Dataset: B. elegans cold water coral lab culture: skeleton-tissue

This methodology section describes this dataset and other closely related datasets collected as part of this study (see "Related Datasets").

Corals (Balanophyllia elegans, LSID=urn:lsid:marinespecies.org:taxname:286920) were collected by divers using blunt-tipped diving knives to remove corals from vertical rock walls at 10-20 m depths. A subset of the corals were immediately frozen for determination of N isotope ratios of tissue and skeleton. Another subset of corals were shipped live overnight to St. Olaf College for the culture experiments. 

For the culture experiment, corals were divided into four groups that were each fed Artemia  (LSID=urn:lsid:marinespecies.org:taxname:480245) nauplii with a different known d15N. The coral tissue was sampled at discrete intervals over the course of the experiment as described below. 

For the starvation experiment, corals were split into two group, starved and unstarved. The starved group was fed once every two weeks and the unstarved corals were fed twice a week. The coral tissue was sampled at discrete intervals throughouth the experiment as described below. 

Once separated from the skeleton, coral tissue was lyophilized and analyzed using a Costech Elemental Analyzer Isotope Ratio Mass Spectrometer. 

Once separated from the coral tissue, the coral skeletons were rinsed and ultrasonicated two times in Milli-Q water, then ultrasonicated in 1% sodium hypochlorite in 20 minute intervals until no tissue remained on the skeleton. The skeletons were then prepared following the methods of Wang et al (2014). The skeleton was ground to a powder using a mortar and pestle, then rinsed with sodium hypochlorite to remove any remaining tissue. The skeletal materials were then dissolved with 4N hydrochloric acid, then oxidized to nitrate by autoclaving in a basic potassium persulfate solution. Skeletal material was oxidized in tandem with standards of glutamine reference material USGS-40 and USGS-41. The samples were then analyzed by Gas Chromatography- Isotope Ratio Mass Spectrometry using the denitrifier method (Sigman et al., 2001). In brief, the denitrifier method uses the denitfrying bacteria Pseudomonas chlororaphis f. sp. aureofaciens to convert nitrate to nitrous oxide. P. aureofaciens was grown in media amended with 10mM nitrate in stoppered glass bottles for 7-10 days before being harvested and resuspended in nitrate free media. Three milliliters of resuspended bacteria was allocated to 20mL headspace vials which were sparged with dinitrogen gas for 6 hours. Nitrate sample solutions were injected into vials (target of 20nmol nitrogen for seawater samples and 7nmol for skeletal matrix samples) and incubated overnight to allow for the complete conversation of nitrate to nitrous oxide. The nitrous oxide was extracted and purified using a Thermo Gas Bench II with a GC Pal autosampler and dual cold traps and analyzed on a Thermo Advantage continuous flow isotope ratio mass spectrometer. Analyzes were referenced to N2O injected from a pure gas cylinder and standardized through comparison potassium nitrate reference materials International Atomic Energy Agency Nitrate (IAEA-N3) and the isotopic nitrate reference material from the United States Geological Survey 34 (USGS-34).

Artemia nauplii samples were stored frozen then lyophilized prior to analysis on the Elemental Analyzer Isotope Ratio Mass Spectrometer. 

Nitrate samples were collected with Van-Doren Sampler and filtered with pre-combusted glass fiber filters (GF/F, 0.7uM nominal pore size). The nitrate concentrations were determined using reduction to nitrous oxide in hot vanadium III solution followed by chemiluminescence detection of nitrous oxide on a Teledyne chemiluminescence NOx analyzer Model T200. The nitrogen and oxygen isotopes of nitrate were analyzed with the denitrifier method on an IRMS (described above).

Suspended particulate organic matter was collected with a Van-Doren Sampler and then collected on pre-combusted GF/F. The filters were lyophilized prior to analysis on an EA-IRMS.

Net tow material was collected with plankton nets with mesh sizes ranging from 80uM, 120uM, and 150uM. The net tow material was filtered and collected on a pre-combusted GF/F which was lyophilized prior to analysis on the EA.

Hydrologic depth profiles were characterized with a CastAway-CTD profiler.