Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods

Abstract

The use of denaturing gradient gel electrophoresis (DGGE) and traditional culture-depending methods for examining the bacterial community of traditional cassava starch fermentation were investigated. It appeared that DGGE profiles of total DNA of cassava dough exhibited 10 distinguishable bands. In contrast, DGGE fingerprints of bacteria recovered from enrichment cultures of fermented dough gave variable profiles containing fewer bands. Bands corresponding to five bacterial species detected by direct PCR-DGGE of total DNA from of cassava dough were also observed in DGGE patterns of enrichment cultures. Eighteen strains were isolated from cultures selected on the basis of their DGGE banding patterns. Assessment of bacterial identification by 16S rDNA sequence similarity revealed that band comigration implied sequence identity. Comparison of 16S rDNA sequences of excised DGGE bands and recovered pure culture isolates with those in GENBANK and the RDP databases revealed that representative bacteria of fermented cassava dough were Lactobacillus and Pediococcus species as well as species of Clostridium, Propionibacterium and Bacillus. Some Lactobacillus species detected in dough samples by sequence analysis of DGGE bands were not recovered in any of the five culture media and conditions used. On the other hand, some species recovered as pure cultures from enrichments were not detected by direct DGGE analysis of total bacterial DNA from cassava dough. Our results provide evidence of the necessity to combine both culture-dependent and culture-independent methods for better description of microbial communities in indigenous cassava starch fermentations.