Reactivation and pilot-scale application of long-term storage denitrification biofilm based on flow cytometry

The work provides a method on the basis of flow cytometry to evaluate the performance of denitrification biofilm during the preservation, reactivation and pilot-scale operation process. The viable cell ratio of denitrification biofilm significantly reduced and further led to the decrease of denitrification capacity after long-term preservation for 5 months. Protein component in tightly bound extracellular polymeric substances (TB-EPS) could serve to enhance microbial adhesion and promote denitrification biofilm formation. With the significant correlation of viable cell ratio and microbial characteristics, 4 °C was more appropriate for preserving denitrification biofilm and conducive to maintain the relatively high denitrification capacity. A maximum denitrification rate of 5.80 gNO₃^--N/m²·d was obtained in pilot-scale anoxic-oxic (AO) process and Dechloromonas became greater prevalence in denitrification suspended carriers. Furthermore, the enrichment of Pseudomonas, Parcubacteria, Acidovorax, Aquabacterium and Unclassified_Flavobacteriaceae enhanced biofilm formation and nutrient conservation. The significantly positive correlation between viable cell ratio and the ratio of nitrate reduction to COD consumption was discovered, and the indices of Chao, ACE, Shannon and Simpson of denitrification biofilm were positively correlated with viable cell ratio, meaning that flow cytometry analysis was reasonable and suitable to evaluate the performances of denitrification biofilm.