N-204. Molecular Detection of Anaerobic Ammonium Oxidizing (ANAMMOX) Bacteria in North Carolina Groundwater Aquifers

M. D. Hirsch, B. Song;
Univ. of North Carolina, Wilmington, NC.

Dissolved inorganic nitrogen (DIN: NH4+, NO3-, and NO2-) is commonly found in groundwater due to anthropogenic inputs such as fertilizer and feedlots. The presence of nitrate/nitrite is of particular concern because nitrite causes blue baby syndrome. Previously, denitrification was considered to be the main removal process of N in groundwater. Anaerobic ammonium oxidation (anammox), a process recently discovered in a wastewater treatment plant, might be involved in groundwater N removal although there is no report for the detection of anammox in groundwater systems. Thus, we conducted molecular analysis to detect anammox bacteria in groundwater samples obtained from underground aquifers of in the piedmont region of North Carolina. Environmental DNA was extracted from the Millipore SterivexTM filter after filtering 1 L of groundwater samples. Anammox bacterial specific PCR amplification detected the presence of anammox bacteria in groundwater samples based on 16s rRNA genes. Phylogenetic analysis of the amplified 16S rRNA genes showed that most of the sequences were closely related to the anammox genus Brocadia, however the sequences from one aquifer formed their own group separate from any of the known anammox genera. In addition, a new molecular detection method for anammox bacteria was developed by targeting hydrazine oxidase (hzoAB) genes, which are essential for the anammox pathway. Using new primers developed in our lab, the hzoAB genes were detected from all of the water samples. Phylogenetic analysis of the hzoAB genes showed that the detected genes were closely related to the genes found in the consortium of Brocadia. We also found a positive correlation between the phylogenies of the 16s rRNA genes and the hzoAB genes. Based on the correlation, we were able confirm the presence of novel anammox bacteria in the groundwater sample where the 16S rRNA genes were not closely related to any of the known anammox bacteria. Therefore, this study provided the first evidence for the presence of anammox bacteria in underground aquifers and demonstrated new molecular methods for anammox bacteria detection based on the hzoAB genes.