Abstract:

NanoSIMS characterization of the spatial distribution of 15N and 13C in samples incubated with tracer 15N2 and 13CH4. These samples were collected in and out (27.783017 -91.507333 and 27.78525 -91.504783) of a bubble plume at GC185 and provide cell-specific measurements of the rates of N2-fixation and CH4 assimilation for comparison with bulk rate measurements. Samples were collected July 16, 2013.

Suggested Citation:

Joe Montoya, Sarah Weber. 2018. EN527/528 nanoSIMS analyses, northern Gulf of Mexico, July 16, 2013. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7V12366

Purpose:

Characterization and identification of the organisms responsible for deep water N2-fixation and methane consumption at natural seeps.

Data Parameters and Units:

All parameters are defined and units provided on the first sheet of the data workbook. A summary listing follows: nanoSIMS rates sheet: NanoSIMS-based rates of nitrogen-fixation and methane assimilation Sample ID: Sample ID for nanoSIMS analyses: dateofanalysis-Carbon/Nitrogen-ROI(region of interest)# Station: Station.event number from cruise EN528 aboard the R/V Endeavor In/Out of plume:Identifies if samples were collected inside or outside of a gas plume. Depth: Depth [meters] Temp: Seawater temperature [degC] Salinity: Seawater salinity [psu] [CH4]: Methane concentration in seawater [µmol L-1] d13CH4: delta-13C signature of seawater methane [‰] BtlVol: Volume of incubation bottles [mL] N2 Spike: Injected volume (at atmospheric pressure) of 15N-labeled nitrogen gas tracer [mL] CH4 Spike: Injected volume (at atmospheric pressure) of 13C-labeled methane gas tracer [mL] IncTime: Incubation time [hours] d15N-PN: delta-15N signature of particulate nitrogen from incubation experiment [‰] d13C-PC: delta-13C signature of particulate carbon from incubation experiment [‰] Aggregate Type: Description of ROIs Size: Size of ROIs as defined by Look@NanoSIMS software (µm) LW ratio: Ratio of ROI length to width ROI Nfix Vat%: Specific rate of nitrogen-fixation derived from ROI ion counts [h-1] ROI Cfix Vat%: Specific rate of methane-assimilation derived from ROI ion counts [h-1] N-Turnover: Nitrogen turnover time; inverse of nitrogen-fixation specific rate [h] C-Turnover: Carbon turnover time; inverse of methane-assimilation specific rate [h] IRMS bulk rates sheet: IRMS-based rates of nitrogen-fixation and methane assimilation Sample ID: Sample ID used by IRMS operator. Station: Station.event number from cruise EN528 aboard the R/V Endeavor In/Out of plume: Identifies if samples were collected inside or outside of a gas plume. Depth: Depth [meters] Temp: Seawater temperature [degC] Salinity: Seawater salinity [psu] [CH4]: Methane concentration in seawater [µmol L-1] d13CH4 : delta-13C signature of seawater methane [‰] BtlVol: Volume of incubation bottles [mL] N2 Spike: Injected volume (at atmospheric pressure) of 15N-labeled nitrogen gas tracer [mL] CH4 Spike: Injected volume (at atmospheric pressure) of 13C-labeled methane gas tracer [mL] IncTime: Incubation time [hours] d15N-PN: delta-15N signature of particulate nitrogen from incubation experiment [‰] d13C-PC: delta-13C signature of particulate carbon from incubation experiment [‰] N-content: Nitrogen content of particulate nitrogen from incubation experiment [µmol] C-content: Carbon content of particulate carbon from incubation experiment [µmol] RhoN-mean: Volumetric rate of nitrogen-fixation, mean from triplicate incubations [nmol L-1 h-h] RhoN-stdev: Standard deviation of volumetric rates of nitrogen fixation [nmol L-1 h-h] RhoC-mean: Volumetric rate of methane-assimilation, mean from triplicate incubations [nmol L-1 h-h]. ND = not detectable RhoC-stdev: Standard deviation of volumetric rates of methane-assimilation [nmol L-1 h-h] Sheets entitled "######x": The title of the sheet matches to the Sample ID listed in "nanoSIMS rates" and represent separate nanoSIMS analyses. All images and graphs were produced using Look@NanoSIMS. The image at the top of each sheet is the ion image summary, containing ion counts of each of the ions analyzed where red = higher counts. The text at the top of each panel designates the ions that were enumerated, with the scale in square brackets [ion counts]: 12C: 12-Carbon 13C: 13-Carbon 16O: 16-Oxygen 12C14N: 12-Carbon + 14-Nitrogen Esi/1AU: Secondary electrons 12C15N: 12-Carbon + 15-Nitrogen 32S: 32-Sulfur The numbers in parentheses in the first panel designate the size of the image [µm] and the number of pixels per image. Below the summary image are: ROI (regions of interest) images showing the distribution of ROIs selected for tracer enrichment analysis for carbon and/or nitrogen. ROIs are sequentially numbered and color-coded based on horizontal position (from left to right). Graphs of statistical comparison of ROI populations using Kruskal-Wallis test to determine significance of 13C or 12C15N enrichment (relative to 12C or 12C14N, respectively). ROI # on the y-axis matches ROI # in the respective ROI image. Site Latitude (decimal degrees) Longitude (decimal degrees) 28.08: 27.783017 -91.507333 28.12: 27.78525 -91.504783

Methods:

Water samples (4.5 L) were amended with 15N2 (10 mL) and 13CH4 (200 microliter), incubated under simulated in situ conditions for 48 h, then terminated by gentle vacuum filtration. Samples were stored frozen for analysis ashore. Bulk rates were measured by continuous flow isotope ratio mass spectrometry using a Micromass Optima IRMS interfaced to a CE NA2500 elemental analyzer at Georgia Tech. Spatial distributions of elements and isotopes were measured at the Leibniz Institute for Baltic Sea Research Warnemünde (Rostock, Germany) using a nanoSIMS 50L instrument (Cameca, Gennevilliers, France). A laser-dissecting microscope (LEICA LMD6500) was used to assess the distribution of biomass on the sample filter, then 5 mm diameter subsamples were excised for analysis. Subsamples were surveyed with the cesium primary ion beam by first performing a chain analysis of 100 x 100 µm measurements and using the total ion counts (TIC) to identify areas that contained potential cell aggregates or other structures. These features were then implanted with a 600 pA primary ion beam in an area of 50*50 µm to reach the steady state of ion formation. These areas (25 x 25 to 48 x 48 µm) were then analyzed with a 1 pA primary ion beam (256 x 256 px, 1 ms/px, 60 planes) and the secondary ions generated (12C–, 13C–, 16O–, 12C14N–, 12C15N–, and 32S–) were collected simultaneously in electron multiplier detectors. Isotopic enrichments were then confirmed with more detailed analyses (10 x 10 to 25 x 25 µm, 1 ms/px, 60 planes).

Instruments:

Micromass Optima IRMS interfaced to a CE NA2500 elemental analyzer. nanoSIMS 50L instrument (Cameca, Gennevilliers, France) for spatial and isotopic analysis. Laser-dissecting microscope (LEICA LMD6500) for identifying targets

Individual Files: