Assessment of bacterial species present in Pasig River and Marikina River soil using 16S rDNA phylogenetic analysis

Abstract

The Pasig River system, which includes its major tributaries, the Marikina, Taguig-Pateros, and San Juan Rivers, is the most important river system in Metro Manila. It is known to be heavily polluted due to the dumping of domestic, industrial and solid wastes. Identification of microbial species present in the riverbed may be used to assess water and soil quality, and can help in assessing the river’s capability of supporting other flora and fauna. In this study, 16S rRNA gene or 16S rDNA sequences obtained from community bacterial DNA extracted from riverbed soil of Napindan (an upstream site along the Pasig River) and Vargas (which is along the Marikina River) were used to obtain a snapshot of the types of bacteria populating these sites. The 16S rDNA sequences of amplicons produced in PCR with total DNA extracted from soil samples as template were used to build clone libraries. Four positive clones were identified from each site and were sequenced. BLAST analysis revealed that none of the contiguous sequences obtained had complete sequence similarity to any known cultured bacterial species. Using the classification output of the Ribosomal Database Project (RDP) Classifier and DECIPHER programs, 16S rDNA sequences of closely related species were collated and used to construct a neighbor-joining phylogenetic tree using MEGA6. Six out of the 8 cloned samples were found to belong to obligate anaerobe species, suggesting that these species live deep within the sediment layer and do not have access to dissolved oxygen. Three species were found to be associated with sulfate-reducing bacteria, which suggests an abundance of sulfur containing compounds in the riverbed. This is the first census report of the Pasig River microbial population using an approach that utilizes 16S rDNA sequences without culture nor isolation of bacteria. Further studies employing multiple composite samples and larger sample sizes are recommended for more comprehensive bacterial taxonomic profiles as well as evaluation of interactions between community members and bacterial response to environmental perturbations. Keywords—16S rDNA, metagenomics, Pasig River system, DNA sequence analysis, molecular phylogeny