Unique stratification of biofilm density in heterotrophic membrane-aerated biofilms: An experimental and modeling...

We consistently find a band of high cell density develop within heterotrophic membrane-aerated biofilms. This study reports and attempts to explain this unique behavior. Biofilm density affects volumetric reaction rates, biofilm growth rates, substrate diffusion, and mechanical behavior. Yet the mechanisms and dynamics of biofilm density development are poorly understood. In this study, a membrane-aerated biofilm, where O₂ was supplied from the base of the biofilm and acetate from the bulk liquid, was used to explore spatial and temporal patterns of density development. Biofilm density was assessed by optical coherence tomography. After inoculation, the biofilm quickly increased in thickness, with a low density throughout. However, as the biofilm reached a stable thickness of around 1000 μm, a high-density layer developed in the biofilm interior. The layer slowly expanded over time. Oxygen microprofiles in the biofilm showed this layer coincided with the most metabolically active zone, resulting from counter-diffusing O₂ and acetate. The formation of this dense layer appeared to be related to changes in growth rates. Initially, high growth rates throughout the biofilm presumably led to fast-growing, low-density biofilms. As the biofilm became thicker, and as substrates became limiting in the biofilm interior, growth rates decreased, resulting in new growth at a higher density. A 1-D mathematical model with variable biofilm density was developed by linking the rates of extracellular polymeric substances (EPS) production to the growth rate. The model captured the initial fast growth at a low density, followed by a slower, substrate-limited growth in the biofilm interior, producing a dense band within the biofilm. Together, these results suggest that low growth rates can lead to high-density zones within the interior of counter-diffusional biofilms. These findings should also be relevant to conventional, co-diffusional biofilms, although differences in density may be less obvious.