Intracellular Nitrate Research Articles

Changes in isotope fractionation during nitrate assimilation by marine eukaryotic and prokaryotic algae under different pH and CO2 conditions

AbstractThe impact of environmental factors on nitrogen (N) and oxygen (O) isotope effects during algal nitrate assimilation causes uncertainty in the field application of sedimentary N isotope records and nitrate isotopes to understand the marine nitrogen cycle. Ocean acidification is predicted to change nitrogen cycling including nitrate assimilation, but how N and O isotope effects during algal nitrate assimilation vary in response to changes in seawater pH and partial pressure CO2 (pCO2) remains unknown. We measured N and O isotope effects during nitrate assimilation and physiological states of the marine diatom Thalassiosira weissflogii and Synechococcus under different pH (8.1 or 7.8) and pCO2 (400 or 800 μatm) conditions. Low pH and/or high pCO2 equally decreased N and O isotope effects during nitrate assimilation by diatoms possibly due to reducing cellular nitrate efflux/uptake ratio and decreased isotope effects for nitrate uptake, whereas they did not affect those by Synechococcus with low intracellular nitrate concentration and limited nitrate efflux. Our results provide compelling experimental evidence showing different changes in N and O isotope effects during nitrate assimilation by marine eukaryotic and prokaryotic phytoplankton at low pH and/or high pCO2. These findings suggest new insight into environmental controls on variability in the isotope effect during algal nitrate assimilation, and have implications for improving a predictive understanding of N and O isotope tools in acidified oceans.

Benthic foraminifera and gromiids from oxygen-depleted environments – survival strategies, biogeochemistry and trophic interactions

Abstract. The oceans are losing oxygen (O2), and oxygen minimum zones are expanding due to climate warming (lower O2 solubility) and eutrophication related to agriculture. This trend is challenging for most marine taxa that are not well adapted to O2 depletion. For other taxa this trend might be advantageous because they can withstand low O2 concentrations or thrive under O2-depleted or even anoxic conditions. Benthic foraminifera are a group of protists that include taxa with adaptations to partly extreme environmental conditions. Several species possess adaptations to O2 depletion that are rare amongst eukaryotes, and these species might benefit from ongoing ocean deoxygenation. In addition, since some foraminifera can calcify even under anoxic conditions, they are important archives for paleoceanographic reconstruction in O2-depleted environments. This paper reviews the current state of knowledge about foraminifera from low-O2 environments. Recent advances in our understanding of specific survival strategies of foraminifera to withstand O2 depletion are summarized and discussed. These adaptations include an anaerobic metabolism, heterotrophic denitrification, symbiosis with bacteria, kleptoplasty and dormancy and have a strong impact on their preferred microhabitat in the sediments, especially the ability of some benthic foraminiferal species to denitrify. Benthic foraminifera also differ regarding their trophic strategies, which has an additional impact on the selection of their microhabitat. For example, some species are strict herbivores that feed exclusively on fresh phytodetritus and live close to the sediment surface, while some species are non-selective detrivores that occupy intermediate to deep infaunal habitats. There is evidence that foraminifers have the capacity to undergo phagocytosis, even under anoxia, and some foraminiferal species which can withstand low-O2 conditions seem to prey on meiofauna. Also, due to their high abundances in O2-depleted environments and their metabolic adaptations, benthic foraminifera are key players in marine nutrient cycling, especially within the marine N and P cycles. This review summarizes the denitrification rates for the species that are known to denitrify and the intracellular nitrate concentrations of the species that are known to intracellularly store nitrate. Finally, equations are provided that can be used to estimate the intracellular nutrient storage and denitrification rates of foraminifera and might be integrated into biogeochemical models.

Characterization and metabolic pathway of Pseudomonas fluorescens 2P24 for highly efficient ammonium and nitrate removal

The ammonium and nitrate removal performance and metabolic pathways of a biocontrol strain, Pseudomonas fluorescens 2P24, were investigated. Strain 2P24 could completely remove 100 mg/L ammonium and nitrate, with removal rates of 8.27 mg/L/h and 4.29 mg/L/h, respectively. During these processes, most of the ammonium and nitrate were converted to biological nitrogen via assimilation, and only small amounts of nitrous oxide escaped. The inhibitor allylthiourea had no impact on ammonium transformation, and diethyl dithiocarbamate and sodium tungstate did not inhibit nitrate removal. Intracellular nitrate and ammonium were detectable during the nitrate and ammonium transformation process, respectively. Moreover, the nitrogen metabolism functional genes (glnK, nasA, narG, nirBD, nxrAB, nirS, nirK, and norB) were identified in the strain. All results highlighted that P. fluorescens 2P24 is capable of assimilatory and dissimilatory nitrate reduction, ammonium assimilation and oxidation, and denitrification.

NIN-like protein 7 transcription factor is a plant nitrate sensor.

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants' primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.

In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress. However, its physiological function is not yet clear. Like many gut pathogens, Vibrio cholerae also faces nitrosative stress, which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae by immunoblotting and LC-ESI-MS/MS proteomic analysis. Our results indicated that in-vivo PTN in V. cholerae was culture media independent. Surprisingly, in-vivo PTN was reduced in V. cholerae proteome under anaerobic or hypoxic condition in a nutrient deprived state. Interestingly, intracellular nitrate content was more than the nitrite content in V. cholerae under anaerobic conditions. Additionally, biochemical measurement of GSH/GSSG ratio, activities of catalase and SOD, ROS and RNS imaging by confocal microscopy confirmed a relative intracellular oxidizing environment in V. cholerae under anaerobic conditions. This altered redox environment favors the oxidation of nitrite which may be generated from protein denitration enriching the intracellular nitrate pool. The cell survival of V. cholerae can finally be facilitated by nitrate reductase (NapA) utilizing that nitrate pool. Our cell viability study using wild type and ΔnapA strain of V. cholerae also supported the role of NapA mediated cell survival under nutrient deprived anaerobic conditions. In spite of having nitrate reductase (NapA), V. cholerae lacks any nitrite reductase (NiR). Hence, in-vivo nitration may provide an avenue for toxic nitrite storage and also may help in nitrosative stress tolerance mechanism preventing further unnecessary protein nitration in V. cholerae proteome.

Ammonium and Sulfate Assimilation Is Widespread in Benthic Foraminifera

Nitrogen and sulfur are key elements in the biogeochemical cycles of marine ecosystems to which benthic foraminifera contribute significantly. Yet, cell-specific assimilation of ammonium, nitrate and sulfate by these protists is poorly characterized and understood across their wide range of species-specific trophic strategies. For example, detailed knowledge about ammonium and sulfate assimilation pathways is lacking and although some benthic foraminifera are known to maintain intracellular pools of nitrate and/or to denitrify, the potential use of nitrate-derived nitrogen for anabolic processes has not been systematically studied. In the present study, NanoSIMS isotopic imaging correlated with transmission electron microscopy was used to trace the incorporation of isotopically labeled inorganic nitrogen (ammonium or nitrate) and sulfate into the biomass of twelve benthic foraminiferal species from different marine environments. On timescales of twenty hours, no detectable 15N-enrichments from nitrate assimilation were observed in species known to perform denitrification, indicating that, while denitrifying foraminifera store intra-cellular nitrate, they do not use nitrate-derived nitrogen to build their biomass. Assimilation of both ammonium and sulfate, with corresponding 15N and 34S-enrichments, were observed in all species investigated (with some individual exceptions for sulfate). Assimilation of ammonium and sulfate thus can be considered widespread among benthic foraminifera. These metabolic capacities may help to underpin the ability of benthic foraminifera to colonize highly diverse marine habitats.

Intracellular nitrate storage by diatoms can be an important nitrogen pool in freshwater and marine ecosystems

Identifying and quantifying nitrogen pools is essential for understanding the nitrogen cycle in aquatic ecosystems. The ubiquitous diatoms represent an overlooked nitrate pool as they can accumulate nitrate intracellularly and utilize it for nitrogen assimilation, dissipation of excess photosynthetic energy, and Dissimilatory Nitrate Reduction to Ammonium (DNRA). Here, we document the global co-occurrence of diatoms and intracellular nitrate in phototrophic microbial communities in freshwater (n = 69), coastal (n = 44), and open marine (n = 4) habitats. Diatom abundance and total intracellular nitrate contents in water columns, sediments, microbial mats, and epilithic biofilms were highly significantly correlated. In contrast, diatom community composition had only a marginal influence on total intracellular nitrate contents. Nitrate concentrations inside diatom cells exceeded ambient nitrate concentrations ∼100–4000-fold. The collective intracellular nitrate pool of the diatom community accounted for <1% of total nitrate in pelagic habitats and 65–95% in benthic habitats. Accordingly, nitrate-storing diatoms are emerging as significant contributors to benthic nitrogen cycling, in particular through Dissimilatory Nitrate Reduction to Ammonium activity under anoxic conditions.

Reactive oxygen species, nitric oxide and plant cell death associated with caspase-like protease activity during somatic embryogenesis in Fraxinus mandshurica

Programmed cell death occurs in browning explants of Fraxinus mandshurica during somatic embryogenesis, but the underlying mechanism is unclear. In this study, single cotyledons of zygotic embryos of F. mandshurica were used as explants. Mitochondrial structure and function, caspase-3-like protease activity, hydrogen peroxide metabolism, and nitric oxide accumulation induced by high concentrations of sucrose and plant growth regulators were studied. The results show that plant growth regulators induced somatic embryogenesis and also promoted explant browning. High sucrose concentrations had similar effects. High concentrations of sucrose and plant growth regulators led to the accumulation of hydrogen peroxide and nitric oxide which induced changes in mitochondrial structure and function such as modifications in mitochondrial morphology, increased membrane permeability, decreased membrane potential, and the release of cytochrome c into the cytoplasm. An increase in caspase-3-like protease activity triggered programmed cell death in some browning explant cells. During somatic embryogenesis there were increased activities of superoxide dismutase, peroxidase, and catalase, which are associated with hydrogen peroxide metabolism and jointly maintain reactive oxygen species levels. Intracellular nitric oxide synthase and nitrate reductase activities were not significantly correlated with nitric oxide content. Instead, intracellular nitric oxide may be derived from non-enzymatic reactions. Our results indicate that hydrogen peroxide and nitric oxide may function as signals, playing key roles in somatic embryogenesis and programmed cell death of explant cells of F. mandshurica. The interaction between nitric oxide and reactive oxygen species determines the occurrence of programmed cell death in explant cells; somatic embryogenesis and programmed cell death are positively regulated by hydrogen peroxide. However, the regulation of nitric oxide is complex.

Effects of temperature on the nitrate reductase activity and growth of Ulva prolifera.

To better understand the effect of temperature on the growth and nitrate reductase activity (NRA) of Ulva prolifera and their relationships, the effects of five different temperatures (10, 15, 20, 25, and 30°C) were investigated in a laboratory setup. In this study, an optimization invitro analysis method for Ulva prolifera NRA was developed. Under different treatments, the NRA, nitrate concentration, pH, the intracellular nitrate and nitrite concentrations, and the POC/PON were evaluated. The results of the invitro analysis method showed it was optimal for the NRA assay when the extraction time was 6min, enzymatic reaction time 30min, volume of phenazine methosulfate (PMS) solution 50μL, NADH concentration 0.36mM, and KNO3 concentration 10mM. The maximal NRA (NRAmax ) appeared on the 2nd day in the 10, 15, and 20°C (low-temperature) groups and on the 1st day in the 25 and 30°C (high-temperature) groups. The algal growth ended earlier at a high temperature, ending after 5d at 30 and 25°C and 7d at 20°C and 9d at 15°C, and the alga at 10°C had been growing during the incubation period. Ulva prolifera cultivated in a range of 10-20°C had a long growth cycle and the NRA decreased with increasing temperature when exceeded 15°C, a positivecorrelation between algal growth and NRA was observed. This study supports NRA is a suitable proxy of the effects of temperature changes on the ability of Ulva prolifera to uptake and metabolize nitrogen nutrients.

Potential foraminiferal nitrate transport in sediments in contact with oxic overlying water

AbstractSedimentary denitrification accounts for more than half of the global marine fixed nitrogen loss, but this removal is often restricted by nitrate diffusive supply in sediments. The results of this study showed that foraminiferal nitrate transport may supply nitrate below the nitrate penetration depth and promote nitrogen removal in sediments in contact with oxic overlying water. Six dominant foraminiferal species in the Yellow Sea sediments accumulated high concentrations of intracellular nitrate (25–68 mM), which is 1000 times higher than the maximum pore water nitrate concentration. Notably, this is the first time that Protelphidium tuberculatum, the most dominant species at almost all stations, is reported to have elevated intracellular nitrate. P. tuberculatum contributes 45% to 92% of the foraminiferal intracellular nitrate pools and plays an important role in foraminiferal nitrate transport. The potential foraminiferal denitrification rates below the nitrate penetration depth induced by foraminiferal nitrate transport ranged from 4 to 117 μmol N m−2 d−1 and contributed 3% to 81% of sedimentary denitrification. The intensity of foraminiferal nitrate transport showed a negative correlation with the nitrate penetration depth and positively correlated with the chlorophyll a (Chl a) and chloroplastic pigment equivalent concentrations, which indicate that shallower redox depth (nitrate penetration depth) and increased food supply (Chl a, chloroplastic pigment equivalent) may promote foraminiferal nitrate transport. The trophic oxygen model was modified by adding the pore water nitrate to explain foraminiferal nitrate transport in various marine environments, and this modified trophic oxygen model predicts the observed high foraminiferal nitrate transport intensity.

Comparison of the modified Monod and Droop function combined with Logistic function for describing algae growth

Microcystis aeruginosa (M. aeruginosa) are algae found in common freshwater blooms in China, and Dunaliella tertiolecta (D. tertiolecta) are economically important marine algae. Understanding of the microbial growth kinetics plays a significant role in the management of M. aeruginosa’s blooms and biodiesel production by D. tertiolecta. This study has shown that the combination of mechanistic models (Logistic and Monod) proved to be efficient in describing relationship between M. aeruginosa growth rates and specific concentrations of total dissolved phosphorus (TDP), orthophosphate (PO4 3--P), total dissolved nitrogen (TDN) and ammonia (NH4 +-N) reasonably with R2=0.28-0.93. Meanwhile, results also show that both PO43--P and NH4 +-N are important forms of TDP and TDN in influencing M. aeruginosa growth. It was also noted that the combination of modified Monod and Logistic functions is suitable for describing specific growth rates of D. tertiolecta versus extracellular nitrate concentrations (R2=0.24-0.72). In terms of the combination of Droop and Logistic functions, it was analysed to better explain the relationships between M. aeruginosa specific growth rates and cellular P and N concentrations (R2=0.41-0.86) as compared to the application of Droop function alone. It is also observed that the relationship between D. tertiolecta specific growth rates and intracellular nitrate concentrations also can be well described by the combination of Logistic and Droop functions. In addition, M. aeruginosa growth was affected by less intracellular P concentrations than intracellular N concentrations. In sum, the combination of modified Monod and Logistic functions and the combination of Droop and Logistic functions all can predict algae growth reasonably well, while the combination of Droop and Logistic functions is slightly better. Meanwhile, it is through these two combinations that two sets of better parameters in modified Monod and Droop functions can be respectively obtained to characterize algal population kinetics with changing nutrient concentrations.

Initial short-term nitrate uptake in juvenile, cultivated Saccharina latissima (Phaeophyceae) of variable nutritional state

Abstract To reach the goal of large-scale seaweed cultivation in Norway, new knowledge concerning commercially important species like the kelp Saccharina latissima is essential. This includes fundamental understanding of physiological mechanisms like nutrient uptake kinetics to better understand its ecological niche and nutritional requirements. The initial short-term nitrate (NO3−) uptake kinetics in cultivated S. latissima juvenile sporophytes were evaluated under nitrogen-saturated and nitrogen-limited conditions. The uptake was measured for concentrations in a gradient from 2 to 18 μM NO3− which is representative of Norwegian coastal waters in the main growth season of winter and early spring. Preconditioning treatments led to internal nitrogen pools (total tissue nitrogen and intracellular nitrate) that were significantly lower for the N-limited than for the N-saturated sporophytes prior to the experiment. Nitrate uptake rates, related to biomass (V) and intracellular nitrogen content (U), were linearly related to the substrate concentrations for both N-limited and N-saturated sporophytes, indicating that S. latissima requires high ambient nitrate concentrations to maintain rapid growth. The sporophytes with deficient internal nitrogen pools exhibited higher uptake rates of NO3− than sporophytes with higher internal pools of nitrogen. Mathematical modelling was used to investigate the temporal development of total tissue nitrogen (QN) based on the nitrogen-specific uptake rates (U) and revealed a near linear response of U to changes in QN. The model also found that a maximum estimated value for QN was only approached after more than 20 days at external NO3- concentrations of 8 μM. These results expand the physiological understanding of cultivated S. latissima and are important for a sustainable upscaling of seaweed farm production.

Latitudinal, seasonal and depth-dependent variation in growth, chemical composition and biofouling of cultivated Saccharina latissima (Phaeophyceae) along the Norwegian coast

The Norwegian coastline covers more than 10° in latitude and provides a range in abiotic and biotic conditions for seaweed farming. In this study, we compared the effects of cultivation depth and season on the increase in biomass (frond length and biomass yield), chemical composition (protein, tissue nitrogen, intracellular nitrate and ash content) and biofouling (total cover and species composition) of cultivated Saccharina latissima at nine locations along a latitudinal gradient from 58 to 69° N. The effects of light and temperature on frond length and biofouling were evaluated along with their relevance for selecting optimal cultivation sites. Growth was greater at 1–2 m than at 8–9 m depth and showed large differences among locations, mainly in relation to local salinity levels. Maximum frond lengths varied between 15 and 100 cm, and maximum biomass yields between 0.2 and 14 kg m−2. Timing of maximum frond length and biomass yield varied with latitude, peaking 5 and 8 weeks later in the northern location (69° N) than in the central (63° N) and southern (58° N) locations, respectively. The nitrogen-to-protein conversion factor (averaged across all locations and depths) was 3.8, while protein content varied from 22 to 109 mg g−1 DW, with seasonality and latitude having the largest effect. The onset of biofouling also followed a latitudinal pattern, with a delayed onset in northern locations and at freshwater-influenced sites. The dominant epibiont was the bryozoan Membranipora membranacea. Our results demonstrate the feasibility of S. latissima cultivation along a wide latitudinal gradient in North Atlantic waters and underscore the importance of careful site selection for seaweed aquaculture.

Nitrate uptake and metabolism in human skeletal muscle cell cultures

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

Anaerobic ammonium oxidation (anammox) and denitrification in Peru margin sediments

The upwelling system of coastal Peru supports very high primary production, contributing to an oxygen deficient zone (ODZ) in subsurface waters and high organic matter deposition rates to underlying sediments. Although anammox and denitrification have been relatively well studied in ODZ waters, few studies have investigated these processes in the underlying sediments. We sampled seven stations over a large geographic area along the Peru margin, spanning a water depth of 100–3240 m. At two of the central shelf stations (100 m and 325 m), we observed Thioploca , with a well-developed mat at the shallowest station (100 m). We measured sediment properties and conducted shipboard 15 N-incubations of homogenized sediments to determine potential rates of anammox and denitrification and potential controlling factors at each station. Diversity of anammox bacteria based on 16S rRNA and hydrazine oxidoreductase ( hzo ) sequences and hzo gene abundances were measured at each station. Overall, organic C content was high across the stations (3–12%), except for two of the deepest stations (~1.5%). Porewater ammonium fluxes and ammonium production rates in shipboard incubations, reflecting sediment organic carbon decomposition rates, were higher at the two central shelf stations compared to the other stations. The range in average potential rates was 2.1–80.4 nmol N cm −3 h −1 for denitrification and 1.8–44.2 nmol N cm −3 h −1 for anammox. The range in relative anammox ( ra ) across stations was 2.6–47.4%, with an average of 34.2%. The lowest ra was found at the shallowest shelf station with Thioploca mats and highest ammonium production rates. The ra jumped up to 45.9% at the station with the next highest ammonium production rates, corresponding to the deeper shelf station (325 m). At the other stations, ra was relatively high (39.6–47.4%), except at one station (16.3%), reflecting similar ammonium production rates due to decomposition across these stations. Anammox bacteria in the Candidatus Scalindua genus were the only anammox bacteria detected in Peru margin sediments based on 16S rRNA or hzo sequences. Copy number of hzo indicated abundant populations of anammox bacteria across the stations. However, hzo copy number did not correlate with anammox rates or ra . Overall, our results suggest that anammox contributes significantly to N 2 production in Peru margin sediments, except in shelf sediments with high decomposition rates and dense Thioploca mats. • Anammox and denitrification rates were generally high in Peru margin sediments. • Intracellular nitrate was a complicating factor in shelf sediments in the study. • Anammox rates were inhibited in the presence of a dense Thioploca mat. • Anammox rates jumped up in the presence of low density Thioploca . • Density and metabolic status of Thioploca may influence anammox rates.

Intracellular nitrate in sediments of an oxygen-deficient marine basin is linked to pelagic diatoms.

Intracellular nitrate is an important electron acceptor in oxygen-deficient aquatic environments, either for the nitrate-storing microbes themselves, or for ambient microbial communities through nitrate leakage. This study links the spatial distribution of intracellular nitrate with the abundance and identity of nitrate-storing microbes in sediments of the Bornholm Basin, an environmental showcase for severe hypoxia. Intracellular nitrate (up to 270 nmol cm-3 sediment) was detected at all 18 stations along a 35-km transect through the basin and typically extended as deep as 1.6 cm into the sediment. Intracellular nitrate contents were particularly high at stations where chlorophyll contents suggested high settling rates of pelagic primary production. The depth distribution of intracellular nitrate matched that of the diatom-specific photopigment fucoxanthin in the upper 1.6 cm and calculations support that diatoms are the major nitrate-storing microbes in these sediments. In contrast, other known nitrate-storing microbes, such as sulfide-oxidizing bacteria and foraminifers, played only a minor role, if any. Strikingly, 18S rRNA gene sequencing revealed that the majority of the diatoms in the sediment were pelagic species. We conclude that intracellular nitrate stored by pelagic diatoms is transported to the seafloor by settling phytoplankton blooms, implying a so far overlooked 'biological nitrate pump'.

Kinetic exploration of intracellular nitrate storage in marine microalgae

ABSTRACTIn this study, a recently developed model accounting for intracellular nitrate storage kinetics was thoroughly studied to understand and compare the storage capacity of Phaeodactylum tricornutum and Amphora coffeaeformis. In the first stage the identifiability of the biokinetic parameters was examined. Next, the kinetic model was calibrated for both microalgal species based on experimental observations during batch growth experiments. Two kinetic parameters were calibrated, namely the maximum specific growth rate and the nitrate storage rate (). A significant difference was observed for the nitrate storage rate between both species. For P. tricornutum, the nitrate storage rate was much higher ( = 0.036 m3 g−1 DW d−1) compared to A. coffeaeformis ( = 0.0004 m3 g−1 DW d−1). This suggests that P. tricornutum has a more efficient nitrate uptake ability and intracellular nitrate storage capacity and also indicates the need for determination of in order to quantify nitrate storage.

Live benthic foraminifera in the Yellow Sea and the East China Sea: vertical distribution, nitrate storage, and potential denitrification

Benthic foraminifera were investigated to determine their vertical distribution, nitrate storage, and potential denitrification in the Yellow Sea (YS) and the East China Sea (ECS). The phytodetritus content and freshness as well as sediment redox conditions were key factors determining the vertical distribution of foraminifera in sediments of the YS and the ECS. The intracellular nitrate (ICNO3) concentrations of Nonionella stella, Hanzawaia nipponica, Bolivina robusta, Cancris auriculus, and Globobulimina pacifica ranged from 3 to 114 mM, which in some cases was several hundred-fold more than the pore water nitrate (PWNO3) concentrations. The genus Hanzawaia was for the first time reported to store ICNO3 (316 +/- 73 pmol ind.(-1); 11 +/- 3 mM). The significant correlation between the ICNO3 concentration and foraminiferal abundance (Pearson correlation: r = 0.401, p < 0.01, n = 60) suggested that foraminifera may have an important role in nitrate storage in the sediments. The foraminiferal intracellular nitrate (FINO3) pool ranged from 9 to 74% of the ICNO3 pool in sediments, indicating that nitrate may be stored by other sediment organisms (e.g. diatoms). The chlorophyll a concentration, chloroplastic pigment equivalents (CPE), and chlorophyll a:phaeopigment ratio were all significantly positively correlated with the ICNO3 concentration (Pearson correlation: r = 0.563, 0.603, and 0.457, respectively; p < 0.01; n = 60), indicating that phytodetritus (e.g. diatoms) might also contribute to sedimentary ICNO3. Potential foraminiferal denitrification rates ranged from 9 to 92 mu mol m(-2) d(-1) in the YS and the ECS, indicating that benthic foraminifera might play a role in sedimentary denitrification.

Sinks and Sources of Intracellular Nitrate in Gromiids.

A substantial nitrate pool is stored within living cells in various benthic marine environments. The fate of this bioavailable nitrogen differs according to the organisms managing the intracellular nitrate (ICN). While some light has been shed on the nitrate carried by diatoms and foraminiferans, no study has so far followed the nitrate kept by gromiids. Gromiids are large protists and their ICN concentration can exceed 1000x the ambient nitrate concentration. In the present study we investigated gromiids from diverse habitats and showed that they contained nitrate at concentrations ranging from 1 to 370 mM. We used 15N tracer techniques to investigate the source of this ICN, and found that it originated both from active nitrate uptake from the environment and from intracellular production, most likely through bacterial nitrification. Microsensor measurements showed that part of the ICN was denitrified to N2 when gromiids were exposed to anoxia. Denitrification seemed to be mediated by endobiotic bacteria because antibiotics inhibited denitrification activity. The active uptake of nitrate suggests that ICN plays a role in gromiid physiology and is not merely a consequence of the gromiid hosting a diverse bacterial community. Measurements of aerobic respiration rates and modeling of oxygen consumption by individual gromiid cells suggested that gromiids may occasionally turn anoxic by their own respiration activity and thus need strategies for coping with this self-inflicted anoxia.

Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates.

Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations >60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2–3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.