Abstract
Nitrogen (N) is an important element in the marine ecosystem that often limits primary production. Cycling of N from organic to inorganic forms occurs through many oxidation states, and is dependent on a variety of microbial activities. Nitrate (NO₃⁻) can be used in several microbial respiratory pathways, including denitrification, anammox, and dissimilatory NO₃⁻ reduction to ammonia (NH₄⁺; DNRA). Denitrification starts with NO₃⁻ reduction to nitrite (NO₂⁻) and ultimately results in N loss as gaseous forms (N₂O and N2). Denitrification can serve as a "purifying" N sink in eutrophic coastal systems, but the intermediate N₂O is a potent greenhouse gas. In contrast, DNRA does not result in N loss from the system. This study investigated the potential for these dissimilatory NO₃⁻ reduction processes in the microbiome of copepods, the most abundant mesozooplankton in the ocean. Several bacteria with genes for dissimilatory NO₃⁻reduction were isolated from copepods collected from coastal North Atlantic Ocean. Draft genome data show that Marinobacter sp. NCT7M contains the entire canonical denitrification pathway with all steps from NO₃⁻ to N₂, while Vibrio sp. NCT10V only contains the pathways for the alternate, periplasmic NO₃⁻ reduction and DNRA. Culture studies with these bacterial strains were conducted in collaboration with researchers at the University of Southern Denmark. Reverse transcriptase-quantitative PCR showed that expression of genes involved in denitrification (napA, narG, nirS, nosZ) occurred under all oxygen conditions, and initial concentrations of NO₃⁻ decreased over time, coinciding with a rise in the expected product of the reduction, NO₂⁻. Both ³⁰N₂ and ²⁹N₂ were generated from 15NO₃⁻, as evidence of denitrification in NCT7M. Unexpectedly, the relative level of expression of the Marinobacter sp. NCT7M denitrification genes and rate of production of N₂ were not dependent on oxygen concentration. However, the lack of nrfC gene expression and no production of ¹⁵NH₄⁺ from ¹⁵NO3- suggested that DNRA was not active in Vibrio sp. NCT10V, although its napA gene was expressed. The results suggest that temperate marine copepods may provide a previously unrecognized habitat for denitrifiers. In addition, Marinobacter sp. NCT7M appears to be an aerobic denitrifier that are rarely considered in marine ecosystem studies.