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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.

The effects of water availability defined as water activity (aw) or matric water potential (ψm) on microbial community dynamics during the start-up operation of mesophilic fed-batch composting (FBC) of household biowaste were studied using commercially available personal composters. The community changes were monitored for 2 months by direct cell counting, quinone profiling, and 16S rRNA gene sequencing of culturable predominant bacteria. The aw level lowered linearly with operation time and reached around 0.95 at the end of operation. During the steady-state period, aw or ψm had a strong positive correlation with moisture content and ubiquinone content and negative correlation with pH, electric conductivity, and partially saturated menaquinone content. Results of 16S rRNA gene-based phylogenetic identification of the predominant isolates as well as of cultureindependent quinone profiling indicated that a drastic population change from ubiquinone-containing members of Proteobacteria to Actinobacteria took place during the overall period of operation. Most of the actinobacterial isolates grew on nutrient agar with an aw of less than 0.98, whereas none of the proteobacterial isolates did. These results suggest that water availability is an important determinant of the population shift from the Proteobacteria to Actinobacteria during the start-up operation of mesophilic fed-batch garbage composting.

A redox dye-staining method using 5-cyano 2,3-ditolyl tetrazolium chloride (CTC) was modified for differential detection of metabolically active gram-positive and gram-negative bacteria. Exponentially growing cells of representative species of the phyla Actinobacteria, Firmicutes and Proteobacteria were stained with CTC in the presence of a substrate mixture, Meldola’s Blue and KCN, and post-treated with acetone. Epifluorescence microscopy and spectrophotometric experiments showed that the acetone treatment resulted in the almost complete extraction of CTC formazan crystals from the gram-negative species, whereas it had little or no effect on CTC-stained gram-positive bacteria. Metabolically active gram-positive and gram-negative bacteria in municipal-sewage activated sludge measured by the modified CTC-staining method with microscopic detection composed 24 and 29% of the total count on average, respectively, both of which corresponded to approximately 74% of the viable gram-positive and gram-negative counts detected with a ViaGram Red + Bacterial Gram Stain and Viability kit. Flow cytometric analyses of the CTC-stained and acetone-extracted activated sludge samples gave similar counts to those measured by epifluorescence microscopy. These results indicate that the modified CTC stain procedure with epifluorescence microscopy or flow cytometry can be used as a simple and rapid method for the estimation of metabolically active populations of gram-positive bacteria and gram-negative bacteria in the environment.