Characterization of microorganisms contributing to denitrification in tile drain biofilters in illinois

Agricultural nitrates are thought to be a primary cause of the large hypoxic zone in the Gulf of Mexico. Subsurface woodchip biofilters developed at the University of Illinois remove on average 50-60% of nitrates from tile drains, and in some cases achieve complete removal during periods of low flow. Soil microorganisms that colonize the biofilters transform aqueous nitrate to inert nitrogen gas by the denitrification pathway. However, beyond this basic mechanism, understanding of how the biofilters function has previously been largely superficial. In particular, characterization of individual species contributing to denitrification and the overall microbial community could enable better monitoring, optimization, and control of biofilters. In this study, the type(s) of organisms contributing to denitrification were characterized using denitrification enzyme assays of woodchip samples under inhibition with either tetracycline (to inhibit bacteria) or cycloheximide (to inhibit fungi). Under tetracycline inhibition, denitrification was reduced almost to zero, suggesting that denitrification is mediated primarily by bacterial populations. However, under cycloheximide inhibition denitrification was also reduced to 30-40% of uninhibited levels. Because fungal denitrification should have resulted in activity in the presence of tetracycline, which was not observed, we hypothesize that the cycloheximide inhibition is indirect. For example, inhibiting fungal metabolism may block fungi from releasing organic substrate molecules to bacteria from the woodchips. Current work is focused on identification of particular species and characterization of the microbial community within biofilters using fluorescence in-situ hybridization (FISH) and automated ribosomal intergenic spacer analysis (ARISA).