Insights into nitrogen fixation below the euphotic zone: trials in an oligotrophic marginal sea and global compilation

Abstract. Nitrogen (N₂) fixation, the energetically expensive conversion of N₂ to ammonia, plays an important role in balancing the global nitrogen budget. Defying historic paradigms, recent studies have detected non-cyanobacterial N₂ fixation in deep, dark oceanic waters. Even low volumetric rates can be significant considering the large volume of these waters. However, measuring aphotic N₂ fixation is an analytical challenge due to the low particulate nitrogen (PN) concentrations. Here, we investigated N₂ fixation rates in aphotic waters in the South China Sea (SCS). To increase the sensitivity of N₂ fixation rate measurements, we applied a novel approach requiring only 0.28 μg N for measuring the isotopic composition of particulate nitrogen. We conducted parallel ¹⁵N₂-enriched incubations in ambient seawater, seawater amended with amino acids and poisoned (HgCl₂) controls, along with incubations that received no tracer additions to distinguish biological N₂ fixation. Experimental treatments differed significantly from our two types of controls, those receiving no additions and killed controls. Amino acid additions masked N₂ fixation signals due to the uptake of added ¹⁴N-amino acid. Results show that the maximum dark N₂ fixation rates (1.28 ± 0.85 nmol N L⁻¹ d⁻¹) occurred within upper 200 m, while rates below 200 m were mostly lower than 0.1 nmol N L⁻¹ d⁻¹. Nevertheless, N₂ fixation rates between 200 and 1000 m accounted for 39 ± 32 % of depth-integrated dark N₂ fixation rates in the upper 1000 m, which is comparable to the areal nitrogen inputs via atmospheric deposition. Globally, we found that aphotic N₂ fixation studies conducted in oxygenated environments yielded rates similar to those from the SCS (< 1 nmol N L⁻¹ d⁻¹), regardless of methods, while higher rates were occasionally observed in low-oxygen (< 62 µM) regions. Regression analysis suggests that particulate nitrogen concentrations could be a predictive proxy for detectable aphotic N₂ fixation in the SCS and eastern tropical south Pacific. Our results provide the first insight into aphotic N₂ fixation in SCS and support the importance of the aphotic zone as a globally-important source of new nitrogen to the ocean.