Unexpectedly high degree of anammox and DNRA in seagrass sediments: Description and application of a revised isotope pairing technique

Understanding the magnitude of nitrogen (N) loss and recycling pathways is crucial for coastal N management efforts. However, quantification of denitrification and anammox by a widely-used method, the isotope pairing technique, is challenged when dissimilatory NO3− reduction to NH4+ (DNRA) occurs. In this study, we describe a revised isotope pairing technique that accounts for the influence of DNRA on NO3− reduction (R-IPT-DNRA). The new calculation procedure improves on previous techniques by (1) accounting for N2O production, (2) distinguishing canonical anammox from coupled DNRA-anammox, and (3) including the production of 30N2 by anammox in the quantification of DNRA. This approach avoids the potential for substantial underestimates of anammox rates and overestimates of denitrification rates in systems where DNRA is a significant NO3− reduction pathway. We apply this technique to simultaneously quantify rates of anammox, denitrification, and DNRA in intact sediments adjacent to a seagrass bed in subtropical Australia. The effect of organic carbon lability on NO3− reduction was also addressed by adding detrital sources with differing C:N (phytoplankton- or seagrass-derived). DNRA was the predominant pathway, contributing 49-74% of total NO3− reduction (mean 0.42 µmol N m−2 h−1). In this high C:N system, DNRA outcompetes denitrification for NO3−, functioning to recycle rather than remove N. Anammox exceeded denitrification (mean 0.18 and 0.04 µmol N m−2 h−1, respectively) and accounted for 64-86% of N loss, a rare high percentage in shallow coastal environments. Owing to low denitrification activity, N2O production was ∼100-fold lower than in other coastal sediments (mean 7.7 nmol N m−2 h−1). All NO3− reduction pathways were stimulated by seagrass detritus but not by phytoplankton detritus, suggesting this microbial community is adapted to process organic matter that is typically encountered. The R-IPT-DNRA is widely applicable in other environments where the characterization of co-existing NO3− reduction pathways is desirable.