|dc.description.abstract||Solvent-producing clostridial strains were used extensively during the first part of this century for the industrial production of acetone and butanol and in the last two decades have again become the focus of investigation due to their potential applications in biotechnology. A starch-fermenting Clostridium species, isolated and patented during World War I and later named Clostridium acetobutylicum, was the first successful industrial strain to be used for the large-scale production of solvents. From the mid-1930's onwards, when molasses became abundant, the acetone-butanol (AB) fermentation process in most countries was switched from utilizing starch-based substrates to this Jess expensive sugar-based substrate. This in turn led to the isolation and patenting of numerous solvent-producing clostridia capable of fermenting molasses, each of which was given a novel species name. However, the majority of these saccharolytic solvent-producing clostridial strains were never scientifically recognized as legitimate species and once the industrial AB fermentation process declined these names fell into disuse. At present the majority of the industrial solvent-producing clostridia held in culture collections around the world tend to be classified as C. acetobutylicum or C. beijerinckii. In recent years however, there has been the growing awareness that there is a considerable degree of heterogeneity amongst strains currently classified as C. acetobutylicum. This has highlighted the need for a re-examination and re-assessment of the taxonomic and phylogenetic relationships of this industrially important group of bacteria.
In this study, 72 solvent-producing clostridial strains, the majority of which are currently classified as C. acetobutylicum, were examined by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. DNA fingerprinting was achieved by digesting genomic bacterial DNA with infrequently cutting restriction endonucleases and resolving the resulting fragments by pulsed-field gel electrophoresis (PFGE). Based on the results obtained from these two approaches the 72 strains could be divided into 17 distinct groups. There was a high degree of correlation between the biotypes and DNA fingerprints within each group indicating that the strains were similar both phenotypically and genotypically. The DNA fingerprints obtained for strains belonging to the same group exhibited high levels of similarity but differed markedly between the 17 groups.
To establish the relationships of the different groups, the partial 16S rRNA gene sequence, corresponding to positions 830-1383 (Escherichia coli numbering), of a prototype strain from each group was determined. This was achieved by polymerase chain reaction (PCR) amplification and direct sequencing of the resultant PCR product using primers designed in this study. DNA sequence analysis of this segment of the 16S rRNA gene provided a relatively quick and reliable method of comparing the prototype strains. The results obtained of a comparative analysis of the partial 16S rRNA gene sequences, indicated that the 17 biotype and DNA fingerprint groups could be assembled into four taxonomic groups (taxonomic groups I-IV). In order to establish more definitive phylogenetic positions for the four taxonomic groups of solvent-producing clostridia, the complete 16S rRNA gene sequences were determined for the strains ATCC 824(T), NCP 262, Nl-4, and NCIMB 8052 which represented the taxonomic groups I, II, III, and IV, respectively. The phylogenetic analysis of the complete 16S rRNA gene sequences revealed that amylolytic strains belonging to taxonomic group I were only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90%- 90.5%). The NCIMB 8052 strain (taxonomic group IV), formerly catalogued as being equivalent to the C. acetobutylicum type strain ATCC 824(T) (taxonomic group I), was found to belong to a different species based on 16S rRNA gene sequence analysis. The NCIMB 8052 strain exhibited 100% level of 16S rRNA gene sequence similarity with the type strain of C. beijerinckii. The strains belonging to taxonomic groups II, III, and IV were much more closely related (levels of sequence similarity, 98.2%- 98.9%). Based on the 16S rRNA gene sequences alone it was not possible to determine whether these three taxonomic groups of saccharolytic solvent-producing clostridia constituted three separate species or were three subgroups belonging to a single species since they exhibited sequence similarities higher than 97%. However, during the course of this study the results obtained by Johnson and Chen (1995) from genomic DNA-DNA hybridization studies of solvent-producing clostridia, established that the three taxonomic groups II, III, and IV were all separate species of saccharolytic solvent-producing clostridia. Their analysis also supports the finding that taxonomic group I consisted of a collection of distantly related amylolytic strains originally designated C. acetobutylicum. The outcome of this study and that of other researchers has indicated that all of the saccharolytic strains presently classified as C. acetobutylicum will need to be reclassified. The names "C. saccharo-butyl-acetonicumliquefaciens," "C. saccharoperbuty/acetonicum," and C. beijerinckii are proposed for the three saccharolytic solvent-producing Clostridium species on the basis of prior usage.||