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1.
Proc Natl Acad Sci U S A ; 116(33): 16448-16453, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346083

RESUMO

Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.

2.
mSystems ; 4(1)2019.
Artigo em Inglês | MEDLINE | ID: mdl-30746496

RESUMO

Two-component sensory (TCS) systems link microbial physiology to the environment and thus may play key roles in biogeochemical cycles. In this study, we surveyed the TCS systems of 328 diverse marine bacterial species. We identified lifestyle traits such as copiotrophy and diazotrophy that are associated with larger numbers of TCS system genes within the genome. We compared marine bacterial species with 1,152 reference bacterial species from a variety of habitats and found evidence of extra response regulators in marine genomes. Examining the location of TCS genes along the circular bacterial genome, we also found that marine bacteria have a large number of "orphan" genes, as well as many hybrid histidine kinases. The prevalence of "extra" response regulators, orphan genes, and hybrid TCS systems suggests that marine bacteria break with traditional understanding of how TCS systems operate. These trends suggest prevalent regulatory networking, which may allow coordinated physiological responses to multiple environmental signals and may represent a specific adaptation to the marine environment. We examine phylogenetic and lifestyle traits that influence the number and structure of two-component systems in the genome, finding, for example, that a lack of two-component systems is a hallmark of oligotrophy. Finally, in an effort to demonstrate the importance of TCS systems to marine biogeochemistry, we examined the distribution of Prochlorococcus/Synechococcus response regulator PMT9312_0717 in metaproteomes of the tropical South Pacific. We found that this protein's abundance is related to phosphate concentrations, consistent with a putative role in phosphate regulation. IMPORTANCE Marine microbes must manage variation in their chemical, physical, and biological surroundings. Because they directly link bacterial physiology to environmental changes, TCS systems are crucial to the bacterial cell. This study surveyed TCS systems in a large number of marine bacteria and identified key phylogenetic and lifestyle patterns in environmental sensing. We found evidence that, in comparison with bacteria as a whole, marine organisms have irregular TCS system constructs which might represent an adaptation specific to the marine environment. Additionally, we demonstrate the biogeochemical relevance of TCS systems by correlating the presence of the PMT9312_0717 response regulator protein to phosphate concentrations in the South Pacific. We highlight that despite their potential ecological and biogeochemical relevance, TCS systems have been understudied in the marine ecosystem. This report expands our understanding of the breadth of bacterial TCS systems and how marine bacteria have adapted to survive in their unique environment.

3.
J Proteome Res ; 18(4): 1461-1476, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30702898

RESUMO

Ocean metaproteomics is an emerging field enabling discoveries about marine microbial communities and their impact on global biogeochemical processes. Recent ocean metaproteomic studies have provided insight into microbial nutrient transport, colimitation of carbon fixation, the metabolism of microbial biofilms, and dynamics of carbon flux in marine ecosystems. Future methodological developments could provide new capabilities such as characterizing long-term ecosystem changes, biogeochemical reaction rates, and in situ stoichiometries. Yet challenges remain for ocean metaproteomics due to the great biological diversity that produces highly complex mass spectra, as well as the difficulty in obtaining and working with environmental samples. This review summarizes the progress and challenges facing ocean metaproteomic scientists and proposes best practices for data sharing of ocean metaproteomic data sets, including the data types and metadata needed to enable intercomparisons of protein distributions and annotations that could foster global ocean metaproteomic capabilities.

4.
Metallomics ; 11(1): 183-200, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30443649

RESUMO

Copper toxicity has been a long-term selection pressure on bacteria due to its presence in the environment and its use as an antimicrobial agent by grazing protozoa, by phagocytic cells of the immune system, and in man-made medical and commercial products. There is recent evidence that exposure to increased copper stress may have been a key driver in the evolution and spread of methicillin-resistant Staphylococcus aureus, a globally important pathogen that causes significant mortality and morbidity worldwide. Yet it is unclear how S. aureus physiology is affected by copper stress or how it adapts in order to be able to grow in the presence of excess copper. Here, we have determined quantitatively how S. aureus alters its proteome during growth under copper stress conditions, comparing this adaptive response in two different types of growth regime. We found that the adaptive response involves induction of the conserved copper detoxification system as well as induction of enzymes of central carbon metabolism, with only limited induction of proteins involved in the oxidative stress response. Further, we identified a protein that binds copper inside S. aureus cells when stressed by copper excess. This copper-binding enzyme, a glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis, is inhibited by copper in vitro and inside S. aureus cells. Together, our data demonstrate that copper stress leads to the inhibition of glycolysis in S. aureus, and that the bacterium adapts to this stress by altering its central carbon utilisation pathways.

5.
Environ Microbiol ; 20(8): 3109-3126, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30117243

RESUMO

Diatoms are a highly successful group of photosynthetic protists that often thrive under adverse environmental conditions. Members of the genus Pseudo-nitzschia are ecologically important diatoms which are able to subsist during periods of chronic iron limitation and form dense blooms following iron fertilization events. The cellular strategies within diatoms that orchestrate these physiological responses to variable iron concentrations remain largely uncharacterized. Using a combined transcriptomic and proteomic approach, we explore the exceptional ability of a diatom isolated from the iron-limited Northeast Pacific Ocean to reorganize its intracellular processes as a function of iron. We compared the molecular responses of Pseudo-nitzschia granii observed under iron-replete and iron-limited growth conditions to those of other model diatoms. Iron-coordinated molecular responses demonstrated some agreement between gene expression and protein abundance, including iron-starvation-induced-proteins, a putative iron transport system and components of photosynthesis and the Calvin cycle. Pseudo-nitzschia granii distinctly differentially expresses genes encoding proteins involved in iron-independent photosynthetic electron transport, urea acquisition and vitamin synthesis. We show that P. granii is unique among studied diatoms in its physiology stemming from distinct cellular responses, which may underlie its ability to subsist in low iron regions and rapidly bloom to outcompete other diatom taxa following iron enrichment.


Assuntos
Diatomáceas/genética , Diatomáceas/metabolismo , Ferro/metabolismo , Diatomáceas/classificação , Diatomáceas/isolamento & purificação , Transporte de Elétrons , Oceano Pacífico , Fotossíntese , Proteômica , Transcriptoma
6.
Environ Microbiol ; 19(6): 2348-2365, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28371229

RESUMO

Marine Synechococcus thrive over a range of light regimes in the ocean. We examined the proteomic, genomic and physiological responses of seven Synechococcus isolates to moderate irradiances (5-80 µE m-2 s-1 ), and show that Synechococcus spans a continuum of light responses ranging from low light optimized (LLO) to high light optimized (HLO). These light responses are linked to phylogeny and pigmentation. Marine sub-cluster 5.1A isolates with higher phycouribilin: phycoerythrobilin ratios fell toward the LLO end of the continuum, while sub-cluster 5.1B, 5.2 and estuarine Synechococcus with less phycouribilin fell toward the HLO end of the continuum. Global proteomes were highly responsive to light, with > 50% of abundant proteins varying more than twofold between the lowest and highest irradiance. All strains downregulated phycobilisome proteins with increasing irradiance. Regulation of proteins involved in photosynthetic electron transport, carbon fixation, oxidative stress protection (superoxide dismutases) and iron and nitrogen metabolism varied among strains, as did the number of high light inducible protein (Hlip) and DNA photolyase genes in their genomes. All but one LLO strain possessed the photoprotective orange carotenoid protein (OCP). The unique combinations of light responses in each strain gives rise to distinct photophysiological phenotypes that may affect Synechococcus distributions in the ocean.


Assuntos
Transporte de Elétrons/genética , Fotossíntese/genética , Ficobilinas/metabolismo , Ficoeritrina/metabolismo , Synechococcus/genética , Synechococcus/fisiologia , Urobilina/análogos & derivados , Adaptação Ocular , Ciclo do Carbono/genética , Luz , Estresse Oxidativo/genética , Ficobilissomas/metabolismo , Filogenia , Proteômica , Synechococcus/isolamento & purificação , Urobilina/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(50): 14237-14242, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27911777

RESUMO

Nearly all iron dissolved in the ocean is complexed by strong organic ligands of unknown composition. The effect of ligand composition on microbial iron acquisition is poorly understood, but amendment experiments using model ligands show they can facilitate or impede iron uptake depending on their identity. Here we show that siderophores, organic compounds synthesized by microbes to facilitate iron uptake, are a dynamic component of the marine ligand pool in the eastern tropical Pacific Ocean. Siderophore concentrations in iron-deficient waters averaged 9 pM, up to fivefold higher than in iron-rich coastal and nutrient-depleted oligotrophic waters, and were dominated by amphibactins, amphiphilic siderophores with cell membrane affinity. Phylogenetic analysis of amphibactin biosynthetic genes suggests that the ability to produce amphibactins has transferred horizontally across multiple Gammaproteobacteria, potentially driven by pressures to compete for iron. In coastal and oligotrophic regions of the eastern Pacific Ocean, amphibactins were replaced with lower concentrations (1-2 pM) of hydrophilic ferrioxamine siderophores. Our results suggest that organic ligand composition changes across the surface ocean in response to environmental pressures. Hydrophilic siderophores are predominantly found across regions of the ocean where iron is not expected to be the limiting nutrient for the microbial community at large. However, in regions with intense competition for iron, some microbes optimize iron acquisition by producing siderophores that minimize diffusive losses to the environment. These siderophores affect iron bioavailability and thus may be an important component of the marine iron cycle.


Assuntos
Ferro/metabolismo , Água do Mar/análise , Água do Mar/microbiologia , Sideróforos/metabolismo , Adaptação Fisiológica , Disponibilidade Biológica , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Genes Bacterianos , Ferro/farmacocinética , Ligantes , Oceano Pacífico , Filogenia , Microbiologia da Água
8.
Nat Commun ; 6: 8155, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26327191

RESUMO

Nitrogen fixation rates of the globally distributed, biogeochemically important marine cyanobacterium Trichodesmium increase under high carbon dioxide (CO2) levels in short-term studies due to physiological plasticity. However, its long-term adaptive responses to ongoing anthropogenic CO2 increases are unknown. Here we show that experimental evolution under extended selection at projected future elevated CO2 levels results in irreversible, large increases in nitrogen fixation and growth rates, even after being moved back to lower present day CO2 levels for hundreds of generations. This represents an unprecedented microbial evolutionary response, as reproductive fitness increases acquired in the selection environment are maintained after returning to the ancestral environment. Constitutive rate increases are accompanied by irreversible shifts in diel nitrogen fixation patterns, and increased activity of a potentially regulatory DNA methyltransferase enzyme. High CO2-selected cell lines also exhibit increased phosphorus-limited growth rates, suggesting a potential advantage for this keystone organism in a more nutrient-limited, acidified future ocean.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Evolução Biológica , Fixação de Nitrogênio/efeitos dos fármacos , Oscillatoria/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Dióxido de Carbono/farmacologia , Fixação de Nitrogênio/fisiologia , Oceanos e Mares , Oscillatoria/crescimento & desenvolvimento , Oscillatoria/fisiologia , Fósforo/metabolismo
9.
Proc Natl Acad Sci U S A ; 112(32): 9944-9, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26216989

RESUMO

Marine Synechococcus are some of the most diverse and ubiquitous phytoplankton, and iron (Fe) is an essential micronutrient that limits productivity in many parts of the ocean. To investigate how coastal and oceanic Atlantic Synechococcus strains acclimate to Fe availability, we compared the growth, photophysiology, and quantitative proteomics of two Synechococcus strains from different Fe regimes. Synechococcus strain WH8102, from a region in the southern Sargasso Sea that receives substantial dust deposition, showed impaired growth and photophysiology as Fe declined, yet used few acclimation responses. Coastal WH8020, from the dynamic, seasonally variable New England shelf, displayed a multitiered, hierarchical cascade of acclimation responses with different Fe thresholds. The multitiered response included changes in Fe acquisition, storage, and photosynthetic proteins, substitution of flavodoxin for ferredoxin, and modified photophysiology, all while maintaining remarkably stable growth rates over a range of Fe concentrations. Modulation of two distinct ferric uptake regulator (Fur) proteins that coincided with the multitiered proteome response was found, implying the coastal strain has different regulatory threshold responses to low Fe availability. Low nitrogen (N) and phosphorus (P) availability in the open ocean may favor the loss of Fe response genes when Fe availability is consistent over time, whereas these genes are retained in dynamic environments where Fe availability fluctuates and N and P are more abundant.


Assuntos
Ecossistema , Ferro/farmacologia , Synechococcus/fisiologia , Oceano Atlântico , Proteínas de Bactérias/metabolismo , Geografia , Fotossíntese/efeitos dos fármacos , Synechococcus/efeitos dos fármacos
10.
Proteomics ; 15(20): 3521-31, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26097212

RESUMO

Proteomics has great potential for studies of marine microbial biogeochemistry, yet high microbial diversity in many locales presents us with unique challenges. We addressed this challenge with a targeted metaproteomics workflow for NtcA and P-II, two nitrogen regulatory proteins, and demonstrated its application for cyanobacterial taxa within microbial samples from the Central Pacific Ocean. Using METATRYP, an open-source Python toolkit, we examined the number of shared (redundant) tryptic peptides in representative marine microbes, with the number of tryptic peptides shared between different species typically being 1% or less. The related cyanobacteria Prochlorococcus and Synechococcus shared an average of 4.8 ± 1.9% of their tryptic peptides, while shared intraspecies peptides were higher, 13 ± 15% shared peptides between 12 Prochlorococcus genomes. An NtcA peptide was found to target multiple cyanobacteria species, whereas a P-II peptide showed specificity to the high-light Prochlorococcus ecotype. Distributions of NtcA and P-II in the Central Pacific Ocean were similar except at the Equator likely due to differential nitrogen stress responses between Prochlorococcus and Synechococcus. The number of unique tryptic peptides coded for within three combined oceanic microbial metagenomes was estimated to be ∼4 × 10(7) , 1000-fold larger than an individual microbial proteome and 27-fold larger than the human proteome, yet still 20 orders of magnitude lower than the peptide diversity possible in all protein space, implying that peptide mapping algorithms should be able to withstand the added level of complexity in metaproteomic samples.


Assuntos
Biomarcadores , Metagenoma , Filogenia , Proteoma/genética , Variação Genética , Humanos , Oceanos e Mares , Prochlorococcus/genética , Especificidade da Espécie , Synechococcus/genética
11.
Proc Natl Acad Sci U S A ; 112(4): 1173-8, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25587132

RESUMO

Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name "Candidatus Nitrosopelagicus brevis" str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.


Assuntos
Archaea , Proteínas Arqueais , Regulação da Expressão Gênica em Archaea/fisiologia , Proteoma , Proteômica , Microbiologia da Água , Sequência de Aminoácidos , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/biossíntese , Proteínas Arqueais/genética , Metagenômica , Dados de Sequência Molecular , Oceanos e Mares , Proteoma/biossíntese , Proteoma/genética
12.
Science ; 345(6201): 1173-7, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25190794

RESUMO

Marine primary productivity is strongly influenced by the scarcity of required nutrients, yet our understanding of these nutrient limitations is informed by experimental observations with sparse geographical coverage and methodological limitations. We developed a quantitative proteomic method to directly assess nutrient stress in high-light ecotypes of the abundant cyanobacterium Prochlorococcus across a meridional transect in the central Pacific Ocean. Multiple peptide biomarkers detected widespread and overlapping regions of nutritional stress for nitrogen and phosphorus in the North Pacific Subtropical Gyre and iron in the equatorial Pacific. Quantitative protein analyses demonstrated simultaneous stress for these nutrients at biome interfaces. This application of proteomic biomarkers to diagnose ocean metabolism demonstrated Prochlorococcus actively and simultaneously deploying multiple biochemical strategies for low-nutrient conditions in the oceans.


Assuntos
Nitrogênio/metabolismo , Fósforo/metabolismo , Prochlorococcus/metabolismo , Proteínas/metabolismo , Água do Mar/microbiologia , Estresse Fisiológico , Biomarcadores/metabolismo , Cobalto/metabolismo , Ferro/metabolismo , Oceano Pacífico , Proteômica/métodos
13.
ISME J ; 6(5): 951-60, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22134648

RESUMO

Nitrogen can be a limiting macronutrient for carbon uptake by the marine biosphere. The process of denitrification (conversion of nitrate to gaseous compounds, including N(2) (nitrogen gas)) removes bioavailable nitrogen, particularly in marine sediments, making it a key factor in the marine nitrogen budget. Benthic foraminifera reportedly perform complete denitrification, a process previously considered nearly exclusively performed by bacteria and archaea. If the ability to denitrify is widespread among these diverse and abundant protists, a paradigm shift is required for biogeochemistry and marine microbial ecology. However, to date, the mechanisms of foraminiferal denitrification are unclear, and it is possible that the ability to perform complete denitrification is because of the symbiont metabolism in some foraminiferal species. Using sequence analysis and GeneFISH, we show that for a symbiont-bearing foraminifer, the potential for denitrification resides in the endobionts. Results also identify the endobionts as denitrifying pseudomonads and show that the allogromiid accumulates nitrate intracellularly, presumably for use in denitrification. Endobionts have been observed within many foraminiferal species, and in the case of associations with denitrifying bacteria, may provide fitness for survival in anoxic conditions. These associations may have been a driving force for early foraminiferal diversification, which is thought to have occurred in the Neoproterozoic era when anoxia was widespread.


Assuntos
Bactérias/metabolismo , Desnitrificação , Foraminíferos/microbiologia , Nitratos/metabolismo , Bactérias/classificação , DNA Bacteriano/genética , Sedimentos Geológicos/microbiologia , Hibridização in Situ Fluorescente , Isótopos de Nitrogênio/metabolismo , Filogenia , Simbiose
14.
Science ; 333(6047): 1282-5, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21798895

RESUMO

The ocean is an important global source of nitrous oxide (N(2)O), a greenhouse gas that contributes to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be the primary sources of marine N(2)O, but the isotopic signatures of N(2)O produced by these processes are not consistent with the marine contribution to the global N(2)O budget. Based on enrichment cultures, we report that archaeal ammonia oxidation also produces N(2)O. Natural-abundance stable isotope measurements indicate that the produced N(2)O had bulk δ(15)N and δ(18)O values higher than observed for ammonia-oxidizing bacteria but similar to the δ(15)N and δ(18)O values attributed to the oceanic N(2)O source to the atmosphere. Our results suggest that ammonia-oxidizing archaea may be largely responsible for the oceanic N(2)O source.


Assuntos
Amônia/metabolismo , Archaea/metabolismo , Óxido Nitroso/metabolismo , Água do Mar/microbiologia , Archaea/enzimologia , Bactérias/metabolismo , Meios de Cultura , Desnitrificação , Modelos Lineares , Dados de Sequência Molecular , Nitrificação , Isótopos de Nitrogênio , Oxirredução , Isótopos de Oxigênio , Oceano Pacífico
15.
Anal Chem ; 83(5): 1850-6, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21302935

RESUMO

The bacterial conversion of aqueous nitrate (NO(3)(-)) to nitrous oxide (N(2)O) for isotopic analysis has found widespread use since its introduction (Sigman, D. M.; Casciotti, K. L.; Andreani, M.; Galanter, M.; Böhlke, J. K. Anal. Chem.2001, 73, 4145-4153; Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Böhlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). The bacterial strain Pseudomonas aureofaciens (ATTC no. 13985) was shown to convert NO(3)(-) to N(2)O while retaining both N and O isotopic signatures, and automation of the isotopic analysis of N(2)O greatly increased the throughput of the method (Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Böhlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). Continued development of the denitrifier method has led to increased precision and throughput of NO(3)(-) isotopic analysis. Presented here are several recent procedural modifications and the demonstration of their effectiveness.


Assuntos
Bactérias/metabolismo , Isótopos/análise , Nitratos/análise , Nitratos/metabolismo
16.
Anal Chem ; 81(1): 184-92, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19032028

RESUMO

We describe a new method for analysis of the nitrogen isotopic composition of sedimentary porphyrins. This method involves separation and purification of geoporphyrins from sediment samples using liquid chromatography and HPLC, oxidation of the nitrogen within porphyrin-enriched fractions using a two-step process, and isotopic analysis of the resulting nitrate using the denitrifier method. By analysis of these degradation products of chlorophylls, we are able to measure an isotopic signature that reflects the nitrogen utilized by primary producers. The high sensitivity of the denitrifier method allows measurement of small samples that contain low concentrations of porphyrins. Extraction of only 50 nmol of nitrogen (nmol N) allows the following five analyses to be made (each on approximately 10 nmol N): nitrogen concentration, an assessment of potential contamination by nonporphyrin N, and three replicate isotopic measurements. The measured values of delta15N have an average analytical precision of +/-0.5 per thousand (1sigma) and an average contribution from Rayleigh fractionation of 0.7 per thousand from incomplete oxidation of porphyrin N to nitrate. The overall method will enable high-resolution records of delta15N values to be obtained for geological and ecological applications.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Sedimentos Geológicos/química , Nitrogênio/análise , Porfirinas/isolamento & purificação , Técnicas de Química Analítica/métodos , Clorofila/análise , Clorofila/química , Clorofila A , Fósseis , Nitratos/análise , Nitratos/química , Óxido Nítrico/análise , Óxido Nítrico/química , Nitrogênio/química , Isótopos de Nitrogênio/análise , Oxirredução , Porfirinas/análise , Porfirinas/química
17.
Anal Chem ; 79(6): 2427-36, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17295443

RESUMO

Nitrite is a central intermediate in the nitrogen cycle and can persist in significant concentrations in ocean waters, sediment pore waters, and terrestrial groundwaters. To fully interpret the effect of microbial processes on nitrate (NO3-), nitrite (NO2-), and nitrous oxide (N2O) cycling in these systems, the nitrite pool must be accessible to isotopic analysis. Furthermore, because nitrite interferes with most methods of nitrate isotopic analysis, accurate isotopic analysis of nitrite is essential for correct measurement of nitrate isotopes in a sample that contains nitrite. In this study, nitrite salts with varying oxygen isotopic compositions were prepared and calibrated and then used to test the denitrifier method for nitrite oxygen isotopic analysis. The oxygen isotopic fractionation during nitrite reduction to N2O by Pseudomonas aureofaciens was lower than for nitrate conversion to N2O, while oxygen isotopic exchange between nitrite and water during the reaction was similar. These results enable the extension of the denitrifier method to oxygen isotopic analysis of nitrite (in the absence of nitrate) and correction of nitrate isotopes for the presence of nitrite in "mixed" samples. We tested storage conditions for seawater and freshwater samples that contain nitrite and provide recommendations for accurate oxygen isotopic analysis of nitrite by any method. Finally, we report preliminary results on the equilibrium isotope effect between nitrite and water, which can play an important role in determining the oxygen isotopic value of nitrite where equilibration with water is significant.

18.
Anal Chem ; 78(7): 2377-81, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16579623

RESUMO

A new method has been developed for the oxygen isotopic analysis of water utilizing the exchange of oxygen atoms with nitrite followed by conversion of nitrite to nitrous oxide by hydrazoic acid. This method is analogous to that employing carbon dioxide equilibration in that the analyte is added as a reagent to the water sample and exchanges oxygen isotopes with the water. In this procedure, however, the equilibration is more rapid and less sensitive to changes in ambient temperature, as the exchange is terminated by conversion of nitrite to nitrous oxide prior to analysis. The nitrous oxide is then analyzed using an on-line purge and trap system coupled to an isotope ratio mass spectrometer. This method makes use of previously established methods for nitrite isotopic analysis and commercially available equipment. It has high throughput, relatively low standard deviation (0.1 per thousand or better), and small sample size (down to 100 microL), is simple, and is applicable to both fresh- and seawater samples.


Assuntos
Isótopos de Oxigênio/análise , Água do Mar/análise , Água/análise , Azidas/química , Nitritos/química , Óxido Nitroso/química , Oxigênio/química , Reprodutibilidade dos Testes , Água do Mar/química , Sensibilidade e Especificidade , Temperatura Ambiente , Água/química
19.
Anal Chem ; 77(17): 5589-95, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16131070

RESUMO

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

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