Little Lagoon is a shallow coastal lagoon that is tidally connected to the Gulf of Mexico but has no riverine inputs. The water in the lagoon is replenished solely from precipitation and groundwater inputs primarily on the East end (Su et al. 2012). Because of the rapid development in Baldwin County, a large amount of NO3- enters the Little Lagoon system through SGD (Murgulet & Tick 2008). In this region, there can be rapid changes in the depth to groundwater (Fig. 4.1 inset) and episodic SGD inputs to the lagoon (Su et al.2013). Within the lagoon, three sites were selected (East, Mouth, and West) to represent the gradient that exists across the lagoon from the input of groundwater. Sites were sampled on a near-monthly basis from February 2012 to February 2013.

DNRA 
Approximately 1 L of outflow water was collected from the inflow water and each core forDNRA analysis. Appropriate sample volume was determined after NH4 + nutrient analysis and expected atom % enrichment. δ15N-NH4 + was measured in samples, constructed blanks, and standards that bracketed the NH4 + concentration of the samples following a modified ammonium diffusion procedure (Holmes et al. 1998) that collects NH4 + dissolved in water by converting NH4 + to NH3 under basic conditions and then traps the NH3 on an acidified glass fiber filter. Non diffused standards were prepared according to Stark and Hart (1996) to account for blank corrections. After 15N analysis on a Europa Scientific SL-2020 system (Stable Isotope Lab, Utah State University), DNRA was calculated from the production rate of 15NH4 + (p15NH4 +) during the incubation according to Christensen et. al (2000): (7) where is the production of 15N-NH4 + and D14 and D15 are the denitrification rates of 14N-NO3 - and 15N-NO3 -, respectively. This assumes that DNRA takes place in the same sediment layers as denitrification and that the 15N labeling of NO3 - being reduced to NH4 + equals the 15N labeling of NO3 - being reduced to N2 (Christensen et al. 2000).

Denitrification and anammox from slurry assays

Volumetric rates of denitrification, anammox, and the relative contribution of anammox to gross N2 production were determined from sediment slurry incubations. Slurry rates for depth-integrated sediments (0-50 mm) were prepared in Exetainers (Thamdrup & Dalsgaard 2002) with artificial seawater (ASW) (70.2g NaCl, 3.0g KCl, 49.4 g MgSO4*7H2O, 5.8g CaCl2*2H2O L-1) constructed at a salinity of 52 and diluted with deionized water to match the salinity of each site. After dilution, homogenized sediment from 0 to 50 mm was added to the ASW and the incubation bottle was sparged with N2 and amended with 100 μmol L-1 Na15NO3 - (99 atom %). Sediment slurry was dispensed to 12 ml Exetainers, yielding approximately 1 ml of sediment and 11 ml ASW with no headspace. For each site, 12 vials total were incubated with three vials stopped at time points 0 to 36 h. Incubations were stopped by adding 250 μL of ZnCl2 and resealing the vials without headspace. Denitrification and anammox rates in slurries were calculated according to equations 5 and 6 described below. 

Excess 29N2 and 30N2 concentrations for intact core and slurry incubations were calculated from dissolved 29N2:28N2 and 30N2:28N2 measured using a MIMS. Rates of excess 29N2 (p29) and 30N2 (p30) production were calculated from the flux calculation described above. Rates of ambient 14N2 production (p14) in core incubations with 15NO3 - tracer addition were determined as (Nielsen 1992, Risgaard-Petersen et al. 2003): 

(1) p14 = 2 x r14 ± [p29 + p30 ± (1 - r16)]

The 14N:15N ratio of NO3 - undergoing reduction to N2 (r14) was determined as follows: 

(2) r14 = [R29 x (1 - ra) - ra] x (2 - ra) ^-1

where R29 was the ratio of p29 to p30 determined for the cores and ra was the relative contribution of anammox to gross N2 production determined in vial slurry incubations. Gross denitrification and anammox rates within intact sediment cores with 15NO3 - tracer addition were calculated as follows: 

(3) denitrification = p14 ± (1 - ra)

(4) anammox = p14 ± ra

Denitrification stimulated by the added 15N-NO3 - (D15) was calculated from the classical IPT (Nielsen 1992) and these amended rates are a measure of the denitrification capacity under field conditions when NO3 - is not limiting. 

Rates of denitrification and anammox in vial slurry incubations with 15NO3 - amendments were calculated from the equations of Thamdrup and Dalsgaard (2002): (5) (6) where FN was the fraction of 15N in NO3 -. For months when anammox slurry incubations were not performed (August and November 2012), p14 is calculated as D14 from the IPT (Nielsen 1992). Potential denitrification and anammox rates were converted to an areal basis using the wet weight of the sediment in the slurry. All rates and fluxes pertaining to N species in this study were normalized to one atom N.

Additional methodology can be found in:

Bernard, Rebecca & Mortazavi, Behzad & A. Kleinhuizen, Alice. (2015). Dissimilatory nitrate reduction to ammonium (DNRA) seasonally dominates NO3− reduction pathways in an anthropogenically impacted sub-tropical coastal lagoon. Biogeochemistry. 125. 47-64. 10.1007/s10533-015-0111-6.