MICROBIAL DIVERSITY IN KUSAYA GRAVY

Microbial population in kusaya gravy from Niijima Island was analyzed by grouping of functional characteristics and genetic identification f rom 16S rRNA genes (16S rDNA). Viable conunts in TSSY medium supplemen ted with 0.1% thioglycolate and 0.3% agar using the MPN method were de termined as 10(11)/ml, which was the highest value compared with previ ous reports on kusaya gravy. Each of denitrifying, nitrate reducers, s ulfate reducers and proteolytic bacteria were estimated as about 10(9) /ml, and starch hydrolyzing and lactic acid producing bacteria were es timated as less than 10(3)/ml. Viable counts on TSA medium, which were superior to BPG and B(6)P(6)G media, were 10(8) CFU/ml. The 16S rDNA were amplified from kusaya gravy by PCR without cultivation of baceria , cloned, and sequenced. Sequence analysis of the cloned partial 16S r DNA indicated that the major bacteria in kusaya gravy were Bacteroides -Cytophaga group bacteria, wall-less spirochaeta-like bacteria, uniden tified bacteria, and so on. The results of genetic identification did not coincide with those of cultural methods; i.e., most of the above b acteria have not been isolated by plate methods so far, and Corynebact erium and Peptococcuceae which were representative organisms in the pl ate surface method under aerobic or anaerobic conditions, respectively , were not detected by the PCR method. These results indicated that th ere are diverse organisms which are unknown and unculturable in kusaya gravy.