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Effluent discharged from water reclamation facilities (WRFs) contains dissolved organic nitrogen, termed effluent dissolved organic nitrogen (EDON), that subsequently enters coastal waterways. It is still unclear at what rate EDON can be taken up by microbial communities relative to other nitrogen (N) substrates. Bench-scale sequencing batch reactors (SBRs), used to mimic WRFs, were supplied with 15N-labeled ammonium (15NH4+) to produce 15N-labeled EDON (EDO15N) that was subsequently used to measure uptake rates along a salinity gradient of the York River, Virginia, USA, in the spring and summer. Although NH4+ dominated influent N pools, only a small fraction (4.1%) of EDON was produced from NH4+ microbial assimilation in biological treatment processes. When added as a short-term (4-h) tracer, the EDO15N was taken up by estuarine microbes at rates 0.01–0.434 μmol N L−1 h−1, which are similar to rates of NH4+ and nitrate uptake. When added to 48-h bioassays, EDON stimulated phytoplankton growth more at the lower salinity (0–8‰) sites (8.5–13.8 μg Chl a L−1) than at the higher salinity (20‰) site (up to 0.4 μmol Chl a L−1). The microbes in the 0.7–5 μm size fraction had significantly higher EDO15N uptake rates than the larger size fraction (e.g., > 5 μm, p

The Arctic is warming at a rate nearly twice the global average, leading to thawing permafrost, increased coastal erosion, and enhanced delivery of riverine terrestrially-derived dissolved organic matter (tDOM) to coastal waters. This humic-rich tDOM has the ability to attenuate light required for photosynthesis and stimulate heterotrophic growth by supplying a source of labile organic carbon. Due to tDOM's high carbon to nitrogen (C : N) ratio, additional nitrogen is required for microorganisms to utilize this excess carbon for growth, thus exacerbating competition between autotrophs and heterotrophs for limiting nutrients and potentially reducing primary production. The effect of Arctic tDOM additions on nitrate uptake by two microplankton size fractions in the coastal Chukchi Sea was quantified using 15N tracer methods. To assess the biogeochemical vs. spectral impacts of tDOM, the uptake incubations were amended with either tDOM or light attenuating films that mimic light absorption by the tDOM. Nitrate uptake and primary production rates in the larger, predominantly phytoplankton, size fraction generally decreased with increasing tDOM additions. The change in light attenuation alone accounted for a ∼ 50% reduction in nitrate uptake. Responses in the smaller size fraction varied seasonally with tDOM additions stimulating uptake in spring and suppressing it in summer. The largest variation in summer nitrate uptake can be explained by the shared effect of biogeochemistry and light attenuation. Therefore, large increases in tDOM delivery currently occurring and predicted to increase in the coastal Arctic, could reduce primary production, broadly impact nitrogen and carbon cycling, and affect higher trophic levels.

Biogeochemical rate processes in the Arctic are not currently well constrained, and there is very limited information on how rates may change as the region warms. Here we present data on the sensitivity of ammonium (NH4+) uptake and nitrification rates to short-term warming. Samples were collected from the Chukchi Sea off the coast of Barrow, Alaska, during winter, spring, and summer and incubated for 24 h in the dark with additions of 15NH4+ at −1.5, 6, 13, and 20°C. Rates of NH4+ uptake and nitrification were measured in conjunction with bacterial production. In all seasons, NH4+ uptake rates were highest at temperatures similar to current summertime conditions but dropped off with increased warming, indicative of psychrophilic (i.e., cold-loving) microbial communities. In contrast, nitrification rates were less sensitive to temperature and were higher in winter and spring compared to summer. These findings suggest that as the Arctic coastal ecosystem continues to warm, NH4+ assimilation may become increasingly important, relative to nitrification, although the magnitude of NH4+ assimilation would be still be lower than nitrification.

How Arctic climate change might translate into alterations of biogeochemical cycles of carbon (C) and nitrogen (N) with respect to inorganic and organic N utilization is not well understood. This study combined 15 N uptake rate measurements for ammonium, nitrate, and urea with 15 N- and 13 C-based DNA stable-isotope probing (SIP). The objective was to identify active bacterial and archeal plankton and their role in N and C uptake during the Arctic summer and winter seasons. We hypothesized that bacteria and archaea would successfully compete for nitrate and urea during the Arctic winter but not during the summer, when phytoplankton dominate the uptake of these nitrogen sources. Samples were collected at a coastal station near Barrow, AK, during August and January. During both seasons, ammonium uptake rates were greater than those for nitrate or urea, and nitrate uptake rates remained lower than those for ammonium or urea. SIP experiments indicated a strong seasonal shift of bacterial and archaeal N utilization from ammonium during the summer to urea during the winter but did not support a similar seasonal pattern of nitrate utilization. Analysis of 16S rRNA gene sequences obtained from each SIP fraction implicated marine group I Crenarchaeota (MGIC) as well as Betaproteobacteria , Firmicutes , SAR11, and SAR324 in N uptake from urea during the winter. Similarly, 13 C SIP data suggested dark carbon fixation for MGIC, as well as for several proteobacterial lineages and the Firmicutes . These data are consistent with urea-fueled nitrification by polar archaea and bacteria, which may be advantageous under dark conditions.

To investigate nutrient release from decaying fish carcasses as a potential source fueling blooms of the toxic dinoflagellate, Karenia brevis (C.C. Davis) G. Hansen & O. Moestrup this study evaluated the type and quantity of nutrients (nitrogen (N), phosphorus (P), and carbon (C)) released from decaying fish using both wet chemistry and compound specific (Fourier transform ion cyclotron resonance mass spectrometry, FT-ICR MS) techniques. A 3 day bioassay experiment was then conducted to examine how eutrophic (Charlotte Harbor, CH) and oligotrophic (Blue Water, BW) microbial communities in the eastern Gulf of Mexico (GoM) respond to fish-derived nutrients (FDN) relative to other in situ N sources. Decaying fish released a suite of compounds, primarily in the form of ammonium (NH 4 + ); release rates ranged from 37.7 ± 12.6 to 102.3 ± 4.7 μmol N L −1 g −1 wet wt. d −1 . Phosphate release was 13.9 ± 2.8 to 21.4 ± 2.9 μmol P L −1 g −1 wet wt. d −1 . A total of 3515 organic compounds were produced, which were dominated by lipid and protein-like compounds. In the bioassay, FDN were utilized by microbial communities collected from both CH and BW, with complete drawdown of fish-derived NH 4 + in the CH treatment in conjunction with a 3-fold increase in Chl a . FDN provided the necessary nutrients to alleviate P limitation in the BW treatment, resulting in a 13-fold increase in Chl a . Results from this experiment indicate FDN are important N and P sources to the microbial community along the eastern GoM and have important implications for maintenance of associated ichthyotoxic K. brevis blooms.

The West Florida Shelf (WFS) encompasses a range of environments from inshore estuarine to offshore oligotrophic waters, which are frequently the site of large and persistent blooms of the toxic dinoflagellate, Karenia brevis. The goals of this study were to characterize the nitrogen (N) nutrition of plankton across the range of environmental conditions on the WFS, to quantify the percentage of the plankton N demand met through in situ N regeneration, and to determine whether planktonic N nutrition changes when high concentrations of Karenia are present. In the fall of 2007, 2008, and 2009 we measured ambient nutrient concentrations and used stable isotope techniques to measure rates of primary production and uptake rates of inorganic N (ammonium, NH4+, and nitrate, NO3−), and organic N and carbon (C; urea and amino acids, AA) in estuarine, coastal, and offshore waters, as well as coastal sites with Karenia blooms present. In parallel, we also measured rates of in situ N regeneration – NH4+ regeneration, nitrification, and photoproduction of NH4+, nitrite and AA. Based on microscope observations, ancillary measurements, and previous monitoring history, Karenia blooms sampled represented three bloom stages – initiation in 2008, maintenance in 2007, and late maintenance/stationary phase in 2009. Nutrient concentrations were highest at estuarine sampling sites and lowest at offshore sites. Uptake of NH4+ and NO3− provided the largest contribution to N nutrition at all sites. At the non-Karenia sites, in situ rates of NH4+ regeneration and nitrification were generally sufficient to supply these substrates equal to the rates at which they were taken up. At Karenia sites, NO3− was the most important N substrate during the initiation phase, while NH4+ was the most important N form used during bloom maintenance and stationary phases. Rates of NH4+ regeneration were high but insufficient (85 ± 36% of uptake) to support the measured NH4+ uptake at all the Karenia sites although nitrification rates far exceeded uptake rates of NO3−. Taken together our results support the “no smoking gun” nutrient hypothesis that there is no single nutrient source or strategy that can explain Karenia's frequent dominance in the waters where it occurs. Consistent with other papers in this volume, our results indicate that Karenia can utilize an array of inorganic and organic N forms from a number of N sources.

This study represents the most comprehensive assessment of kinetic parameters for Karenia brevis to date as it encompasses natural populations sampled during three different bloom years in addition to cultured strains under controlled conditions. Nitrogen (N) uptake kinetics for ammonium (NH 4 + ), nitrate (NO 3 − ), urea, an amino acid mixture, individual amino acids (glutamate and alanine), and humic substrates were examined for the toxic red tide dinoflagellate, K. brevis , during short term incubations (0.5–1 h) using 15 N tracer techniques. Experiments were conducted using natural populations collected during extensive blooms along the West Florida Shelf in October 2001, 2002, and 2007, and in cultured strains (CCFWC 251 and CCFWC 267) obtained from the Florida Fish and Wildlife Institute culture collection. Kinetic parameters for the maximum uptake velocity ( V max ), half-saturation concentration ( K s ), and the affinity constant ( α ) were determined. The affinity constant is considered a more accurate indicator of substrate affinity at low concentrations. K. brevis took up all organic substrates tested, including N derived from humic substances. Uptake rates of the amino acid mixture and some NO 3 − incubations did not saturate even at the highest substrate additions (50–200 μmol N L −1 ). Based upon the calculated α values, the greatest substrate preference was for NH 4 + followed by NO 3 − ≥ urea, humic compounds and amino acids. The ability of K. brevis to utilize a variety of inorganic and organic substrates likely helps it flourish under a wide range of nutrient conditions from bloom initiation in oligotrophic waters offshore to bloom maintenance near shore where ambient nutrient concentrations may be orders of magnitude greater.

Dissolved organic nitrogen (DON) produced by the nitrogen-fixer Trichodesmium sp. has the potential to serve as a nitrogen source for the red tide dinoflagellate Karenia brevis. Dis- solved organic matter (DOM) from laboratory cultures of Trichodesmium sp. was isolated, concen- trated and then supplied as a nutrient source to K. brevis cells collected from the Gulf of Mexico. K. brevis abundance increased immediately after Trichodesmium sp. cellular exudate (TCE) addition, allowing the population to double within the first 24 h. There was rapid and complete utilization of the TCE DON as well as ~89% of the TCE dissolved organic phosphorus (DOP). Additionally, ter- minal restriction fragment length polymorphism (TRFLP) was used to assess the bacterial commu- nity response to the addition of TCE . The number of bacterial operational taxonomic units (OTUs) initially increased after the TCE DOM addition, but decreased as K. brevis reached its maximum abundance. Electrospray ionization mass spectrometry (ESI-MS) and Fourier transform ion cy- clotron resonance mass spectrometry (FT-ICR MS) were used to chemically characterize the DOM. Approximately 25% of compounds disappeared within the first 24 h, corresponding to the greatest increase in K. brevis abundance. Using FT-ICR MS, 391 DON and 219 DOP potentially bioavailable compounds were characterized. The bioavailable DON compounds were highly re- duced and 44% had molar ratios indicative of lipid or protein-like compounds. The changes in DON concentration and compound composition show that Tricho desmium sp. provides a sufficient source of nitrogen to directly or indirectly support K. brevis blooms.

In advanced wastewater treatment plants that achieve high levels of nitrogen (N) removal, up to one-third of the N in effluent is organic, herein referred to as effluent organic N (EON). While we know that inorganic N is highly labile, it is unclear what fraction of EON is bioavailable. In this study, we demonstrate the utility of a method that can be used to examine the reactivity of EON in natural receiving waters to better understand both the ecosystem response and the potential bioavailability of EON. The technique is suitable for analyzing polar organic matter in natural waters; electrospray ionization coupled with Fourier transform mass spectrometry. Bioassays were performed on samples collected at the end of the biological process from two wastewater treatment plants achieving advanced N removal. The samples were concentrated, and then added to natural water samples collected from the oligohaline James River, a major tributary of the Chesapeake Bay. Our results demonstrate that while the lignin-like fraction of the effluent dissolved organic matter (some of which contains N) was conserved, a large portion of aliphatic and aromatic compounds containing N was removed (79–100%) during incubations, while other compounds were produced. Furthermore, the two effluents exhibited differences in the degree of degradation and type of degradation, which can be related both to the various processes employed in the two WWTPs and the dramatic differences in the type of influent they received. These findings suggest that EON is highly reactive in the natural environment and that simple assays examining net consumption or production of bulk dissolved organic N pools are inadequate for assessing the bioavailability of EON.

Crustacean zooplankton produce dissolved organic matter (DOM) and inorganic nutrients via sloppy feeding, excretion, and fecal pellet leaching. These different mechanisms of the release of metabolic products, however, have never been individually isolated. Our study was designed to determine the relative importance of these different modes on release of dissolved organic carbon (DOC), ammonium (NH4+), and urea from Acartia tonsa calanoid copepods feeding on the diatom Thalassiosira weissflogii. Excretion and sloppy feeding were the dominant modes of DOC production (80 and 20% of total DOC release, respectively) and NH4+ release (93 and 7% of total NH4+ release, respectively). Urea, however, was predominately produced via sloppy feeding and fecal pellet leaching (25% and 62% of total urea release, respectively). Urea contributed 20% of total measured nitrogen (TMN; NH4+ + urea) released from copepods, and constituted 100% of TMN released via fecal pellet leaching, 47% of TMN released via sloppy feeding, and only 3.5% of TMN released via excretion. TMN release was > 100% of copepod body N d− 1, resulting in low DOC:TMN release ratios (4.1 for sloppy feeding, 2.1 for cumulative release of sloppy feeding, excretion, and fecal pellet leaching). Our results suggest that the mechanism of release plays an important role in the amount of different forms of DOM, NH4+, and urea available to bacteria and phytoplankton.

Harmful algal blooms (HABs), including toxic species, have been increasing in frequency, range, and duration over the past several decades. The effect of a harmful or toxic algal diet on zooplankton nutrient regeneration, however, has not been previously examined. In this study, we determined the effects of non-bloom and bloom concentrations of non-toxic and toxic cultures of HAB species Prorocentrum minimum and Karlodinium veneficum on grazing and production of dissolved organic carbon (DOC), nitrogen (DON), and phosphorus (DOP), and inorganic nutrients, ammonium (NH4+) and phosphate (PO43−), by the copepod Acartia tonsa and the heterotrophic dinoflagellate Oxyrrhis marina. Ingestion rates of grazers were significantly higher while feeding on bloom algal concentrations compared to non-bloom algal concentrations, but were always below 1% body C d−1 for A. tonsa (ingestion rate range of 0.5–31 ng C individual−1 d−1) and below 2% body C d−1 for O. marina (range of 0.1–8.8 pg C individual−1 d−1). However, rates of inorganic nutrient and dissolved organic matter (DOM) release, when detected, were always >100% of carbon (C), nitrogen (N), and phosphorus (P) ingested. Additionally, the quantity and forms (organic vs. inorganic) of nutrients released by zooplankton were significantly different between non-toxic and toxic algal treatments, and typically higher grazer DOM release occurred while feeding on toxic algal cultures. DOM was the only detected form of nutrient released from O. marina, and DON and DOP were significant portions of total dissolved N and P released for A. tonsa feeding on toxic K. veneficum (69–84% and 73%, respectively). Low grazing rates on all algal diets used in our study, regardless of cell concentration or toxicity, likely resulted in starvation and subsequent catabolism of grazer body tissue. The potential for additional factors affecting variable grazer nutrient release between toxic and non-toxic algal treatments, including algal nutrient quality and direct toxic effects, are discussed. Our results suggest these grazers may not be capable of controlling bloom formation of these HAB species, and that nutrient cycling dynamics in the coastal ocean may change with increases in the presence of harmful and toxic algal blooms.

Uptake of inorganic and organic nitrogen (N) by phytoplankton and bacteria was inves- tigated during a mesocosm study conducted in Raunefjord, Norway in April 2005. One mesocosm was fertilized with nitrate and phosphate at a ratio of 16:1 and maintained in the light, while one un- amended light mesocosm served as a control. Dissolved nutrients, phytoplankton and bacterial bio- mass, and phytoplankton community composition were monitored throughout the 26 d experiment. Uptake of 15 N-labeled ammonium and nitrate, and dual-labeled ( 15 N and 13 C) urea and dissolved free amino acids (DFAA) was measured for phytoplankton and bacteria using 2 methods: size fractiona- tion into 0.2-0.8 and >0.8 μm size classes, and flow cytometric sorting based on chlorophyll autoflu- orescence. Prior to fertilization, dissolved inorganic N concentrations were low and comprised ~5% of total dissolved N. Added nitrate was completely utilized in the amended mesocosm within 10 d, stimulating a large bloom of colonial Phaeocystis pouchetii. Ammonium contributed over half of total measured N uptake by phytoplankton and bacteria in both enclosures, while nitrate and urea each supplied roughly 10 to 25%. Overall, DFAA were a negligible N source to phytoplankton but con- tributed 11% to total bacterial N uptake. Bacterial uptake represented a significant portion of total uptake of all N forms, especially urea and DFAA. Comparison of the 2 methods for measuring phyto- plankton versus bacterial uptake demonstrates how the use of 0.8 μm filters can lead to significant overestimation of phytoplankton N uptake due to the retention of bacterial biomass.

Little is known about the relative importance of inorganic and organic nitrogen (N) sources in fueling production of phytoplankton versus heterotrophic bacteria on the continental shelf. This issue was addressed during two diel experiments conducted in the Mid-Atlantic Bight at the Long-term Ecosystem Observatory, LEO-15, off southern New Jersey. Uptake of 15 N-labeled ammonium (NH 4 + ), nitrate (NO 3 − ), and nitrite (NO 2 − ), and dual-labeled ( 15 N and 13 C) urea and dissolved free amino acids was measured in water taken from the surface and bottom mixed layers approximately every 4 h over two 24 h periods in July 2002. Two methods were used to quantify 15 N uptake rates: (1) traditional filtration into various phytoplankton and bacterial size classes, and (2) flow cytometric (FCM) sorting of autotrophic cells based on the presence of chlorophyll autofluorescence. Due to a strong pycnocline, the nutrient composition was quite distinct between the surface and bottom mixed layers. Dissolved organic N (DON) comprised >99% of the total dissolved N (TDN) pool in surface waters, whereas the bottom-water TDN pool was roughly divided between NH 4 + , NO 3 − , and DON. Urea was the dominant N form used by all fractions at the surface, and although phytoplankton >3 μm was responsible for most of the urea uptake, bacterial use was detected using stable isotopes and also suggested by ure C sequence analysis. The majority of ure C sequences recovered from the 0.2–0.8 μm fraction belonged to members of the Alphaproteobacteria (46%), whereas those of the 0.8–3.0 μm size class consisted primarily of Cyanobacteria (70%). In contrast to the surface, N uptake in the bottom layer was dominated by NH 4 + . The bacterial fraction was responsible for 20–49% of the size-fractionated NH 4 + and NO 3 − uptake in surface samples and 36–93% at the bottom. These results suggest that organic N, such as urea, is a viable source of N nutrition to phytoplankton forced to compete with heterotrophic bacteria for limited inorganic N.

Total maximum daily loads for nitrogen (N) are currently being established for the Chesapeake Bay watershed. While we know inorganic N is bioavailable in the environment and therefore its input contributes to cultural eutrophication, the bioavailability of organic N is unclear. Using bioassay experiments, we examined the impact of effluent-derived organic nitrogen (EON) from wastewater treatment plants on natural water samples collected along an estuarine/salinity gradient within the lower Chesapeake Bay watershed. All of the inorganic N and between 31% and 96% of the EON was removed during biotic bioassays within the first 2 days. Further, there was substantial abiotic reactivity of effluent N when it was added to natural water samples. Results demonstrate that organic and inorganic N in effluent is removed to support the growth of microbial communities. These are the first results aimed at assessing the reactivity of EON in natural waters along an estuarine/salinity gradient.

Two different approaches to measuring phytoplankton nitrogen (N) use were compared in late summer 2004 along the main axis of Chesapeake Bay. Uptake of 15N-labeled ammonium and nitrate and dual-labeled (15N and 13C) urea and dissolved free amino acids (DFAA) were measured in surface water samples from upper, mid, and lower bay stations. Two distinct methods were used to assess the relative uptake of N substrates by phytoplankton and correct for bacterial artifacts: (1) traditional filtration using Whatman glass fiber (GF/F) filters and (2) flow cytometric (FCM) sorting of chlorophyll-containing cells. The concentration of dissolved inorganic N (DIN) decreased with distance south along the bay, whereas dissolved organic N (DON) concentrations were relatively constant. Absolute N uptake rates measured using the traditional approach exceeded those of FCM-sorted phytoplankton, thereby suggesting the possibility of bacterial “contamination.” Ammonium was the dominant N form used throughout the transect, although FCM-sorted phytoplankton relied more on urea and DFAA as the ratio of DON/DIN increased toward the bay mouth. Overall, ammonium comprised 74 ± 17%, urea 10 ± 9%, DFAA 9 ± 7%, and nitrate 7 ± 12% of total measured N uptake by phytoplankton. Results suggest that bacteria relied primarily on DFAA and ammonium for N nutrition but also used N from urea at a rate similar to that of phytoplankton, whereas bacterial nitrate uptake was insignificant. On average, phytoplankton uptake of ammonium, urea, and DFAA was overestimated by 61%, 53%, and 135%, respectively, as a result of bacterial retention on GF/F filters.

Large "blooms" of ctenophores (Mnemiopsis leidyi) and scyphomedusae (Chrysaora quinquecirrha) occur throughout the York River, a sub-estuary of Chesapeake Bay. These gelatinous zooplankton blooms can influence carbon (C) and nutrient cycling through excretion of dissolved organic matter (DOM), and inorganic nitrogen (N) and phosphorus (P). We measured dissolved organic carbon, nitrogen and phosphorus (DOC, DON and DOP), ammonium (NH + 4 ) and phosphate (PO 3- 4 ) released by M. leidyi and C. quinquecirrha in the laboratory and estimated their contribution to in situ DOC and inorganic pools. Both species released high amounts of DOC compared with DON and DOP DOM released by Mnemiopsis was C-rich with higher DOC:DON (29:1) compared with the Redfield ratio (6.6C:1N). Daily turnover of DOC and DON in ctenophores was high (25.2% of body C and 18.3% of body N), likely due to mucus production. In contrast, individual Chrysaora released DOC and DON similar to Redfield stoichiometry, but daily turnover of these compounds was low (

The marine nitrogen (N) cycle is a complex network of biological transformations in different N pools. The linkages among these different reservoirs are often poorly understood. Traditional methods for measuring N uptake rely on bulk community properties and cannot provide taxonomic information. 15 N-based stable isotope probing (SIP), however, is a technique that allows detection of uptake of individual N sources by specific microorganisms. In this study we used 15 N SIP methodology to assess the use of different nitrogen substrates by Synechococcus spp. and diatoms on the west Florida shelf. Seawater was incubated in the presence of 15 N-labeled ammonium, nitrate, urea, glutamic acid, and a mixture of 16 amino acids. DNA was extracted and fractionated using CsCl density gradient centrifugation. Quantitative PCR was used to quantify the amounts of Synechococcus and diatom DNA as a function of density, and 15 N tracer techniques were used to measure rates of N uptake by the microbial community. The ammonium, nitrate, urea, and dissolved primary amine uptake rates were 0.077, 0.065, 0.013, and 0.055 μmol N liter −1 h −1 , respectively. SIP data indicated that diatoms and Synechococcus spp. actively incorporated N from [ 15 N]nitrate, [ 15 N]ammonium, and [ 15 N]urea. Synechococcus also incorporated nitrogen from [ 15 N]glutamate and 15 N-amino acids, but no evidence indicating uptake of labeled amino acids by diatoms was detected. These data suggest that N flow in communities containing Synechococcus spp. and diatoms has more plasticity than the new-versus-recycled production paradigm suggests and that these phytoplankters should not be viewed strictly as recycled and new producers, respectively.

A mesocosm experiment was conducted during the spring of 2003 in Raunefjord, west Norway. Inorganic nutrients (16 µmol l -1 nitrate, 1 µmol l -1 phosphate) were added to 2 enclosures with 10% of those concentrations (1.6 µmol l -1 nitrate, 0.1 µmol l -1 phosphate) added daily thereafter; a third unamended mesocosm was used as a control. Nitrogen (N) (ammonium, nitrate, urea and amino acid) uptake rates for >0.8 µm (largely composed of phytoplankton) and 0.2 to 0.8 µm (largely composed of bacteria) size classes were measured, as well as nutrient, chlorophyll, phytoplankton and microzooplankton concentrations. The nutrient additions initiated a process resulting in a large bloom of Phaeocystis pouchetii colonies. There was a 2.5 wk lag, during which a smaller bloom of phototrophic flagellates, followed by diatoms, formed in all mesocosms; diatoms increased until sili- cic acid was depleted. After the flagellate and diatom bloom dissipated, the mesocosms were depleted of inorganic N. Dissolved organic N (DON) remained constant in all mesocosms during the flagellate and diatom bloom but increased with the onset of the large P. pouchetii bloom, largely due to release of urea and amino acids, which made up >93% of the DON pool at the end of the experi- ment. Uptake rates of urea often accounted for the largest percentage of total N uptake in all meso- cosms for both the >0.8 µm and 0.2 to 0.8 µm size classes, generally providing the largest percentage (up to 88%) of bacterial N demand of any substrate. This was particularly true during the Phaeocys- tis bloom when urea uptake rates were up to 65 times greater than the other N substrates. We observed a switch from new production, when nitrate uptake dominated during the diatom bloom, to regenerated production during the Phaeocystis bloom. The present study adds to the growing body of evidence that organic N is a substantial source of N for both phytoplankton and bacteria.

Bacterioplankton nutrient metabolism in the Eastern Tropical North Pacific (ETNP) was assessed using specific activities of intracellular nitrogen (N) assimilation enzymes and hydrolytic ectoenzymes during amendment experiments, mesocosms, and diel studies of in situ rates. Glutamine synthetase (GS) and assimilatory nitrate reductase (ANR) were used to investigate N bioavailability, alkaline phosphatase (AP) to assess phosphorous (P) bioavailability and β-glucosidase (β-Glu) to detect shifts in the use of labile dissolved organic carbon (DOC). Conditions regulating activity of each enzyme were tested using incubations of

Biological nutrient removal is a process commonly used in water resource recovery facilities to reduce dissolved inorganic nitrogen (DIN) concentrations in effluent; this process is less effective at removing all of the effluent dissolved organic nitrogen (EDON). The goal of this study was to investigate the fate of EDON after it undergoes the disinfection process and enters receiving waters. The authors quantified the abiotic effects of effluent exposure to sunlight, increased salinity, and a combination of the two factors. Effluent dissolved organic nitrogen showed significant breakdown during the disinfection process (UV and chlorine) and when exposed to sunlight and increasing salinity. Approximately 7% of the EDON was transformed to DIN and dissolved primary amines after exposure to 9 hours of sunlight and a salinity increase from 0 to 33. The production of DIN and primary amines should be taken into account when considering sources of labile nitrogen to aquatic ecosystems.

Dissolved organic nitrogen (DON) and carbon (DOC) often dominate the dissolved nitrogen and organic carbon fluxes from rivers, yet they are not considered to affect coastal water quality because of their assumed refractory nature. The objective of this study was to quantify DON and DOC bioavailability to bacteria in 9 rivers on the east coast of the United States during a 6 d dark bioassay experiment. Water was collected from the freshwater portion of a forest stream in New Jer- sey (Forest 17a), and from the Bass (New Jersey), Delaware (New Jersey), Hudson (New York), Altamaha (Georgia), Savannah (Georgia), Pocomoke (Maryland), Choptank (Maryland), and Peconic (New York) Rivers during base-flow conditions. DON concentrations ranged from 1 to 35 µM and comprised 8 to 94% of the total dissolved nitrogen (TDN) in these rivers. Bioassay results indicate that 23% (±4) of the DON (2 ± 1 µM) was bioavailable in all the rivers except the Bass and Pocomoke, where no DON consumption was measured. Of the TDN consumed by bacteria, DON comprised 43% (±6), demonstrating that DON is an important nitrogen source for bacteria. In contrast, only 4% (±1) of DOC (12 ± 3 µM), was bioavailable in the 9 rivers. Percent-wise, 8 times more DON was consumed relative to DOC in 6 of the rivers, demonstrating that DON cycles faster than DOC. Overall, our study demonstrates that DON is an important part of the TDN pool that needs to be incorporated into coastal nitrogen loading budgets because it is bioavailable on the order of days.

Humic substances were isolated from the Satilla (GA), Altamaha (GA), and York (VA) rivers via the commonly used XAD-8 extraction technique. Humics were also formed in the laboratory by allowing Spartina alterniflora plants to humify under controlled laboratory conditions over the course of 1 year; the S. alterniflora had been grown with 15N-enriched ammonium (NH4+) in the sediment such that the humics produced were enriched with 15N. The chemical characteristics of the natural and laboratory-produced humic substances were evaluated in a number of ways. Chemical shifts in the atomic carbon: nitrogen (C:N) ratio were monitored after exposing natural humic substances to environmentally relevant NH4+ concentrations. Structural characteristics of natural humics and laboratory-produced humics were determined using Fourier Transform Infrared (FTIR) spectroscopy, molecular weight (MW) size fractionation, and humic 15N isotope enrichment. Results indicate that first, humic substances become more fulvic-like in character as aging time increased in the laboratory. Second, as humic aging time increased (from 1 week to 6 months), the humic-N becomes more enriched with 15N. Third, the C:N ratio in the low MW size fraction of the humics decreased by more than half during the year of sampling (from 37 to 16). These results suggest that the structure and chemical composition of humic compounds change significantly during formation. Lastly, exposure of humic substances to environmentally relevant levels of NH4+ caused a significant decrease in humic C:N ratios. This result suggests that loss of ammonium during extraction using XAD can cause an underestimation of humic-N content such that humic substances in the environment are likely more N-rich than previously thought.

On the basis of mass balance calculations performed for nitrogen (N) uptake experiments in the Southern California Bight (SCB), it has been suggested that a significant portion of dissolved inorganic N (DIN) uptake results in the production of dissolved organic N (DON). To investigate this process, the fate of ammonium (NH 4 + ) and nitrate (NO 3 − ) uptake was quantified within the euphotic zone at three coastal stations in the SCB using 15 N tracer techniques. Several trends in the fate of DIN and the production of DON were observed. First, production of particulate N (PN), from both NH 4 + and NO 3 − , was quantitatively more important in near surface waters, while DON release dominated within the nitracline. Second, the percentage of gross N uptake released as DON was generally higher when NO 3 − , rather than NH 4 + , was the substrate. Third, the percentage of N released as DON was higher at night, relative to the day. Fourth, rates of DON release were significantly correlated to NH 4 + regeneration, suggesting that similar mechanisms are responsible for both processes—presumably grazing. The results of this study indicate that the DON pool is a sink for DIN uptake on the time scale of hours. One implication of this finding is that new production estimates based on 15 NO 3 − uptake rates will likely underestimate particle flux out of the surface layer because the rate of NO 3 − uptake is underestimated due to loss of DO 15 N during the incubation. On time scales of months to years, however, the N that is taken up as NO 3 − and released as DON will likely contribute to export flux via incorporation of the dissolved phase during seasonal mixing into sinking particles or transport. The export of DON on these time scales argues for the use of gross uptake rates to calculate f -ratios.

As part of an integrated study of the regulation of carbon fixation in the offshore Mississippi River plume, we measured the rates of 15 N-labeled ammonium and nitrate uptake in the surface plume waters from offshore to nearshore along the plume axis towards the Mississippi Delta. Concentrations of nitrate in the plume ranged from 0.19 to 2.5 µM with the highest concentrations primarily in the shoreward stations, while ammonium ranged from 0.17 to 0.44 µM, showing little spatial variability. Rates of ammonium uptake ranged from 16.5 to 260 nM h -1 , and showed a strong trend of increasing values from offshore towards the Mississippi Delta. In contrast, nitrate uptake rates ranged from 3.2 to 25 nM h -1 . The high rates of ammonium uptake in the presence of low ammo- nium concentrations and elevated nitrate was made possible by elevated rates of ammonium re- generation that exceeded ammonium uptake by 1.7 to 5.7-fold in the plume. The plume exhibited relatively low f-ratios and also contained elevated levels of Synechococcus as determined by flow cytometry and high levels of form IA (α -cyanobacterial) rbcL transcripts. These data suggest that a major portion of the carbon fixation observed in the offshore Mississippi River plume represents recycled production supported by high rates of ammonium regeneration.

We examined the temporal and spatial variability of urea concentrations and urea uptake and regeneration rates collected on cruises along the longitudinal axis of the Chesapeake Bay between 1972 and 1998. Interannually, mean Bay-wide surface urea concentrations ranged between 0.49 and 0.91 μg-at N l−1 with a nearly 50% decrease in surface concentrations observed between 1988 and 1998. Concentrations of urea from samples collected within ∼1 m of the bottom were generally higher and much more varable than surface samples. Seasonally, two different patterns were observed in mean Bay-wide surface urea concentrations. Urea concentrations from near surface waters exhibited a clear summer peak for 1988 through 1994, while for 1973 and 1996 to 1998 a distinct winter-spring peak in concentration was observed. Urea concentrations from deeper waters showed a similar seasonal trend each year with peak concentrations measured in spring. Spatially, urea concentrations in the surface waters decreased in a conservative-type pattern from 0.91 μg-at N I−1 at the freshwater end member to 0.46 μg-at N I−1 at the ocean end member. Mean Bay-wide surface urea uptake rates displayed a seasonal pattern throughout the data set with maximum uptake rates (up to 0.33 μg-at N I−1 h−1) consistently observed during summer. Mean Bay-wide surface regeneration rates were highest but most variable during fall (1.63±0.82 μg-at N I−1 h−1). Mean urea uptake and regeneration rates displayed opposing spatial trends along the axis of the Bay with uptake rates being lowest in the North Bay where regeneration rates were highest. The average temporal and spatial patterns of urea concentration in Chesapeake Bay appear to reflect a balance between external inputs and internal biological recycling.

We compared the persulfate (PO), ultraviolet (UV), and high temperature oxidation (HTO) methods used to analyze total dissolved nitrogen (TDN) concentrations in aquatic samples to determine whether the three methods differed in terms of standard parameters (blanks, limits of detection and linearity, and precision) or in oxidation efficiency of standard compounds and field samples of varying salinity. The TDN concentrations of several N-containing standard compounds, as well as a humic mixture and a suite of field samples collected from the Sargasso Sea, Chesapeake Bay and an aquaculture pond were determined with the three methods. The PO method had the highest percent recoveries for the range of labile and refractory standard compounds tested (93±13). The HTO method yielded recoveries of 87±14; recoveries increased to 91±10 under optimized conditions. The standard UV method, with 30% H2O2 as the oxidant, was found to be highly variable, producing the lowest percent recoveries (71±21); the oxidation efficiency of the UV method increased substantially in subsequent trials (91±12), when the PO reagent was used in place of H2O2. In the field sample comparison, the PO, UV with PO reagent, and HTO method produced similar results (slopes of the Model II regression lines comparing them ranged from 1.00 to 1.05 with r2≥0.99). The standard UV method, however, produced concentrations 5% to 40% lower than the other methods. Analysis of the spectra emitted by the UV lamp used in this study suggests that variations in the UV spectra reaching the sample may have caused the reduced efficiencies. The poor recovery of some standard compounds with each of the methods suggests that concentrations of TDN, and subsequently DON, measured in the field with any of the methods will likely be underestimated to some degree depending on the composition of the TDN pool at that time. With careful attention to detail, however, the PO and HTO methods can provide reproducible results consistent with each other. The standard UV method, however, was found to be highly unpredictable in practice.

N tracer techniques were used to measure rates of NH and NO uptake, dissolved organic nitrogen (DON) 12 43 release resulting from both ammonium (NH ) and nitrate (NO ) uptake, and NH regeneration at discrete depths 12 1 434 throughout the euphotic zone in Monterey Bay, California, at a site, H3, outside the plume of direct upwelling influence. In March 1993, 94% of the inorganic nitrogen taken up was NH , and the primary fate of nitrogen 1 4 uptake was particle production. In September 1993, NH and NO uptake were more in balance, and the primary 12 43 fate of nitrogen uptake was DON. We suggest that grazing was an important mechanism resulting in DON release in March and that a combination of grazing and a more physiologically stressed phytoplankton population produced the higher observed rates of DON release in September. During both cruises, the percentage of nitrogen released as DON increased with depth, suggesting that deeper in the water column, a smaller percentage of the nitrogen taken up is incorporated into sinking particles. Based on these data, we suggest that the DON pool acts as an intermediate between DIN assimilation and the net formation of particles for export and will thus affect carbon flow in Monterey Bay.

To define the role of dissolved organic nitrogen (DON) in Chesapeake Bay (USA) nitrogen cycling, we performed experiments in the mesohaline Chesapeake Bay dunng 3 seasons: May, during the mid to late spring bloom, August, when patchy summer blooms often develop, and October, after the fall overturn. Every 3 h for 30 h we measured ambient nitrogen concentrations and used "N tracers to determine uptake and regeneration rates of NH,' and urea, uptake rates of NO,(NO; was measured in October only), and rates of DON release due to NH,' uptake. Autotrophic production and dissolved organic carbon (DOC) release were determined sunultaneously using 'v tracer techniques to compare with nitrogen flux rates We found that first, the water column nitrogen demand exceeded nitrogen regeneration in May but n~ t rogen regeneration was over 3 times greater than the nitrogen demand in October. Second, mean rates of autotrophic DON release were highest in May but heterotrophic release was more important in summer and fall. Third, dunng all 3 studies, the C N ratio of DOC to DON release rates was 3.4 to 4.5, indicating release of nitrogen-rich compounds. Release of dissolved organic matter (DOM) \nth a low C:N ratio was corroborated by an observed increase in DON concentrations from 7 to 4 2 pg-at. N I-' from May to August and a concomitant decrease in the C . N ratio of the ambient DOM pool from 36.6 to 8.8. These data suggest that Chesapeake Bay IS primarily autotrophic In the spring, but becomes progressively more heterotrophic later in the year. These data also suggest that DON is an Important intermediate in this transition and serves as a link between the allochthonous nitrogen-based autotrophic production In the spring and the autochthonous nitrogen-based heterotrophic system in the summer and fall.

DISSOLVED organic material in marine and freshwater ecosystems constitutes one of the Earth's largest actively cycled reservoirs for organic matter1. The bacterially mediated turnover of chemically identifiable, low-molecular-mass components of this pool has been studied in detail for nearly three decades, but these compounds constitute less than 20% of the total reservoir2. In contrast, little is known about the fate of the larger, biologically more refractory molecules—including humic substances—which make up the bulk of dissolved organic matter. Here we report results from bacterial bioassays and photochemical studies indicating that exposure to sunlight causes dissolved organic matter to release nitrogen-rich compounds that are biologically available, thus enhancing the bacterial degradation of humic substances. We demonstrate that ammonium is among the nitrogenous compounds released and is produced most efficiently by ultraviolet wavelengths. Photochemical release of ammonium from dissolved organic matter has important implications for nitrogen availability in many aquatic ecosystems, including nitrogen-limited high-latitude environments and coastal oceans, where inputs of terrestrial humic substances are high.

In oceanic, coastal, and estuarine environments, an average of 25 to 41 percent of the dissolved inorganic nitrogen (NH(4) (+) and NO(3) (-)) taken up by phytoplankton is released as dissolved organic nitrogen (DON). Release rates for DON in oceanic systems range from 4 to 26 nanogram-atoms of nitrogen per liter per hour. Failure to account for the production of DON during nitrogen-15 uptake experiments results in an underestimate of gross nitrogen uptake rates and thus an underestimate of new and regenerated production. In these studies, traditional nitrogen-15 techniques were found to underestimate new and regenerated production by up to 74 and 50 percent, respectively. Total DON turnover times, estimated from DON release resulting from both NH(4) (+) and NO(3) (-) uptake, were 10 +/- 1, 18 +/- 14, and 4 days for oceanic, coastal, and estuarine sites, respectively.

In nitrogen uptake studies with 15N tracer techniques, a common observation is that the 15N label added at the start of the experiment is not fully recovered at the end of the experiment in the particulate and dissolved N pools measured. We have made direct measurements of the 15N content of the traditionally measured NH4+ and particulate N pools, as well as the dissolved organic N (DON) and bacterial pools in studies conducted in Chesapeake Bay, the Caribbean Sea, and the Choptank River (a subestuary of Chesapeake Bay). We found that the fate of the “missing 15N” differs from eutrophic to oligotrophic waters and is dependent on the length of incubation, the dissolved inorganic N substrate used, and the length of time the sample was contained before the 15N uptake experiment. In general, it appears that it is more important to include the 15N that enters the DON and bacterial pools to achieve a 15N mass balance in more oligotrophic ecosystems and when longer (>1 h) incubations are used.

Nitrogen is an essential element for all life forms and undergoes various transformation processes mostly mediated by microbes along biogeochemical gradients in the water column and in sediments. Along estuarine gradients a variety of regulating factors such as salinity or nutrient concentrations change rapidly. These processes are summarized and the interactions among processes are presented. A focus lies on dissolved organic nitrogen as an important component for the nutrition of bacteria and phytoplankton. Moreover, organic nitrogen mediates the transport of nitrogen from primary producers into the microbial food web. Most studies summarized here focus on nitrogen in temperate estuaries, but we also present a summary of nitrogen processes in tropical mangrove ecosystems.

The dissolved organic nitrogen (DON) pool in marine waters contains a diverse mixture of compounds. It is therefore difficult to accurately estimate planktonic uptake of DON using the limited number of radiolabeled compounds commercially available. We describe a method to estimate DON uptake rates using 15N-labeled DON recently released from phytoplankton. To make 15N-labeled DON, we incubated surface water with 15NH 4 + and then isolated the DON, including any recently released DO15N, with ion retardation resin. This DON was then added to a freshly collected water sample from the same environment to quantify the rate of DON uptake. The technique was applied to investigate rates of DON uptake relative to inorganic nitrogen in the mesohaline Chesapeake Bay during May 1990 and August 1991. The May experiment took place after the spring bloom, and rates of DON uptake [ranging from 0.31 to 0.53 μg-atom (μg-at) Nl-1 h-1] often exceeded rates of NH 4 + and NO 3 - uptake combined. The rates of DON uptake at this time were higher than estimated bacterial productivity and were not correlated with bacterial abundance or bacterial productivity. They were, however, correlated with rates of NO 3 - uptake. In May, we estimate that only 7 to 32% of DON uptake was a result of urea utilization. In contrast, in August, when regenerated nutrients predominate in Chesapeake Bay, rates of DON uptake (ranging from 0.14 to 0.51 μg-atom Nl-1 h-1) were an average of 50% of the observed rates of NH 4 + uptake. Consistent with the May experiment, rates of DON uptake were not correlated with bacterial production. A sizable fraction of DON uptake, however, appeared to be due to urea utilization; rates of urea uptake, measured independently, were equivalent to an average of 74% of the measured rates of DON uptake. These findings suggest that, during both periods of study, at least a fraction of the measured DON uptake may have been due to utilization by phytoplankton.

The goal of this study was to investigate three potential ways that the soluble organic nitrogen (N) fraction of wastewater treatment plant (WWTP) effluents, termed effluent organic N (EON), could contribute to coastal eutrophication--direct biological removal, photochemical release of labile compounds, and salinity-mediated release of ammonium (NH4+). Effluents from two WWTPs were used in the experiments. For the bioassays, EON was added to water from four salinities (approximately 0 to 30) collected from the James River (VA) in August 2008, and then concentrations of N and phosphorus compounds were measured periodically over 48 h. Bioassay results, based on changes in DON concentrations, indicate that some fraction of the EON was removed and that the degree of EON removal varied between effluents and with salinity. Further, we caution that bioassay results should be interpreted within a broad context of detailed information on chemical characterization. EON from both WWTPs was also photoreactive, with labile NH4+ and dissolved primary amines released during exposure to sunlight. We also present the first data that demonstrate that when EON is exposed to higher salinities, increasing amounts of NH4+ are released, further facilitating EON use as effluent transits from freshwater through estuaries to the coast.

We present a new method for separation of dissolved organic nitrogen (DON) from dissolved inorganic nitrogen (DIN) in marlne samples which permlts the measurement of DO'*N production during DII5N uptake and regeneration experiments. Ion retardat~on resin was used to separate DI1'N from DOI'N and the isolated D0I5N was subsequently analyzed by mass spectrometry Variat~on in D0I5N atom % enrichment in duplicate samples, determined w ~ t h the ion retardation column method, was less than 4"0 . We also separated the low n~olecular weight (LMW) DO"N from total D015N using ultrafiltration and found the ratio of these variables to be a useful index in determining what release processes were likely occurring in a given sample. Examples are presented from 2 types of experiments conducted in Chesapeake Bay waters: first, a 6 h time-course of D015N release from both "NH,' and I5NO3uptake, and second, a series of short-term (0.5 h) release measurements from 15NH,* uptake over the course of a day. In the former, total DO1% release rates resulting from 15NH,* uptake were several-fold higher than those resulting from IsNO3uptake due to extremely low rates of incorporat~on of "NO? into cellular organic material. The release of LMW DOI'N resulting from I5NH,+ uptake decreased from 78 to 21 % of total D0I5N release during the 6 h incubation which suggests that processes other than direct release by phytoplankton (e.g. sloppy feeding, cell lysis) likely became more important as the incubation progressed In the latter expenment, LMW DO'% release was 0 to 16 % of total D0I5N release, which indicates that, in this case, also, processes other than direct release by phytoplankton were dominating the flux of DON from phytoplankton.

The study of nitrogen (N) cycling requires sensitive and accurate methods to measure various N compounds. This chapter reviews existing methods to analyze dissolved and particulate fractions of N with an emphasis on those methodologies that are most commonly used or represent the state of the art, noting that N analysis does indeed seem to be an art at times. Many techniques are temperamental at best and it is not uncommon to find small fetishes devoted to keeping a given mass spectrometer running. The first main section of the review describes techniques to measure the concentrations of N pools, followed by various approaches to chemically characterize organic N fractions. The last major section reviews techniques to measure N-fluxes, including uptake, release, nitrification, denitrification, and N2 fixation. It largely provides the perspective of water column studies; most of the methods, however, could also be used more broadly to analyze sediments, particles, or pore water samples. The difference between a given method for these sample types is principally one of the detection limit required and matrix specific analytical challenges. Analyzing water column samples can be difficult because concentrations can be very low. While concentrations are generally much higher in sediment pore waters, extracting the necessary volume of pore water can be challenging. With respect to rate measurements, most of the techniques outlined could be applied in both the water column and the sediment, and several largely sedimentary rate measurements are specifically discussed below. Tracer approaches in sediments can be especially problematic because the substrate is so heterogeneous that getting uniform mixing of the tracer can be impossible.

Publisher Summary This chapter reviews the forms and sources of regenerated nitrogen (N), both inorganic and organic, to describe the various processes that result in N regeneration and to survey N regeneration rates in estuarine, coastal, and open ocean systems. It specifically focuses on water column N regeneration. Sediments are discussed from the perspective that they serve as a source of N to the water column. Nitrogen is regenerated in both inorganic and organic forms. The inorganic forms include ammonium (NH 4+) nitrate (NO3–), and nitrite (NO2– ). Organic forms include urea, dissolved free amino acids nucleic acids, and a broad suite of dissolved combined amino acids and proteins, as well as other largely unidentified components of the dissolved organic nitrogen pool including enzymes. To measure regeneration rates bioassays or 15N tracer methods are used. With the bioassay method, the accumulation of a compound is measured over time. Bioassays have the advantage that they are relatively easy to perform. However, they can only provide net release rates, will only produce a rate at all if the release rate is greater than any uptake rates, and can suffer from bottle affects if long incubations are needed to see a signal. This discussion concludes with a brief survey of methods and recommendations for future research.

Relative to inorganic nitrogen, concentrations of dissolved organic nitrogen (DON) are often high, even in regions believed to be nitrogen-limited. The persistence of these high concentrations led to the view that the DON pool was largely refractory and therefore unimportant to plankton nutrition. Any DON that was utilized was believed to fuel bacterial production. More recent work, however, indicates that fluxes into and out of the DON pool can be large, and that the constancy in concentration is a function of tightly coupled production and consumption processes. Evidence is also accumulating which indicates that phytoplankton, including a number of harmful species, may obtain a substantial part of their nitrogen nutrition from organic compounds. Ongoing research includes ways to discriminate between autotrophic and heterotrophic utilization, as well as a number of mechanisms, such as cell surface enzymes and photochemical decomposition, that could facilitate phytoplankton use of DON components.

14 páginas, 8 tablas, 1 figura.-- Jonathan H. Sharp ... et al., Routine determination of dissolved organic nitrogen (DON) is performed in numerous laboratories around the world using one of three families of methods: UV oxidation (UV), persulfate oxidation (PO), or high temperature combustion (HTC). Essentially all routine methods measure total dissolved nitrogen (TDN) and calculate DON by subtracting the dissolved inorganic nitrogen (DIN). While there is currently no strong suggestion that any of these methods is inadequate, there are continuing suspicions of slight inaccuracy by UV methods. This is a report of a broad community methods comparison where 29 sets (7 UV, 13 PO, and 9 HTC) of TDN analyses were performed on five samples with varying TDN and DIN concentrations. Analyses were done in a “blind” procedure with results sent to the first author. With editing out one set of extreme outliers (representing 5 out of 145 ampoules analyzed), the community comparability for analyzing the TDN samples was in the 8–28% range (coefficient of variation representing one standard deviation for the five individual samples by 28 analyses). When DIN concentrations were subtracted uniformly (single DIN value for each sample), the comparability was obviously worse (19–46% cv). This comparison represents a larger and more diverse set of analyses, but the overall comparability is only marginally better than that of the Seattle workshop of a decade ago. Grouping methods, little difference was seen other than inconclusive evidence that the UV methods gave TDN values for several of the samples higher than HTC methods. Since there was much scatter for each of the groups of methods and for all analyses when grouped, it is thought that more uniformity in procedures is probably needed. An important unplanned observation is that variability in DIN analyses (used in determining the final analyte in most UV and PO methods) is essentially as large as the variability in the TDN analyses. This exercise should not be viewed as a qualification exercise for the analysts, but should instead be considered a broad preliminary test of the comparison of the families of methods being used in various laboratories around the world. Based on many independent analyses here, none of the routinely used methods appears to be grossly inaccurate, thus, most routine TDN analyses being reported in the literature are apparently accurate. However, it is not reassuring that the ability of the international community to determine DON in deep oceanic waters continues to be poor. It is suggested that as an outgrowth of this paper, analysts using UV and PO methods experiment and look more carefully at the completeness of DIN conversion to the final analyte and also at the accuracy of their analysis of the final analyte. HTC methods appear to be relatively easy and convenient and have potential for routine adoption. Several of the authors of this paper are currently working together on an interlaboratory comparison on HTC methodology., This research was partially supported by NSF grants OCE 96-33296 and OCE 00-82238 to JHS

This chapter describes the dynamics of dissolved organic nitrogen (DON). DON is that subset of the dissolved organic matter (DOM) pool that contains nitrogen (N). From the perspective of a microorganism, this is where the action is—one-stop shopping for N, carbon (C), and energy. Research in DON, however, has lagged far behind that of the larger dissolved organic carbon (DOC) pool. This situation is primarily the result of the substantial analytical challenges inherent in DON research: (1) DON exists in substantially lower concentrations than DOC, (2) multiple chemical analyses are required for a single DON measurement, (3) inorganic N removal is a nightmarish undertaking, and (4) unless one has an easy access to a nuclear reactor manufacturing short-lived 13 N, he or she must be content with labor-intensive stable isotopes rather than the quicker and more sensitive radiotracers. Measurements of DON concentrations have become a routine component of many studies. Nonetheless, because of space limitations DON concentrations in lakes, streams, or groundwater—with some exceptions—are not included in studies.

In the ocean, biologically active elements undergo continuous cycling between the biota, the water column, and the atmosphere. Biological processes and the resultant air/sea exchange of atmospherically active gases are closely modulated by the availability and distribution of key elements. Such processes have been traditionally measured by incubation of representative microbial communities and tracking of end-product appearance or substrate disappearance. The recent advent of molecular techniques allows for the quantification of DNA and the RNA messenger responsible for the synthesis of the enzymes catalyzing specific biochemical processes. However, there is little information on how levels of gene expression for natural populations of micro-organisms correlate with biogeochemical processes.