Name: determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays
Uploaded: 2013-10-01T09:00:00
Description: Watch this Scientific Journal Video about Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays at JoVE.com

Midler til aspekter af dette arbejde blev leveret af forskellige kilder, herunder USA Department of Agriculture Specifik samarbejdsaftale 58-6408-1-595 og National Science Foundation (tildeling 1.049.911).

Kolorimetrisk analyse af Extracellular enzymaktivitet i Jordbund og sediment 1.. Forberedelse af underlaget og Buffer Løsninger til Farvemåletekniske Analyser af enzymaktivitet <ol><li> Forbered 50 mM acetatpuffer (pH 5,0-5,5) ved at blande 50 ml 0,1 M eddikesyre (2,87 ml iseddike i 500 ml vand), 150 ml 0,1 M natriumacetat og 200 ml destilleret H2O PH justeres til 5,0-5,5 med 0,1 M eddikesyre, hvis nødvendigt. </li><li> Der fremstilles en opløsning af 1 M natriumhydroxid (NaOH)...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+organic+phosphorus.">Soil organic phosphorus.</a> Advances in agronomy. 29, 83-113 (1977).</li><li>Sinsabaugh, R. L., Moorhead, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resource+allocation+to+extracellular+enzyme+production:+a+model+for+nitrogen+and+phosphorus+control+of+litter+decomposition.">Resource allocation to extracellular enzyme production: a model for nitrogen and phosphorus control of litter decomposition.</a> Soil biology and biochemistry. 26, 1305-1311 (1995).</li><li>Tabatabai, M. A., Bremner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+p-nitrophenyl+phosphate+for+assay+of+soil+phosphatase+activity.">Use of p-nitrophenyl phosphate for assay of soil phosphatase activity.</a> Soil biology and biochemistry. 1, 301-307 (1969).</li><li>Parham, J. A., Deng, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+quantification+and+characterization+of+&#946;-glucosaminidase+activity+in+soil.">Detection, quantification and characterization of &#946;-glucosaminidase activity in soil.</a> Soil biology and biochemistry. 32, 1183-1190 (2000).</li><li>Kuperman, R. G., Carreiro, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+heavy+metal+concentrations,+microbial+biomass+and+enzyme+activities+in+a+contaminated+grassland+ecosystem.">Soil heavy metal concentrations, microbial biomass and enzyme activities in a contaminated grassland ecosystem.</a> Soil biology and biochemistry. 29, 179-190 (1997).</li><li>Olander, L. P., Vitousek, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+soil+phosphatase+and+chitinase+activity+by+N+and+P+availability.">Regulation of soil phosphatase and chitinase activity by N and P availability.</a> Biogeochemistry. 49, 175-190 (2000).</li><li>Jackson, C. R., Vallaire, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+salinity+and+nutrient+enrichment+on+microbial+assemblages+in+Louisiana+wetland+sediments.">Effects of salinity and nutrient enrichment on microbial assemblages in Louisiana wetland sediments.</a> Wetlands. 29, 277-287 (2009).</li><li>Hoppe, H. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+exoenzymatic+activities+in+the+ecology+of+brackish+water:+measurements+by+means+of+methylumbelliferyl-substrates.">Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl-substrates.</a> Marine ecology progress series. 11, 299-308 (1983).</li><li>Somville, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+and+study+of+substrate+specificity+of+exoglucosidase+activity+in+eutrophic+water.">Measurement and study of substrate specificity of exoglucosidase activity in eutrophic water.</a> Applied and environmental microbiology. 48, 1181-1185 (1984).</li><li>Freeman, C., Liska, G., Ostle, N. J., Jones, S. E., Lock, 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=The+use+of+fluorogenic+substrates+for+measuring+enzyme+activity+in+peatlands.">The use of fluorogenic substrates for measuring enzyme activity in peatlands.</a> Plant and soil. 175, 147-152 (1995).</li><li>Sinsabaugh, R. L., Findlay, S., Franchini, P., Fischer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+analysis+of+riverine+bacterioplankton+production.">Enzymatic analysis of riverine bacterioplankton production.</a> Limnology and oceanography. 42, 29-38 (1997).</li><li>Marx, M. -. C., Wood, M., Jarvis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microplate+fluorometric+assay+for+the+study+of+enzyme+diversity+in+soils.">A microplate fluorometric assay for the study of enzyme diversity in soils.</a> Soil biology and biochemistry. 33, 1633-1640 (2001).</li><li>Jackson, C. R., Weeks, A. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+particle+size+on+bacterial+community+structure+in+aquatic+sediments+as+revealed+by+16S+rRNA+gene+sequence+analysis.">Influence of particle size on bacterial community structure in aquatic sediments as revealed by 16S rRNA gene sequence analysis.</a> Applied and environmental microbiology. 74, 5237-5240 (2008).</li><li>Canion, A. K., Ochs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+population+dynamics+of+freshwater+armored+dinoflagellates+in+a+small+lake+in+Mississippi.">The population dynamics of freshwater armored dinoflagellates in a small lake in Mississippi.</a> Journal of freshwater ecology. 20, 617-626 (2005).</li><li>Sinsabaugh, R. L., Lauber, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+soil+enzyme+activity+at+global+scale.">Stoichiometry of soil enzyme activity at global scale.</a> Ecology letters. 11, 1252-1264 (2008).</li><li>Jackson, C. R., Foreman, C. M., Sinsabaugh, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+enzyme+activities+as+indicators+of+organic+matter+processing+rates+in+a+Lake+Erie+coastal+wetland.">Microbial enzyme activities as indicators of organic matter processing rates in a Lake Erie coastal wetland.</a> Freshwater biology. 34, 329-342 (1995).</li><li>Jackson, C. R., Vallaire, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+activity+and+decomposition+of+fine+particulate+organic+matter+in+a+Louisiana+cypress+swamp.">Microbial activity and decomposition of fine particulate organic matter in a Louisiana cypress swamp.</a> Journal of the north american benthological society. 26, 743-753 (2007).</li><li>Jackson, C. R., Liew, K. C., Yule, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+changes+with+depth+in+microbial+communities+in+a+tropical+Malaysian+peat+swamp+forest.">Structural and functional changes with depth in microbial communities in a tropical Malaysian peat swamp forest.</a> Microbial ecology. 57, 402-412 (2009).</li><li>Rietl, A. J., Jackson, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+ecological+restoration+practices+of+prescribed+burning+and+mechanical+thinning+on+soil+microbial+enzyme+activities+and+leaf+litter+decomposition.">Effects of the ecological restoration practices of prescribed burning and mechanical thinning on soil microbial enzyme activities and leaf litter decomposition.</a> Soil biology and biochemistry. 50, 47-57 (2012).</li><li>Smart, K. A., Jackson, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+scale+patterns+in+microbial+extracellular+enzyme+activity+during+leaf+litter+decomposition+in+a+stream+and+its+floodplain.">Fine scale patterns in microbial extracellular enzyme activity during leaf litter decomposition in a stream and its floodplain.</a> Microbial ecology. 58, 591-598 (2009).</li></ol>

Bestemmelse af aktiviteten af en række mikrobielle ekstracellulære enzymer i jord og sedimenter kan give nyttig indsigt i satserne for næringsstof mineralisering og organisk stof behandling 17. Men jordbunden kan variere i deres fugtniveau, så det er vigtigt at standardisere aktivitet i jorden tørvægt. Dette kræver et ekstra tørring trin (typisk af to dage) end blot at måle enzymaktivitet. Således i modsætning til assays af enzymaktivitet i vandprøver, der giver nær øjeblikkelige resultater, på...

Mikroorganismer, såsom bakterier og svampe finde næringsstoffer og kulstof fra komplekse organiske forbindelser gennem produktion af ekstracellulære enzymer. Disse enzymer typisk hydrolysere polymerer i mindre underenheder, der kan tages i cellen. Derfor, ved et økologisk niveau disse mikrobielle ekstracellulære enzymer er ansvarlig for meget af næringsstof mineralisering og nedbrydning af organisk materiale, der forekommer i naturlige miljøer. Enzymer, såsom cellobiohydrolase (CBH), og β-glucosidase er vigtige for cellulose nedbrydning og arbejde i fællesskab at katalysere hydrolyse af cellulose til glucose 1,2, der tilvejebringer en anvendelige carbonsubstrat til mikrobiel optagelse og assimilation. Enzymet fosfatase frigiver opløselige uorganiske fosfat grupper fra organofosfater, hovedsagelig mineraliserende fosfat og gøre den tilgængelig til brug for de fleste organismer 3. Andre enzymer, såsom N-acetylglucosaminidase (NAGase) er important i kitin nedbrydning og kan gøre både kulstof og kvælstof til rådighed for mikrobiel erhvervelse 4.. En af de nærmere vilkår for analysen af mikrobiel ekstracellulær enzymaktivitet i naturlige miljøer er brugen af kunstige p-nitrophenyl (p NP) forbundne substrater, en tilgang, der oprindeligt blev udviklet til at detektere jord fosfataseaktivitet 5.. Denne tilgang er baseret på påvisning af et farvet slutprodukt, p-nitrophenol, som frigives, når den kunstige substrat hydrolyseres af det relevante enzym. P-nitrophenol kan efterfølgende kvantificeres kolorimetrisk ved at måle dets absorbans på omkring 400-410 nm. Denne metode er siden blevet anvendt til påvisning af andre enzymer, såsom NAGase 6, og har været anvendt i forskellige undersøgelser ser på mikrobiel ekstracellulære enzymaktivitet i jord og sedimenter 7-9. En alternativ fremgangsmåde, der var originally udviklet til at vurdere ekstracellulær glucosidaseaktivitet i vandmiljøer 10,11 gør brug af 4-methylumbelliferon (MUB) i tilknytning substrater. Slutproduktet frigivet (4-methylumbelliferon) er meget fluorescerende og kan påvises ved hjælp af et fluorometer med en excitation / emission indstillingen omkring 360/460 nm. En række MUB forbundne kunstige substrater er til rådighed, tillader fluorometrisk måling af aktiviteten af mindst lige så mange enzymer (fx β-glucosidase, cellobiohydrolase, Nagase, phosphatase), som kan analyseres ved hjælp af p NP-substrat kolorimetrisk procedure. Andre mikrobielle ekstracellulære enzymer, såsom protein-nedbrydende leucinaminopeptidase, kan analyseres fluorometrisk under anvendelse af 7-amino-4-methylcoumarin (COU) i tilknytning substrater. Både MUB-og DA-forbundne substrater er blevet anvendt til at bestemme enzymaktivitet i forskellige terrestriske og akvatiske prøver 12,13. Mens tidligere undersøgelser har descrIbed fluorometriske eller kolorimetrisk mikroplade tilgange til at bestemme ekstracellulær enzymaktivitet 14, og der er behov for en klar præsentation af, hvordan til at gennemføre sådanne analyser. Her vi demonstrere procedurer for højt gennemløb mikroplade teknikker til analyse af ekstracellulære enzymaktivitet i jord og sedimenter ved hjælp af kolorimetrisk p NP-bundne substrater tilgang og i naturlige farvande ved hjælp af fluorescerende MUB-bundne substrater teknik. Vi fokuserer på måling af aktiviteterne i β-glucosidase, NAGase, og phosphatase som disse enzymer kan bindes til carbon, nitrogen og fosfor cykling, hhv. Imidlertid kan de her beskrevne procedurer kan anvendes til måling af andre ekstracellulære enzymer ved hjælp af forskellige kunstige substrater.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS AND MATERIALS</td>
		 </tr>
		 <tr>
			<td>Glacial acetic acid</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sodium acetate</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sodium hydroxide</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>p-Nitrophenol</td>
			<td>Fisher</td>
			<td>BP612-1</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>p-Nitrophenyl (pNP)-phosphate</td>
			<td>Sigma</td>
			<td>N3234</td>
			<td>pNP-substrate</td>
		 </tr>
		 <tr>
			<td>pNP-&beta;-glucopyranoside</td>
			<td>Sigma</td>
			<td>N7006</td>
			<td>pNP-substrate</td>
		 </tr>
		 <tr>
			<td>pNP-&beta;-N-acetylglucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>pNP-substrate</td>
		 </tr>
		 <tr>
			<td>Clear 96-well microplates</td>
			<td>Fisher</td>
			<td>12-563-301</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>96-well deep well blocks</td>
			<td>Costar</td>
			<td>3958</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>Aluminum weigh pans</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sterile 15 ml centrifuge tubes</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sterile 50 ml centrifuge tubes</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td></td>
		 </tr>
		 <tr>
			<td>4-Methylumbelliferyl (MUB)-phosphate</td>
			<td>Sigma</td>
			<td>M8883</td>
			<td>MUB-substrate</td>
		 </tr>
		 <tr>
			<td>4-MUB-glucopyranoside</td>
			<td>Sigma</td>
			<td>M3633</td>
			<td>MUB-substrate</td>
		 </tr>
		 <tr>
			<td>4-MUB-N-acetylglucosaminide</td>
			<td>Sigma</td>
			<td>M2133</td>
			<td>MUB-substrate</td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Black 96-well microplate</td>
			<td>Costar</td>
			<td>3792</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipette reservoir</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge rotor</td>
			<td>Eppendorf</td>
			<td>A-4-81</td>
			<td>For microplates/deep-well blocks</td>
		 </tr>
		 <tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>Microplate fluorometer</td>
			<td>BioTek</td>
			<td>FLx 800</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>8-channel pipettor</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		</tbody>
 </table>

determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays

Meget af næringsstofomsætning og kulstof forarbejdning i naturlige miljøer det sker gennem aktiviteten af ​​ekstracellulære enzymer frigivet af mikroorganismer. Således kan måling af aktiviteten af ​​disse ekstracellulære enzymer giver indsigt i satserne for økosystemet niveau processer, såsom organisk stof nedbrydning eller kvælstof og fosfor mineralisering. Analyser af ekstracellulær enzymaktivitet i miljøprøver involverer typisk udsætte prøverne kunstige kolorimetriske eller fluorometriske substrater og sporing hastigheden af ​​substrat hydrolyse. Her beskriver vi mikroplatte baserede metoder for disse procedurer, der tillader analyse af et stort antal prøver inden for en kort tidsramme. Prøverne får lov til at reagere med kunstige substrater i mikroplader med 96 brønde eller dyb brønd mikroplade blokke, og enzymaktiviteten efterfølgende bestemt ved absorption eller fluorescens af det resulterende slutprodukt anvendelse af en typisk mikroplade reader eller fluorometer. Sådanne high throughput procedurer ikke blot lette sammenligninger mellem rumligt forskellige steder eller økosystemer, men også i væsentlig grad reducere omkostningerne ved sådanne analyser ved at mindske de samlede reagens nødvendige per prøve mængder.

Jord og akvatiske sedimenter typisk have mærkbare niveauer af ekstracellulær enzym aktivitet som følge af vedhæftede mikrobielle samfund (biofilm) vokser på overfladen af partikler. Figur 3 viser, hvordan denne aktivitet skifter afhængigt af størrelsen af partikler opnået fra overfladesedimentet en tredje For strøm i det nordlige Mississippi, USA. En tidligere undersøgelse har vist, at de bakterielle samfund på partikler sediment fra denne strøm kan opdeles i tre forskellige grupper baseret ...

Watch this Scientific Journal Video about Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays at JoVE.com

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

Mikroplade baserede procedurer er beskrevet for det kolorimetriske eller fluorometriske analyse af ekstracellulært enzymaktivitet. Disse procedurer giver mulighed for hurtig analyse af en sådan aktivitet i stort antal miljøprøver inden for et overskueligt tidsrum.

Bestemmelse af Mikrobiel Extracellular enzymaktivitet i Vand, Jord og sediment ved hjælp af High Throughput Mikropladekrav Analyser

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

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Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

Udvikling af Sulfidogenic Slam fra marine sedimenter og Trichlorethylen Reduktion i en opadgående strømning Anaerob Slam Blanket Reactor

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High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

High-throughput screening af Modstridigheden Variationer i lignocelluloseholdige biomasse: Total Lignin lignin Monomerer og enzymatisk Sugar Frigivelse

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

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We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

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Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

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The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

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

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Metoder til Soil Resampling at overvåge ændringer i de kemiske Koncentrationer af skovbundens

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...