Biofilm thickness matters: Deterministic assembly of different functions and communities in nitrifying biofilms

ABSTRACTMicrobial biofilms are important in natural ecosystems and in biotechnological applications. Biofilm architecture influences organisms’ spatial positions, who their neighbors are, and redox gradients, which in turn determine functions. We ask if and how biofilm thickness influences community composition, architecture and functions. But biofilm thickness cannot easily be isolated from external environmental factors. We designed a metacommunity system in a wastewater treatment plant, where either 50 or 400 µm thick nitrifying biofilms were grown simultaneously on biofilm carriers in the same reactor. Model simulations showed that the 50 µm biofilms could be fully oxygenated whereas the 400 µm biofilms contained anaerobic zones. The 50 and 400 µm biofilms developed significantly different communities. due to deterministic factors were stronger than homogenizing dispersal forces in the reactor, despite the fact that biofilms experienced the same history and external conditions. Relative abundance of aerobic nitrifiers was higher in the 50 µm biofilms, while anaerobic ammonium oxidizers were more abundant in the 400 µm biofilms. However, turnover was larger than the nestedness component of between-group beta-diversity, i.e. the 50 µm biofilm was not just a subset of the thicker 400 µm biofilm with reduced taxa richness. Furthermore, the communities had different nitrogen transformation rates. The study shows that biofilm thickness has a strong impact on community composition and ecosystem function, which has implications for biotechnological applications, and for our general understanding of biofilms.IMPORTANCEMicroorganisms colonize all surfaces in water and form biofilms. Diffusion limitations form steep gradients of energy and nutrient sources from the water phase into the deeper biofilm parts, influencing community composition through the biofilm. Thickness of the biofilm will affect diffusion gradients, and is therefore presumably important for biofilm composition. Since environmental factors determine thickness, studies of how thickness influences biofilm functions and community assembly, have been difficult to perform. We studied biofilms for wastewater treatment with fixed thicknesses of 50 and 400 µm during otherwise similar conditions and history. Despite growing in the same wastewater reactor, 16S rRNA gene sequencing and confocal microscopy showed the formation of two different communities, performing different ecosystem functions. Using statistical methods, we show for the first time, how biofilm thickness influences community assembly. The results help our understanding of the ecology of microbial biofilms, and in designing engineered systems based on ecological principles.