chamber). Consequently, the flow rate determines to which extent a biofilm is in a chemostat-like setting (flow rate > 0, e.g., in a flow chamber) with a constant rate of substrate flux into and metabolite flux out of a biofilm or in a batch culture type condition (flow rate = 0), such as in laboratory 96-well plates, where initial high substrate concentrations are converted into accumulating metabolite concentration during the biofilm development (Fig. 1A, B). A laminar flow also exerts shear stress onto a biofilm, and thereby controls the activity of the biofilm cells (van Loosdrecht et al. 2002).

While laminar flow transports nutrients to the boundary layer (i.e., the layer of fluid in the immediate vicinity of a bounding surface; see Fig. 1), molecular diffusion transports nutrients between the laminar layers in the bulk liquid and between the boundary layer and the biofilm (Fig. 1A). There is evidence that bulk liquid flow through a biofilm is negligible, if present at all, and that net transport processes within biofilms are basically limited by diffusion from the surrounding boundary layer to the interior of developed biofilm. Because of the chemostat-like conditions, diffusion of a continuous supply of substrates at low concentrations, which are found in many natural environments, can support a growing biofilm very well. On the other hand, in the absence of a bulk liquid flow or mixing, i.e., in a static system, high substrate concentrations are required to be initially present in order to promote growth of a biofilm (Fig. 1B). Also here, molecular diffusion will control cellular physiology but to a quite different extent than in hydrodynamic systems. As a consequence, the concentration of chemicals, their gradients, and the distribution of such gradients across a biofilm are different in static biofilms systems relative to hydrodynamic systems, and with that the physiology of biofilm cells.

Figure 2 illustrates the dramatic effect of transport limitation on the extent of formation, the structure, and on the relative position of subpopulations within a biofilm. After identical initial conditions, transport-limited biofilm populations are on different trajectories relative to unlimited biofilm environments. Besides the overall dramatically reduced rate of increase in biofilm biomass, transport-limited biofilms exhibit significantly more structure, indicative of local chemical gradients and associated heterogeneous single-cell physiology (Picioreanu et al. 2001; van Loosdrecht et al. 2002). Notably, there is less mixing of phenotypically identical subpopulations in transport-limited biofilms (Fig. 2).

How do physical conditions such as transport by diffusion, including its limitation, determine the physiology of biofilm cells? Under initial conditions in a hydrodynamic systems (Figs. 1A, 2), when only few cells adhere to the substratum surface as an interspersed monolayer, molecular diffusion along the concentration gradient between the boundary layer and the cells causes mass transfer, thereby enabling cells to grow at high rate (Fig. 3). It should be noted that not all cells grow equally rapidly, and very few do not grow at all, thereby setting

Physiology of Microbes in Biofilms No transport limitation

Physiology of Microbes in Biofilms No transport limitation

  1. 2 Simulation of clonal growth of biofilm populations in non-transport-limited (upperpanel) and in transport-limited (lower panel) biofilms (images kindly provided by Cristian Picioreanu after Picioreanu et al. 1998). The two genetically and physiologically identical subpopulations are indicated in green and yellow, respectively. Note the difference in biofilm biomass at day 8 in both types of biofilm, as well as the distinct architecture of biofilms. Note also the enhanced retention of areas of clonal populations in transport-limited biofilms. See text for details.
  2. 2 Simulation of clonal growth of biofilm populations in non-transport-limited (upperpanel) and in transport-limited (lower panel) biofilms (images kindly provided by Cristian Picioreanu after Picioreanu et al. 1998). The two genetically and physiologically identical subpopulations are indicated in green and yellow, respectively. Note the difference in biofilm biomass at day 8 in both types of biofilm, as well as the distinct architecture of biofilms. Note also the enhanced retention of areas of clonal populations in transport-limited biofilms. See text for details.

subpopulations on to different physiological trajectories (Fig. 3b). Using fusions of gfp, encoding unstable GFP, to the ribosomal RNA promoter rrnB, studies of Pseudomonas putida biofilms developing in flow chambers revealed heterogeneous expression of growth activity (Sternberg et al. 1999; Christensen et al. 1999). While isolated cells exhibited similar Gfp fluorescence, once these cells had grown into flat clusters, cells at the periphery and exposed to the bulk liquid flow exhibited higher growth activity compared to cells in the center of clusters. Stimulation of growth of those inactive cells upon addition of a more readily metabolizable substrate indicates the importance of specific catabolic substrates in the physiology of a microorganism (Sternberg et al. 1999).

As the substrate concentration in the bulk liquid remains constant and the biomass in the biofilm increases, the rate of substrate consumption increases in the biofilm and with that the concentration gradient between the bulk and the biofilm. The increased concentration gradient, in turn, leads to an increased substrate flux into the biofilm (Fig. 3). When the rate of substrate (or more precisely, of the growth-limiting compound) consumption in the biofilm exceeds the net rate of flux into the biofilm, the biofilm becomes transport-limited (Fig. 3c) and growth slows down according to the cellular Monod growth kinetics (Fig. 3d). There is still a net growth of the biofilm population, although no longer at high rate

(Fig. 3d). Because of the increase in cell mass within the biofilm, the entire biofilm becomes metabolically and physiologically stratified: as a consequence of microbial growth under diffusion-limited conditions and of unequal diffusion in the biofilm, the growth rate becomes highly variable between cells, with a few bulk

Transport limitation

Transport limitation c

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