Activity and Phylogenetic Composition of Proteolytic Bacteria in Mesophilic Fed-batch Garbage Composters

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Crude extracts of extracellular enzymes were prepared from a solid waste-compost mixture (SCM) taken from two mesophilic fed-batch composting (FBC) reactors for garbage treatment, and were tested for protease activity with azocasein as the substrate. The protease activity was highest at a reaction temperature of 70°C and remained relatively high even at 90°C. The optimum pH for the activity was 7 to 10. There were a significant negative correlation between the protease activity and the moisture content in the FBC reactors when the extracts were prepared. Seventy-six strains of proteolytic bacteria were isolated quantitatively by the agar-plating method and identified phylogenetically by 16S rRNA gene sequencing. Most of the isolates were assigned to members of the phyla Actinobacteria, Bacteroidetes, and Firmicutes, especially those of the genera Bacillus, Cellulosimicrobium, and Ornithinococcus and an unaffiliated Cytophaga-like group. Inhibitor and zymography experiments showed that the proteolytic activities of the isolates belonging to the above-noted Gram-positive genera as well as of the SCM sample were inhibited by phenylmethylsulfonyl fluoride and that the isolates of Bacillus, Cellulosimicrobium, and the Cytophaga-like group excreted a protease similar to those from the SCM sample in electrophoretic mobility. The results of this study suggest that bacteria belonging to the phyla Actinobacteria and Firmicutes and producing alkaline serine protease play primary roles in protein digestion in the mesophilic FBC process.