Combined Use of Cyanoditolyl Tetrazolium Staining and Flow Cytometry for Detection of Metabolically Active Bacteria in a Fed-batch Composting Process

Abstract

Microbial community dynamics with metabolically active bacteria during the start-up operation of a personal fed-batch composting (FBC) reactor were studied. The FBC reactor was loaded daily with household garbage for 2 months. Metabolically active bacteria were monitored by the redox-dye-staining method using 5-cyano-2,3-ditoryl tetrazolium chloride (CTC), and the fluorescent formazans thus produced were detected by epifluorescence microscopy and flow cytometry (FCM). Microscopic CTC-positive (CTC+) counts accounted for 75-84% of the direct total count during the first week of operation and 19-35% thereafter. Slightly higher CTC+ counts were obtained by FCM. Culture-independent approaches by quinone profiling and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes showed that a drastic population change from ubiquinone-containing members of the Proteobacteria to the Actinobacteria took place during the overall period of operation. The PCR-DGGE analysis of FCM-sorted CTC+ cells supported this observation but gave different major clones from those detected in the total community in some cases. These results suggest that metabolically active bacteria as measured by CTC staining are not always predominant in the FBC process.