The biofilm characteristics of a novel three-phase reactor, the circulating bed reactor (CBR), were studied using industrial prototype fed with primary and secondary settled effluent in conditions of tertiary N and secondary C+N nitrification. The results showed a high nitrification rate close to the intrinsic values for N and C+N conditions: up to 2 and 0.6 kgN-NH4 m−3 d−1, or 1.88±0.26 and 0.22±0.07 gN g−1 PR d−1, respectively. The application of an integrated approach for biofilm analysis enabled the better understanding of biofilm dynamics. The biofilm remained relatively thin, below 100 μm, indicating an effective control of the biofilm development. Protein, measured by the conventional colometric method and pyrolysis-GC-MS, was the major fraction accounting for up to 35% of the biomass dry weight and 58% of the biopolymer content. The polysaccharide's fraction remained very low (<3%). The ribosomal RNA probes analysis confirmed the predominance of bacterial cells in the CBR biofilm (80-86% of bacteria versus the universal probe) showing a high proportion of nitrifying bacteria accounting for up to 50% and 27% in the N and C+N removal respectively. Nitrosomonas predominated in tertiary nitrification whereas carbon input led to the appearance of other ammonia oxidizers. This particular composition was characterized by a high state of oxidation of the biomass, expressed by the low COD/DW ratio of about 0.85. In conclusion, it can be stated that this new three-phase bioreactor ensures a high nitrification rate through an effective biofilm control promoting the development of bacterial cells, especially nitrifying bacteria, and minimizing exopolysaccharides production.

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