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. Preparação de pratos, slides e incubação Chambers <ol><li> Prepare o seguinte material para cada microcosmo: um grande plástico Petri fundo prato (d = 10 cm), um modificado (veja o passo 1.2) plástico pequeno Petri fundo prato (d = 5 cm) com tampas e uma contagem de slides de câmara com três cavidades. </li><li> Pegue o número desejado de Petri partes inferiores prato (d = 5 cm). Retire parte da borda com uma serra para caber exatamente um slide (26 mm comprimento da aresta). Para esterilizar o sistema, embeber fundo do prato de Petri e tampas em etanol a 70% O / N e secá-las para, pelo menos, 2 horas numa câmara de fluxo de luz ultravioleta. Armazenar as placas de Petri em um recipiente de plástico hermeticamente fechado, o qual também tinha sido exposta à luz UV durante 2 h. </li><li> Limpar o número desejado de lâminas com etanol a 70% e ar-secá-las. Enrole slides em folha de alumínio e colocá-los em uma mufla durante 5 horas a 450 ° C, para remover eventuais contaminações. </li&gt; <li> Prepare câmaras de incubação de vidro com tampas. Limpe com 70% de etanol e expor câmaras abertas à luz UV por pelo menos 2 horas em uma câmara de fluxo. NOTA: dessecadores grandes (diâmetro de cerca de 30 cm) com tampas podem ser utilizadas para esta finalidade; cada secador pode abrigar até dez configurações microcosmo. </li></ol> 2. Preparação de Mídia e Cultura <ol><li> Para executar 20 microcosmos paralelas, obter 100 ml de meio de triptona de levedura modificada (MTY) 14, cerca de 150 ml de meio mínimo 16 21C (MM), 5 ml de meio mínimo de agar (MMA; 0,5%, w / v), três placas de agar de dextrose de batata (PDA; 1,5%, w / v) para P. ultimum, quatro placas em branco, uma placa de PDA contendo 2 mg L-1 de ciclo-heximida, quatro placas de agarose (3%, w / v) contendo 0,3 g ml -1 de carvão activado e 40 mg de FLU, em porções de 2 mg. </li><li> Prepare MTY contendo 50 mg L-1 de canamicina (canamicina é required para manter o plasmídeo repórter eGFP) e MM contendo 10 mM de acetato de cultivo bacteriano. Use MM sem acetato para preparar 0,5% MMA para a imobilização de bactérias nos microcosmos e despeje placas (a cada 20 ml) de 1,5% PDA para o cultivo de Pythium ultimum e microcosmo setups. </li><li> Inocular algumas hifas de P. ultimum em três placas de PDA fresco e incuba-se no escuro a 25 ° C durante cerca de 72 horas. Em seguida, garantir que as placas são cobertas completamente por micélio fresco e macio. </li><li> Inicie duas culturas de B. sartisoli RP037-MCHE em 50 ml de MTY de um estoque de glicerol. Incubar a 30 ° C com 230 rpm movimento frasco rotativo S / N. </li><li> Cultura Medir a densidade óptica (DO) a 578 nm e centrifugar a 1000 xg durante 10 min (para B. sartisoli RP037-MCHE, o OD 578 esperado é de cerca de 1,0 e as células estão em fase exponencial). Elimine o sobrenadante e re-suspender as célulasem uma quantidade apropriada de MM para atingir uma OD 578 de 0,4. Inocular 20 ml de MM contendo 50 mg L-1 canamicina com 400 ul das células ressuspensas. Incubar a 30 ° C com 230 rpm movimento frasco rotativo S / N. </li><li> Medir OD 578 da cultura em MM (por B. sartisoli RP037-MCHE, o OD 578 esperado é de cerca de 0,2 e as células estão em fase exponencial precoce). Cultura Centrifugar a 2.000 x g durante 10 minutos e re-suspender as células em uma quantidade apropriada de MM para atingir uma DO de 0,4. Misturar 50 ul da suspensão celular com 500 ul de quente, MMA líquido em tubos de 2 ml e armazenar a 50 ° C, até utilização. Use 150 ul de células no MMA para cada microcosmo. </li><li> Verter em placas de gel de agarose que contém carvão activado. Derreter de agarose (3%; w / v) em água duplamente destilada. Misture bem com carvão ativado (0,3 g ml -1) e despeje a mistura em pratos de plástico de Petri(D = 10 cm). </li></ol> 3. Microcosmo Montagem NOTA:. A configuração completa microcosmo está representado esquematicamente na Figura 1 Por favor, consulte a Figura 1 para todas as seguintes etapas individuais. Os passos seguintes são descrevendo a preparação de uma amostra (SAM) com vetores de transporte de micélio e três configurações de controle diferentes (Con Air, CON NEG, CON POS). Um resumo de todos os setups diferentes podem ser encontrados na Tabela 1. <ol><li> Tome número desejado de pratos e slides de Petri e expô-los à luz UV sob uma câmara de fluxo para 30 min. </li><li> Coloque a placa de Petri pequena dentro da grande e encaixar o slide com a abertura da pequena placa de Petri. Pegue uma amostra da suspensão de células quente no MMA e adicionar 150 mL na cavidade meio de um slide. Repita este passo até as cavidades médias de todos os slides sãopreenchido com o MMA. Esperar 5 min para o MMA para solidificar. </li><li> Use uma rolha broca um centímetro para perfurar para fora manchas circulares de uma placa de PDA em branco. Coloque um adesivo de uma distância de 2 mm junto ao MMA dentro da pequena placa de Petri. Adicionar esta para promover o crescimento micelial na configuração (PDA 3 na Figura 1). </li><li> Corte remendos PDA curvas como barreiras mecânicas. Use uma parte do fundo pequena placa de Petri limpo e esterilizado e pressioná-lo em uma placa de PDA. Usar uma espátula para cortar outro anel menor a uma distância de 0,5 cm no agar que resulta em um anel de PDA. <ol><li> Corte um pedaço, que se encaixa exatamente na abertura da pequena placa de Petri na configuração do microcosmo e colocá-lo dentro do fosso na parte superior do slide. Assegure-se que a distância entre o MMA e a barreira PDA circular é de cerca de 2 mm. Feche a tampa da pequena placa de Petri pressionando-o suavemente na barreira PDA. </li><li> Repita os passos 3.4 e 3.4.1 com uma placa PDA contendo 2 g L -1de cicloheximida. NOTA: Esta é a configuração de controle para o transporte a bordo (CON AIR) para as células Bioreporter, desde mycelia são inibidos pela cicloheximida e não crescer demais MMA. </li></ol></li><li> Use uma rolha broca um centímetro para perfurar para fora manchas circulares de uma placa de PDA em branco. Use outro 0,5 centímetros cortiça broca para cortar a metade do primeiro patch. Inserir o PDA-anel pequeno resultante a uma distância de 1 mm junto à barreira PDA (PDA 1 na Figura 1). </li><li> Adicionar 2 mg de FLU para o buraco de PDA 1. Use de 1 cm de cortiça broca para cortar manchas circulares de placas de PDA cobertas de P. ultimum e colocar um adesivo (PDA 2 na Figura 1) para baixo sobre a parte inferior do anel de PDA de modo que o tapete micelar está voltado para os cristais de FLU. <ol><li> Repita o passo anterior, sem adição de cristais gripe. NOTA: Esta é a configuração de controle negativo (CON NEG) para determinar fundofluorescência de células Bioreporter. </li></ol></li><li> Use uma rolha broca um centímetro para perfurar para fora manchas circulares do agar contendo carvão ativado. Coloque quatro sistemas em cada configuração microcosmo para diminuir ainda mais a concentração FLU gasoso (Figura 1). </li><li> Prepare um controlo positivo (CON POS). Adicione alguns cristais gripe para 200 mL de suspensão de células MMA e colocá-lo em uma cavidade de um slide de vazio. </li><li> Coloque uma placa de Petri (d = 10 cm), com um pouco de pó de carvão activado na parte inferior da câmara de incubação para minimizar a concentração de GRIPE gasoso na câmara. Além disso, coloque 4-5 50 ml copos com água estéril no fundo, para manter a umidade alta na câmara. <ol><li> Transferir configurações microcosmos em câmaras de incubação e incubar a 25 ° C durante 96 h. Colocar as amostras aleatoriamente nas diferentes câmaras de crescimento para excluir efeitos de localização. NOTA: Este tempo de incubação aplonas para o P. oomiceto ultimum. Poderia ser diferente para outros organismos. </li></ol></li></ol> 4. Confocal Laser Scanning Microscopy <ol><li> O ideal é usar uma configuração CLSM com microscópio vertical. NOTA: Pode ser equipado com lasers convencionais 488, 561 e 633 linhas nm para excitação ou com uma fonte de laser branco tunable oferta por exemplo,. </li><li> Utilize as seguintes definições para a coleta de imagens: Excitação: 490 nm (eGFP) e 585 nm (mCherry) na intensidade do laser apropriado (excitação simultânea); gamas de emissão: 500-550 nm (eGFP) e 605-650 nm (mCherry); lente objetiva: 63X NA 0,9 immersible água (devido à sua longa distância de trabalho); tamanho do passo para Z-stacks: 0,5 m. </li><li> Abrir configuração microcosmos e usar um bisturi para remover PDA 1, 2, 3 e a barreira PDA. Coloque uma lamela de vidro no topo do MMA e pressione-o suavemente para remover as bolhas de ar. Mount slides no palco microscópio. Adicionar uma gotícula de água na parte superior do poe tampa de deslizamento e usar as configurações mCherry para encontrar células Bioreporter na amostra. </li><li> Obter uma visão geral da amostra e otimizar a relação sinal-ruído, utilizando um brilho-over-under tabela de pesquisa para o vermelho (mCherry) e verde (eGFP) canal. Escolha uma posição aleatória na amostra para analisar Bioreporter fluorescência. NOTA: hifas pode mostrar autofluorescência na gama de emissão Bioreporter (por exemplo, a autofluorescência verde) 17. Certifique-se de selecionar áreas sem hifas se este for o caso. </li><li> Defina e registre-z pilhas para cada posição no verde (eGFP) eo (mCherry) canal vermelho. Evitar o branqueamento da amostra por exposição prolongada à luz ou luz laser de epifluorescência. <ol><li> Repita o passo anterior para dez aleatórios, posições distribuídas uniformemente. </li></ol></li><li> Repita todas as etapas usando as mesmas configurações para todas as amostras da pista de testes (SAM), CON AIR, CON NEG e CON POS. </li></ol&gt; Análise 5. Imagem NOTA: Para analisar vermelho (mCherry) e verde (eGFP) fluorescência na z-stacks gravadas, entre outras opções a ImageJ software livre 18 (http://rsb.info.nih.gov/ij/download.html) pode ser utilizado. <ol><li> Certifique-se de instalar o plugin logi_tool (http://downloads.openmicroscopy.org/bio-formats/4.4.10/). </li><li> Abrir arquivo em ImageJ. Selecione &quot;canais Dividido&quot; e perto do canal verde. Use a macro &#39;área mCherry&#39; fornecido como arquivo suplementar código para quantificação da área de fluorescência vermelha no z-stack. Ajustar as definições preferidas no macro (indicado em negrito). <ol><li> A saída é uma tabela com os pixels medidos acima do limite (e acima do tamanho mínimo definido) na pilha z. NOTA: A soma de todos os valores é o número total de pixels vermelhos (área) na pilha. </li></ol></li><li> Abrir arquivo em ImageJ. Selecione &quot;canais Dividido&quot; e perto canal vermelho. Use a macro<html> 216; eGFP int &#39;fornecido como arquivo de código complementar para quantificar a intensidade de fluorescência verde na pilha z. Ajustar as definições preferidas no macro (indicado em negrito). <ol><li> A saída é uma tabela com as intensidades médias e área de todos os objetos verdes acima do limite (e acima do tamanho mínimo definido) em z-stack. Para calcular a intensidade total de pixels verdes na pilha, multiplique cada intensidade pela área correspondente dizer. </li></ol></li><li> Calcula-se a indução eGFP relativa (eGFP rel): <img alt="Equação 1" src="/files/ftp_upload/52334/52334eq1.jpg" width="300px" /> (1) Observação: Uma vez que é expresso constitutivamente mCherry, o valor resultante de eGFP rel é uma medida relativa para a quantidade de eGFP expressa por uma certa quantidade de células. Por favor, consulte a Tabela 3 para obter mais informações sobre o cálculo do eGFP rel. </li></ol>tle &quot;&gt; 6. Quantificação Chemical NOTA: configurações microcosmos também pode ser usado para realizar a quantificação de quantidades química GRIPE translocados com ou sem um dissipador de contaminante abiótico. <ol><li> Enrole 15 ml frascos de vidro, 1 ml frascos GC / MS e inserções em folha de alumínio. Encha evaporação pratos com Na 2 SO 4; colocar tudo em forno de mufla durante 5 h a 450 ° C para remover possíveis contaminações. Guarde o Na 2 SO 4 dentro de um secador ativado. </li><li> Cortar PDMS fibras de vidro revestidas de 0,5 cm de comprimento. Lave fibras agitando quatro vezes 2 horas em metanol fresco. Repita as etapas de lavagem mais quatro vezes com água ultrapura e armazenar fibras limpas em água ultrapura num frasco de vidro selado. </li><li> Prepare a 1,5 L de tolueno / acetona (3: 1, v / v) contendo 10 mg L -1 fenantreno-10 d. </li><li> Siga as etapas 1, 2 e 3 do protocolo. Prepare microcosmos sem bioucélulas eporter estudar transporte FLU (SAM (-) e CON AIR (-)) e com células Bioreporter (SAM) para verificar a degradação FLU. <ol><li> Opcionais (sem células Bioreporter): Coloque três PDMS revestidas de fibras de vidro (geralmente utilizados para experiências SPME) como pia contaminante artificial e quantificável dentro MMA do SAM (-) usando uma pinça fina. </li><li> Retire as fibras de vidro usando uma pinça fina e transferir cada um em um GC / MS frasco com insert. </li><li> Adicionar 200 mL de tolueno e agitar verticalmente S / N e analisar através de GC / MS. Use as configurações definidas no Quadro 2. </li></ol></li><li> Transferir MMA a partir da cavidade do meio para um frasco de vidro de 15 ml e adicionar cerca de três espátulas de Na 2 SO 4. Misture bem com uma espátula e adicione 10 ml de solvente. Vortex da mistura e agitar horizontalmente O / N no escuro. <ol><li> Transferir 1001; l de amostra em GC / MS frasco com insert. Adicionar 10 ul de acenaftileno-d 08 (10 mg L-1) e analisar através de GC / MS usando o mesmo programa como descrito em 6.4.3. </li></ol></li></ol>

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The authors declare that they have no competing financial interest.

A configuração microcosmo apresentado mostrou adequado para estudar a biodisponibilidade de substâncias químicas separadas espacialmente para degradar organismos após a absorção e transporte por micélio. Transporte em fase gasosa potencial de compostos voláteis parcialmente é impedido e células Bioreporter bacterianas podem ser visualizados sem elaborada a preparação da amostra e, portanto, com o mínimo de perturbação do sistema sensível. Ao mesmo tempo, a análise química da amostra pode ser facilment...

O solo é densamente povoada por uma vasta gama de microorganismos tais como bactérias 1,2. No entanto, as condições deste habitat são desafiadoras, especialmente em termos de disponibilidade de água 3. Bactérias permanentemente precisa procurar condições ótimas em ambientes heterogêneos 4, mas a ausência de filmes contínuos de água está resultando em mobilidade restrita 5 impedindo-os a se espalhar livremente. Além disso, as taxas de difusão dos solutos (por exemplo, nutrientes) são reduzidos sob condições não saturadas 6. Assim, as bactérias e os nutrientes são muitas vezes fisicamente separados e acessibilidade nutriente é limitada 3. Como conseqüência, um vetor de transporte de compostos químicos que não exige uma fase contínua de água pode ajudar a superar essas limitações. De fato, muitos microorganismos como fungos e oomycetes desenvolveram uma forma de crescimento filamentosas que lhes permite crescer através de poros cheios de ar e atingindo assim mobilizing também física nutrientes 7 e 8 carbonáceos substâncias separadas por longas distâncias. Eles podem até atuar como vetores de transporte biológicos que entregam açúcares e outras fontes de energia para as bactérias 9. Absorção e transporte de organismos micélio também foi mostrado para os poluentes orgânicos hidrofóbicos, tais como hidrocarbonetos aromáticos policíclicos (HAP) em Pythium ultimum 10 ou em fungos micorrízicos arbusculares 11. Desde PAH são contaminantes solúveis em água onipresentes e mal 12 no solo, transporte mediado por micélio pode ajudar a aumentar a biodisponibilidade de contaminantes para os potenciais degradadores bacterianas. Considerando que o montante total de transporte de contaminantes pode ser quantificada diretamente por meios químicos 10, biodisponibilidade de contaminantes transportados por micélio de bactérias que degradam e outros organismos não podem ser avaliados facilmente. O protocolo a seguir apresenta um método para avaliar o impacto da mycelia sobre a biodisponibilidade de contaminantes bacterianos para degradadores de forma direta; ele permite que a coleta de informações sobre o impacto espaço-temporal de contaminantes nos ecossistemas microbianos. Nós descrevemos como configurar um sistema de microcosmo insaturada elaborado imitando as interfaces ar-água no solo, ligando uma fonte pontual PAH fisicamente separado com bactérias Bioreporter HAP-degradante através de vectores de transporte de micélio. Como o transporte pelo ar é excluído, o efeito do transporte micelial-baseado em PAH biodisponibilidade para as bactérias podem ser estudados de forma isolada. Em mais detalhe, foram aplicados três anéis HAP fluoreno, o organismo micelial Pythium ultimum e o Bioreporter bactéria Burkholderia sartisoli RP037-MCHE 13 nas configurações descritas microcosmos. A bactéria B. sartisoli RP037-MCHE foi originalmente construído para estudar os fluxos fenantreno para a célula 14 e expressa a proteína verde fluorescente melhorada (eGFP), como resultado do fluxo de PAHa célula, enquanto que a fluorescência vermelha mCherry é expressa constitutivamente. Informações detalhadas sobre a construção repórter é dada pelo Tecon et al. 13 Em testes preliminares, a bactéria não revelaram natação e apenas capacidade swarming muito lento. Era capaz de migrar lentamente em hifas de Pythium ultimum, quando aplicado como uma suspensão densa no topo das hifas. Uma vez que as bactérias foram embebidas em agarose na seguinte protocolo, a migração em hifas não ocorreu. Usando a microscopia confocal por varrimento laser (MCVL), as bactérias Bioreporter pode ser visualizado directamente em microcosmos e expressão de eGFP pode ser quantificada em relação à quantidade de células (proporcional ao sinal de mCherry) com a ajuda do software ImageJ. Isto permite comparar qualitativamente a biodisponibilidade em diferentes cenários (isto é, maior ou menor). FLU foi encontrado para ser biodisponível após o transporte micelial por P. ultimum (isto é,foi maior do que no controlo negativo). Além disso, o protocolo descreve como para quantificar a quantidade total de transporte mediado por micélio através de meios químicos e para verificar a biodisponibilidade de contaminantes usando fibras de vidro revestidas com silicone (fibras de SPME) em microcosmos idênticos. Resultados usando esta configuração microcosmo têm sido publicados e discutidos para a combinação de P. ultimum, fluoreno e B. sartisoli RP037-MCHE 15. Aqui, o foco recai sobre a descrição do método detalhado e a identificação de potenciais armadilhas do protocolo para fornecer esse conhecimento para os potenciais novos pedidos. Outras aplicações podem envolver vários fungos, espécies de bactérias (por exemplo, de locais contaminados), e outros contaminantes (por exemplo, pesticidas) ou contaminante de alimentação (por exemplo, solos envelhecidos).

<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.

Os resultados aqui apresentados já foram publicados anteriormente 15. Por favor, consulte o artigo para discussão mecanicista e ambiental detalhado. Após a gravação de imagem via CLSM, a projeção de intensidade máxima pode ser realizada utilizando o respectivo software microscópio ou ImageJ para ganhar uma primeira impressão visual da amostra e os controles (Figura 2). Mais tarde, os conjuntos de dados podem ser projetadas de forma diferente, a fim de mo...

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

Um ensaio de todo Bioreporter celular com Burkholderia sartisoli RP037-MCHE foi desenvolvido para detectar frações de um contaminante orgânico (ou seja, fluoreno) disponível para a degradação bacteriana após o transporte ativo por micélio ponte poros cheios de ar em um sistema modelo insaturada água.

Uma Abordagem Bioreporter celular todo avalie Transportes e biodisponibilidade de contaminantes orgânicos em água insaturadas Sistemas

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 visualizações.  Helmholtz Centre for Environmental Research - UFZ.  Um ensaio de todo Bioreporter celular com Burkholderia sartisoli RP037-MCHE foi desenvolvido para detectar frações de um contaminante orgânico (ou seja, fluoreno) disponível para a degradação bacteriana após o transporte ativo por micélio ponte poros cheios de ar em um sistema modelo insaturada água. 

Vídeo: 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.

EPA Método 1615. Medição de Enterovirus e Norovirus Ocorrência na água pela cultura e RT-qPCR. I. Recolha de amostras de vírus

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

Ambiente

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.

A Volume Bioassay pequeno para avaliar Bacteriana / fitoplâncton Co-cultura utilizando água-Pulse-Amplitude Modulada (água-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.

EPA Método 1615. Medição de Enterovirus e Norovirus Ocorrência na água por cultura e RT-qPCR. Parte III. Detecção de vírus por 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.

Monólito escavação para Coupled Hidrológicos, geoquímicos e microbiológicos Investigações

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.

Implementação de Emissões de sistemas portáteis de medição (PEMS) para o Regulamento de Emissões-condução Real (RDE) na 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.

Vegetated Sistemas de Tratamento para Remoção de Contaminantes Associados à Toxicidade de Águas Superfícies na Agricultura e Escorrências Urbanas

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.

Medição e mapeamento de padrões de erosão e deposição relacionadas às concentrações de carbonato de solo sob gestão agrícola

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.

Melhorando a espectroscopia de infravermelho de caracterização da matéria orgânica do solo com subtrações espectrais

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.

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

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

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.

Projeto de microtrat e preparação de amostras de plantas e solo para 15análises de nitrogênio

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. ...