We performed shotgun metagenomic sequencing (on Illumina HiSeq 2500 platform) of DNA extracted from water samples collected from freshwater ponds located across studied urbanization gradients. Quality trimmed and assembled scaffolds were used for gene prediction and were further summarized into a gene-catalog of ~10 million genes. This gene catalog was annotated against multiple databases including KEGG, Pfam, TIGRFAM and eggNOG to gain multi-view insights into functional responses of microbes to urbanization. Gene abundances were calculated by mapping metagenomic reads on the gene catalog. Molecular trait matrices of relative frequencies of KEGG pathways and modules associated with biogeochemical cycles, pesticide degradation and other relevant environmental pathways were created and their responses to urbanization (defined as percentage built-up area around studied ponds), abiotic and biotic variables and land-use variables were determined.
The resulting gene catalog of more than 10 million microbial genes, annotated against multiple databases is an extremely valuable resource, which will be useful for researchers studying microbial responses to anthropogenic stress in freshwater habitats. We found significant differences in nitrogen fixation pathways among urban and rural ponds with rural ponds showing a higher abundance of nif D genes. A marked difference in pathway of chemoautotrophic oxidation of sulfur compounds was noticed along the studied gradient of urbanization. In addition, we were also able to detect other metabolic processes relevant to environmental microbes, which varied between ponds situated in highly urbanized areas (built-up area more than 15%) and rural areas (built-up area less than 3%), namely ascorbate and aldarate metabolism, caprolactam degradation, selenocompound metabolism, folate biosynthesis, linoleic acid metabolism, biosynthesis of unsaturated fatty acids & pentose and glucouronate interconversion. We also found a significant difference in the abundance of a gene (K14541: urea carboxylase / allophanate hydrolase) involved in the atrazine degradation pathway between rural and urban ponds. Majority of these metabolic pathways were influenced by local factors rather than regional factors. Together, these results indicate strong differences in metabolic potential of microbial communities across various levels of urbanization and point towards the fact that microbial communities predominantly respond to land use changes on a local scale. This is important in policy decisions, as it informs us on the scale at which land use must be managed for optimum ecosystem functioning.
