Biz USGS ve Vermont Su Kaynakları ve Lake Araştırmaları Merkezi finansman sağlamak için teşekkür ederiz.

1. Fosfor Ekstraksiyon <ol><li> 0.25 M sodyum hidroksit (NaOH, 40,00 g / mol) + 0.05 M Ethylenediaminetetraacetic asit (EDTA, 292,24 g / mol) 1L bir çözüm hazırlayın. </li><li> 30 ml, 50 ml konik tüp ıslak veya kuru toprak / örnek 2 g NaOH + EDTA çözeltisi ekleyin. </li><li> Ajitasyon ile oda sıcaklığında 16 saat süreyle inkübe edin. </li><li> 3 dakika boyunca 3000 xg santrifüjleyin. </li></ol> 2. Örnek Özü pH Ayarı <ol><li> 0.1 mL kısım 1.5 ml mikro santrifüj tüpüne aktarın. </li><li> 2,5 M asetik asit 0.017 ml, 0.144 ml 0.4 M asetat tamponu (sodyum asetat% 70.5 [CH 3 COONa, 82,03 g / mol] ve asetik asit% 29.5, pH 5.0), ve 0,739 mL deiyonize su. </li><li> Ayarlanan özü son hacim 1 ml olacak ve nihai pH 5.0 olacak. </li></ol> 3. Enzim Stok Çözüm Hazırlığı <ol><li> 1 L 0,1 M sodyum asetat (CH 3 COONa, 82,03 g / mol) solüsyonu, pH 5.0 hazırlayın. </li><li> Son konsantrasyonları her 0.5 Birimler / mL böyle bir adım 3.1 &#39;de hazırlanan sodyum asetat tamponunda 2 x 10 ml asit fosfataz stok çözelti hazırlayın. Asit fosfataz Triticum aestivum (GP, genel olarak 0.5 U mg-1 katı, EC 3.1.3.2 buğday tohumu) Tip I Asit fosfataz Tip IV-S Solanum tuberosum (patates, PP, genel olarak 4.6 U mg-1 katı, EC 3.1.3.2) * Faaliyet Üniteleri, 1 Adet 37 saatte çözüm ortofosfat 1 micromole kurtuluş olarak tanımlanan tedarikçi ° C ile belirtilir </li><li> Biri mL deiyonize su şimdi bu 4 saklanabilir ° C ileride kullanmak için sulandırın Penicillium citrinum (NP1, genellikle 500 U mg -1 katı mantarlar) nükleaz P1 (EC 3.1.30.1) liyofilize. </li><li> Final konsantrasyonu 2.5 Birimler / mL NP1 böyle bir adım 3.2 &#39;de hazırlanan 10 ml tüpler biri haline Pipet NP1 tersini birkaç kez hafifçe karıştırın. </li><li> 30 dakika boyunca iki adet 10 ml enzim çözümleri 3000 xg santrifüjleyin. </li></ol> 4. P Kalibrasyon Eğrisi ve Kontroller <ol><li> 1 mM potasyum fosfat 1 L (KH2PO4, 174,18 g / mol) stok solüsyonu hazırlayın. </li><li> 1.5 ml santrifüj tüpüne 1 mM potasyum fosfat stok solüsyonu 1 ml ekleyin ve yedi 0,5 mL seri dilüsyonları gerçekleştirmek. </li><li> 20 nmol-0.625 nmol P. arasında değişen 7 dilüsyonları böylece ilk tüp atın </li><li> Her tüp yinelenen bir standart 96-plaka H - Satır B 80 mcL aktarın. </li><li> 80 mcL sodyum asetat tamponu (bölüm 3.1) Bir satır sütun 11 ve 12-0 nmol P kalibrasyon noktası. </li><li> Her sütun 11 ve 12 şu anda 8 referans örnekleri 0 nmol 20 nmol inorganik fosfat içerir. </li><li> Sütun 10 aşağıdaki denetimleri içerecektir: <ol><li> Sütun 10 ilk üç kuyulara 40μL PP + GP enzim çözeltisi + 40 mcL sodyum asetat tamponu (bölüm 3.1) ekleyin. </li><li> 40 mcL PP + GP + NP1 enzim solüsyonu + önümüzdeki üç kuyu için 40 mcL sodyum asetat tamponu ekleyin. </li><li> 10 nmol glukoz-6 fosfat (C 6 H 11 Na 2 O 9 P &amp;bullet; XH 2 O, 304.1 g / mol) + iyi biri ve final de 40 mcL PP + GP enzim çözeltisi içeren 40 mcL sodyum asetat çözeltisi ekle Sütun 10 eklemek enzim çözeltisi yerine sodyum asetat çözeltisi. </li></ol></li></ol> 5. Örnek + Enzim Kuluçka <ol><li> Her numune, test edilen standart bir 96-plaka 1 satır ilk 9 kuyu yer alacak. </li><li> Bölüm 2 kuyu 1-9 pH ayarlı örnek özler 40 mcL dağıtın 8 satır. </li><li> Tüm örnekler dağıtıldıktan sonra * PP + GP enzim sütun çözüm 1-3, PP + GP + NP1, sütunlar 4-6 ve sütun 7-9 sodyum asetat tamponu (Bölüm 3.1 hazırlanan). Dağıtmak için bir çok kanallı pipet kullanın * Bu adım, tüm örneklerde eşdeğer inkübasyon süresi sağlamak için hızlı bir şekilde yapılmalıdır. </li><li> Kapak, 37 ° 96-iyi, bir kapak ve inkübe örnekleri ile levha + enzim çözümleri, kontrol ve kalibrasyon eğrisi tam 1 saat ° C </li></ol> 6. Yayımlanan ve Arka Plan İnorganik P Kolorimetrik Ölçümü <ol><li> 50 mL deiyonize su aşağıdaki çözümleri her hazırlayın: Çözüm A *: 0,1 M askorbik asit (C6H8O6, 176,12 g / mol) + 0,5 M trikloroasetik asit (Cl3CCOOH, 163,39 g / mol) Çözelti B: 0.01 M amonyum molibdat ((NH4) 2MoO4, 196,01 g / mol) Çözüm: C: 0.1 M sodyum sitrat (HOC (COONa) (CH2COONa) 2 2H2O, 294,10 g / mol) + 0.2 M sodyum arsenat (NaAsO2, 129,91 g / mol) +% 5 buzul asetik asit (CH3CO2H 60.05 g / mol) Çözüm bir günde hazır olmalıdır. </li><li> Çözüm SDS 25 mcL 100 mcL Çözüm, Çözüm B ve 50 mcL ve tüm kuyulara Çözüm C 20 mcL 96-iyi plyedik. Bir çok kanallı pipet ile hızlı bir şekilde yapın. </li><li> Plaka Kapak ve oda sıcaklığında 30 dakika inkübe edin. </li><li> Ayarlanabilir herhangi bir mikro-plaka okuyucu 850 nm&#39;de absorbansı ölçülür. </li></ol> 7. P Bileşiklerin Sınıflandırılması <ol><li> Bir elektronik tablo uygulaması (örn. Microsoft Excel) ham veri İthalat. </li><li> X ekseni üzerinde 0 ile 40 nmol P ve y-ekseni üzerinde yinelenen absorbans ölçümleri ortalaması: inorganik fosfor kalibrasyon eğrisi çizilir. </li><li> Lineer regresyon yapın ve en uygun hat denklemi bulmak. </li><li> Sütunlar 1-3: Doğrudan kalibrasyon geçerli eğrisi bu arka plan inorganik P. </li><li> Sütunlar 4-6: Ortalama üç nüsha örnek değerler ve brüt absorbans-P hidrolize P monoesters türetilmiştir denetimleri (enzim çözüm + arka plan inorganik P) dışarı çıkarmak </li><li> - Bu hidrolize P diesters P Sütunlar 7-9: sütun 4-6 çıkarmak absorbans değerleri hariç 7.5 ile aynı. </li><li> Kalibrasyon denklemi uygulanarak tarafından yayımlanan nmol P absorbans verileri dönüştürmek. </li><li> nmol P mg P / kg orijinal toprak geri ilgili olabilir yayınladı. Alternatif olarak, her P sınıfı oranlarda değerlendirilmesi, aynı zamanda veri analizi için yararlı bir yaklaşım olabilir. </li></ol> 8. Temsilcisi Sonuçlar: Kolorimetrik kimya sonra 96-plaka hızlı bir görsel denetim prosedürü doğru olup olmadığı ipuçları sunacak. Kontrol etmeniz gereken ilk şey, sıvı seviyesi, yan profil, plaka tarama her bir kuyu. Tüm kuyularda reaktiflerin tam olarak 275 mcL olmamalıdır. Sonraki üç nüsha örnek kuyuların renk, görsel olarak incelemek ve kalibrasyon kuyu yinelenen. Bu teknik çoğaltmak aynı gölge mavi olmalıdır. Sonraki glukoz-6 fosfat içeren iki kuyu için kalibrasyon eğrisi uygulamak ve doğrulamak toplam P kullanılarak hesaplanan değerler emin olun, ICP-OES ya da alternatif bir yöntem kullanarak ölçülen toplam çıkarılan P sonra inorganik P. 10 nmol yayınladı protokol çıkarılan P miktarı aşmayın. Not: Çizelgede Dikkatli veri yönetimi kantitatif hatalarına karşı korunmaya yardımcı olur. Böylece bir şablon oluşturarak, bir defada bir çok sayı ile ilgili olacak harcanan zaman geçirmiş olacak. <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="Şekil 1" /> Şekil 1. 8 örnekleri (satırlar AH) ve bir kalibrasyon eğrisi (sütunlar 11 ve 12) sonuçları gösteren 96 plaka. Kontroller sütun 10. Sütun 1-3 ve 4-6 arasındaki renk yoğunluğunu arttırmak hidrolize diester P bileşikler nedeniyle, sütun 4-6 ve 7-9 arasında, hidrolize monoester P bileşikleri nedeniyle. <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="Şekil 2" /> Şekil 2, yüksek verimli enzimatik hidroliz kullanarak Vermont toprak örneği 0.25 M NaOH-0.05 M EDTA özü P sınıfları Dağılımı . Hata çubukları standart sapma, n = 3 göstermektedir.

<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>

Doğası gereği, küçük hacimlerde kullanarak hızlı bir yöntem büyük dikkat gerektirir. Bu nedenle en kritik adımlar plaka üzerine pipetleme çözümler içeren bu. Doğru, ve en önemlisi, tutarlı bir pipet tekniği bu testin başarısı için çok önemlidir. NaOH-EDTA çıkarma P Birçok örnekte, en çok üç sınıfa karakterize olmasını sağlayacaktır: ortofosfat, P monoester diester P. Topraklar, gübrenin, sediment veya NaOH-EDTA ekstrakte P karakterize edilebilir içeren herhangi bir diğer çevresel numune . Çevre P formları mutlaka istikrarlı değildir ve örnek bir ordu araştırmacı istihdam gerek kalmadan tehlikeye önce bu tekniği örnekleri karakterize sağlayacaktır. Bu testte, örneklerin çok sayıda işlenecek uygundur. Reaktif ihtiyaçları ve yönetilebilir bir seviyede (örneğin 1,5 mL mikrosantrifüj tüpler yerine 50 ml cam şişeler) alanı gereksinimleri küçülttüm. Bu uyum, aynı zamanda atık üretimi sınırlar.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </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> Asetik sirke asidi </td><td> Sigma-Aldrich </td><td> 242853 </td></tr><tr><td> Sodyum Asetat </td><td> Sigma-Aldrich </td><td> S2889 </td></tr><tr><td> Buğday asit fosfataz </td><td> Sigma-Aldrich </td><td> P3627 </td></tr><tr><td> Patates asit fosfataz </td><td> Sigma-Aldrich </td><td> P1146 </td></tr><tr><td> Nükleazlar P1 </td><td> Sigma-Aldrich </td><td> N8630 </td></tr><tr><td> Potasyum Fosfat </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> Glukoz-6 Fosfat </td><td> Sigma-Aldrich </td><td> G7250 </td></tr><tr><td> SDS </td><td> Sigma-Aldrich </td><td> L4390 </td></tr><tr><td> Askorbik asit </td><td> Sigma-Aldrich </td><td> A5960 </td></tr><tr><td> TCA </td><td> Sigma-Aldrich </td><td> T9159 </td></tr><tr><td> Amonyum molibdat </td><td> Sigma-Aldrich </td><td> A1343 </td></tr><tr><td> Sodyum Sitrat </td><td> Sigma-Aldrich </td><td> S1804 </td></tr><tr><td> Sodyum arsenat </td><td> Sigma-Aldrich </td><td> S9663 </td></tr></tbody></table>

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

Birçok organik fosfor (P) molekülleri türleri, çevre örnekleri 1 mevcuttur . Geleneksel P ölçümleri kolorimetrik reaktifler 2,3 ile reaksiyona girmeyen bu yana, bu organik P bileşikler algılamaz . Enzimatik hidroliz (EH) 4,5 çevre örneklerinde organik P formları doğru olarak tanımlamak için gelişmekte olan bir yöntemdir . Bu yöntem, yalnızca Fosfor-31 Nükleer Manyetik Rezonans Spektroskopisi (31 P-NMR), pahalı ve uzman teknik İÖ6 gerektirir yöntemi doğruluk uydurma . Biz bir mikroplaka okuyucu sistemi 7 fosfor üç sınıf (monoester P diester P ve inorganik P) ölçme yeteneğine sahip bir enzimatik hidroliz yöntemi adapte var. Bu yöntem, toprak, sediment, gübresi ve, konsantre, su örnekleri P türlerini ölçmek için, hızlı, doğru, uygun fiyatlı ve kullanıcı dostu araçları ile araştırmacılar sağlar. Bu standart bir laboratuarda yapılabilir, organik P formları ve enzim-labilite ölçmek için sadece yüksek verimli bir yöntemdir. Ortaya çıkan veriler sistemi besin içeriği ve ötrofikasyon potansiyeli inceleyen bilimciler daha yakından görmemizi sağlar.

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

Bu protokol, ölçmek ve sınıflandırmak P monoesters, P diesters ve inorganik P. olarak toprak fosfor standart bir laboratuvarda tek seferde 96 numune ölçülen olabilir mikroplaka okuyucu kullanır enzimatik hidroliz yüksek verimli bir yöntem açıklanır.

Çevre Örneklerinde Organik P Yüksek Verim Ölçme ve Sınıflandırma

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

Research

JoVE Journal

Biology

12.6K Görüntüleme.  University of Vermont. Bu protokol, ölçmek ve sınıflandırmak P monoesters, P diesters ve inorganik P. olarak toprak fosfor standart bir laboratuvarda tek seferde 96 numune ölçülen olabilir mikroplaka okuyucu kullanır enzimatik hidroliz yüksek verimli bir yöntem açıklanır.

Video: 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.

Yüksek Verimli Mikroplate Tahliller kullanarak Waters, Topraklar ve tortulları Mikrobiyal Ekstrasellüler Enzim Aktivitesi Belirlenmesi

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

Çevre

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.

Potansiyel Toprak Ekstrasellüler Enzim Aktivitelerinin Yüksek verim Florimetrik Ölçme

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.

Göl tortulları Laboratuvar belirlenen Fosfor Flux İç Fosfor Yükleniyor bir tedbir olarak

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.

Ekstraksiyon ve Topraklarda Mikrobiyal Fosfolipid Yağ Asitleri Analizi

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.

Fosfonat adsorpsiyon taneli ferrik minyatürize fosfor belirleme yöntemini kullanarak hidroksit üzerine belirlemek için en iyi duruma getirilmiş yordamı

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.

Karmaşık doğal organik madde karışımları karakterize için tek işlem hacmi tamamlayıcı yüksek çözünürlüklü analitik teknikler

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.

Ücret belirlenmesi- ve Polyfluorinated kimyasal türler ile kombine hedef ve hedef tarama olmayan yüksek çözünürlüklü kütle spektrometresi iş akışı

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.

Su kalite değerlendirmesi için laboratuar Microcosms fosfor serbest ölçme

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.

Arabidopsis'te Osmotik Stres Sinyali Profili Çıkarmak için Fosfoproteomik Strateji

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

Biyokimya

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.

Bakterilerde inorganik polifosfat için raporlaması

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 ...