High-precision measurement of phenylalanine and glutamic acid δ15 N by coupling ion-exchange chromatography and purge-and-trap continuous-flow isotope ratio mass spectrometry.

RATIONALE: Nitrogen isotopic compositions (δ15 N) of source and trophic amino acids (AAs) are crucial tracers of N sources and trophic enrichments in diverse fields, including archeology, astrobiochemistry, ecology, oceanography, and paleo-sciences. The current analytical technique using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) requires derivatization, which is not compatible with some key AAs. Another approach using high-performance liquid chromatography-elemental analyzer-IRMS (HPLC/EA/IRMS) may experience coelution issues with other compounds in certain types of samples, and the highly sensitive nano-EA/IRMS instrumentations are not widely available. METHODS: We present a method for high-precision δ15 N measurements of AAs (δ15 N-AA) optimized for canonical source AA-phenylalanine (Phe) and trophic AA-glutamic acid (Glu). This offline approach entails purification and separation via high-pressure ion-exchange chromatography (IC) with automated fraction collection, the sequential chemical conversion of AA to nitrite and then to nitrous oxide (N2 O), and the final determination of δ15 N of the produced N2 O via purge-and-trap continuous-flow isotope ratio mass spectrometry (PT/CF/IRMS). RESULTS: The cross-plots of δ15 N of Glu and Phe standards (four different natural-abundance levels) generated by this method and their accepted values have a linear regression slope of 1 and small intercepts demonstrating high accuracy. The precisions were 0.36‰-0.67‰ for Phe standards and 0.27‰-0.35‰ for Glu standards. Our method and the GC/C/IRMS approach produced equivalent δ15 N values for two lab standards (McCarthy Lab AA mixture and cyanobacteria) within error. We further tested our method on a wide range of natural sample matrices and obtained reasonable results. CONCLUSIONS: Our method provides a reliable alternative to the current methods for δ15 N-AA measurement as IC or HPLC-based techniques that can collect underivatized AAs are widely available. Our chemical approach that converts AA to N2 O can be easily implemented in laboratories currently analyzing δ15 N of N2 O using PT/CF/IRMS. This method will help promote the use of δ15 N-AA in important studies of N cycling and trophic ecology in a wide range of research areas.

Effects of concentrated poultry operations and cropland manure application on antibiotic resistant Escherichia coli and nutrient pollution in Chesapeake Bay watersheds.

Manure from poultry operations is typically applied to nearby cropland and may affect nutrient loading and the spread of antibiotic resistance (ABR). We analyzed the concentrations of nitrogen and phosphorus and the occurrence of ABR in Escherichia coli (E. coli) and extra-intestinal pathogenic E. coli isolates from streams draining 15 small (<19 km2) watersheds of the Chesapeake Bay with contrasting levels of concentrated poultry operations. Total nitrogen and nitrate plus nitrite concentrations increased with poultry barn density with concentrations two and three times higher, respectively, in watersheds with the highest poultry barn densities compared to those without poultry barns. Analysis of N and O isotopes in nitrate by mass spectrometry showed an increase in the proportion of 15N associated with an increase in barn density, suggesting that the nitrate associated with poultry barns originated from manure. Phosphorus concentrations were not correlated with barn density. Antibiotic susceptibility testing of putative E. coli isolates was conducted using the disk diffusion method for twelve clinically important antibiotics. Of the isolates tested, most were completely susceptible (67%); 33% were resistant to at least one antibiotic, 24% were resistant to ampicillin, 13% were resistant to cefazolin, and 8% were multi-drug resistant. Resistance to three cephalosporin drugs was positively associated with an index of manure exposure estimated from poultry barn density and proportion of cropland in a watershed. The proportion of E. coli isolates resistant to cefoxitin, cefazolin, and ceftriaxone, broad-spectrum antibiotics important in human medicine, increased by 18.9%, 16.9%, and 6.2%, respectively, at the highest estimated level of manure exposure compared to watersheds without manure exposure. Our results suggest that comparisons of small watersheds could be used to identify geographic areas where remedial actions may be needed to reduce nutrient pollution and the public health risks of ABR bacteria.

Kelp beds and their local effects on seawater chemistry, productivity, and microbial communities.

Kelp forests are known as key habitats for species diversity and macroalgal productivity; however, we know little about how these biogenic habitats interact with seawater chemistry and phototroph productivity in the water column. We examined kelp forest functions at three locales along the Olympic Peninsula of Washington state by quantifying carbonate chemistry, nutrient concentrations, phytoplankton productivity, and seawater microbial communities inside and outside of kelp beds dominated by the canopy kelp species Nereocystis luetkeana and Macrocystis pyrifera. Kelp beds locally increased the pH, oxygen, and aragonite saturation state of the seawater, but lowered seawater inorganic carbon content and total alkalinity. Although kelp beds depleted nitrate and phosphorus concentrations, ammonium and dissolved organic carbon (DOC) concentrations were enhanced. Kelp beds also decreased chlorophyll concentrations and carbon fixed by phytoplankton, although kelp carbon fixation more than compensated for any difference in phytoplankton production. Kelp beds entrained distinct microbial communities, with higher taxonomic and phylogenetic diversity compared to seawater outside of the kelp bed. Kelp forests thus had significant effects on seawater chemistry, productivity and the microbial assemblages in their proximity. Thereby, the diversity of pathways for carbon and nitrogen cycling was also enhanced. Overall, these observations suggest that the contribution of kelp forests to nearshore carbon and nitrogen cycling is greater than previously documented.

A Ti(III) reduction method for one-step conversion of seawater and freshwater nitrate into N2 O for stable isotopic analysis of 15 N/14 N, 18 O/16 O and 17 O/16 O.

RATIONALE: The nitrogen and oxygen (δ15 N, δ18 O, and δ17 O values) isotopic compositions of nitrate (NO3 - ) are crucial tracers of nutrient nitrogen (N) sources and dynamics in aquatic systems. Current methods such as bacterial denitrification or Cd-azide reduction require laborious multi-step conversions or toxic chemicals to reduce NO3 - to N2 O for 15 N and 18 O isotopic analyses by isotope ratio mass spectrometry (IRMS). Furthermore, the 17 O composition of N2 O cannot be directly disentangled using IRMS because 17 O contributes to mass 45 (15 N). METHODS: We describe a new one-step chemical conversion method that employs Ti(III) chloride to reduce nitrate to N2 O gas in septum sample vials. Sample preparation takes only a few minutes followed by a 24-h reaction producing N2 O gas (65-75% recovery) which partitions into the headspace. The N2 O headspace was measured for 15 N, 18 O and 17 O by IRMS or laser spectrometry. RESULTS: IRMS and laser spectrometric analyses gave accurate and reproducible N and O isotopic results down to 50 ppb (3.5 µM) NO3 -N, similar in precision to the denitrifier and Cd-azide methods. The uncertainties for dissolved nitrate reference materials (USGS32, USGS34, USGS35, IAEA-NO3 ) were ±0.2‰ for δ15 N values and ±0.3‰ for δ18 O values using IRMS. For laser-based N2 O isotope analyses the results were similar, with an δ17 O uncertainty of ±0.9‰ without any need for 15 N correction. CONCLUSIONS: Advantages of the Ti(III) reduction method are simplicity, low cost, and no requirement for toxic chemicals or anaerobic bacterial cultures. Minor corrections may be required to account for sample nitrate concentration variance and potential chemical interferences. The Ti(III) method is easily implemented into laboratories currently using N2 O headspace sampling apparatus. We expect that the Ti(III) method will promulgate the use of N and O isotopes of nitrate in important studies of nutrient dynamics and pollution in a wide range of aquatic ecosystems.

Copepod-Associated Gammaproteobacteria Respire Nitrate in the Open Ocean Surface Layers.

Microbial dissimilatory nitrate reduction to nitrite, or nitrate respiration, was detected in association with copepods in the oxygenated water column of the North Atlantic subtropical waters. These unexpected rates correspond to up to 0.09 nmol N copepod-1 d-1 and demonstrate a previously unaccounted nitrogen transformation in the oceanic pelagic surface layers. Genes and transcripts for both the periplasmic and membrane associated dissimilatory nitrate reduction pathways (Nap and Nar, respectively) were detected. The napA genes and transcripts were closely related with sequences from several clades of Vibrio sp., while the closest relatives of the narG sequences were Pseudoalteromonas spp. and Alteromonas spp., many of them representing clades only distantly related to previously described cultivated bacteria. The discovered activity demonstrates a novel Gammaproteobacterial respiratory role in copepod association, presumably providing energy for these facultatively anaerobic bacteria, while supporting a reductive path of nitrogen in the oxygenated water column of the open ocean.

Single cell genomic and transcriptomic evidence for the use of alternative nitrogen substrates by anammox bacteria.

Anaerobic ammonium oxidation (anammox) contributes substantially to ocean nitrogen loss, particularly in anoxic marine zones (AMZs). Ammonium is scarce in AMZs, raising the hypothesis that organic nitrogen compounds may be ammonium sources for anammox. Biochemical measurements suggest that the organic compounds urea and cyanate can support anammox in AMZs. However, it is unclear if anammox bacteria degrade these compounds to ammonium themselves, or rely on other organisms for this process. Genes for urea degradation have not been found in anammox bacteria, and genomic evidence for cyanate use for anammox is limited to a cyanase gene recovered from the sediment bacterium Candidatus Scalindua profunda. Here, analysis of Ca. Scalindua single amplified genomes from the Eastern Tropical North Pacific AMZ revealed genes for urea degradation and transport, as well as for cyanate degradation. Urease and cyanase genes were transcribed, along with anammox genes, in the AMZ core where anammox rates peaked. Homologs of these genes were also detected in meta-omic datasets from major AMZs in the Eastern Tropical South Pacific and Arabian Sea. These results suggest that anammox bacteria from different ocean regions can directly access organic nitrogen substrates. Future studies should assess if and under what environmental conditions these substrates contribute to the ammonium budget for anammox.

N and O isotope (δ15 Nα , δ15 Nß , δ18 O, δ17 O) analyses of dissolved NO3- and NO2- by the Cd-azide reduction method and N2 O laser spectrometry.

RATIONALE: The nitrogen and oxygen (δ15 N, δ18 O, δ17 O) isotopic compositions of NO3- and NO2- are important tracers of nutrient dynamics in soil, rain, groundwater and oceans. The Cd-azide method was used to convert NO3- or NO2- to N2 O for N and triple-O isotopic analyses by N2 O laser spectrometry. A protocol for laser-based headspace isotope analyses was compared with isotope ratio mass spectrometry. Lasers provide the ability to directly measure 17 O anomalies which can help discern atmospheric N sources. METHODS: δ15 N, δ18 O and δ17 O values were measured on N/O stable isotopic reference materials (IAEA, USGS) by conversion to N2 O using the Cd-azide method and headspace N2 O laser spectrometry. A 15 N tracer test assessed the position-specific routing of N to the α or ß positions in the N2 O molecule. A data processing algorithm was used to correct for isotopic dependencies on N2 O concentration, cavity pressure and water content. RESULTS: NO3- /NO2- nitrogen is routed to the 15 Nα position of N2 O in the azide reaction; hence the δ15 Nα value should be used for N2 O laser spectrometry results. With corrections for cavity pressure, N2 O concentration and water content, the δ15 NαAIR , δ18 OVSMOW and δ17 OVSMOW values (‰) of international reference materials were +4.8 ± 0.1, +25.9 ± 0.3, +12.7 ± 0.2 (IAEA NO3 ), -1.7 ± 0.1, -26.8 ± 0.8, -14.4 ± 1.1 (USGS34) and +2.6 ± 0.1, +57.6 ± 1.2, +51.2 ± 2.0 (USGS35), in agreement with their values and with the isotope ratio mass spectrometry results. The 17 O excess for USGS35 was +21.2 ± 9‰, in good agreement with previous results. CONCLUSIONS: The Cd-azide method yielded excellent results for routine determination of δ15 N, δ18 O and δ17 O values (and the 17 O excess) of nitrate or nitrite by laser spectrometry. Disadvantages are the toxicity of Cd-azide chemicals and the lack of automated sampling devices for N2 O laser spectrometers. The 15 N-enriched tracer test revealed potential for position-specific experimentation of aqueous nutrient dynamics at high 15 N enrichments by laser spectrometry, but exposed the need for memory corrections and improved spectral deconvolution of 17 O.

Steady-State Oxygen Isotope Effects of N2O Production in Paracoccus denitrificans.

Knowledge of isotopic discrimination, or fractionation, by denitrifying bacteria can benefit agricultural fertilizer management, wastewater treatment, and other applications. However, the complexity of N transformation pathways in the environment and the sensitivity of denitrification to environmental conditions warrant better isotopic distinction between denitrification and other processes, especially for oxygen isotopes. Here, we present a dataset of δ18O measurements in continuous culture of Paracoccus denitrificans. The authors hope that it will be useful in further studies of N2O in the environment.

Endangered Right Whales Enhance Primary Productivity in the Bay of Fundy.

Marine mammals have recently been documented as important facilitators of rapid and efficient nutrient recycling in coastal and offshore waters. Whales enhance phytoplankton nutrition by releasing fecal plumes near the surface after feeding and by migrating from highly productive, high-latitude feeding areas to low-latitude nutrient-poor calving areas. In this study, we measured NH4+ and PO43- release rates from the feces of North Atlantic right whales (Eubalaena glacialis), a highly endangered baleen whale. Samples for this species were primarily collected by locating aggregations of whales in surface-active groups (SAGs), which typically consist of a central female surrounded by males competing for sexual activity. When freshly collected feces were incubated in seawater, high initial rates of N release were generally observed, which decreased to near zero within 24 hours of sampling, a pattern that is consistent with the active role of gut microflora on fecal particles. We estimate that at least 10% of particulate N in whale feces becomes available as NH4+ within 24 hours of defecation. Phosphorous was also abundant in fecal samples: initial release rates of PO43- were higher than for NH4+, yielding low N/P nutrient ratios over the course of our experiments. The rate of PO43- release was thus more than sufficient to preclude the possibility that nitrogenous nutrients supplied by whales would lead to phytoplankton production limited by P availability. Phytoplankton growth experiments indicated that NH4+ released from whale feces enhance productivity, as would be expected, with no evidence that fecal metabolites suppress growth. Although North Atlantic right whales are currently rare (approximately 450 individuals), they once numbered about 14,000 and likely played a substantial role in recycling nutrients in areas where they gathered to feed and mate. Even though the NH4+ released from fresh whale fecal material is a small fraction of total whale fecal nitrogen, and recognizing the fact that the additional nitrogen released in whale urine would be difficult to measure in a field study, the results of this study support the idea that the distinctive isotopic signature of the released NH4+ could be used to provide a conservative estimate of the contribution of the whale pump to primary productivity in coastal regions where whales congregate.

NC10 bacteria in marine oxygen minimum zones.

Bacteria of the NC10 phylum link anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophy pathway. A niche for NC10 in the pelagic ocean has not been confirmed. We show that NC10 bacteria are present and transcriptionally active in oceanic oxygen minimum zones (OMZs) off northern Mexico and Costa Rica. NC10 16S rRNA genes were detected at all sites, peaking in abundance in the anoxic zone with elevated nitrite and methane concentrations. Phylogenetic analysis of particulate methane monooxygenase genes further confirmed the presence of NC10. rRNA and mRNA transcripts assignable to NC10 peaked within the OMZ and included genes of the putative nitrite-dependent intra-aerobic pathway, with high representation of transcripts containing the unique motif structure of the nitric oxide (NO) reductase of NC10 bacteria, hypothesized to participate in O2-producing NO dismutation. These findings confirm pelagic OMZs as a niche for NC10, suggesting a role for this group in OMZ nitrogen, methane and oxygen cycling.

The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements.

We report on the contamination of commercial 15-nitrogen (15N) N2 gas stocks with 15N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. 15N2 gas is used to estimate N2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled 15N2 into organic matter. However, the microbial assimilation of bioavailable 15N-labeled N2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N2 fixation rates. 15N2 gas procured from three major suppliers was analyzed for the presence of these 15N-contaminants. Substantial concentrations of 15N-contaminants were detected in four Sigma-Aldrich 15N2 lecture bottles from two discrete batch syntheses. Per mole of 15N2 gas, 34 to 1900 µmoles of 15N-ammonium, 1.8 to 420 µmoles of 15N-nitrate/nitrite, and ≥21 µmoles of 15N-nitrous oxide were detected. One 15N2 lecture bottle from Campro Scientific contained ≥11 µmoles of 15N-nitrous oxide per mole of 15N2 gas, and no detected 15N-nitrate/nitrite at the given experimental 15N2 tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles 15N-nitrous oxide per mole 15N2, and trace concentrations of 15N-ammonium and 15N-nitrate/nitrite. 15N2 gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the 15N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N2 fixation assays suggests that the degree of detected 15N-ammonium contamination could yield inferred N2 fixation rates ranging from undetectable, <0.01 nmoles N L(-1) d(-1), to 530 nmoles N L(-1) d(-1), contingent on experimental conditions. These rates are comparable to, or greater than, N2 fixation rates commonly detected in field assays. These results indicate that past reports of N2 fixation should be interpreted with caution, and demonstrate that the purity of commercial 15N2 gas must be ensured prior to use in future N2 fixation rate determinations.

Bait and the susceptibility of American lobsters Homarus americanus to epizootic shell disease.

Shell disease (SD) has been observed in lobster populations for almost a hundred years, but recently, rates of an epizootic form of shell disease (ESD) have increased in the southern New England (USA) area. A large proportion of fish in the diet of American lobsters Homarus americanus has been linked to increased rates of SD. Therefore, the use of fish as lobster bait may be linked to increased ESD rates in lobsters. Lobsters from the western portion of Martha's Vineyard, MA (41 degrees N, 71 degrees W), were randomly divided into 3 groups of 16 and exposed to dietary treatments (100% herring; 48% crab, 48% blue mussel and 4% plant matter; or 50% herring, 24% crab, 24% mussel, 2% plant matter) to determine if lobster tissue delta15N levels reflected diet. The results of the feeding experiment confirmed that differences in diet are observed in the delta15N levels of lobster muscle tissue. The delta15N levels of tissue samples from 175 wild lobsters with varying degrees of ESD were unrelated to ESD severity but did indicate lobsters were eating large amounts of fish (bait). This result does not support the speculation that fish used as bait is contributing to ESD outbreaks in portions of the southern New England area.

Some challenges of an "upside down" nitrogen budget--science and management in Greenwich Bay, RI (USA).

When nutrients impact estuarine water quality, scientists and managers instinctively focus on quantifying and controlling land-based sources. However, in Greenwich Bay, RI, the estuary opens onto a larger and more intensively fertilized coastal water body (Narragansett Bay). Previous inventories of nitrogen (N) inputs to Greenwich Bay found that N inputs from Narragansett Bay exceeded those from the local watershed, suggesting that recent efforts to reduce local watershed N loads may have little effect on estuarine water quality. We used stable isotopes of N to characterize watershed and Narragansett Bay N sources as well as the composition of primary producers and consumers throughout Greenwich Bay. Results were consistent with previous assessments of the importance of N inputs to Greenwich Bay from Narragansett Bay. As multiple N sources contribute to estuarine water quality, effective management requires attention to individual sources commensurate with overall magnitude, regardless of the political complications that may entail.

Amino-group-specific natural abundance nitrogen isotope ratio analysis in amino acids.

Amino acid (AA) nitrogen (N) stable isotope ratio analysis has found a wide variety of important applications including indication of the trophic level of an organism, tracing N transfer within food webs, and monitoring of AA resynthesis during heterotrophic microbial reworking of organic matter. Despite its utility, the current methodology is difficult to employ consistently for natural abundance level precision. Here, we report a new and robust method for high-precision N-compound-specific isotope analysis (N-PCIA) for single-N-containing AAs and N-position-specific isotope analysis (N-PSIA) for poly-N AAs. First the amino-N in AAs was liberated and oxidized to NO2(-) by hypochlorite at high pH. The NO2(-) produced was then quantified colorimetrically with excess hypochlorite quenched using arsenite. Subsequently, buffered azide was used to reduce NO2(-) to N2O for isotope ratio analysis using a purge-and-trap isotope ratio mass spectrometer. In the case of glycine delta15N, the average precision was SD = 0.3 per thousand. Reaction yields and labeling experiments show that this oxidation reaction is highly specific, targeting the alpha-amino group (peptide-N) of most poly-N AAs. This permits specific determination of the delta15N of peptide-N in arginine, tryptophan, and histidine. In the case of lysine, however, the side-chain amino group was found to be partially labile to hypochlorite oxidation. Using isotope fractionation factors estimated from single-N analogues of lysine, the intramolecular delta15N of lysine was calculated by mass balance, and this generally agreed with results for the same sample material analyzed by a previously published enzymatic method. Our method has the advantages of being relatively rapid, robust, and applicable to all poly-N AAs. We have also found it to work well for determining total delta15N of amino-N in complex sample matrices that have not been susceptible to previous approaches.

Isotopic apportionment of atmospheric and sewage nitrogen sources in two Connecticut rivers.

We used the dual isotope approach to identify sources of nitrate (NO3-) to two mixed land-use watersheds draining to Long Island Sound. In contrastto previous work, we found that sewage effluent NO3- was not consistently enriched in 15N. However, these effluents followed a characteristic denitrification line in delta15N-delta18O space, which could be used as a source signature. We used this signature, together with those of atmospheric deposition and microbial nitrification, to calculate ranges of possible contributions from each of these sources. These estimates are unaffected by any denitrification that may have taken place in soils or streams. Our estimates for atmospheric nitrogen only include unprocessed atmospheric deposition, i.e., NO3-that is not taken up in watershed soils before being delivered to rivers. Using this method, the contribution of atmospheric NO3- could be assessed with good precision and was found to be very low at all our sampling sites during baseflow. During a moderate storm event, atmospheric deposition contributed up to approximately 50% of stream NO3-, depending on the site, with the sites that experienced more stormflow showing a greater contribution of atmospheric NO3-. Our estimates of sewage contribution generally had too large a range to be useful.

Sensitive measurement of NH4+ 15N/14N (delta 15NH4+) at natural abundance levels in fresh and saltwaters.

We report a new method for determining the 15N/14N of NH4+ at natural abundance level in both freshwater and seawater. NH4+ is first quantitatively oxidized to NO2- by hypobromite (BrO-) at pH approximately 12. After the addition of sodium arsenite to consume excess BrO-, yield is verified by colorimetric NO2- determination. NO2- is further reduced to N2O using a 1:1 sodium azide and acetic acid buffer solution using previously established procedures. The product N2O is then analyzed for isotopic composition using a continuous flow purge and cryogenic trap system coupled to an isotope ratio mass spectrometer. Reliable delta 15N values (standard deviation is 0.3 per thousand or better) are obtained over an NH4+ concentration range of 0.5-10 microM using 20 mL volumes of either freshwater or seawater samples. Higher concentration samples are readily diluted to lower concentration. Preexisting NO2- is removed by treatment with sulfanilic acid. There is no interference from any of the nitrogen-containing compounds tested except short-chain aliphatic amino acids (i.e., glycine) which typically are present at very low environmental concentrations. As compared to published methods, our approach is more robust, readily applicable at low concentrations and small sample volumes, and requires less time for preparation and analysis.

Methods for measuring denitrification: diverse approaches to a difficult problem.

Denitrification, the reduction of the nitrogen (N) oxides, nitrate (NO3-) and nitrite (NO2-), to the gases nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2), is important to primary production, water quality, and the chemistry and physics of the atmosphere at ecosystem, landscape, regional, and global scales. Unfortunately, this process is very difficult to measure, and existing methods are problematic for different reasons in different places at different times. In this paper, we review the major approaches that have been taken to measure denitrification in terrestrial and aquatic environments and discuss the strengths, weaknesses, and future prospects for the different methods. Methodological approaches covered include (1) acetylene-based methods, (2) 15N tracers, (3) direct N2 quantification, (4) N2:Ar ratio quantification, (5) mass balance approaches, (6) stoichiometric approaches, (7) methods based on stable isotopes, (8) in situ gradients with atmospheric environmental tracers, and (9) molecular approaches. Our review makes it clear that the prospects for improved quantification of denitrification vary greatly in different environments and at different scales. While current methodology allows for the production of accurate estimates of denitrification at scales relevant to water and air quality and ecosystem fertility questions in some systems (e.g., aquatic sediments, well-defined aquifers), methodology for other systems, especially upland terrestrial areas, still needs development. Comparison of mass balance and stoichiometric approaches that constrain estimates of denitrification at large scales with point measurements (made using multiple methods), in multiple systems, is likely to propel more improvement in denitrification methods over the next few years.

Chemical conversion of nitrate and nitrite to nitrous oxide for nitrogen and oxygen isotopic analysis in freshwater and seawater.

We present a novel method for nitrogen and oxygen natural isotopic abundance analysis of nitrate and nitrite of seawater and freshwater at environmental concentrations. The method involves the reduction of nitrate to nitrite using spongy cadmium with further reduction to nitrous oxide using sodium azide in an acetic acid buffer. For separate nitrite analysis, the cadmium reduction step is simply bypassed. Nitrous oxide is purged from the water sample and trapped cryogenically using an automated system with subsequent release into a gas chromatography column. The isolated nitrous oxide is then analyzed on a continuous flow isotope ratio mass spectrometer via an open split. This paper describes the basic protocol and reaction conditions required to obtain reproducible natural abundance level nitrogen and oxygen isotopic ratios from nitrate, nitrite, or both, and the results obtained to support these conclusions. A standard deviation less than 0.2 per thousand for nitrogen and 0.5 per thousand for oxygen was found for nitrate samples ranging in concentration from 40 to 0.5 microM (better for nitrite), with a blank of 2 nmol for 50-mL samples. Nitrogen and oxygen isotopic fractionation and oxygen atom exchange were consistent within each batch of analysis. There was no interference from any seawater matrixes. Only one other method published to date can measure the nitrate oxygen isotopic abundance in seawater and none that do so for nitrite alone in the presence of nitrate. This method may prove to be simpler, faster, and obtain isotopic information for lower concentrations of nitrate and nitrite than other methods.

Southern Ocean iron enrichment experiment: carbon cycling in high- and low-Si waters.

The availability of iron is known to exert a controlling influence on biological productivity in surface waters over large areas of the ocean and may have been an important factor in the variation of the concentration of atmospheric carbon dioxide over glacial cycles. The effect of iron in the Southern Ocean is particularly important because of its large area and abundant nitrate, yet iron-enhanced growth of phytoplankton may be differentially expressed between waters with high silicic acid in the south and low silicic acid in the north, where diatom growth may be limited by both silicic acid and iron. Two mesoscale experiments, designed to investigate the effects of iron enrichment in regions with high and low concentrations of silicic acid, were performed in the Southern Ocean. These experiments demonstrate iron's pivotal role in controlling carbon uptake and regulating atmospheric partial pressure of carbon dioxide.

The effect of millennial-scale changes in Arabian Sea denitrification on atmospheric CO2.

Most global biogeochemical processes are known to respond to climate change, some of which have the capacity to produce feedbacks through the regulation of atmospheric greenhouse gases. Marine denitrification-the reduction of nitrate to gaseous nitrogen-is an important process in this regard, affecting greenhouse gas concentrations directly through the incidental production of nitrous oxide, and indirectly through modification of the marine nitrogen inventory and hence the biological pump for CO2. Although denitrification has been shown to vary with glacial-interglacial cycles, its response to more rapid climate change has not yet been well characterized. Here we present nitrogen isotope ratio, nitrogen content and chlorin abundance data from sediment cores with high accumulation rates on the Oman continental margin that reveal substantial millennial-scale variability in Arabian Sea denitrification and productivity during the last glacial period. The detailed correspondence of these changes with Dansgaard-Oeschger events recorded in Greenland ice cores indicates rapid, century-scale reorganization of the Arabian Sea ecosystem in response to climate excursions, mediated through the intensity of summer monsoonal upwelling. Considering the several-thousand-year residence time of fixed nitrogen in the ocean, the response of global marine productivity to changes in denitrification would have occurred at lower frequency and appears to be related to climatic and atmospheric CO2 oscillations observed in Antarctic ice cores between 20 and 60 kyr ago.