Methodology

Sediment samples were collected near Smith Island, VA in Smith Island Bay. Smith Island is one of the southern barrier islands of the Delmarva Peninsula and part of the Virginia Coastal Reserve Long Term Ecological Research site. A restored oyster reef, isolated on a mudflat, clam bed used as part of an aquaculture lease and subtidal sand flat were established as sampling locations. Studies were conducted seasonally in August 2013, April 2014, July 2014, and November 2014. At each event, samples were taken for sediment biogeochemical flux incubations and sediment physico-chemical properties (sediment organic matter, benthic algal biomass, porewater nutrients).

For sediment flux incubations, triplicate sediment cores (9.5cm i.d. X 10 cm sediment depth) were collected by hand at each of the three locations: restored oyster reefs, clam aquaculture beds and subtidal bare sediments located approximately 50m from the reef and aquaculture. Samples from the oyster reef were collected directly adjacent to the reef. For the clam aquaculture samples, the predator exclusion net was taken off prior to sample collection. Associated infauna were not removed from any of the samples (i.e. live clams were included in the clam aquaculture cores). On average there were 4 clams per core with a mean biomass density of 493 ± 42.94g DW m-2. However, oyster reef cores did not contain live oysters. Water chemistry was assessed with a YSI and ~170 L of water was collected from Smith Island Bay for use in the continuous flow core incubations. Samples were also collected for dissolved nutrient analysis.

Upon collection, sediment cores and water were transported to an environmental chamber set to in situ temperature at The Virginia Institute of Marine Science (VIMS) in Gloucester Point, VA. Once at VIMS, cores were submerged in site water to mimic high tide (saturated) conditions and held in the dark for 12-16 hrs. The following day each core was sealed with gas tight lids equipped with an inflow and outflow port and incubated in a continuous flow system. Cores were acclimated for 24 hours before sampling to allow the system to reach steady state. Samples for dissolved nutrients and gasses were collected from the outflow port of each sediment core three times over the course of 24 hours after an initial pre-incubation period. A bypass line that flowed directly into the sample vial was used to determine the concentration of dissolved constituents entering the cores. This line also accounted for tubing and pump effects on water chemistry.

Isotopic enrichment experiments were conducted to determine rates of denitrification. After the initial sampling period, the reservoir water was enriched with 15N-NaNO3 (98atm%) to a final concentration of ~100 μmol L-1. Samples were collected for dissolved gas analysis three times after the initial 24-hour acclimation period after the reservoir was enriched. The concentration of 29N2 and 30N2 was measured using a membrane inlet mass spectrometer (MIMS). The details of methods are published in Smyth et al. (2017).

Nutrients and isotopic composition of N2 and NH4+ were used to calculate the fluxes and rates as reported in Smyth et al. (2017).