The quinone profiles of microbial biomass samples from wastewater and natural aquatic environments were studied. A soil sample was also analyzed for comparison. Attempts were made to perform numerical analyses on these profiles by using three parameters, the microbial divergence index (MDq), the bioenergetic divergence index (BDq), and the dissimilarity index (D), and the algorithm of the neighbor-joining method was used to make clustering trees based on D value matrix data. For these purposes, a new personal computer program named "BioCLUST" was developed. The neighbor-joining dendrograms were useful for the grouping of different microbial communities based on D value matrix data. Also, the communities could be classified effectively by plotting MDq data against BDq values. The numerical analysis indicated that sewage activated sludges formed a tight cluster independent of the location of plants and scale of reactors. This was also the case for influent raw sewage. Sewage microbial mats and river water were located in a group with the raw sewage cluster as the sister group. The soil and lake showed their respective independent lines. The use of our new program should facilitate microbial community analyses based on quinone patterns.

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The direct total count of bacteria and the concentration of isoprenoid quinones were measured in 82 samples from different aquatic and soil environments. The geometric mean of the total count (TC)/total quinine (TQ) ratios obtained with different environments ranged from 1.1 to 2.2´10 3 cells fmol -1 . When the concentration of the total respiratory quinone (TRQ), i.e., ubiquinones and menaquinones, were taken into account, the geometric mean of the TC/TRQ ratios fell into a range of 2.2 to 4.1´10 3 cells fmol -1 . A high positive correlation was noted between TC and TQ (r 2 =0.9864) or TRQ (r 2 =0.9990). Based on the relationship between TC and TRQ, 1 nmol of the total quinone was estimated to be equivalent to 2.5´10 9 cells of bacteria on average. These results indicate that the concentration of the respiratory quinones can be used as a good measure of bacterial counts and biomass in the environment.

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Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.