Significance of the spatial distribution of microbial species in mixed‐population biofilms

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Abstract
Dynamic changes in spatial microbial distribution in mixed‐population biofilms resulting from interspecies competition between heterotrophs and nitrifiers were experimentally investigated using a microslicer technique. Biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. The results showed that variation in the carbon loading rate (CLR) and the composition of the feed substrate resulted in rapid and substantial changes in the spatial distribution of nitrifiers in mixed population biofilms within a week, which significantly influenced the NH4‐N removal rate. Heterotrophs were more successful than nitrifiers in acquiring dissolved oxygen and space and dominated throughout the biofilms, especially in the surface biofilm. Nitrifiers were therefore diluted in the surface biofilm and mainly found in the inner biofilm. The fraction differences of NH4‐oxidizers and NO2‐oxidizers over the biofilm depth were about 2 orders and 3–4 orders of magnitude at CLR=0.8 and 1.6 g‐C m‐2 d‐1 respectively, indicating a steep spatial gradient of nitrifiers. Although total areal density of NH4‐oxidizers in the biofilms were in the same order, the NH4‐N flux decreased by about 9% and 24% after one month at CLR=0.8 and 1.6 g‐C m‐2 d‐1 respectively. This decrease of NH4‐N flux was attributed to the extent of the stratified spatial distribution of NH4‐oxidizers. Heterotrophs lie near the surface and act as a diffusion barrier, resulting in an increase in internal dissolved oxygen and NH4‐N diffusion resistance for NH4‐oxidizers. These results suggest that simplifying or neglecting the spatial distribution of microbial species can lead to substantial errors in biofilm kinetic parameters determined from measured substrate removal rates. For implication in reactor design, the extent of the spatial distribution of microbial species in a biofilm is especially important for interpretation of nitrification efficiency in the presence of organic matter.

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