Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Project Information
• Funding

These data were collected during July 2020 from a sandy subterranean estuary (STE) located in Gloucester Point, Virginia, USA. A pole-corer was used to collect triplicate sediment cores roughly 50cm in length from the mid tide line of the beach during low tide. Cores were sectioned into ten centimeter increments. Porewater was collected from surface water and piezometers placed at 10cm increments to the same depth of 50cm.  Core sections were used in isotope tracer incubations with 15N-labeled substrates to measure potential nitrification rates. Briefly, 10 g of the composited sediment from each 10 cm section were used in sediment slurry incubation experiments with 40mL of porewater from the same depth interval. Samples were incubated for 0, 6, and 12 hours in in 100mL HDPE bottles and amended with 5 atom% 15 µM 15NH4+ (Cambridge Isotope) at the measured in situ temperature. After the incubation period, samples were spun down, porewater was decanted and filtered with a 0.45 µm syringe filter (Whatman GE) and were frozen (-20 °C) until analysis.  Porewater nutrient concentrations were analyzed for NOx (nitrate + nitrite), nitrite, and ammonium with a Lachat autoanalyzer. The isotopic enrichment of nitrate  and nitrite products were measured  using a gas bench isotope ratio mass spectrometer (IRMS, Delta V Plus, Thermo Fisher Scientific, Waltham, MA) using the bacterial reduction method. A culture of Pseudomonas aureofaciens reduced NOx in collected samples to N2O. The isotopic composition of the N2O was measured with isotope-ratio mass spectrometry (IRMS) (Sigman et al., 2001). 

Instruments used: Lachat QuikChem 8000 automated ion analyzer Lachat In- struments, Milwaukee, WI, USA; detection limits for NO3−, NH4+, and PO43− are 0.20, 0.36, and 0.16 μM, respectively. Gas bench isotope ratio mass spectrometer (IRMS) Delta V Plus, Thermo Fisher Scientific, Waltham, MA.

NSF Award Abstract:
Nitrogen is an important nutrient that maintains high coastal ecosystem productivity. Yet excess nitrogen delivery can cause serious water quality deterioration including harmful algal blooms, fish kills, and oxygen free dead zones. Numerous nitrogen transformations regulate the balance between nitrogen delivery and nitrogen removal in coastal environments and the majority of these reactions occur in sediments where seawater passes through the subsurface and mixes with groundwater transported from uplands. This mixing zone, referred to as the subterranean estuary, is characterized by very different geochemistry than either the seawater above it or the groundwater below it. Thus, it has the potential to host a variety of unique reactions that affect nitrogen availability to the overlying water. Scientists from the College of William and Mary, Virginia Institute of Marine Science (VIMS), and the University of Connecticut (UConn) propose to examine the importance of a cryptic nitrogen cycle, a novel and potentially widespread nitrogen cycling process in the subterranean estuary. The cryptic nitrogen cycle comprises anoxic ammonium oxidation to nitrite (anoxic nitrification) coupled with anaerobic ammonium oxidation (anammox) or denitrification producing harmless dinitrogen gas. The proposed project represents highly transformative science because it has the potential to change the current paradigm detailing operation of the biogeochemical nitrogen cycle in anoxic environments. Occurrence of the cryptic nitrogen cycle would have broad implications for the nitrogen budget of terrestrial and groundwater systems and the coastal ocean. Characterization of the cryptic nitrogen cycle will allow us to better understand interactions among the nitrogen, metals, and sulfur cycles, and potential impacts of ongoing human modification of coastal environments. Educational contribution of this project focuses on graduate and undergraduate student training. Two graduate students at VIMS and UConn will receive interdisciplinary training in microbiology, molecular ecology, and biogeochemistry while several undergraduates recruited through the VIMS REU (Research Experience for Undergraduates) Program and the UConn marine science programs will also participate in the project. In addition, three summer undergraduate interns will be recruited from Hampton University, a historically Black college, and trained to enhance minority education and research in marine science. Public outreach will be achieved through popular venues such as VIMS Marine Science Day, and the VIMS After Hours Public Lecture Series at VIMS. Tobias at UConn also provides educational contributions and outreach efforts through the UConn Marine Scholars and Early College Experience programs and an exhibit at Mystic Aquarium.

A cryptic nitrogen cycle is proposed as a new process coupling anoxic nitrification to microbial nitrogen removal pathways such as anammox and denitrification. Unlike anammox, which refers to the oxidation of ammonium by nitrite to form dinitrogen (N2) gas, anoxic nitrification occurs by oxidation of ammonium in the absence of oxygen using other common chemical oxidants such as metal oxides (namely, Fe and Mn) or sulfate, abundant in many marine and coastal systems. The thermodynamic favorability of these reactions relies on coupling nitrite formed via these oxidants with anammox or denitrification. Due to the coupling, nitrite will not accumulate or be measurable in anoxic marine systems. Thus, a cryptic N cycle responsible for nitrite production can occur as a novel N transforming process in anoxic environments, serve as a vital link to N2 production, and attenuate N loads discharging from a subterranean estuary (STE). Preliminary results from a STE in the York River Estuary located in Virginia showed substantial N2 production, representing removal of 50-75% of the fixed groundwater N, in ferruginous and sulfidic zones where neither nitrite nor nitrate were detectable. Stable isotope incubation experiments using the 15N tracer and molecular analysis of microbial communities suggest that coupled anoxic nitrification and anammox processes are the dominant N2 production pathways rather than canonical denitrification in the STE. Therefore, coupled anoxic nitrification-anammox in coastal groundwater may be a major unrecognized sink for fixed nitrogen at the land-sea interface. In addition to coastal groundwater, the cryptic N cycle has potential importance in anoxic zones and ocean basins. This proposal focuses on the STE because geochemical conditions there appear optimal for the proposed reactions to occur, and our preliminary data show strong evidence for a cryptic N cycle. The proposed work uses a combined geochemical, 15N isotope tracer and microbiological approach to evaluate environmental controls on the cryptic N cycle as well as to estimate its contribution to reduction of fixed N fluxes to the coastal ocean. Four approaches are proposed: (1) Field characterization of anoxic nitrification reactions and associated microbial communities in a subterranean estuary; (2) Laboratory incubation experiments to identify hotspots of the cryptic N cycle; (3) Controlled microcosm experiments to determine geochemical controls on anoxic nitrification; and (4) in situ assessment of anoxic nitrification to estimate the importance of the cryptic N cycle in a coastal aquifer.