Sandy seepage faces as bioactive nitrate reactors: Biogeochemistry, microbial ecology and metagenomics

Subterranean estuaries are highly dynamic in processing dissolved inorganic nitrogen (DIN). Here we investigate DIN turnover in surface sediments (0-20 cm depth) at the higher, medium and lower intertidal of a seepage face, i.e., the outer “mouth” of the subterranean estuary, during four consecutive seasons in Sanggou Bay, China. Throughout the studied period, ammonium (NH₄⁺) and nitrite (NO₂⁻) concentrations in the sampled porewaters did not vary significantly with depth or season. In contrast, peaks in porewater nitrate (NO₃⁻) concentration and decreases in δ¹⁵N-NO₃⁻ and δ¹⁸O-NO₃⁻ were observed in the 15-20 cm depth (bottom) sediment, particularly during summer and autumn. Coupled with NO₃⁻ production, the sediment total nitrogen was also markedly peaking in the bottom layer of the studied seepage face. Together with abundant heterotrophic microbes in the sediment, this NO₃⁻ accumulation was linked to a reaction chain including organic matter decomposition, ammonification and nitrification. During winter, porewater enrichment in total nitrogen occurred closer to the surface of the seepage face but triggered also active NO₃⁻ production. This pattern reinforced the importance of pelagic organic matter supply on NO₃⁻ production. In the shallower depths of the seepage face (<12 cm), active net NO₃⁻ removal occurred except in winter. The isotopic fractionation (δ¹⁵N-NO₃⁻ and δ¹⁸O-NO₃⁻) and metagenomic results revealed denitrification as the main pathway for NO₃⁻ reduction. Biological assimilation from benthic primary producers may also consume a fraction of NO₃⁻ at the sediment water interface. Both NO₃⁻ production and removal significantly varied in magnitude with season (−13.6 to 6.2 nmol cm⁻³ h⁻¹). Substrate supply was the key driver for nitrate cycling, as evidenced by the high NO₃⁻ production rate in spring by comparison to autumn. The highest NO₃⁻ turnover rates were found in summer, suggesting the combined influence of advection rates and sediment microbiota composition. In spite of active removal (peak NO₃⁻ removal capability: 61%), a significant amount of NO₃⁻ was still transported from the seepage face into the bay waters. The magnitude of NO₃⁻ fluxes ranged from 312 to 476 kg N d⁻¹, accounting for approximately 15% of the total exogenous NO₃⁻ loading into the bay. NO₃⁻ isotopic fingerprint revealed chemical fertilizer as the main source of terrestrial NO₃⁻ in SGD, highlighting the importance of land use to coastal system nitrogen budgets.