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1. Extração de fósforo <ol><li> Prepare uma solução de 1L de 0,25 M de hidróxido de sódio (NaOH, 40,00 g / mol) + 0,05 M de ácido etilenodiaminotetracético (EDTA, 292,24 g / mol). </li><li> Adicionar 30 mL da solução de NaOH + EDTA a 2 g de solo úmido ou seco / amostra em um tubo cônico de 50 mL. </li><li> Incube com agitação por 16 h à temperatura ambiente. </li><li> Centrifugar a 3000 xg por 3 minutos. </li></ol> 2. Ajuste de pH da amostra Extract <ol><li> Transferência de uma alíquota de 0,1 mL para um tubo de 1,5 mL micro centrífuga. </li><li> Adicionar 0,017 mL de ácido acético 2,5 M, 0,144 mL de 0,4 M tampão acetato (70,5% de acetato de sódio [CH 3 COONa, 82,03 g / mol] e 29,5% de ácido acético, pH 5,0) e 0,739 mL de água deionizada. </li><li> O volume final do extrato ajustado será de 1 mL eo pH final será 5,0. </li></ol> 3. Enzima Preparação Solução stock <ol><li> Prepare 1 L de 0,1 M de acetato de sódio (CH 3 COONa, 82,03 g / mol) solução, pH 5.0. </li><li> Prepare 2 x 10 mL de ácido soluções estoque fosfatase no buffer de acetato de sódio preparada na etapa 3,1 tal que as concentrações finais são cada 0,5 Unidades / mL. Fosfatase ácida Tipo I de Triticum aestivum (gérmen de trigo; GP, geralmente 0,5 mg sólida U-1, EC 3.1.3.2) Fosfatase ácida Tipo IV-S de Solanum tuberosum (batata; PP, geralmente 4,6 mg U-1 sólido, EC 3.1.3.2) * Unidades de actividade são especificados pelo fornecedor, onde uma unidade é definida como a libertação de um micromole de ortofosfato para a solução por hora a 37 ° C. </li><li> Reconstituir liofilizado nuclease P1 (CE 3.1.30.1) de Penicillium citrinum (fungos; NP1, geralmente 500 U mg -1 sólido) em um mL de água deionizada, isso agora pode ser armazenado a 4 ° C para uso futuro. </li><li> Pipeta NP1 em um dos 10 tubos mL preparada no passo 3,2 tais que a concentração final é de 2,5 Unidades / mL NP1; misture suavemente por inversão várias vezes. </li><li> Centrifugar as duas soluções enzimáticas 10 mL 3000 xg por 30 minutos. </li></ol> 4. P curva de calibração e controles <ol><li> Prepare 1 L de 1 mM fosfato de potássio (K2HPO4, 174,18 g / mol) solução estoque. </li><li> Adicionar 1 mL da solução estoque 1 mM fosfato de potássio a um tubo de centrífuga de 1,5 mL e 0,5 mL realizar sete diluições em série. </li><li> Descartar o primeiro tubo para que você tenha sete diluições variando de 20 nmol-0.625 nmol P. </li><li> Transferência de 80 mL de cada tubo em duplicado para linhas B - H de um padrão em placas de 96 poços. </li><li> Adicionar 80 mL de tampão acetato de sódio (ponto 3.1) com a linha A de colunas 11 e 12 este é o ponto de calibração 0 nmol P. </li><li> Cada coluna 11 e 12 agora contém oito amostras de referência de 0 a 20 nmol de fosfato inorgânico nmol. </li><li> Coluna 10 conterá os seguintes controles: <ol><li> Adicionar 40μL PP + solução enzimática GP + 40 mL de tampão de acetato de sódio (ponto 3.1) para os três primeiros poços na coluna 10. </li><li> Adicionar 40 mL PP + GP + solução enzimática NP1 + 40 mL de tampão de acetato de sódio para os próximos três poços. </li><li> Adicionar 40 mL de acetato de sódio contendo 10 glicose-6-fosfato nmol (C 6 H 11 O 9 Na 2 P 2 O &amp;bullet; xH, 304,1 g / mol) + 40 mL PP + solução enzimática para um GP bem e para o bem-finais em solução Coluna 10 acetato de sódio em vez de adicionar solução enzimática. </li></ol></li></ol> 5. Amostra Enzima + Incubação <ol><li> Cada amostra que está sendo testado vai ocupar os primeiros 9 poços em uma linha de uma placa padrão de 96 poços. </li><li> Distribuir 40 mL de extratos com pH ajustado amostra da Seção 2 para poços 09/01 em até 8 linhas. </li><li> Depois de todas as amostras foram distribuídas * use uma pipeta multicanal para distribuir PP + solução enzimática GP às colunas 1-3, PP + GP + NP1 às colunas 4-6 e tampão acetato de sódio (preparado na seção 3.1) para colunas 7-9. * Esta etapa deve ser realizada rapidamente para assegurar todas as amostras de obter tempo de incubação equivalente. </li><li> Cobrir a placa de 96 poços com uma tampa e incubar amostras + soluções enzimáticas, controles e curva de calibração exatamente 1 hr a 37 ° C. </li></ol> 6. Medida colorimétrica de Lançamentos e P inorgânicos Background <ol><li> Prepare 50 mL de cada uma das seguintes soluções em água deionizada: Solução A *: 0,1 M de ácido ascórbico (C6H8O6, 176,12 g / mol) + ácido tricloroacético 0,5 M (Cl3CCOOH, 163,39 g / mol) Solução B: 0,01 M molibdato de amônio ((NH4) 2MoO4, 196,01 g / mol) Solução C: 0,1 M citrato de sódio (HOC (COONa) (CH2COONa) 2 2H2O, 294,10 g / mol) + 0,2 arseniato de sódio M (NaAsO2, 129,91 g / mol) + 5% de ácido acético glacial (CH3CO2H, 60,05 g / mol) * A solução deve ser preparado diariamente. </li><li> Adicionar 25 ml de solução SDS, 100 ml de solução A, 20 mL de solução B e 50 mL de C e solução para todos os poços na pl de 96 poçoscomeu. Fazer isso rapidamente com uma pipeta multicanal. </li><li> Cobrir a placa e incubar 30 minutos à temperatura ambiente. </li><li> Medir a absorvância a 850 nm em qualquer leitor de micro-placa ajustável. </li></ol> 7. Classificação de Compostos P <ol><li> Importação de dados brutos em um aplicativo de planilha (por exemplo, Microsoft Excel). </li><li> Traçar a curva de calibração de fósforo inorgânico: 0-40 nmol P sobre o eixo-x ea média de medições de absorbância duplicada no eixo-y. </li><li> Executar uma regressão linear e encontrar a equação da linha de melhor ajuste. </li><li> Colunas 1-3: Diretamente aplicar a curva de calibração, isto é fundo P. inorgânicos </li><li> Colunas 4-6: Média de valores da amostra triplicado e subtrair controles (solução enzimática + P inorgânico de fundo) do bruto absorbância-P este é derivado de mono-ésteres hidrolisáveis ​​P. </li><li> Colunas 7-9: O mesmo que 7,5, exceto os valores de absorbância subtrair colunas 4-6 - este é P de diésteres hidrolisável P. </li><li> Converter os dados de absorvância em nmol P liberado pela aplicação da equação de calibração. </li><li> nmol P liberado pode ser relacionado para mg P / kg de solo original. Alternativamente, avaliando as proporções de cada classe P também pode ser uma abordagem útil para a análise de dados. </li></ol> 8. Resultados representativos: Uma rápida inspeção visual da placa de 96 poços após a química colorimétrico vai oferecer pistas sobre se ou não o procedimento foi realizado corretamente. A primeira coisa a verificar é o nível de líquido em cada poço, verificando o perfil lado do prato. Deve haver exatamente 275 mL de reagentes em todos os poços. Em seguida, inspecionar visualmente a cor dos poços de amostras duplicadas triplicado e poços de calibração. Estas técnicas devem ser replicar o mesmo tom de azul. Em seguida aplicar a curva de calibração para os dois poços contendo glicose-6-fosfato e verificar que eles lançaram todos os nmol 10 de P. Após inorgânicos P total extraído foi medido utilizando ICP-OES ou um método alternativo, verifique se o P total valores calculados utilizando este protocolo não excedam a quantidade de P que foi extraído. Nota: o gerenciamento de dados cuidadosa na planilha vai ajudar a proteger contra erros quantitativos. Você estará lidando com um monte de números de uma vez, criando assim um modelo será tempo bem gasto. <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="Figura 1" /> Figura 1. A placa de 96 poços mostrando resultados para 8 amostras (linhas AH) e uma curva de calibração (colunas 11 e 12). Controles estão na coluna 10. Aumentar a intensidade de cor entre as colunas 1-3 e 4-6 é devido ao hidrolisado compostos monoester P, entre as colunas 4-6 e 7-9 é devido a compostos de P diéster hidrolisada. <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="Figura 2" /> Figura 2. Distribuição das classes P em NaOH 0,05 M-0,25 M EDTA extrato de uma amostra de solo Vermont usando alto rendimento de hidrólise enzimática. Barras de erro indicam o desvio padrão, 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>

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Por sua própria natureza, um método rápido utilizando pequenos volumes requer muito cuidado. Portanto, os passos mais críticos são aqueles que envolvem soluções de pipetagem para a placa. Precisas e, mais importante, a técnica da pipeta consistentes são essenciais para o sucesso deste ensaio. A extração NaOH-EDTA permitirá que a maior parte do P em amostras de muitos para ser caracterizada em três classes: ortofosfato, P monoéster e solos diéster P., adubos, sedimentos ou qualquer outra amostra do ambiente que contém NaOH-EDTA-extraível P pode ser caracterizada . Formas de P em amostras ambientais não são necessariamente estáveis ​​e esta técnica vai garantir amostras são caracterizadas antes de amostras são comprometidos sem a necessidade de empregar um exército de pesquisadores. Este ensaio é especialmente adequado quando um grande número de amostras a serem processadas. As necessidades de reagentes e requisitos de espaço foram reduzidas a um nível administrável (por exemplo, tubos de 1,5 mL de microcentrífuga em vez de 50 frascos de vidro mL). Esta adaptação também limita a produção de resíduos.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </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> Ácido acético glacial </td><td> Sigma-Aldrich </td><td> 242853 </td></tr><tr><td> Acetato de sódio </td><td> Sigma-Aldrich </td><td> S2889 </td></tr><tr><td> Trigo fosfatase ácida </td><td> Sigma-Aldrich </td><td> P3627 </td></tr><tr><td> Fosfatase ácida de batata </td><td> Sigma-Aldrich </td><td> P1146 </td></tr><tr><td> Nuclease P1 </td><td> Sigma-Aldrich </td><td> N8630 </td></tr><tr><td> Fosfato de potássio </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> Glicose-6-fosfato </td><td> Sigma-Aldrich </td><td> G7250 </td></tr><tr><td> SDS </td><td> Sigma-Aldrich </td><td> L4390 </td></tr><tr><td> Ácido ascórbico </td><td> Sigma-Aldrich </td><td> A5960 </td></tr><tr><td> TCA </td><td> Sigma-Aldrich </td><td> T9159 </td></tr><tr><td> Molibdato de amônio </td><td> Sigma-Aldrich </td><td> A1343 </td></tr><tr><td> Citrato de sódio </td><td> Sigma-Aldrich </td><td> S1804 </td></tr><tr><td> Arseniato de sódio </td><td> Sigma-Aldrich </td><td> S9663 </td></tr></tbody></table>

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

Muitos tipos de compostos orgânicos de fósforo (P) moléculas existem em amostras ambientais 1. P medições tradicionais não detectam estes compostos orgânicos P, uma vez que não reagem com reagentes colorimétricos 2,3. Hidrólise enzimática (EH) é um método emergente para caracterizar com precisão formas de P orgânico em amostras ambientais 4,5. Este método só é superado em precisão por Espectroscopia de fósforo-31 de Ressonância Magnética Nuclear (31 P-NMR), um método que é caro e requer especializados training6 técnica. Nós adaptamos um método de hidrólise enzimática capaz de medir a três classes de fósforo (P monoéster, P diéster e P inorgânico) para um sistema de leitor de microplacas 7. Este método fornece aos pesquisadores um meio rápido, preciso, acessível e de fácil utilização para medir espécies P nos solos, sedimentos, adubos e, se concentrou, amostras aquáticas. Este é o único método de alto rendimento para medir as formas e enzima labilidade de P orgânico que pode ser realizado em um laboratório padrão. Os dados resultantes fornece insights para os cientistas que estudam o conteúdo do sistema de nutrientes e potencial de eutrofização.

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

Este protocolo descreve um método de alto rendimento da hidrólise enzimática que utiliza um leitor de microplacas para medir e classificar o fósforo do solo como P monoésteres, diésteres de P inorgânico e P. Até 96 amostras podem ser medidos de uma só vez em um laboratório padrão.

High-Throughput medição e classificação de P orgânico em amostras ambientais

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

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12.6K visualizações.  University of Vermont. Este protocolo descreve um método de alto rendimento da hidrólise enzimática que utiliza um leitor de microplacas para medir e classificar o fósforo do solo como P monoésteres, diésteres de P inorgânico e P. Até 96 amostras podem ser medidos de uma só vez em um laboratório padrão.

Vídeo: 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.

Determinação da Microbial Extracelular Enzyme actividade nas águas, solos e sedimentos, utilizando alta taxa de transferência de microplaca Ensaios

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

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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.

De alta capacidade Fluorometric Medição do Potencial dos Solos extracelulares atividades enzimáticas

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.

Determinados pelo Laboratório Fósforo Flux do Lago Sedimentos como medida de Fósforo Interno Carregando

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.

Extração e Análise de Microbial Phospholipid Fatty Acids em solos

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.

Procedimento otimizado para determinar a adsorção de fosfonatos para Granular hidróxido férrico, usando um método de determinação de fósforo miniaturizado

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-produção complementares técnicas analíticas de alta resolução para caracterização de misturas complexas de matéria orgânica Natural

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.

Identificação por- e espécies químicas perfluorados com um fluxo de trabalho combinada alvo e não alvo-triagem de alta resolução de espectrometria de massa

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.

Medição da liberação de fósforo em microcosmos laboratoriais para avaliação da qualidade da água

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.

Estratégia fosfoproteômica para traçar sinalização de estresse osmótico em Arabidopsis

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

Bioquímica

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.

Análise para inorgânico polifosfato em bactérias

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 ...