MOLECULAR QUANTIFICATION OF IN SITU EXPRESSION OF FUNCTIONAL GENES IN ANAMMOX AND DENITRIFYING COMMUNITIES IN THE CAPE COD AQUIFER

In Cape Cod, MA, land dumping of over 8 billion gallons of treated sewage from 1936 to 1995 by the Massachusetts Military Reservation served as the source of a contamination plume within the Cape Cod aquifer. The plume has been suboxic to anoxic and contains high concentrations of nitrate (NO₃^-) and ammonium (NH₄⁺). In order to determine in situ activities of anammox and denitrification removing N from the aquifer, ¹⁵NO₂^- tracer tests were conducted and gene expression of anammox and denitrifying communities were quantified through qPCR of cDNA for hzo (hydrazine oxidoreductase) and nosZ (nitrous oxide reductase) genes, respectively. Tracer Test 1 was conducted at an altitude within the aquifer where NH₄⁺ concentrations were 10 µM. In the experiment, transcriptional expression of anammox and denitrification genes was evident before the injected NO₂^- arrived at the sampling location, but increased in the presence of the tracer, reaching a maximum expression level before the NO₂^- concentration reached a peak. In Tracer Test 2, which was conducted in a zone in which background NH₄⁺ concentrations were low, there was little to no background expression of genes before the arrival of NO₂^-. Both N removal genes reached high levels of expression concurrent with or after the tracer, NO₂^-, concentration reached a maximum. It is surmised that transcriptional expression of hzo and nosZ genes is already occurring where high levels of NH₄⁺ are present within the aquifer, and if NO₂^- is delivered within the aquifer system, both processes quickly respond and increase transcriptional expression. Thus, NO₂^- availability is found to be key in enhancing the expression of the N removal processes, anammox and denitrification, in a nitrogen-contaminated aquifer.