PI-141:

Physiological responses of Actinobacteria to water stress during fed-batch composting as determined by cultural, trasncriptomic, and proteomic analyses

Our previous study has shown that microorganisms in fed-batch composting (FBC) processes under steady-state conditions, of which Actinobacteria are the major constituents, are highly cultivable and yield much larger numbers of colonies on agar plates than expected from direct viable counts as measured by a tetrazolium reduction assay using CTC (Takebayashi et al., M&E 22:279 [2007]). This phenomenon is a puzzle, because the CTC assay has been believed to be able to detect metabolically active microorganisms with covering the underestimation by plating to count viable microbes. The main purpose of this study was to confirm whether that FBC reactors under steady-state conditions produce much higher plate counts than CTC-positive (CTC+) counts and to find out a plausible explanation for this. To address this subject, we focused on the effects of water activity (a_w) stress on metabolic activity of Actiobacteria as measured by CTC staining and transcriptomic and proteomic analyses. An FBC reactor was operated for 70 days with daily loading of household biowaste. During this period of operation, a_w lowered linearly with time, and plate counts of bacteria were 3.1-fold higher than CTC+ counts on average at the fully acclimated stage. When actinobacterial isolates as the predominant cultivable bacteria from the FBC process were grown under a_w stress, there were no significant differences in the culturability by plate counting among the cultures, whereas relative CTC+ counts decreased with decreasing a_w. Transcriptomic and proteomic analyses of a representative isolate showed that many of the genes involved in cellular metabolism and genetic information processing were downregulated by aw stress. These results suggest that, in low a_w manure compost, metabolic activity of Actinobacteria is temporarily reduced to such a level as hardly reacting with CTC, but they are still viable and easily recoverable by exposure to culture media.

keywords:compost,*Actinobacteria*,water activity,CTC reduction,transcriptomics,proteomics