Bacteria In Activated Waste-Water Through Culture-Independent Molecular Techniques

Uncultivable bacteria, those that resist cultivation, represent an estimated 99% of environmental microbial communities. These uncultivable bacteria are being classified using culture-independent molecular techniques such as cloning and phylogenetic analysis based on the sequence of the 16S rRNA gene. Numerous novel bacterial lineages have been discovered in the last 10 years through such approach. Our goal was to characterize the diversity of bacteria in wastewater through a phylogenetic tree. We hypothesized that novel 16S rRNA gene sequences would be found in samples taken from the secondary tanks of a wastewater treatment plant. To identify those novel sequences we performed PCR with broad-range primers targeting 16S rDNA, cloned, sequenced and used bioinformatics for its analysis. We used the NCBI Genbank to blast 100 sequences and acquire their known affiliation based on 90% of maximum identity. Sequence analysis at the phylum level resulted in 85% of Proteobacteria, 6% of Bacteroidetes, 6% of Actinobacteria and the final 3% composed of Acidobacteria, Firmicutes and Tenericutes each. These results were compared to a second set of blasted sequences, which were also abundant in Proteobacteria. We concluded that the Proteobacteria phylum dominates an environment such as wastewater. With this type of research we are capable of describing how different groups of bacteria are organized in a specific type of environment. We are currently in the process of generating a phylogenetic tree with our unique sequences.