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

Abstract

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.

Water Availability Is a Critical Determinant of a Population Shift from Proteobacteria to Actinobacteria during Start-Up Operation of Mesophilic Fed-Batch Composting

“…In addition, bacterial strains isolated previously from FBC systems (strains TUT1003 to TUT1096 and TUT1200 to TUT1296) 12,25) were used. For this test, PBYG agar plates having different levels of aw were prepared by mixing with different concentrations of PEG 300 solution.…”

“…To confirm the universality of the aforementioned phenomena, several bacterial strains previously isolated from different FBC processes and identified with a wide variety of genera of Actinobacteria, Firmicutes, and Proteobacteria 12,25) were also tested for growth response to low aw. Combined data on the phylogenetic positions, quinone systems, and growth response to low a w of these isolates are shown in Fig.…”

“…Therefore, there may be a need to consider a characteristic microbiological basis for the mesophilic FBC process different from that for the conventional composting system. In recent years, the FBC process for the biotreatment of garbage and food waste has been studied from a microbiological as well as kinetic point of view 1,8,[12][13][14]17,[22][23][24][25][26][27]31) . In previous studies on mesophilic FBC reactors for garbage treatment, we found that a population shift from members of the phylum Proteobacteria to those of the phylum Actinobacteria took place during the start-up period of operation 12,23,25) .…”

“…Moisture is an additional critical ecological determinant in the FBC process as well as in the conventional batch composting system 14,17,25) . It has been shown that the solid waste-compost mixture (SCM) in FBC reactors operating under steady-state conditions has 30-40% moisture 12,23,25) , at which the microorganisms present exhibited the highest protease activity 25) .…”

“…It has been shown that the solid waste-compost mixture (SCM) in FBC reactors operating under steady-state conditions has 30-40% moisture 12,23,25) , at which the microorganisms present exhibited the highest protease activity 25) . Biologically, water availability is more critical than moisture.…”