79NISR RESEARCH GRANT
2002 Research Grant

Discovery and Microbiological Analyses of a Novel Biorecycling System of Garbage that Proceeds with Participation ofLactic-acid Bacteria as Predominant Species
Toru NAKAYAMA
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
Research aims
We found a novel system of biorecycling (composting)of garbage that is sustained under thermoacidophilic conditions (at pH 3.5–6.5 and 50–70°C) for prolonged periods oftime (more than 2 years). This is in striking contrast with
the nature of known accelerated high-temperature composting processes, which generally proceed under slightly alkaline conditions (pH 7.5–9). This process was also found toemit only low levels of odor, in contrast to other known
composting processes. Thus, due to its sustainability andlow level of odor emission, the system may be a low-maintenance process that could overcome the problems associated with conventional accelerated-composting processes.
In the present study, we analyzed microflora of the processby polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescent in situ hybridization (FISH) to show the participation of lactic acid bacteria
during this novel composting process.
Methods
The composting apparatus used in this study had amaximum capacity of 5 kg (wet wt) of garbage a day. Prior
to the first addition of the substrate (garbage), starter microorganisms (0.5 g, wet wt.) that had been maintained as amixed culture of thermoacidophilic and thermoacido-tolerant soil bacteria and base material (cedar-wood saw dust,
32 liters) were mixed in a vessel by running the machine for
1 h without any substrate. Excess water in the fresh garbage
was drained using a basket, and a specific quantity of thesubstrate was added daily to the vessel of the composting
system. Typical contents of added substrates were food refuse and raw residual materials generated during cooking.
Basically, all edible materials, fruit rinds, animal skins, fishbones, and eggshells are good substrates for the composting
system. However, large animal bones, shells, and wood materials were not used as substrates for composting. Throughrepeated additions and composting of the substrate, the volume of the product compost in the vessel gradually increased. At appropriate times, the excess compost was removed to maintain the volume of the compost in the vessel
at less than 27 liters. PCR-DGGE analysis of the compostmicroflora was carried out using PCR primers 520F (GTG
CCA GCM GCC GCG G), 1400R (ACG GGC GGT GTG

TRC), and GC1400R (CGC CCG GGG CGC GCC CCG
GGC GGG GCG GGG GCA CGG GGG GAC GGG CGGTGT GTR C). FISH analysis of the compost microflora
were carried out using the fluorescence-labeled oligonucleotide probes EUB338-FITC [5-(fluo)GCT GCC TCC
CGT AGG AGT-3] and LAB-Cy3 [5-(Cy3)GGT ATT
AGC AYC TGT TTC CA-3]. Organic acid analysis of thecompost samples were done using a Photal CAPI-3200 microcapillary electrophoresis system (Otsuka Eectronics).
Results
The pH and temperature of the compost were in theranges of 5.5–5.6 and 60–65°C, respectively, during theprocess throughout the composting process monitored, indicating that the composting proceeds under thermoacidophilic conditions. Figure 1 shows the results of PCRDGGE analysis of the compost microflora using universal
primers designed for specific amplification of 16S (or 18S)rDNA sequences. Several major bands (1204A-D, Fig. 1)
were amplified and were analyzed for their nucleotide sequences. The results showed that all of these DNAs have
16S rDNA sequences of the genus Lactobacillus, suggesting that lactobacilli should predominantly occur in the compost to play an important role for biodegradation of organic
wastes even under thermoacidophilic conditions. To morequantitatively evaluate the participation of lactic acid bacteria during the composting, FISH analysis of the compost
microflora was also carried out. The results showed that thecompost contained 4.6107 microbial cells per g compost,
44–54% of which were specifically hybridized with the lactic acid-specific probe LAB-Cy3. These results showed that
lactic acid bacteria were indeed predominant species of thecompost ecosystem. The organic acid analysis showed theoccurrence of high levels (up to 850 ppm) of lactic acid andacetic acid in the compost, suggesting that lactic fermentation should take place during the process. The compost extract inhibited the growth of Escherichia coli (not shown),
implying the existence of bacteriocin-like substances in thecompost.
Conclusion
The present composting process showed a stable microbial ecosystem with lactic-acid bacteria being predomi-

80Fig. 1.

PCR-DGGE analysis of the composting process.

nant species. These lactic-acid bacteria should probably bethermotolerant or thermophilic species that have originallybeen present in garbage and have survived under thermoacidophilic conditions of the process. It is likely that thegrowth of microorganisms that may hamper the compostprocess is severely inhibited by acidic conditions due to lactic fermentation, high-temperature conditions, and bacteriocin produced by the habitat bacteria, and this may accountfor the observed stability of the process.