The Diversity of Nitrogen-Cycling Microbial Genes in a Waste Stabilization Pond Reveals Changes over Space and Time that Is Uncoupled to Changing Nitrogen Chemistry.

Nitrogen removal is an important process for wastewater ponds prior to effluent release. Bacteria and archaea can drive nitrogen removal if they possess the genes required to metabolize nitrogen. In the tropical savanna of northern Australia, we identified the previously unresolved microbial communities responsible for nitrogen cycling in a multi-pond wastewater stabilization system by measuring genomic DNA and cDNA for the following: nifH (nitrogen fixation); nosZ (denitrification); hzsA (anammox); archaeal AamoA and bacterial BamoA (ammonia oxidation); nxrB (nitrite oxidation); and nrfA (dissimilatory NO3 reduction to NH3). By collecting 160 DNA and 40 cDNA wastewater samples and measuring nitrogen (N)-cycling genes using a functional gene array, we found that genes from all steps of the N cycle were present and, except for nxrB, were also expressed. As expected, N-cycling communities showed daily, seasonal, and yearly shifts. However, contrary to our prediction, probes from most functional groups, excluding nosZ and AamoA, were different between ponds. Further, different genes that perform the same N-cycling role sometimes had different trends over space and time, resulting in only weak correlations between the different functional communities. Although N-cycling communities were correlated with wastewater nitrogen levels and physico-chemistry, the relationship was not strong enough to reliably predict the presence or diversity of N-cycling microbes. The complex and dynamic response of these genes to other functional groups and the changing physico-chemical environment provides insight into why altering wastewater pond conditions can result an abundance of some gene variants while others are lost.