Contributors | Affiliation | Role |
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Popp, Brian N. | University of Hawaiʻi at Mānoa (SOEST) | Principal Investigator, Contact |
Hollibaugh, James T. | University of Georgia (UGA) | Co-Principal Investigator |
Wallsgrove, Natalie J. | University of Hawaiʻi at Mānoa (SOEST) | Scientist |
Rauch, Shannon | Woods Hole Oceanographic Institution (WHOI BCO-DMO) | BCO-DMO Data Manager |
Sample Collection. Samples were collected on the Antarctic continental shelf and slope west of the Antarctic Peninsula within the PAL-LTER sampling domain (http://pal.lternet.edu/) during summer (cruise dates 30 Dec 2017 through 12 Feb 2018; sampling dates 5 Jan to 4 Feb 2018) from the ARSV Laurence M Gould (LMG 1801, PAL-LTER cruise 26, DOI: 10.7284/907858). Sampling focused on three or 4 depths at each station chosen to represent the Antarctic Surface Water (ASW, 0 -34 m depth), the Winter Water (WW, the water column temperature minimum, generally between 35 and 174 m) the Circumpolar Deep Water (CDW, 175-1000 m) and slope water (SLOPE, >1000 m, generally ~10 m above the bottom at deep stations on the slope, 2500-3048m). Water samples were collected from Niskin bottles (General Oceanics Inc., Miami, FL, USA) into opaque 2 L HDPE plastic bottles or into aged, acid-washed, sample-rinsed 250 ml polycarbonate bottles (Nalge) completely filled (~270 mL) directly from Niskin bottles as soon as possible after the rosette was secured on deck. Subsequent processing took place in an adjacent laboratory.
Samples for DNA analysis were taken from the 2 L opaque HDPE bottles and were filtered under pressure through 0.22 um pore size Sterivex GVWP filters (EMD Millipore, Billerica, MA, USA) using a peristaltic pump. Residual seawater was expelled from the filter using a syringe filled with air, then ~1.8 ml of lysis buffer (0.75 M sucrose, 40 mM EDTA, 50 mM Tris, pH 8.3) was added to the filter capsule, which was capped and placed in a -20 °C freezer. The frozen samples were aggregated into Ziploc Freezer Bags and transferred to a -80 °C freezer for the remainder of the cruise and for shipping to the laboratory.
Two samples of the Sterivex filtrate (40 mL each into new 50 mL disposable centrifuge tubes, VWR, rinsed 3x with sample) were frozen immediately at -20 °C, then aggregated into Ziploc Freezer Bags and transferred to a -80 ° freezer for the remainder of the cruise and for shipping to the laboratory. These were used for subsequent determination of 1) urea concentration and 2) the natural abundance of 15N in the nitrite plus nitrate pools (15NOₓ hereinafter). An additional sample of the Sterivex filtrate was stored in a polycarbonate bottle at 4 °C for subsequent onboard determination of ammonia concentration by the Holmes et al (1999) o-phthaldialdehyde method and nitrite concentration by the diazo-coupling method (Strickland and Parsons 1972). Technical difficulties encountered during onboard analysis resulted in the loss of ammonium and nitrite data for some samples.
Samples for DNA and chemical analyses were shipped on dry ice from Punta Arenas, Chile to the Hollibaugh laboratory at the University of Georgia. Upon arrival they were stored in a -80 °C freezer until analyzed. Samples for 15N analysis were shipped on dry ice from Punta Arenas, Chile to the Popp laboratory at the University of Hawaii. Upon arrival they were stored in a -40 °C freezer until analyzed.
Nitrogen oxidation rates. Oxidation rates of N supplied as ammonium, nitrite, urea and putrescine (1,4 diaminobutane) were measured in ~48 h incubations using 15N-labeled substrates (>98 at% 15N, Cambridge Isotope Laboratories, Tewksbury,MA, USA) added within ~1 hr of sample collection to yield ~44 nM amendments (Santoro et al., 2010; Beman et al., 2012). Labeled substrates were added to duplicate bottles that were placed in cardboard boxes and incubated in the dark in a Percival incubator (Perry, IA, USA). Incubation temperature was recorded at 5-minute time steps with HOBO TidBit data loggers (Onset Computer Corp., Bourne MA, Figure 1) placed in bottles of filtered seawater incubated in cardboard boxes identical to those used for experiments (see the "Incubator_Temperature.xlsx" Supplemental File). Incubations were terminated after ~48 hr by decanting 40 mL subsamples from each bottle into new, sample rinsed, 50 mL polypropylene centrifuge tubes that were immediately frozen at -80 °C. Water in these tubes was used for subsequent analysis of 15NOₓ. The natural abundance of 15N in NOₓ was taken as the initial (time = 0) value for calculating the amount of 15N oxidized to nitrate or nitrite during the incubations.
Chemical analyses. Urea content was determined by the diacetyl monoxime method (Rahmatullah and Boyde 1980, Mulvenna and Savidge 1992). Subsamples from samples that were also used to determine oxidation of 15N supplied as putrescine were sent to Dr. X. Mou’s laboratory at Kent State University where they were analyzed to determine polyamine and DFAA content as described previously (Lu et al 2014).
15N in nitrite plus nitrate. The 15NOₓ in samples was measured using the ‘denitrifier method’ (Sigman et al., 2001) with Pseudomonas aureofaciens as described in Popp et al. (1995), Dore et al. (1998) and Beman et al. (2011). The nitrous oxide produced was analyzed using a Gas Bench II coupled to a MAT 252 mass spectrometer following the recommendations of Casciotti et al. (2002). Typically nineteen samples plus one sample duplicate was analyzed along with duplicate reference materials USGS 32, USGS 34 and USGS 35 (or NIST 3), which were used to normalize the measured d15N values to AIR. In addition, a laboratory reference solution made from analytical grade NaNO₃ with d15N value (-52.2‰) that was known through extensive characterization using NIST/USGS reference materials was also analyzed in duplicate with each batch of 19 samples.
We calculated oxidation rates from d15N enrichment of the NOₓ pool in the bottles at the ends of the incubations compared to the initial value in the unamended seawater sample ("natural abundance"). We assumed that the d15N of naturally occurring ammonia, urea and putrescine is the same as that of N in bulk organic matter, and that the d15N value of nitrite is our samples is -30 o/oo as reported by Smart et al. (2015). Samples with low or no activity sometimes yielded negative rates because the d15NOₓ "natural abundance" value for that sample was greater than the d15NOₓ value of amended sample. We analyzed control samples consisting of filtered seawater taken at the beginning of the cruise, amended with 15NO₂, then immediately frozen at -80 °C, or "time zero" samples from time course experiments performed throughout the cruise, to determine the contribution of autooxidation or isotope exchange to the apparent rate of nitrite oxidation. These samples were treated with sulfamic acid to remove unreacted 15NO₂ (Granger and Sigman 2009). The analysis indicated that about 7.9% of the 15N supplied as NO₂ had been converted to 15NO₃ by the time we analyzed the samples. We also performed an independent chemical analysis of nitrite and nitrate (Strickland and Parsons 1972) in the (nominally) 0.125 mM 15NO₂ working stock solution a few months after the cruise. This analysis indicated that about 14.3% of the of the nitrite plus nitrate in the stock was nitrate. Because this stock had been handled and shipped separately from the cruise samples, we used 7.9% as the best estimate of the amount of 15N label converted to nitrate. We have incorporated corrections for this reaction into rates calculated from field data.
We ran time-course incubations with samples from 2 or 3 depths at 34 stations to verify that oxidation rates did not change significantly during incubations, for example, due to substrate depletion or changes in the population of ammonia oxidizers. These experiments were set up in 250 ml polycarbonate bottles as above. Two bottles were sampled at each time point over time courses of 72 to 96 h. These data are presented in Table 1 (PDF; see Supplemental Files). AO rates determined from the slope of linear regressions of the data from a given sample were compared to rates determined from samples taken at the 48 hr time point. We performed analogous experiments to examine the effect on N oxidation rates of variation in incubation temperature and in substrate concentration.
Summary Statistics for d15NOx (mean, median, maximum, minimum, and standard error of d15NOx measurements grouped by the water mass depth ranges) are provided in the Supplemental File d15NOx_Summary_Statistics.JPG.
Precision. Analytical uncertainty in d15N values was determined from duplicate analyses of USGS reference materials, our laboratory reference solution and samples analyzed in duplicate and ranged from 0.36‰ to 0.56‰ (Table 1 (PDF) Supplemental File). All rate measurements were also performed in duplicate (biological replicates) and their uncertainty is also presented in Table 1. Accuracy was determined based on isotope analysis of the laboratory reference solution, which was not used to normalize the isotopic results of samples and was found to be 0.42‰ (at% 15N = 0.00019, n = 56).
Rate calculations. We integrated the data we collected to calculate oxidation rates of N supplied as ammonia and urea as described in Popp et al. (1995), Dore et al. (1998) and Beman et al. (2011). We used ammonium concentration data from shipboard analyses. Nitrite + nitrate concentrations were determined by PAL-LTER personnel and were obtained from their database. Urea concentrations were measured on samples shipped frozen to the University of Georgia (Hollibaugh lab). Chemical data needed for rate calculations were not available for some samples so we substituted water mass averages determined from other samples taken on the cruise.
We determined the limits of detection and precision of nutrient analyses as follows. The precision of nitrate plus nitrite analyses run by LTER personnel (https://oceaninformatics.ucsd.edu/datazoo/catalogs/pallter/datasets/27) was reported to be 100 nM. The precision and limit of detection of putrescine (polyamine) analysis is given in Lu et al. (2018) as 1 nM. We determined the precision of ammonium, urea and nitrite analyses as the mean standard deviation of replicate (2 or 3) analyses of a given sample. They are: ammonium, 65 nM; urea, 10 nM; and nitrite, 70 nM. The limits of detection were taken as 1.96 times the precision of the relevant measurements.
We ran Monte Carlo simulations to estimate the precision and the limits of detection of rate measurements. The models incorporated the estimates of precision given above and the means of the measured in situ concentrations of the reactants, the mean in situ concentration of NOₓ (from PAL-LTER data), the precision of the measured d15NOₓ in the experiments at the beginning (natural abundance) and end of the incubations. We ran 10,000 trials using random numbers generated with population means and standard deviations (assuming normally distributed variance and produced using an Excel spreadsheet in the EasyFit® app) ) equivalent to the test values. The standard deviation of the 10,000 rates calculated in this manner was taken as an overall estimate of the precision of the rates we report. The results of these models are summarized in Supplemental Table 3. Our estimates of the precision of the rate measurements (oxidation of 15N supplied as ammonium urea, putrescine or nitrite to 15NOₓ, nmol L-1 d-1) are: ammonia, 2.18; urea 0.31; putrescine, 0.51; nitrite, 4.6, for relative standard deviations (RSD; ((standard deviation/mean)*100)) of: 15.3%; 11.3%; 8.2% and 32%, respectively, of the calculated rates. Model runs, which also tested the sensitivity of the calculated rates to the accuracy of estimates of some variables, are summarized in Supplemental Table 3 (.xlsx file), which also includes the equations used to calculate rates.
BCO-DMO Processing:
- renamed fields to comply with BCO-DMO naming conventions;
- 2021-03-16: replaced with data file received on 2021-03-14; converted latitude and longitude values to negative; formatted date/time field to ISO8601 format.
- 2021-04-08: replaced data file with copy received on 2021-03-18.
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Natural_Abundance.csv (Comma Separated Values (.csv), 10.88 KB) MD5:714a72620ea0494fad6565f48ea2cdb3 Primary data file for dataset ID 842944 |
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Control_Experiments.xlsx (Octet Stream, 24.22 KB) MD5:c459cd733a5d700b7f0ba4d486fa3a48 This spreadsheet reports the results of experiments conducted to 1) test the linearity of time course incubations; 2) examine the response of oxidation rates to substrate concentration; or 3) examine the response of nitrification to incubation temperature.
Column Headers; Data Description; Units
Incubation Duration, hours; Time after adding 15N-labeled substrate; hours
NH4 (or urea or nitrite) oxidized; Amount of the 15N-labeled substrate oxidized to 15NOx; nM
15N added as NH4 (or urea or nitrite); The concentration of 15N-labeled substrate in each replicate bottle after amendment; nM
Rate nmol L-1 d-1; The rate at which the N from ammonia, urea or nitrite substrate was oxidized to NOx, nmol of substrate N oxidized; L-1 d-1
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d15NOx_Summary_Statistics.JPG (JPEG Image (.jpg), 29.25 KB) MD5:2212b9c01f0358d7485cdfce918a4ad8 Summary Statistics for d15NOx. Reports mean, median, maximum, minimum and standard error of d15NOx measurements grouped by the water mass depth ranges indicated.
"ASW" is "Antarctic Surface Water,"
"WW" is "Winter Water,"
"CDW" is "Circumpolar Deep Water,"
"Slope" is a sample taken off the continental shelf, either from deep basins or on the continental slope. Depth ranges are given, units are parts per thousand (o/oo or per mil). |
Incubator_Temperature.xlsx (Octet Stream, 95.68 KB) MD5:78c979e751830154cb5b53107574700f This spreadsheet reports the temperature in the incubator used in our measurements of N oxidation rates. Data were collected using a HOBO TidBit logger (Logger S/N: 20211709, Sensor S/N: 20211709) placed in a bottle of seawater identical to those used in the experiments that was placed in a cardoard box at the back and middle shelf of the incubator.
Column Headers; Data Description; Units
Date Time; Date and time of the measurement reported as local time (GMT-05:00), MM/DD/YYY HH:MM format, 24 hr clock.
Temperature °C; Water temperature in the monitored bottle; °C
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Table 1. Uncertainty in nitrogen isotope analyses of nitrite plus nitrate filename: Table1.pdf (Portable Document Format (.pdf), 121.62 KB) MD5:b696069f0d93658380747d19cba7f94c Uncertainty (standard deviation) in nitrogen isotope analyses of nitrite plus nitrate in seawater samples. Associated with data from the project "Collaborative Research: Chemoautotrophy in Antarctic Bacterioplankton Communities Supported by the Oxidation of Urea-derived Nitrogen". |
Table 3. Summary of Monte Carlo Models filename: Table3_Summary_of_Monte_Carlo_Models.xlsx (Octet Stream, 18.32 KB) MD5:7568936f47c4065fbd9037822462ed36 Summary of the results of Monte Carlo simulations to estimate precision of 15N oxidation rate measurements. NOTE: The cells in this file are not "locked" so that the formulae can be read. Cells are linked so that a change to one of them affects others.
This file is associated with data from the project "Collaborative Research: Chemoautotrophy in Antarctic Bacterioplankton Communities Supported by the Oxidation of Urea-derived Nitrogen". |
Parameter | Description | Units |
Event_Log_Number | Sequential numbers keyed to the bridge log of activities | unitless |
Cast_Start_Time_GMT | Date and time of day for beginning CTD cast = sample collection; 24-hour clock; formatted to ISO8601 standard (UTC/GMT): YYYY-MM-DDThh:mmZ | unitless |
Latitude | Latitude in decimal degrees (negative values = South) | degrees North |
Longitude | Longitude in decimal degrees (negative values = West) | degrees East |
Station_Description | PAL-LTER category for the station | unitless |
LTER_Grid_Station | Station location on the PAL-LTER sampling grid (http://pal.lternet.edu) | unitless |
Sample_Depth | Depth sampled in meters | meters (m) |
d15N | d15N value of the NO2 + NO3 in the sample as determined by the denitrifier method, units of parts per thousand (o/oo or per mil). Replicate determinations are retained but were averaged for use in calculating oxidation rates. | parts per thousand (o/oo or per mil) |
d18O | d18O value of the NO2 + NO3 in the sample as determined by the denitrifier method, units of parts per thousand (o/oo or per mil). | parts per thousand (o/oo or per mil) |
Analytical_Batch | Samples were run in batches of 20 samples with 10 standards, this column identifies the batch that included a specific sample | unitless |
Dataset-specific Instrument Name | Percival incubator |
Generic Instrument Name | In-situ incubator |
Generic Instrument Description | A device on a ship or in the laboratory that holds water samples under controlled conditions of temperature and possibly illumination. |
Dataset-specific Instrument Name | Gas Bench II coupled to a MAT 252 mass spectrometer |
Generic Instrument Name | Isotope-ratio Mass Spectrometer |
Generic Instrument Description | The Isotope-ratio Mass Spectrometer is a particular type of mass spectrometer used to measure the relative abundance of isotopes in a given sample (e.g. VG Prism II Isotope Ratio Mass-Spectrometer). |
Dataset-specific Instrument Name | Niskin bottles (General Oceanics Inc., Miami, FL, USA) |
Generic Instrument Name | Niskin bottle |
Generic Instrument Description | A Niskin bottle (a next generation water sampler based on the Nansen bottle) is a cylindrical, non-metallic water collection device with stoppers at both ends. The bottles can be attached individually on a hydrowire or deployed in 12, 24, or 36 bottle Rosette systems mounted on a frame and combined with a CTD. Niskin bottles are used to collect discrete water samples for a range of measurements including pigments, nutrients, plankton, etc. |
Dataset-specific Instrument Name | HOBO TidBit data loggers |
Generic Instrument Name | Temperature Logger |
Generic Instrument Description | Records temperature data over a period of time. |
Website | |
Platform | ARSV Laurence M. Gould |
Start Date | 2017-12-30 |
End Date | 2018-02-12 |
Description | Additional cruise information is available from the Rolling Deck to Repository (R2R): https://www.rvdata.us/search/cruise/LMG1801
Cruise DOI: 10.7284/907858 |
NSF Award Abstract:
Part 1: The project addresses fundamental questions regarding the role of nitrification (the conversion of ammonium to nitrate by a two-step process involving two different guilds of microorganisms: ammonia- and nitrite-oxidizers) in the Antarctic marine ecosystem. Specifically, the project seeks to evaluate the contribution of primary production supported by the energy in nitrogen compounds to the overall supply of organic carbon to the food web of the Southern Ocean. Previous measurements indicate that nitrification could contribute about 9% to primary production supporting the Antarctic food web on an annual basis, but those measurements did not include the additional production associated with nitrite oxidation. Additionally, the project will aim to determine the significance of the contribution of other sources of nitrogen, (specifically organic nitrogen and urea released by other organisms) to nitrification because these contributions may not be assessed by standard protocols. Such work will aid in better understanding the basis of the energy for the Antarctic marine ecosystems on an annual basis as well as better aid in understanding the energetics of the ecosystem in times and places where primary production based on light energy is limited (i.e. during the polar night or under sea ice cover).
This project will result in training a postdoctoral researcher and provide undergraduate students opportunities to gain hand-on experience with research on microbial geochemistry. The Palmer Long Term Ecological Research (LTER) activities have focused largely on the interaction between ocean climate and the marine food web affecting top predators. Relatively little effort has been devoted to studying processes related to the microbial geochemistry of nitrogen cycling, yet these are a major themes at other LTER sites. This work will contribute substantially to understanding an important aspect of nitrogen cycling and bacterioplankton production in the study area. The team will be working synergistically and be participating fully in the education and outreach efforts of the Palmer LTER, including making highlights of the findings available for posting to their project web site and participating in any special efforts they have in the area of outreach.
Part 2: The proposed work will quantify oxidation rates of 15N supplied as ammonium, urea and nitrite, allowing the estimation of the contribution of urea-derived N and complete nitrification (ammonia to nitrate) to chemoautotrophy and bacterioplankton production in Antarctic coastal waters. The project will compare these estimates to direct measurements of the incorporation of 14C into organic matter in the dark for an independent estimate of chemoautotrophy. The team aims to collect samples spanning the water column: from surface water (~10 m), winter water (50-100 m) and circumpolar deep water (>150 m); on a cruise surveying the continental shelf and slope west of the Antarctic Peninsula in the austral summer of 2018. Other samples will be taken to measure the concentrations of nitrate, nitrite, ammonia and urea, for qPCR analysis of the abundance of relevant microorganisms, and for studies of related processes. The project will rely on collaboration with the existing Palmer LTER to ensure that ancillary data (bacterioplankton abundance and production, chlorophyll, physical and chemical variables) will be available. The synergistic activities of this project along with the LTER activities will provide a unique opportunity to assess chemoautotrophy in context of the overall ecosystem's dynamics- including both primary and secondary production processes.
Funding Source | Award |
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NSF Office of Polar Programs (formerly NSF PLR) (NSF OPP) | |
NSF Office of Polar Programs (formerly NSF PLR) (NSF OPP) |