Subsurface drains (tile drains) used to augment drainage in agricultural fields serve as a major pathway for agricultural nitrate pollution to enter surface waters. Used primarily in the Midwestern United States, nitrates from tile drainage systems contribute to eutrophication within the Gulf of Mexico, ultimately leading to the formation of the Gulf of Mexico hypoxic zone. One cost-effective solution for reducing the quantity of nitrate entering surface waters is the denitrifying biofilter. A typical denitrifying biofilter consists of a woodchip-filled trench inline with the drainage tile; woodchips provide a carbon substrate to the microorganisms that convert nitrate to nitrogen gas through the denitrification pathway. Research to date has focused on applying traditional engineering approaches to improve biofilter performance and reliability. Although previous work has produced valuable results related to the selection of appropriate biofilter media, and optimization of operational parameters, denitrifying biofilters still perform somewhat unpredictably. Therefore, in this work we sought to understand how environmental and management factors affect the microbial communities responsible for biofilter functional. To do so, we employed two different approaches. First, in our spatial study we sampled one biofilter over the course of an afternoon in 2007 to determine how total and denitrifying bacterial communities varied by depth, transect, and position along a transect. Second, in our temporal study we sampled three biofilters over two years, January 2009 – December 2010, to determine how total bacterial, denitrifying bacterial, and fungal communities correlated with environmental and management variables over time. Total bacterial community structure was analyzed by Automated Ribosomal Intergenic Spacer Analysis (ARISA), denitrifying bacteria community structure was determined by Terminal Restriction Fragment Length Polymorphism (T-RFLP) of nosZ (one of the nitrous oxide reductase genes), and fungal ARISA (FARISA) was used to determine fungal community structure. Spatial and temporal results from the biofilter studies have provided valuable insight into how microbial communities, essential to the functionality of the denitrifying biofilter, vary over space and time. Results from our spatial study indicate that the composition of the total bacterial community varied by depth and sampling transect, but not by sampling position along a transect. Denitrifying bacteria community composition, unlike total bacteria, showed little variance by depth, transect, or sampling position. Results from our temporal study indicate that depth and season were two of the most important factors influencing the structure of total bacterial, fungal, and denitrifying bacterial communities within all three biofilters. Correspondence analysis results suggest that microbial community structuring by depth may have been driven by moisture and temperature gradients. In addition to depth, microbial community composition was influenced by seasonal factors within all three denitrifying biofilters. For 2009 and 2010 bi-annual seasonal variation was observed for samples collected in January – June or July – December. Results from correspondence analysis suggest that seasonality was likely driven by moisture, water flow, and temperature. In addition to observing patterns in community composition related to depth and season, we were able to identify small subsets of the total bacterial, denitrifying, and fungal populations that were either influential in shaping the overall community structure, were correlated to strong biofilter performance, or both. The application of denitrifying biofilters in tile drain networks shows the promise of significantly reducing anthropogenic inputs of nitrogen into aquatic ecosystems. By developing an understanding of how microbial population dynamics, environmental parameters, and management factors relate to biofilter performance, reliability, stability and resilience, the effectiveness and viability of the denitrifying biofilter as a treatment technology will ultimately be enhanced.
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