Wir danken der USGS und die Vermont Water Resources and Lake Studies Center für die Bereitstellung von Mitteln.

1. Phosphorus Extraction <ol><li> Bereiten Sie eine 1L Lösung von 0,25 M Natronlauge (NaOH, 40,00 g / mol) + 0,05 M Ethylendiamintetraessigsäure (EDTA, 292,24 g / mol). </li><li> 30 ml der NaOH + EDTA-Lösung bis 2 g des nassen oder getrockneten Boden / Probe in einem 50 mL konischen Rohr. </li><li> Inkubieren unter Rühren für 16 h bei Raumtemperatur. </li><li> Centrifuge 3000 xg für 3 Minuten. </li></ol> 2. Probenextrakt pH-Regelung <ol><li> Übertragen einer 0,1 ml Aliquot auf einem 1,5 mL Mikro-Zentrifugenröhrchen. </li><li> Add 0.017 mL 2,5 M Essigsäure, 0,144 ml 0,4 M Acetatpuffer (70,5% von Natriumacetat [CH 3 COONa, 82,03 g / mol] und 29,5% Essigsäure, pH 5,0) und 0,739 ml deionisiertem Wasser. </li><li> Das Endvolumen eingestellt Extrakt wird 1 mL und der endgültige pH-Wert 5,0 liegen. </li></ol> 3. Enzyme Stammlösung Vorbereitung <ol><li> Bereiten Sie 1 L von 0,1 M Natriumacetat (CH 3 COONa, 82,03 g / mol)-Lösung, pH 5,0. </li><li> Bereiten Sie 2 x 10 ml saure Phosphatase Stammlösungen in der Natrium-Acetat-Puffer in Schritt 3.1, so dass Endkonzentrationen je 0,5 Einheiten / ml vorbereitet sind. Saure Phosphatase Typ I aus Triticum aestivum (Weizenkeime, GP, in der Regel 0,5 U mg-1 fest, EC 3.1.3.2) Saure Phosphatase Typ IV-S aus Solanum tuberosum (Kartoffel, PP, in der Regel 4,6 U mg-1 fest, EC 3.1.3.2) * Einheiten der Aktivität werden durch den Lieferanten, wobei 1 Unit als die Befreiung von 1 Mikromol Orthophosphat, um die Lösung pro Stunde bei 37 ° C definiert angegeben </li><li> Lyophilisierte Nuklease P1 (EG 3.1.30.1) aus Penicillium citrinum (Pilze, NP1, in der Regel 500 U mg -1 fest) in einem mL VE-Wasser-dieser kann nun bei 4 ° C gelagert werden für eine spätere Verwendung. </li><li> Pipette NP1 in einem der 10 ml-Röhrchen in Schritt 3.2, so dass die Endkonzentration 2,5 Einheiten / mL NP1 ist bereit, sanft durch mehrmaliges Wenden mischen. </li><li> Centrifuge die beiden 10 mL Enzymlösungen 3000 xg für 30 Minuten. </li></ol> 4. P Calibration Curve and Controls <ol><li> Bereiten Sie 1 l 1 mM Kaliumphosphat (K2HPO4, 174,18 g / mol) Stammlösung. </li><li> 1 ml der 1 mM Kaliumphosphat-Stammlösung in ein 1,5 ml Zentrifugenröhrchen und führen sieben 0,5 ml Verdünnungsreihen. </li><li> Entsorgen Sie die erste Röhre, so dass Sie 7 Verdünnungen von 20 nmol-0,625 nmol P. haben </li><li> Transfer-80 ul aus jedem Röhrchen in zweifacher Ausfertigung, um Zeilen B - H eines Standard-96-Well-Platte. </li><li> Add 80 ul von Natrium-Acetat-Puffer (Abschnitt 3.1) zu A der Spalten 11 und 12-dies ist die 0 nmol P Kalibrierpunkt. Reihe </li><li> Jede Spalte 11 und 12 nun enthält 8 Referenzproben von 0 nmol bis 20 nmol anorganischem Phosphat. </li><li> Spalte 10 enthält die folgenden Steuerelemente: <ol><li> Add 40 ul PP + GP Enzymlösung + 40 ul Natriumacetatpuffer (Abschnitt 3.1) zu den ersten drei Brunnen in Spalte 10. </li><li> Zugabe von 40 ul PP + GP + NP1 Enzymlösung + 40 ul Natriumacetatpuffer auf die nächsten drei Brunnen. </li><li> Zugabe von 40 ul Natriumacetatlösung mit 10 nmol Glucose-6-Phosphat (C 6 H 11 Na 2 O 9 P &amp;bullet; xH 2 O, 304,1 g / mol) + 40 ul PP + GP Enzymlösung auf eine gut und die endgültige gut in Spalte 10 add Natriumacetatlösung statt Enzymlösung. </li></ol></li></ol> 5. Sample + Enzyminkubation <ol><li> Jede Probe getestet wird belegen die ersten 9 Vertiefungen in 1 Reihe mit einem Standard 96-Well-Platte. </li><li> Verteilen Sie 40 ul pH-eingestellten Sample Auszüge aus Kapitel 2 in die Vertiefungen 1-9 in bis zu 8 Zeilen. </li><li> Nachdem alle Proben verteilt wurden * Verwendung einer Mehrkanal-Pipette PP + GP Enzymlösung Spalten 1-3, PP + GP + NP1 Spalten 4-6 und Natriumacetatpuffer (hergestellt in Abschnitt 3.1), Spalten 7-9. Verteilen * Dieser Schritt muss schnell durchgeführt werden, um sicherzustellen, alle Proben erhalten gleichwertige Inkubationszeit. </li><li> Decken Sie die 96-Well-Platte mit einem Deckel und inkubieren Proben + Enzymlösungen, Kontrollen und Kalibrierkurve genau 1 Stunde bei 37 ° C. </li></ol> 6. Kolorimetrische Messung der Released und Hintergrund Anorganische P <ol><li> Bereiten Sie 50 mL von jeder der folgenden Lösungen in VE-Wasser: Lösung A *: 0,1 M Ascorbinsäure (C6H8O6, 176,12 g / mol) + 0,5 M Trichloressigsäure (Cl3CCOOH, 163,39 g / mol) Lösung B: 0,01 M Ammoniummolybdat ((NH4) 2MoO4, 196,01 g / mol) Lösung C: 0,1 M Natriumcitrat (HOC (COONa) (CH2COONa) 2 2H2O, 294,10 g / mol) + 0,2 M Natriumarsenat (NaAsO2, 129,91 g / mol) + 5% Eisessig (CH3CO2H, 60,05 g / mol) * Lösung A muss täglich zubereitet werden. </li><li> Fügen Sie 25 ul Lösung SDS, 100 ul Lösung A, 20 ul Lösung B und 50 ul und der Lösung C in alle Vertiefungen in den 96-Loch-plaß. Tun Sie dies schnell mit einer Mehrkanal-Pipette. </li><li> Die Platte abdecken und inkubieren 30 Minuten bei Raumtemperatur. </li><li> Die Absorption bei 850 nm in einer abstimmbaren Mikro-Platten-Lesegerät. </li></ol> 7. Klassifikation der P-Verbindungen <ol><li> Import von Rohdaten in ein Tabellenkalkulationsprogramm (zB Microsoft Excel). </li><li> Plot der anorganischen Phosphor-Eichkurve: 0 bis 40 nmol P auf der x-Achse und den Mittelwert der doppelten Absorptionsmessungen auf der y-Achse. </li><li> Führen Sie eine lineare Regression und finden Sie die Gleichung der Trendlinie. </li><li> Spalten 1-3: Direkt bewerben die Eichkurve, das ist Hintergrund anorganischen P. </li><li> Spalten 4-6: Durchschnittliche dreifacher Ausfertigung Abtastwerte und subtrahieren Kontrollen (Enzymlösung + Hintergrund anorganischen P) von den Brutto-Absorption, das ist P aus hydrolysierbaren P Monoester abgeleitet. </li><li> Spalten 7-9: Gleiche wie 7,5 bis subtrahieren Absorptionswerte aus den Spalten 4-6 - das ist P aus hydrolysierbaren P Diester. </li><li> Convert Absorption Daten nmol P durch die Anwendung der Kalibrierung Gleichung freigegeben. </li><li> nmol P freigesetzt werden können zurück zu mg P / kg des ursprünglichen Bodens bezogen werden. Alternativ kann die Beurteilung Anteile der einzelnen P-Klasse auch ein nützlicher Ansatz für die Analyse von Daten. </li></ol> 8. Repräsentative Ergebnisse: Eine schnelle visuelle Überprüfung der 96-Well-Platte nach dem kolorimetrischen Chemie bietet Anhaltspunkte, ob das Verfahren korrekt durchgeführt wurde. Das erste, was zu überprüfen ist das Niveau der Flüssigkeit in jede Vertiefung durch das Scannen der Seite Profil der Platte. Es sollte genau 275 ul von Reagenzien in alle Vertiefungen werden. Als nächstes Sichtprüfung der Farbe des dreifachen Probenvertiefungen und doppelte Kalibrierung Brunnen. Diese technischen replizieren sollten die gleichen Blauton sein. Anschließend eine Eichkurve, die beiden Brunnen, die Glucose-6-Phosphat und überprüfen sie veröffentlicht alle 10 nmol von anorganischen P. Nach insgesamt extrahierten P gemessen wurde mittels ICP-OES oder eine alternative Methode, sicherzustellen, dass die Gesamt-P berechneten Werte mit dieser Protokoll nicht den Betrag übersteigen, von P, extrahiert wurde. Hinweis: Eine sorgfältige Datenverwaltung in der Tabelle wird der Hut vor quantitativen Fehlern helfen. Sie werden mit viel Zahlen zu tun auf einmal so eine Vorlage erstellen, wird die Zeit gut verbracht werden. <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ein 96-Well-Platte, welche die Ergebnisse für 8 Proben (Reihen AH) und eine Eichkurve (Spalten 11 und 12). Kontrollen sind in Spalte 10. Farbintensität zu erhöhen zwischen den Spalten 1-3 und 4-6 ist darauf zurückzuführen, hydrolysiert Monoester P-Verbindungen, zwischen den Spalten 4-6 und 7-9 ist darauf zurückzuführen, hydrolysiert Diester P-Verbindungen. <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Verteilung der P-Klassen in einer 0,25 M NaOH-0.05 M EDTA-Extrakt eines Vermont Bodenprobe mit High-Throughput-enzymatische Hydrolyse. Fehlerbalken zeigen die Standardabweichung, n = 3.

<ol>
	<li>Turner, B. L., Frossard, E., Baldwin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organic phosphorus in the environment. , (2005).</li><li>Murphy, J., Riley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+single+solution+method+for+determination+of+phosphate+in+natural+waters.">A modified single solution method for determination of phosphate in natural waters.</a> Anal. Chim. Acta. 27, 31-36 (1962).</li><li>Dick, W. A., Tabatabai, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+orthophosphate+in+aqueous+solutions+containing+labile+organic+and+inorganic+phosphorus+compounds.">Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds.</a> J. Environ. Qual. 6, 82-85 (1977).</li><li>He, Z., Toor, G. S., Honeycutt, C. W., Sims, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+hydrolysis+approach+for+characterizing+labile+phosphorus+forms+in+dairy+manure+under+mild+assay+conditions.">An enzymatic hydrolysis approach for characterizing labile phosphorus forms in dairy manure under mild assay conditions.</a> Bioresour. Technol. 97, 1660-1668 (2006).</li><li>He, Z., Honeycutt, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+molybdenum+blue+method+for+orthophosphate+determination+suitable+for+investigating+enzymatic+hydrolysis+of+organic+phosphates.">A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates.</a> Commun. Soil Sci. Plant Anal. 36, 1373-1383 (2005).</li><li>Cade-Menun, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+phosphorus+in+environmental+and+agricultural+samples+by+31P+nuclear+magnetic+resonance+spectroscopy.">Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy.</a> Talanta. 66, 359-371 (2005).</li><li>Johnson, N. R., Hill, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+species+composition+of+poultry+manure-amended+soil+using+high-throughput+enzymatic+hydrolysis.">Phosphorus species composition of poultry manure-amended soil using high-throughput enzymatic hydrolysis.</a> Soil Sci. Soc. Am. J. 74, 1786-1791 (2010).</li></ol>

Von Natur erfordert eine schnelle Methode mit kleinen Volumina großer Sorgfalt. Deshalb ist die wichtigste Schritte sind solche, die Pipettieren Lösungen auf den Teller. Präzise, ​​und vor allem konsistente Pipette Technik sind wesentlich für den Erfolg dieses Tests. Die NaOH-EDTA-Extraktion wird es den meisten P in vielen Proben in drei Klassen charakterisiert werden: Orthophosphat, Monoester und Diester P P. Böden, Dünger, Sedimente oder andere umweltschonende Probe, die NaOH-EDTA-extrahierbare P charakterisiert werden kann enthält . P bildet in Umweltproben sind nicht unbedingt stabil und diese Technik wird sichergestellt, Proben charakterisiert werden, bevor Proben, ohne dass ein Heer von Forschern beschäftigen, sind gefährdet. Dieser Test eignet sich besonders, wenn eine große Anzahl von Proben bearbeitet werden sollen. Das Reagenz muss und Platzbedarf wurden bis auf ein erträgliches Maß (zB 1,5 ml Mikrozentrifugenröhrchen anstatt 50 mL Glaskolben) skaliert. Diese Anpassung auch Grenzen von Abfällen.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> NaOH </td><td> Sigma-Aldrich </td><td> S8045 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> EDS </td></tr><tr><td> Eisessig </td><td> Sigma-Aldrich </td><td> 242853 </td></tr><tr><td> Natriumacetat </td><td> Sigma-Aldrich </td><td> S2889 </td></tr><tr><td> Weizen Acid Phosphatase </td><td> Sigma-Aldrich </td><td> P3627 </td></tr><tr><td> Potato Acid Phosphatase </td><td> Sigma-Aldrich </td><td> P1146 </td></tr><tr><td> Nuclease P1 </td><td> Sigma-Aldrich </td><td> N8630 </td></tr><tr><td> Kaliumphosphat </td><td> Sigma-Aldrich </td><td> P2222 </td></tr><tr><td></td><td> Sigma-Aldrich </td><td></td></tr><tr><td></td><td> Sigma-Aldrich </td><td></td></tr><tr><td> Glucose-6-Phosphat </td><td> Sigma-Aldrich </td><td> G7250 </td></tr><tr><td> SDS </td><td> Sigma-Aldrich </td><td> L4390 </td></tr><tr><td> Askorbinsäure </td><td> Sigma-Aldrich </td><td> A5960 </td></tr><tr><td> TCA </td><td> Sigma-Aldrich </td><td> T9159 </td></tr><tr><td> Ammoniummolybdat </td><td> Sigma-Aldrich </td><td> A1343 </td></tr><tr><td> Natriumzitrat </td><td> Sigma-Aldrich </td><td> S1804 </td></tr><tr><td> Natriumarsenat </td><td> Sigma-Aldrich </td><td> S9663 </td></tr></tbody></table>

high-throughput measurement and classification of organic p in environmental samples

Viele Arten von organischen Phosphor (P)-Moleküle existieren in Umweltproben 1. Traditionelle P Messungen nicht erkennen diese organischen P-Verbindungen, da sie nicht mit kolorimetrischen Reagenzien 2,3 reagieren. Die enzymatische Hydrolyse (EH) ist ein aufstrebendes Verfahren zur genauen Charakterisierung organischer P bildet in Umweltproben 4,5. Diese Methode ist nur in der Genauigkeit von Phosphor-31 Nuclear Magnetic Resonance Spectroscopy (31 P-NMR)-eine Methode, die teuer ist und erfordert spezielle technische training6 übertrumpft. Wir haben eine enzymatische Hydrolyse-Methode für die Messung von drei Klassen von Phosphor (P Monoester, Diester P und anorganischen P) zu einem Mikroplatten-Reader System 7 angepasst. Diese Methode bietet den Forschern eine schnelle, präzise, ​​kostengünstig und anwenderfreundlich bedeutet P Spezies in Böden, Sedimenten, Dünger und wenn konzentriert, aquatischen Proben zu messen. Dies ist die einzige High-Throughput-Verfahren zur Messung der Formen und Enzym-Labilität von organischen P, die in einem Standard-Labor durchgeführt werden kann. Die resultierenden Daten einen Einblick für Wissenschaftler, System Nährstoffgehalt und Eutrophierungspotenzial.

Watch this Scientific Journal Video about High-Throughput Measurement and Classification of Organic P in Environmental Samples at JoVE.com

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Dieses Protokoll beschreibt eine High-Throughput-Verfahren zur enzymatischen Hydrolyse, dass ein Mikroplatten-Reader nutzt, um zu messen und zu klassifizieren Boden Phosphor als P Mono-, Di-P und anorganischen P. Bis zu 96 Proben auf einmal in einem Standard-Labor gemessen werden.

High-Throughput Measurement and Classification of Organic P in Umweltproben

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

high-throughput-measurement-classification-organic-p-environmental

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.6K Aufrufe.  University of Vermont. Dieses Protokoll beschreibt eine High-Throughput-Verfahren zur enzymatischen Hydrolyse, dass ein Mikroplatten-Reader nutzt, um zu messen und zu klassifizieren Boden Phosphor als P Mono-, Di-P und anorganischen P. Bis zu 96 Proben auf einmal in einem Standard-Labor gemessen werden.

Serie: High-Throughput Measurement and Classification of Organic P in Environmental Samples

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Bestimmung mikrobieller extrazelluläre Enzymaktivität in Wasser, Böden und Sedimente mit hohem Durchsatz Mikrotiterplatten-Assays

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Forschung

Umwelt

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer&#39;s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays,&#160;but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

Hochdurchsatz-Fluorometric Messung der Boden Mögliche Extrazelluläre Enzymaktivitäten

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Labor-Phosphor bestimmt Flux von Seesedimenten als Maß der internen Phosphorbelastung

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Extraktion und Analyse von Microbial Phospholipidfettsäuren in Böden

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

Optimierte Verfahren zur Bestimmung der Adsorption von Phosphonaten auf Granular Ferric Hydroxid mit einer miniaturisierten Phosphor Entschlossenheit Methode

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Single-Durchsatz ergänzende hochauflösende Analysetechniken für die Charakterisierung von komplexen natürlichen organischen Substanz Mischungen

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Identifizierung von pro- und Polyfluorinated chemische Spezies mit einem kombinierten gezielte und nicht gezielte Screening hochauflösende Massenspektrometrie-Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

Messung der Phosphorfreisetzung in Labormikrokosmen zur Wasserqualitätsbewertung

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Phosphoproteomic Strategie zur Profilierung osmotischer Stresssignalisierung bei Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Biochemie

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Prüfung für anorganisches Polyphosphat in Bakterien

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...