Table of Contents

• Dataset Description
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
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HC Data - Ammonia-nitrite-nitrate assays

OPA ammonia assay
Prepare all solutions in acid-washed (0.1% HCl), triple-rinsed (MilliQ H2O) brown HDPE bottles. Prepare stock solutions with freshly drawn ultra-pure deionized Milli-Q water. For the OPA solution use ethanol of the highest purity available.

SOLUTIONS:
Sodium sulfite solution: Dissolve 1 g sodium sulfite in 125 ml MilliQ water.
 
Borate buffer solution: Dissolve 40 g disodium tetraborate decahydrate in 1000 ml MilliQ water. If necessary, filter the solution to remove turbitity. The solution is stable.

OPA solution: Dissolve 2 g of standard grade o-phtaldialdehyde (P- 1378 Sigma) in 50 ml of ethanol in the dark.

Ammonium chloride stock solution (10mM): Dissolve 535 mg dried NH4Cl in 1000 ml of MilliQ water.

WORKING REAGENT:
In a HDPE bottle mix 1000 ml of borate buffer with 50 ml of OPA solution and 5 ml of sodium sulfite. This reagent is light-sensitive. Let the reagent age at least one day before use. Store dark for at least 2 month.
 
PROTOCOL:
Prepare 2 µM ammonium standard in a 250ml HDPE bottle: Add 40 µl of ammonium chloride stock solution (10 mM) to 200 ml Milli-Q water.
 
Prepare standard curve as follows:
Standard [nM] MilliQ water 2 µM NH4Cl
0 80 0
0 80 0
10 79.6 0.4
25 79 1
500 60 20
1000 40 40
2000 0 80
 
Fill 80 ml of water sample into a 250-ml HDPE bottle.
Add 20 ml of working reagent to all samples and standards.
Incubate for accurately 30 min at 65°C and cool to room temperature.
Analyze fluorescence intensity in Turner fluorometer with CDOM/Ammonia kit.
 
Reference:
Holmes, R. M., A. Aminot, R. Kerouel, B. A. Hooker and B. J. Peterson (1999). A simple and precise method for measuring ammonium and marine and freshwater ecosystems. Canadian Journal of Fisheries and Aquatic Sciences 56(10): 1801-1809. Keroul & Aminot (1997) Fluorometric determination of ammonia in sea and estuarine waters by direct segmented flow analysis. Marine Chemistry 57: 265-275.
 
 
Nitrite Determination
REAGENTS:
Sulfanilamide reagent: Add 100 ml conc. HCl to 700ml MilliQ water
Add 10g Sulfanilamide, stir to dissolve.
Make up volume to 1000 ml
Store at 4?C in the dark, stable for several weeks.
 
Naphthylethylenediamine (NED) reagent: Dissolve 1 g of N-(1-Naphthyl)-ethylendiamin-dihydrochloride in 1000 ml of MilliQ water. Store at 4?C in the dark, stable for several weeks.
 
Nitrite standard stock solution (10 mM): Dissolve 0.690 g dried NaNO2 in 1000 ml MilliQ water.
 
PROCEDURE:
Prepare 2 µM nitrite standard in a 250ml HDPE bottle: Add 40 µl of nitrite standard stock solution (10 mM) to 200 ml artificial seawater (SCM salts).
 
Prepare nitrite standard curve as follows:
Standard [nM] SCM salts [ml] 2 µM Nitrite Standard [ml]
0 100 0
0 100 0
10 99.5 0.5
25 98.75 1.25
500 75 25
1000 50 50
2000 0 100
 
Fill 100 ml of sample water into 250ml HDPE bottles.
Add 2 ml of sulfanilamide reagent, swirl and let stand for 1 min.
Add 2 ml of NED reagent and swirl again.
Incubate for 10 to 15 min.
Read absorbance at 540nm in a 5cm cuvette
 
Grasshoff, Kremling, Erhard (1999) Methods of Seawater Analysis, 3 edn. Wiley-VCH
 
Nitrate Assay for Seawater
Deionized water and standards
For preparing standard and reagent solutions use deionized water purified by a distilling unit followed by the Millipore Synergy 185 Water System that produces water with 18 MΩ resistance.
 
REAGENTS:
2% (w/v) resorcinol solution: prepare fresh daily by dissolving 2.0 g of ACS reagent grade resorcinol crystals in 100 ml Milli-Q water.
 
Concentrated sulfuric acid
Nitrate stock standard solution (10 mM): Dissolve 0.85 g NaNO3 in 1 L of MilliQ water. Store stock solutions in a 1L polyethylene bottle at 4 °C in a refrigerator.
Prepare working standard solutions from serial dilutions of stock solution with artificial sea water (SCM media – 26 g NaCl, 5 g MgSO4•7H2O, 5 g MgCl2•6H2O, 1.5 g CaCl2•2H2O, 0.1 g KBr in 1 L Milli-Q water).
 
PROCEDURE:
Prepare 40 µM nitrate standard in a 250ml HDPE bottle by adding 400 µl of nitrate standard stock solution (10 mM) to 99.6 ml of artificial seawater (SCM media).
 
Prepare nitrate standard curve as follows:
Standard [µM] SCM salts [ml] 40 µM Nitrate Standard [ml]
0 20 0
0 20 0
0.25 19.875 0.125
0.5 19.75 0.25
10 15 5
20 10 10
40 0 20
 
Transfer 20.0 ml of a seawater sample or standard into a 250 ml brown PE bottle.
Add 2.4 ml of 2% resorcinol solution and swirl to mix the resorcinol with the sample.
Carefully add 20.0 ml of concentrated sulfuric acid, close the bottle with its lid and then gently swirl to mix the solution. Let the bottles stand for 30 min.
Place the bottles in a water bath until it reaches room temperature.
Measure the absorbance of the sample at 505 nm in a 5 cm cuvette against a blank with acidified seawater.
 
Reference:
Zhang, J.Z. & Fischer, C.J., 2006. A simplified resorcinol method for direct spectrophotometric determination of nitrate in seawater. Mar. Chem. 99, 220-226.

BCO-DMO Processing Notes
Generated from original .xls file "Ingalls_HC_Data for NODC.xls" contributed by Anitra Ingalls

BCO-DMO Edits
- Multiple sheet spreadsheet converted to individual, single sheet spreadsheets by cruise
- Cruise Id standardized to R2R Catalog
- Cruise Id and station metadata added to each data record
- Parameters edited to conform to BCO-DMO parameter naming convention
- "Date (euro)" removed
- Decimal data values padded to consistent decimal places as reported
- "nd" (no data) value inserted in blank cells

Project Summary
Recent advances in molecular microbial ecology have overturned canonical paradigms of the marine nitrogen cycle. Estimates of global nitrogen fixation are regularly revised upward, the non-traditional bacterial denitrification pathway known as anammox is now thought to be responsible for a significant portion of global denitrification, and the discovery of ammonia-oxidizing Archaea necessitates a reevaluation of the contribution of traditional nitrifying bacteria to the global nitrogen cycle. While environmental gene sequencing and geochemical studies were critical to these discoveries, much of our understanding could not have been gained without the aid of studies on representative organisms in pure culture. Since their discovery in 1992, the ecological role of mesophilic marine Archaea has remained a mystery due in large part to the lack of a cultured representative.

We now have a mesophilic marine Crenarchaea in culture along with several lines of evidence that this and many other pelagic marine Crenarchaea oxidize ammonia to obtain the energy needed to sustain autotrophic carbon fixation. The distribution of marine Crenarchaea and their genes encoding ammonia-oxidizing enzymes, suggests that these organisms are responsible for the oxidation of a significant portion of the ocean's reduced nitrogen pools.

Here we propose to begin to better understand the physiological capabilities, distribution and quantitative significance of ammonia-oxidizing Crenarchaea. Our group is uniquely positioned to launch a comprehensive set of studies that will use cutting edge techniques to answer the following questions:
1) What factors control the rate and efficiency of Archaeal ammonia-oxidation?

2) What is the relative role of Bacteria and Archaea in ammonia-oxidation in the marine environment?

3) How can biomarkers be used to detect and assess the physiological status of living ammonia-oxidizing Bacteria and Archaea?

Our study uniquely combines culture work, molecular biology, organic geochemistry and field investigations into one of the first studies of the role of marine Crenarchaea in the biogeochemical cycling of nitrogen.