Dominant heterocyclic composition of dissolved organic nitrogen in the ocean: A new paradigm for cycling and persistence.

Marine dissolved organic nitrogen (DON) is one of the planet's largest reservoirs of fixed N, which persists even in the N-limited oligotrophic surface ocean. The vast majority of the ocean's total DON reservoir is refractory (RDON), primarily composed of low molecular weight (LMW) compounds in the subsurface and deep sea. However, the composition of this major N pool, as well as the reasons for its accumulation and persistence, are not understood. Past characterization of the analytically more tractable, but quantitatively minor, high molecular weight (HMW) DON fraction revealed a functionally simple amide-dominated composition. While extensive work in the past two decades has revealed enormous complexity and structural diversity in LMW dissolved organic carbon, no efforts have specifically targeted LMW nitrogenous molecules. Here, we report the first coupled isotopic and solid-state NMR structural analysis of LMW DON isolated throughout the water column in two ocean basins. Together these results provide a first view into the composition, potential sources, and cycling of this dominant portion of marine DON. Our data indicate that RDON is dominated by 15N-depleted heterocyclic-N structures, entirely distinct from previously characterized HMW material. This fundamentally new view of marine DON composition suggests an important structural control for RDON accumulation and persistence in the ocean. The mechanisms of production, cycling, and removal of these heterocyclic-N-containing compounds now represents a central challenge in our understanding of the ocean's DON reservoir.

Optimization of the LLNL/CAMS gas-accepting ion source and 1 MV compact AMS for natural abundance radiocarbon analysis of CO2.

The Lawrence Livermore National Laboratory - Center for Accelerator Mass Spectrometry (LLNL/CAMS) 1 MV AMS system was converted from a biomedical AMS instrument to a natural abundance 14C spectrometer. The system is equipped with a gas-accepting hybrid ion source capable of measuring both solid (graphite) and gaseous (CO2) samples. Here we describe a series of experiments intended to establish and optimize 14CO2 measurement capabilities at natural abundance levels. A maximum instantaneous ionization efficiency of 8 % was achieved with 3 % CO2 in helium at a flow rate of approximately 220 µL/min (3.5 µg C/min). For modern materials (e.g., OX I) we measured an average of 240 ± 50 14C counts/µg C, equivalent to a total system efficiency of approximately 3 %. Experimental CO2 samples with F14C values ranging from 0.20 to 1.05 measured as both graphite and directly as CO2 gas produced equivalent values with an average offset of < 2σ.

Conversion of the LLNL/CAMS 1 MV biological AMS system to a semi-automated natural abundance 14C spectrometer: system optimization and performance evaluation.

The Lawrence Livermore National Laboratory - Center for Accelerator Mass Spectrometry compact 1 MV biomedical accelerator mass spectrometer was repurposed and optimized for the semi-automated radiocarbon measurement of natural abundance environmental samples. Substantial efforts were made to greatly improve instrument precision and develop semi-automation capabilities for unattended operation. Here we present results from 15 months of routine system operation and evaluate the system performance based on 30 sample wheels measured with directly comparable operating conditions over 7 months from August 2019 to March 2020. Unattended operation was enabled through software that tracks specific error conditions and can initiate a complete instrument shutdown when specific criteria were met. The average measurement precision was found to be 2.7 ± 0.7 ‰ based on repeated measurements of OX I standards. Accuracy was assessed with measurements of standard materials with known 14C-content, spanning 0.5 to 1.5 modern, and by comparison to split samples measured with the 10 MV FN AMS system. We also assessed sample size and age limitations using 14C-free materials, finding that we can routinely analyze samples as small as 300 µg C and less than 33000 years without the need for size-specific correction protocols.

Radiocarbon Analysis of Individual Amino Acids: Carbon Blank Quantification for a Small-Sample High-Pressure Liquid Chromatography Purification Method.

Compound-specific radiocarbon analysis (CSRA) of amino acids (AAs) is of great interest as a proxy for organic nitrogen (N) cycling rates, dating archeological bone collagen, and investigating processes shaping the biogeochemistry of global N reservoirs. However, recoverable quantities of individual compounds from natural samples are often insufficient for radiocarbon ((14)C) analyses (<50 µg C). Constraining procedural carbon (C) blanks and their isotopic contributions is critical for reporting of accurate CSRA measurements. Here, we report the first detailed quantification of C blanks (including sources, magnitudes, and variability) for a high-pressure liquid chromatography (HPLC) method designed to purify individual AAs from natural samples. We used pairs of AA standards with either modern (M) or dead (D) fraction modern (Fm) values to quantify MC and DC blanks within several chromatographic regions. Blanks were determined for both individual and mixed AA standard injections with peak loadings ranging from 10 to 85 µg C. We found 0.8 ± 0.4 µg of MC and 1.0 ± 0.5 µg of DC were introduced by downstream sample preparation (drying, combustion, and graphitization), which accounted for essentially the entire procedural blank for early eluting AAs. For late-eluting AAs, higher eluent organic content and fraction collected volumes contributed to total blanks of 1.5 ± 0.75 µg of MC and 3.0 ± 1.5 µg of DC. Our final measurement uncertainty for 20 µg of C of most AAs was ±0.02 Fm, although sample size requirements are larger for similar uncertainty in late-eluting AAs. These results demonstrate the first CSRA protocol for many protein AAs with uncertainties comparable to the lowest achieved in prior studies.

High-precision measurement of phenylalanine δ15N values for environmental samples: a new approach coupling high-pressure liquid chromatography purification and elemental analyzer isotope ratio mass spectrometry.

RATIONALE: Compound-specific isotope analysis of individual amino acids (CSI-AA) is a powerful new tool for tracing nitrogen (N) source and transformation in biogeochemical cycles. Specifically, the δ(15)N value of phenylalanine (δ(15)N(Phe)) represents an increasingly used proxy for source δ(15)N signatures, with particular promise for paleoceanographic applications. However, current derivatization/gas chromatography methods require expensive and relatively uncommon instrumentation, and have relatively low precision, making many potential applications impractical. METHODS: A new offline approach has been developed for high-precision δ(15)N measurements of amino acids (δ(15)N(AA)), optimized for δ(15)N(Phe) values. Amino acids (AAs) are first purified via high-pressure liquid chromatography (HPLC), using a mixed-phase column and automated fraction collection. The δ(15)N values are determined via offline elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). RESULTS: The combined HPLC/EA-IRMS method separated most protein AAs with sufficient resolution to obtain accurate δ(15)N values, despite significant intra-peak isotopic fractionation. For δ(15)N(Phe) values, the precision was ±0.16‰ for standards, 4× better than gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS; ±0.64‰). We also compared a δ(15)N(Phe) paleo-record from a deep-sea bamboo coral from Monterey Bay, CA, USA, using our method versus GC/C/IRMS. The two methods produced equivalent δ(15)N(Phe) values within error; however, the δ(15)N(Phe) values from HPLC/EA-IRMS had approximately twice the precision of GC/C/IRMS (average stdev of 0.27‰ ± 0.14‰ vs 0.60‰ ± 0.20‰, respectively). CONCLUSIONS: These results demonstrate that offline HPLC represents a viable alternative to traditional GC/C/IMRS for δ(15)N(AA) measurement. HPLC/EA-IRMS is more precise and widely available, and therefore useful in applications requiring increased precision for data interpretation (e.g. δ(15)N paleoproxies).