Dataset: Saanich Inlet field and laboratory methods related to experimental rate data acquired aboard the R/V John Strickland during two cruises in August, 2021 and June, 2022

Sample collection

For all rate experiments, water was sampled through gas-tight Viton tubing into vials or glass bottles filling from the bottom with three times overflow. Vials or bottles were closed bubble-free with deoxygenated butyl rubber stoppers and kept cool/dark until return to the laboratory. Collection vessels included 20-mL glass vials (2021) or 250-mL glass serum bottles (2022) for N₂O production experiments, 500-mL glass bottles for N₂O consumption, anaerobic ammonia oxidation, denitrification, dissimilatory nitrite reduction to ammonium, and nitrite oxidation experiments, and 250-mL glass serum bottles for dark carbon fixation experiments.

Experimental set-up

Laboratory set up for N₂O production: In 2021, a 2-mL N₂ headspace was introduced in each 20-mL vial before it underwent 2 cycles composed of 30 seconds of vigorous shaking followed by 30 seconds of flushing with N₂. Each vial was then amended with one of three ¹⁵N-labeled tracer solutions (¹⁵NH₄⁺/¹⁴NO₂^-, ¹⁵NO₂^-/¹⁴NH₄⁺, ¹⁵NO₃^-/¹⁴NH₄) to quantify rates of N₂O production from NH₄⁺ oxidation and NO₃^- and NO₂^- reduction. Each tracer solution was added to six vials for a 2 µmol·L^-1 increase in the concentration of both the ¹⁵N and ¹⁴N  substrates. In 2022, a helium headspace was added to 250-mL bottles followed immediately by purging of the water for 30 minutes with helium. Each bottle was then amended with one of the three tracer solutions and the liquid dispensed into replicate 12-mL exetainer vials for incubation. Each vial was injected with 1µM unlabeled ¹⁴N₂O to stem the loss of ¹⁵N-labeled N₂O to other processes (2022 only). In both years, vials were incubated in the dark at in situ temperature and were terminated at 0 and 12 hours (2021) or 0, 9, and 18 hours (2022) by injection of saturated mercuric chloride.

Laboratory set up for N₂O consumption: A headspace was introduced followed immediately by purging of the water for 30 minutes with helium. Water was dispensed with > 2 volumes overflow into 12-mL exetainer vials that were immediately closed with deoxygenated chlorobutyl rubber septa. Each vial received a 2-mL helium headspace before undergoing 2 cycles composed of 30 seconds of vigorous shaking followed by 30 seconds of flushing with helium.  Each vial was then injected with ¹⁵N₂O (1 µM) and incubated in the dark at in situ temperature, and were terminated at four time points, 0, 6, 12 and 24 hours, by injection of saturated mercuric chloride.

Laboratory set up for anaerobic ammonia oxidation, denitrification, dissimilatory nitrite reduction to ammonium, and nitrite oxidation: A headspace was introduced and ¹⁵NO₂ added (5 µM) followed immediately by purging of the water for 30 minutes with helium. Water was dispensed with > 2 volumes overflow into 12-mL exetainer vials that were immediately closed with deoxygenated chlorobutyl rubber septa. Each vial received a 2-mL helium headspace before undergoing 2 cycles composed of 30 seconds of vigorous shaking followed by 30 seconds of flushing with helium.  Vials were then incubated in the dark at in situ temperature, and were terminated at four time points, 0, 6, 12 and 24 hours, by injection of saturated mercuric chloride.

Laboratory set up for dark carbon fixation: ¹³C-DIC was introduced into each serum bottle and a 20-mL helium headspace inserted, before undergoing 2 cycles composed of 30 seconds of vigorous shaking followed by 30 seconds of flushing with helium.  A sample of the headspace was placed into a 3 mL glass vial, and preserved with mercuric chloride, headspace free for labeling percentage determination (averaged 20 %). Serum bottles were then incubated in the dark at in situ temperature, and duplicate bottles were terminated at two time points 0 and 24 hours by filtration onto pre-combusted glass fiber filters (GF-75). Filters were subsequently oven dried and stored at room temperature until analysis.