Funding by the German Environmental Foundation (DBU) is acknowledged. The authors thank Ute Kuhlicke for technical help with CLSM analysis and Birgit W&#252;rz, Rita Remer, and Jana Reichenbach for skilled experimental help. The authors would particularly like to thank Prof. Jan Roelof van der Meer and Dr. Robin Tecon for fruitful discussion and providing the bioreporter strain. It contributes to the &#8216;Chemicals in the Environment&#8217; (CITE) research program of the Helmholtz Association.

1. Preparazione di piatti, Scivoli e incubazione Chambers <ol><li> Preparare il seguente materiale per ogni microcosmo: un grande plastico Petri fondo piatto (d = 10 cm), uno modificato (vedi punto 1.2) piccolo plastica Petri fondo piatto (d = 5 cm) con coperchi e un conteggio vetrino da camera con tre cavità. </li><li> Prendete il numero desiderato di Petri parti inferiori piatto (d = 5 cm). Rimuovere parte della tesa con una sega per adattarsi esattamente una diapositiva (26 mm di bordo). Per sterilizzare il sistema, immergere Petri fondo piatto e coperchi in etanolo al 70% O / N e asciugare per almeno 2 ore in un armadio di flusso ai raggi UV. Conservare le piastre di Petri in un contenitore di plastica ermeticamente sigillato, che era stato anche esposto a luce UV per 2 h. </li><li> Pulire il numero desiderato di diapositive con il 70% di etanolo e asciugare all&#39;aria. Avvolgere diapositive in carta stagnola e metterli in un forno a muffola per 5 ore a 450 ° C per rimuovere eventuali contaminazioni. </li&gt; <li> Preparare camere di incubazione di vetro con coperchi. Pulire con il 70% di etanolo e esporre le camere aperte a luce UV per almeno 2 ore in un armadio di flusso. NOTA: grandi essiccatori (diametro circa 30 cm) con coperchi può essere utilizzato per questo scopo; ogni essiccatore può ospitare fino a dieci configurazioni microcosmo. </li></ol> 2. Preparazione di mezzi e culture <ol><li> Per eseguire 20 microcosmi paralleli, ottenere 100 ml di modifica medio triptone-lievito (MTY) 14, circa 150 ml di 21C minima media 16 (MM), 5 ml di minima agar (MMA, 0,5%, w / v), tre piastre di agar destrosio di patata (PDA, 1,5%, w / v) per P. ultimum, quattro piastre vuote, una piastra PDA contenente 2 mg L -1 cicloeximide, quattro piastre di agarosio (3%, w / v) contenente 0,3 g ml -1 carbone attivo e 40 mg di influenza in 2 porzioni mg. </li><li> Preparare Mty contenente 50 mg L -1 di kanamicina (kanamicina è required a mantenere il eGFP giornalista plasmide) e MM contenente acetato di 10 mm per la coltivazione batterica. Utilizzare MM, senza acetato per preparare 0,5% MMA per l&#39;immobilizzazione dei batteri nei microcosmi e versare piastre (ogni 20 ml) di 1,5% PDA per la coltivazione di Pythium ultimum e microcosmo configurazioni. </li><li> Seminare alcuni ife di P. ultimum su tre piastre PDA freschi e incubare al buio a 25 ° C per circa 72 ore. Allora sì che le piastre sono ricoperte completamente di micelio fresco e soffice. </li><li> Inizia due culture di B. sartisoli RP037-Mche in 50 ml di MTY da un stock di glicerolo. Incubare a 30 ° C con 230 giri movimento pallone rotante O / N. </li><li> Densità cultura Misura ottica (OD) a 578 nm e centrifugare a 1000 xg per 10 minuti (per B. sartisoli RP037-Mche, l&#39;OD atteso 578 è di circa 1,0 e le cellule sono in fase esponenziale). Gettare il surnatante e risospendere le cellulein una quantità appropriata di MM raggiungere un OD 578 di 0,4. Seminare 20 ml di MM contenenti 50 mg L -1 kanamicina con 400 ml di cellule risospese. Incubare a 30 ° C con 230 giri movimento pallone rotante O / N. </li><li> Misura 578 OD della cultura in MM (per B. sartisoli RP037-Mche, l&#39;OD atteso 578 è di circa 0,2 e le cellule sono in fase iniziale esponenziale). Cultura Centrifugare a 2.000 xg per 10 min e risospendere le cellule in una quantità appropriata di MM a raggiungere un diametro di 0,4. Mescolare 50 ml di sospensione cellulare con 500 ml di caldo, liquido MMA in 2 ml provette e conservare a 50 ° C fino utilizzo. Utilizzare 150 ml di cellule in MMA per ogni microcosmo. </li><li> Versare in piastre di gel di agarosio contenente carbone attivo. Melt agarosio (3%; w / v) in acqua bidistillata. Mescolare accuratamente con carboni attivi (0,3 g ml -1) e versare la miscela in piastre di Petri di plastica(D = 10 cm). </li></ol> 3. Microcosmo di montaggio NOTA:. L&#39;impostazione microcosmo completo è illustrato schematicamente nella figura 1, fare riferimento alla Figura 1 per tutti i successivi singoli passi. Le seguenti operazioni descrivono la preparazione di un campione (SAM) con i vettori di trasporto miceliari e tre differenti configurazioni di controllo (CON AIR, CON NEG, CON POS). Una sintesi di tutte le diverse configurazioni si trovano nella tabella 1. <ol><li> Prendere numero desiderato di piastre di Petri e scivoli ed esporli ai raggi UV sotto un armadietto di flusso per 30 min. </li><li> Posizionare la piccola scatola di Petri all&#39;interno della grande e adattare la diapositiva attraverso la fessura della piccola scatola di Petri. Prendere un campione di sospensione cellulare caldo in MMA e aggiungere 150 ml nella cavità centrale di una diapositiva. Ripetere questa operazione fino a quando le cavità centrali di tutte le diapositive sonopieno di MMA. Attendere 5 min per il MMA solidificare. </li><li> Utilizzare da 1 cm sonda di carotaggio a pugni le patch circolare da una piastra PDA vuoto. Collocare un cerotto ad una distanza di 2 mm accanto MMA all&#39;interno del piccolo piatto Petri. Aggiungere questo per promuovere la crescita del micelio nella configurazione (PDA 3 nella figura 1). </li><li> Tagliare patch curve PDA come barriere meccaniche. Utilizzare una parte inferiore piccola scatola di Petri pulita e sterile e premere in un piatto di PDA. Utilizzare una spatola per tagliare un altro anello più piccolo ad una distanza di 0,5 cm in agar che si traduce in un PDA-ring. <ol><li> Tagliare un pezzo, che si inserisce esattamente nella fessura del piccolo piatto Petri nella configurazione microcosmo e posizionarlo all&#39;interno del gap superiore della diapositiva. Assicurarsi che la distanza tra il MMA e la barriera PDA circolare è circa 2 mm. Chiudere il coperchio della piccola scatola di Petri premendo delicatamente nella barriera PDA. </li><li> Ripetere i punti 3.4 e 3.4.1 con un piatto PDA contenente 2 g L -1di cicloesimide. NOTA: Questa è l&#39;impostazione di controllo per il trasporto per via aerea (CON AIR) verso le cellule marcatura già, poiché miceli sono inibiti dal cicloeximide e non invadere MMA. </li></ol></li><li> Utilizzare da 1 cm sonda di carotaggio a pugni le patch circolari di un piatto PDA vuoto. Utilizzare un altro 0,5 centimetri sonda di carotaggio per tagliare la metà del primo patch. Posizionare la risultante piccola PDA-ring ad una distanza di 1 mm vicino alla barriera PDA (PDA 1 nella Figura 1). </li><li> Aggiungere 2 mg di influenza nel foro di PDA 1. Utilizzare da 1 cm sonda di carotaggio per tagliare le patch circolari da lastre PDA ricoperte di P. ultimum e mettere un cerotto (PDA 2 in Figura 1) inferiore verso il basso sull&#39;anello PDA in modo che il tappetino del micelio sia rivolta verso i cristalli influenza. <ol><li> Ripetere il passaggio precedente, senza l&#39;aggiunta di cristalli di influenza. NOTA: Questa è l&#39;impostazione di controllo negativo (CON NEG) per determinare sfondofluorescenza delle cellule marcatura già. </li></ol></li><li> Utilizzare da 1 cm sonda di carotaggio a pugni le patch circolari dal agar contenente carbone attivo. Mettere quattro patch in ogni configurazione microcosmo di diminuire ulteriormente la concentrazione FLU gassosa (Figura 1). </li><li> Preparare un controllo positivo (CON POS). Aggiungere alcuni cristalli FLU a 200 ml di sospensione cellulare MMA e metterlo in una cavità di una sagoma vuota. </li><li> Inserire un piatto Petri (d = 10 cm) con una certa polvere di carbone attivo sul fondo della camera di incubazione per minimizzare la concentrazione FLU gassosa nella camera. Inoltre, posizionare 04:56 50 ml bicchieri con acqua sterile sul fondo per mantenere alta umidità nella camera. <ol><li> Trasferire configurazioni microcosmo in camere di incubazione e incubare a 25 ° C per 96 ore. I campioni in modo casuale nelle diverse camere di crescita per escludere effetti di localizzazione. NOTA: Questa volta ap incubazioneveli alla oomicete P. ultimum. Potrebbe essere diverso per altri organismi. </li></ol></li></ol> 4. Microscopia confocale a scansione laser <ol><li> Idealmente utilizzare un setup CLSM con il microscopio in posizione verticale. NOTA: Può essere equipaggiato con i laser tradizionali offrendo ad esempio, 488, 561 e 633 linee di nm per l&#39;eccitazione o con una sorgente laser sintonizzabile bianco. </li><li> Utilizzare le seguenti impostazioni per la raccolta di immagini: Eccitazione: 490 nm (eGFP) e 585 nm (mCherry) presso l&#39;intensità del laser appropriata (eccitazione simultanea); gamme di emissione: da 500 a 550 nm (eGFP) e 605-650 nm (mCherry); obiettivo obiettivo: 63X NA 0.9 immersible acqua (a causa della sua lunga distanza di lavoro); passo per z-stack: 0.5 micron. </li><li> Configurazione aperta microcosmo e usare un bisturi per rimuovere PDA 1, 2, 3 e la barriera PDA. Mettere un vetrino di vetro sulla parte superiore del MMA e premerlo delicatamente per rimuovere le bolle d&#39;aria. Mount vetrino sul palco microscopio. Aggiungere una goccia d&#39;acqua sopra thcopertura e di slittamento e utilizzare le impostazioni mCherry per trovare le cellule marcatura già nel campione. </li><li> Prendi una rapida panoramica del campione e ottimizzare rapporto segnale-rumore con un bagliore-over-under tabella di ricerca per il rosso (mCherry) e verde (eGFP) del canale. Scegliete una posizione casuale sul campione da analizzare marcatura già fluorescenza. NOTA: ife può mostrare autofluorescenza nel range di emissione di marcatura già (ad esempio, l&#39;autofluorescenza verde) 17. Assicurarsi di selezionare le aree senza ife se questo è il caso. </li><li> Definire e registrare z-stack per ogni posizione nel verde (eGFP) e la (mCherry) canale rosso. Evitare sbiancamento del campione dalla lunga esposizione alla luce epifluorescenza o luce laser. <ol><li> Ripetere il passaggio precedente per dieci casuali, posizioni uniformemente distribuiti. </li></ol></li><li> Ripetere tutti i passi con le stesse impostazioni per tutti i campioni della pista di prova (SAM), CON AIR, CON NEG e CON POS. </li></ol&gt; Analisi 5. Immagine NOTA: Per analizzare rosso (mCherry) e verde (eGFP) fluorescenza nelle z-stack registrati, tra le altre opzioni del software ImageJ free 18 (http://rsb.info.nih.gov/ij/download.html) può essere utilizzato. <ol><li> Assicurarsi di installare il plugin logi_tool (http://downloads.openmicroscopy.org/bio-formats/4.4.10/). </li><li> Apri il file in ImageJ. Selezionare &quot;canali Split&quot; e vicino canale verde. Utilizzare la macro &#39;zona mCherry&#39; fornito come file di codice aggiuntivo per quantificare area di fluorescenza rossa in z-stack. Regolare le impostazioni preferite nella macro (indicata in grassetto). <ol><li> L&#39;uscita è una tabella con i pixel misurati sopra soglia (e la dimensione minima sopra definita) nello stack z. NOTA: La somma di tutti i valori è il numero totale di pixel rossi (area) nello stack. </li></ol></li><li> Apri il file in ImageJ. Selezionare &quot;canali Split&quot; e vicino canale rosso. Utilizzare la macro<html> 216; eGFP int &#39;fornito come file di codice aggiuntivo per quantificare l&#39;intensità di fluorescenza verde in pila z. Regolare le impostazioni preferite nella macro (indicata in grassetto). <ol><li> L&#39;uscita è un tavolo con intensità media e l&#39;area di tutti gli oggetti verdi sopra soglia (e soprattutto la dimensione minima definita) in z-stack. Per calcolare l&#39;intensità totale di pixel verdi in pila, moltiplicare ciascuna intensità per la superficie corrispondente significa. </li></ol></li><li> Calcolare l&#39;induzione relativo eGFP (eGFP rel): <img alt="Equazione 1" src="/files/ftp_upload/52334/52334eq1.jpg" width="300px" /> (1) NOTA: Poiché mCherry è espresso costitutivamente, il valore risultante di eGFP rel è una misura relativa per la quantità di eGFP espressa da una certa quantità di cellule. Si prega di fare riferimento anche alla Tabella 3 per ulteriori informazioni sul calcolo di eGFP rel. </li></ol>tle &quot;&gt; 6. Quantificazione Chemical NOTA: impostazioni Microcosmo possono anche essere utilizzati per eseguire la quantificazione chimica degli importi INFLUENZA traslocati con o senza un dissipatore contaminante abiotico. <ol><li> Avvolgere 15 fiale ml in vetro, 1 ml GC fiale / MS e inserti in alluminio. Riempire evaporazione piatti con Na 2 SO 4; posizionare tutti in muffola per 5 ore a 450 ° C per rimuovere possibili contaminazioni. Conservare il Na 2 SO 4 all&#39;interno di un essiccatore attivato. </li><li> Tagliare PDMS fibre di vetro rivestito in 0,5 cm di lunghezza. Lavare fibre agitando quattro volte 2 ore in metanolo fresco. Ripetere il lavaggio passaggi altre quattro volte con acqua ultrapura e memorizzare fibre pure in acqua ultrapura in un flaconcino di vetro sigillato. </li><li> Preparare 1,5 L di toluene / acetone (3: 1, v / v) contenente 10 mg L -1 fenantrene-d 10. </li><li> Eseguire i passaggi 1, 2 e 3 del protocollo. Preparare microcosmi senza BIORcellule eporter per studiare il trasporto FLU (SAM (-) e CON AIR (-)) e con le cellule di marcatura già (SAM) per verificare il degrado FLU. <ol><li> Opzionali (senza celle marcatura già): Luogo tre PDMS rivestite in fibra di vetro (di solito utilizzati per esperimenti SPME) come artificiale e quantificabile lavandino contaminante all&#39;interno MMA di SAM (-) utilizzando una pinza sottile. </li><li> Rimuovere fibre di vetro con una pinza sottile e trasferire ognuno in un GC / MS flacone con inserto. </li><li> Aggiungere 200 ml di toluene e scuotere verticalmente O / N e analizzare mediante GC / MS. Utilizzare le impostazioni di cui alla tabella 2. </li></ol></li><li> Trasferire MMA dalla cavità centrale in un flaconcino di vetro da 15 ml e aggiungere circa tre spatole di Na 2 SO 4. Mescolare bene con una spatola e aggiungere 10 ml di solvente. Vortex la miscela e agitare orizzontalmente O / N nel buio. <ol><li> Trasferire 1001; l di campione in GC / MS flacone con inserto. Aggiungere 10 ml di acenaftilene-D 08 (10 mg L -1) e analizzare tramite GC / MS con lo stesso programma, come descritto al punto 6.4.3. </li></ol></li></ol>

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The authors declare that they have no competing financial interest.

La configurazione microcosmo presentato dimostrato adatto per studiare la biodisponibilità di sostanze chimiche spazialmente separate per degradare organismi dopo l&#39;assorbimento e il trasporto da miceli. Potenziale di trasporto del gas in fase di composti volatili parzialmente è impedito e le cellule di marcatura già batteriche può essere visualizzato senza preparazione del campione elaborato e quindi con il minimo disturbo del sistema sensibile. Allo stesso tempo, l&#39;analisi chimica del campione può essere ...

Suolo è densamente popolato da una vasta gamma di microrganismi come batteri 1,2. Tuttavia, le condizioni in questo habitat sono impegnativi, soprattutto in termini di disponibilità di acqua 3. I batteri in modo permanente devono cercare le condizioni ottimali in ambienti eterogenei 4, ma l&#39;assenza di film d&#39;acqua continuo si traduce in mobilità limitata 5 ostacolando loro di diffondersi liberamente. Inoltre, i tassi di diffusione di soluti (ad esempio, nutrienti) sono abbassati in condizioni insaturi 6. Così, batteri e sostanze nutrienti sono spesso separati fisicamente e l&#39;accessibilità dei nutrienti è limitato a 3. Di conseguenza, un vettore di trasporto per i composti chimici che non richiede un acqua-fase continua potrebbe contribuire a superare queste limitazioni. In realtà, molti microrganismi, come funghi e oomiceti hanno sviluppato una forma di crescita filamentosa che permette loro di crescere attraverso pori pieni d&#39;aria e raggiungere in tal modo mobilizing anche fisica nutrienti 7 e 8 sostanze carboniose separato su lunghe distanze. Essi possono anche fungere da vettori di trasporto biologici che forniscono zuccheri e altre fonti di energia ai batteri 9. Assorbimento e trasporto in organismi miceliari è stato anche dimostrato di inquinanti organici idrofobici come gli idrocarburi policiclici aromatici (IPA) in Pythium ultimum 10 o nei funghi micorrizici arbuscolari 11. Dal PAH sono contaminanti solubili in acqua onnipresenti e scarsamente 12 nel suolo, trasporti miceli mediata potrebbe contribuire ad aumentare la biodisponibilità dei contaminanti per i potenziali degraders batteriche. Considerando che l&#39;importo totale del trasporto inquinante può essere quantificato direttamente chimica significa 10, la biodisponibilità dei contaminanti trasportati da miceli a batteri degradanti e altri organismi non può essere valutata facilmente. Il protocollo che segue presenta un metodo per valutare l&#39;impatto di MyCelia sulla biodisponibilità contaminante degraders batteriche in modo diretto; permette la raccolta di informazioni sull&#39;impatto spazio-temporale dei contaminanti sugli ecosistemi microbici. Descriviamo come impostare un elaborato sistema di microcosmo insaturo imitando interfacce aria-acqua nel suolo collegando una sorgente puntiforme PAH separati fisicamente con PAH-degradanti marcatura già batteri tramite vettori di trasporto miceliari. Poiché è escluso il trasporto aereo, l&#39;effetto del trasporto miceliale basata sulla PAH biodisponibilità dei batteri può essere studiata in modo isolato. Più in dettaglio, tre anelli PAH fluorene, l&#39;organismo miceliale Pythium ultimum e la marcatura già batterio Burkholderia sartisoli RP037-Mche 13 sono stati applicati nelle configurazioni microcosmo descritti. Il batterio B. sartisoli RP037-Mche è stato originariamente costruito per studiare flussi fenantrene alla cella 14 ed esprime migliorati proteina fluorescente verde (eGFP) per effetto del flusso di PAHla cella, mentre il rosso fluorescente mCherry è espressa costitutivamente. Informazioni dettagliate sulla costruzione giornalista è data da Tecon et al. 13 Nei test preliminari, il batterio non ha rivelato alcuna nuoto e solo molto lenta capacità sciamatura. Era in grado di migrare lentamente ife di Pythium ultimum quando applicato come una sospensione densa sopra il ife. Poiché i batteri sono stati incorporati in agarosio nella seguente protocollo, migrazione ife non si è verificato. Utilizzando microscopio confocale a scansione laser (CLSM), i batteri marcatura già possono essere visualizzati direttamente in microcosmi ed espressione di eGFP possono essere quantificati in relazione alla quantità di cellule (proporzionale al segnale mCherry) con l&#39;aiuto del ImageJ software. Questo permette di confrontare biodisponibilità qualitativamente in diversi scenari (cioè, superiore o inferiore). FLU è risultato essere biodisponibile dopo trasporto micelio di P. ultimum (cioè,era superiore a quello di un controllo negativo). Inoltre, il protocollo descrive come quantificare l&#39;ammontare totale del trasporto miceli mediato tramite mezzi chimici e verificare la biodisponibilità dei contaminanti con fibre di vetro siliconato (fibre SPME) in microcosmi identici. I risultati con questa configurazione microcosmo sono stati pubblicati e discussi per la combinazione di P. ultimum, fluorene e B. sartisoli RP037-Mche 15. Qui, l&#39;attenzione si concentra sulla descrizione metodo dettagliato e l&#39;individuazione di potenziali insidie ​​del protocollo di fornire questa conoscenza per i potenziali ulteriori applicazioni. Ulteriori applicazioni possono coinvolgere vari fungine, specie batteriche (ad esempio, da siti contaminati), e altri contaminanti (ad esempio, i pesticidi) o contaminante di alimentazione (ad esempio, i terreni di età).

<table border="0" cellpadding="0" cellspacing="0" width="1158">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Reagent/ Equipment</td>
			<td style="width:195px;">Company</td>
			<td style="width:128px;">Catalog Number</td>
			<td style="width:523px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Confocal Microscope</td>
			<td style="width:195px;">Leica</td>
			<td style="width:128px;"></td>
			<td>TCS SP5X, LAS AF - Version 2.6.1; or equivalent CLSM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">GC HP 7890 Series GC and Agilent 5975C MSD</td>
			<td>Agilent</td>
			<td style="width:128px;"></td>
			<td>an equivalent GC/MS may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">GC capillary column J&#38;W 121-5522&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Cork borer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td align="right">12863952</td>
			<td>or any other</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Cover slips</td>
			<td style="width:195px;">Marienfeld</td>
			<td style="width:128px;">107222</td>
			<td>High performance, No.1.5H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">GC/MS insterts</td>
			<td style="width:195px;">WICOM</td>
			<td style="width:128px;">WIC 47080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">GC/MS vials 2 ml</td>
			<td style="width:195px;">WICOM</td>
			<td style="width:128px;">WIC 41150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Lids / septa for screw cap vials</td>
			<td style="width:195px;">DIONEX</td>
			<td>49463 / 049464&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Lids for GC/MS vials</td>
			<td style="width:195px;">WICOM</td>
			<td style="width:128px;">WIC 43948/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Objective Slides</td>
			<td style="width:195px;">Menzel</td>
			<td style="width:128px;"></td>
			<td>ordinary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">PDMS coated glass fibers</td>
			<td style="width:195px;">Polymicro Technologies, Inc.</td>
			<td style="width:128px;"></td>
			<td>V (PDMS) = 13.55 &#177; 0.02 &#181;L m-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Petri Dishes small / big</td>
			<td style="width:195px;">Greiner</td>
			<td style="width:128px;">633-102 / 628-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Screw cap vials 40 ml</td>
			<td style="width:195px;">DIONEX</td>
			<td>48783</td>
			<td>other glass vials may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Screw cap vials 60 ml</td>
			<td style="width:195px;">DIONEX</td>
			<td>48784</td>
			<td>other glass vials may be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">Acenaphthylene d08</td>
			<td style="width:195px;">Dr. Ehrenstorfer</td>
			<td style="width:128px;">C 20510100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Acetone</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">9372.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Activated carbon</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:128px;">242276-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Agarose</td>
			<td style="width:195px;">Carl Roth</td>
			<td align="right">2267.4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Fluorene</td>
			<td style="width:195px;">Fluka</td>
			<td style="width:128px;">46880</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:313px;">Kanamycin sulfate</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">T832.2</td>
			<td>50 mg L-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Methanol</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">P7171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Minimal Medium:</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td>100 mL solution 1 + 25 mL solution 2 + 5 mL solution 3 ad. 1000 mL aqua dest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160; Solution 1</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:313px;">&#160;&#160;&#160; Ammonium sulfate&#160;</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">3746.1</td>
			<td>5 g L-1&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:313px;">&#160;&#160;&#160; Magnesium chloride x 6 H2O</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">2189.1</td>
			<td>1 g L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:313px;">&#160;&#160;&#160; Calcium nitrate x 4 H2O</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">P740.1</td>
			<td>0.5 g L-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160; Solution 2</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:313px;">&#160;&#160;&#160; Disodium phosphate</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">P030.1</td>
			<td>55.83 g L-1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:313px;">&#160;&#160;&#160; Monopotassium phosphate</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">3904.1</td>
			<td>20 g L-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160; Solution 3</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td>pH 6.0</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160; Disodium EDTA</td>
			<td style="width:195px;">MERCK</td>
			<td style="width:128px;">1084180250</td>
			<td>0.8 g L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Iron(II) chloride x 4 H2O</td>
			<td style="width:195px;">MERCK</td>
			<td style="width:128px;">1038610250</td>
			<td>0.3 g L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Cobalt(II) chloride x 6 H2O</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">T889.3</td>
			<td>4 mg L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Manganese(II) chloride x 1 H2O&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">&#160; 4320.2</td>
			<td>10 mg L-1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160; Copper(II) sulfate</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">&#160;P023.1</td>
			<td>1 mg L-1</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">&#160;&#160;&#160; Sodium molybdate x 2 H2O</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">&#160;0274.1</td>
			<td>3 mg L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Zinc chloride</td>
			<td style="width:195px;">MERCK</td>
			<td style="width:128px;">1088160250</td>
			<td>2 mg L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Lithium chloride</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">P007.1</td>
			<td>0.5 mg L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Tin(II) chloride x 2 H2O</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">4473.1</td>
			<td>0.5 mg L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Boric acid</td>
			<td style="width:195px;">Riedel-de-Haen&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:128px;">11606</td>
			<td>1 mg L-1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160; Potassium bromide</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">A137.1</td>
			<td>2 mg L-1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160; Potassium iodide</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">6750.1</td>
			<td>2 mg L-1</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160; Barium chloride</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">4453.1</td>
			<td>0.5 mg L-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">MMA</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td>Minimal medium + agarose 0.2 %</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">Phenanthrene d10</td>
			<td style="width:195px;">Dr. Ehrenstorfer</td>
			<td style="width:128px;">C 20920100</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:313px;">Potato Dextrose Agar:</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td>24 g L-1 broth + bacto-agar 1.5 %; pH 6.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160;&#160;&#160; Potato Dextrose broth</td>
			<td>Difco/ Beckton Dickinson</td>
			<td style="width:128px;">254920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160;&#160;&#160; Bacto-agar</td>
			<td>Difco/ Beckton Dickinson</td>
			<td align="right">214040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium acetate x 3 hydr.</td>
			<td style="width:195px;">Carl Roth</td>
			<td style="width:128px;">6779.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium sulfate</td>
			<td style="width:195px;">MERCK</td>
			<td style="width:128px;">&#160;1066495000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Toluene</td>
			<td style="width:195px;">MERCK</td>
			<td style="width:128px;">1083252500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:313px;">mTY medium:</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td>3 g L-1 yeast extract, 5 g L-1 bacto tryptone and 50 mM NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160;&#160;&#160; Yeast extract</td>
			<td style="width:195px;">Merck</td>
			<td>1037530500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160;&#160;&#160; Tryptone</td>
			<td style="width:195px;">Serva</td>
			<td>4864702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">&#160;&#160;&#160; Sodium chloride</td>
			<td style="width:195px;">Carl Roth</td>
			<td>3957.1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">imageJ with logi tool plugin</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td style="width:523px;">http://rsb.info.nih.gov/ij/download.html and http://downloads.openmicroscopy.org/bio-formats/4.4.10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Pythium ultimum strain 67-1</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td style="width:523px;">Obtained from the lab of Dr. Christoph Keel; Department of Fundamental Microbiology, University of Lausanne, Switzerland</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Burkholderia sartisoli RP037-mChe</td>
			<td style="width:195px;"></td>
			<td style="width:128px;"></td>
			<td style="width:523px;">Obtained from the lab of Prof. Jan Roelof van der Meer; Department of Fundamental Microbiology, University of Lausanne, Switzerland</td>
		</tr>
	</tbody>
</table>

a whole cell bioreporter approach to assess transport and bioavailability of organic contaminants in water unsaturated systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

I risultati presentati qui sono già stati pubblicati in precedenza 15. Consultare l&#39;articolo per la discussione dettagliata meccanicistica e ambientale. Dopo la registrazione dell&#39;immagine mediante CLSM, una sporgenza massima intensità può essere effettuato utilizzando il rispettivo software microscopio o ImageJ per ottenere una prima impressione visiva del campione ei controlli (Figura 2). Più tardi, i set di dati possono essere proiettati in modo div...

Watch this Scientific Journal Video about A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems at JoVE.com

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Un intero test marcatura già cella con Burkholderia sartisoli RP037-Mche è stato sviluppato per rilevare frazioni di un contaminante organico (cioè, fluorene) disponibile per la degradazione batterica dopo trasporto attivo da miceli ponte pori pieni d&#39;aria in un sistema modello insaturo acqua.

A Whole marcatura già cellulare approccio per valutare i trasporti e la biodisponibilità di contaminanti organici in acqua insaturi Sistemi

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

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10.3K Views.  Helmholtz Centre for Environmental Research - UFZ.  Un intero test marcatura già cella con Burkholderia sartisoli RP037-Mche è stato sviluppato per rilevare frazioni di un contaminante organico (cioè, fluorene) disponibile per la degradazione batterica dopo trasporto attivo da miceli ponte pori pieni d'aria in un sistema modello insaturo acqua. 

Video: A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

Metodo EPA 1615. Misura di Enterovirus e Norovirus Evento in acqua per la cultura e RT-qPCR. I. Raccolta di campioni di virus

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

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A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Un piccolo volume Bioassay per valutare batterica / fitoplancton Co-cultura Utilizzando WATER-Pulse-modulata in ampiezza (WATER-PAM) fluorometria

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

Metodo EPA 1615. Misura di Enterovirus e Norovirus Presenza in Water di Cultura e RT-qPCR. Parte III. Rilevamento virus mediante RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Terreno lisimetro scavo per accoppiate idrologici, geochimiche e microbiologiche Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Implementation of Portable Emissions Measurement Systems (PEMS) for the Real-driving Emissions (RDE) Regulation in Europe

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. However, on the road, ambient and driving conditions can vary over a wide range, sometimes causing emissions to be higher than those measured in the laboratory. For this reason, the European Commission has developed a complementary Real-Driving Emissions (RDE) test procedure using the Portable Emissions Measurement Systems (PEMS) to verify gaseous pollutant and particle number emissions during a wide range of normal operating conditions on the road. This paper presents the newly-adopted RDE test procedure, differentiating six steps: 1) vehicle selection, 2) vehicle preparation, 3) trip design, 4) trip execution, 5) trip verification, and 6) calculation of emissions. Of these steps, vehicle preparation and trip execution are described in greater detail. Examples of trip verification and the calculations of emissions are given.

Implementation of Portable Emissions Measurement Systems PEMS for the Real-driving Emissions RDE Regulation in Europe

The European Commission has developed a Real-Driving Emissions (RDE) test procedure to verify pollutant emissions during real-world vehicle operation using the Portable Emissions Measurement Systems (PEMS). This paper presents the experimental procedures required by the newly-adopted RDE test.

Implementazione di sistemi di misura portatili (PEMS) per il regolamento delle emissioni Real-guida (RDE) in Europa

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Sistemi di trattamento vegetale per la rimozione dei contaminanti associati alla tossicità dell&#39;acqua superficiale nell&#39;agricoltura e nella rottura urbana

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Misura e mappatura modelli di erosione e deposizione relazionati al suolo carbonato concentrazioni sotto gestione agricola

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Improving Infrared Spectroscopy Characterization of Soil Organic Matter with Spectral Subtractions

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic bonds that constitute the organic component of soils. However, the relatively low organic matter content of soils (commonly &#60; 5% by mass) and absorbance overlap of mineral and organic functional groups in the mid-infrared (MIR) region (4,000-400 cm-1) engenders substantial interference by dominant mineral absorbances, challenging or even preventing interpretation of spectra for SOM characterization. Spectral subtractions, a post-hoc mathematical treatment of spectra, can reduce mineral interference and enhance resolution of spectral regions corresponding to organic functional groups by mathematically removing mineral absorbances. This requires a mineral-enriched reference spectrum, which can be empirically obtained for a given soil sample by removing SOM. The mineral-enriched reference spectrum is subtracted from the original (untreated) spectrum of the soil sample to produce a spectrum representing SOM absorbances. Common SOM removal methods include high-temperature combustion (&#39;ashing&#39;) and chemical oxidation. Selection of the SOM removal method carries two considerations: (1) the amount of SOM removed, and (2) absorbance artifacts in the mineral reference spectrum and thus the resulting subtraction spectrum. These potential issues can, and should, be identified and quantified in order to avoid fallacious or biased interpretations of spectra for organic functional group composition of SOM. Following SOM removal, the resulting mineral-enriched sample is used to collect a mineral reference spectrum. Several strategies exist to perform subtractions depending on the experimental goals and sample characteristics, most notably the determination of the subtraction factor. The resulting subtraction spectrum requires careful interpretation based on the aforementioned methodology. For many soil and other environmental samples containing substantial mineral components, subtractions have strong potential to improve FTIR spectroscopic characterization of organic matter composition.

SOM underlies many soil functions and processes, but its characterization by FTIR spectroscopy is often challenged by mineral interferences. The described method can increase the utility of SOM analysis by FTIR spectroscopy by subtracting mineral interferences in soil spectra using empirically obtained mineral reference spectra.

Migliorare la caratterizzazione spettroscopia infrarossa di materia organica del suolo con sottrazioni spettrale

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic ...

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.

Singolo-velocità effettiva complementari ad alta risoluzione tecniche analitiche per la caratterizzazione di miscele complesse di sostanza organica naturale

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

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Progettazione di microplot e preparazione di campioni di piante e suolo per 15Analisi dell'azoto

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...