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. Preparation of Dishes, Slides and Incubation Chambers
<ol>
	<li>Prepare the following material for each microcosm: one big plastic Petri dish bottom (d&#160;=&#160;10&#160;cm), one modified (see step 1.2) small plastic Petri dish bottom (d&#160;=&#160;5&#160;cm) with lids and one counting chamber slide with three cavities.</li>
	<li>Take the desired number of Petri dish bottom parts (d&#160;=&#160;5&#160;cm). Remove part of the brim with a saw to exactly fit a slide (26&#160;mm edge length). To sterilize the system, soak Petri dish bottoms and lids in 70% ethanol O/N and dry them for at least 2&#160;hr in a flow cabinet under UV-light. Store the Petri dishes in a tightly sealed plastic container, which had also been exposed to UV-light for 2&#160;h.</li>
	<li>Wipe the desired number of slides with 70% ethanol and air-dry them. Wrap slides in aluminum foil and put them in a muffle furnace for 5&#160;hr at 450&#160;&#176;C to remove possible contaminations.</li>
	<li>Prepare glass incubation chambers with lids. Clean with 70% ethanol and expose open chambers to UV-light for at least 2&#160;hr in a flow cabinet. 
	NOTE: Large desiccators (diameter&#160;around 30&#160;cm) with lids may be used for this purpose; each desiccator may house up to ten microcosm setups.</li>
</ol>
2. Preparation of Media and Cultures
<ol>
	<li>To run 20 parallel microcosms, obtain 100&#160;ml of modified tryptone-yeast medium (mTY)14, about 150&#160;ml of 21C minimal medium16 (MM), 5&#160;ml of minimal medium agar (MMA; 0.5%, w/v), three potato dextrose agar plates (PDA; 1.5%, w/v) for P.&#160;ultimum, four blank plates, one PDA plate containing 2 mg&#160;L-1 cycloheximide, four agarose plates (3%, w/v) containing 0.3&#160;g ml-1 activated carbon and 40&#160;mg of FLU in 2&#160;mg portions.</li>
	<li>Prepare mTY containing 50&#160;mg L-1 of kanamycin (kanamycin is required to maintain the eGFP reporter plasmid) and MM containing 10&#160;mM acetate for bacterial cultivation. Use MM without acetate to prepare 0.5% MMA for the immobilization of bacteria in the microcosms and pour plates (each 20&#160;ml) of 1.5% PDA for cultivation of Pythium ultimum and microcosm setups.</li>
	<li>Inoculate some hyphae of P. ultimum on three fresh PDA plates and incubate in the dark at 25&#160;&#176;C for about 72&#160;hr. Then ensure that the plates are overgrown completely by fresh and fluffy mycelium.</li>
	<li>Start two cultures of B.&#160;sartisoli RP037-mChe in 50&#160;ml of mTY from a glycerol stock. Incubate at 30&#160;&#176;C with 230&#160;rpm rotary flask movement O/N.</li>
	<li>Measure culture optical density (OD) at 578&#160;nm and centrifuge at 1,000 x g for 10&#160;min (for B. sartisoli RP037-mChe, the expected OD578 is about 1.0 and cells are in exponential phase). Discard supernatant and re-suspend the cells in an appropriate amount of MM to reach an OD578 of 0.4. Inoculate 20&#160;ml of MM containing 50&#160;mg L-1 kanamycin with 400&#160;&#181;l of the resuspended cells. Incubate at 30&#160;&#176;C with 230&#160;rpm rotary flask movement O/N.</li>
	<li>Measure OD578 of the culture in MM (for B. sartisoli RP037-mChe, the expected OD578 is about 0.2 and cells are in early exponential phase). Centrifuge culture at 2,000 x g for 10 min and re-suspend the cells in an appropriate amount of MM to reach an OD of 0.4. Mix 50&#160;&#181;l of the cell suspension with 500&#160;&#181;l of warm, liquid MMA in 2&#160;ml tubes and store at 50&#160;&#176;C until usage. Use 150&#160;&#181;l of cells in MMA for each microcosm.</li>
	<li>Pour plates of agarose gel containing activated carbon. Melt agarose (3%; w/v) in double distilled water. Mix thoroughly with activated carbon (0.3&#160;g&#160;ml-1) and pour mixture into plastic Petri dishes (d&#160;=&#160;10&#160;cm).</li>
</ol>
3. Microcosm Mounting
NOTE: The complete microcosm setup is depicted schematically in Figure 1. Please refer to Figure 1 for all following individual steps. The following steps are describing the preparation of a sample (SAM) with mycelial transport vectors and three different control setups (CONAIR, CONNEG, CONPOS). A summary of all different setups can be found in Table 1.
<ol>
	<li>Take desired number of Petri dishes and slides and expose them to UV light under a flow cabinet for 30&#160;min.</li>
	<li>Place the small Petri dish inside the big one and fit the slide through the gap of the small Petri dish. Take one sample of the warm cell suspension in MMA and add 150&#160;&#181;l into the middle cavity of one slide. Repeat this step until the middle cavities of all slides are filled with MMA. Wait 5&#160;min for the MMA to solidify.</li>
	<li>Use a 1&#160;cm cork borer to punch out circular patches from a blank PDA plate. Place one patch at a distance of 2&#160;mm next to the MMA inside the small Petri dish. Add this to promote mycelial growth in the setup (PDA 3 in Figure 1).</li>
	<li>Cut curved PDA patches as mechanical barriers. Use a clean and sterile small Petri dish bottom part and press it into a PDA plate. Use a spatula to cut another smaller ring at a distance of 0.5&#160;cm into the agar which results in a PDA-ring.
	<ol>
		<li>Cut out a piece, which exactly fits into the gap of the small Petri dish in the microcosm setup and place it inside the gap on top of the slide. Ensure that the distance between the MMA and the circular PDA barrier is about 2&#160;mm. Close the lid of the small Petri dish gently pressing it into the PDA barrier.</li>
		<li>Repeat steps 3.4 and 3.4.1 with a PDA plate containing 2&#160;g&#160;L-1 of cycloheximide. 
		NOTE: This is the control setup for airborne transport (CONAIR) towards the bioreporter cells, since mycelia are inhibited by cycloheximide and do not overgrow MMA.</li>
	</ol>
	</li>
	<li>Use a 1&#160;cm cork borer to punch out circular patches of a blank PDA plate. Use another 0.5&#160;cm cork borer to cut out the middle of the first patch. Place the resulting small PDA-ring at a distance of 1&#160;mm next to the PDA barrier (PDA 1 in Figure 1).</li>
	<li>Add 2&#160;mg of FLU into the hole of PDA 1. Use a 1&#160;cm cork borer to cut out circular patches from PDA plates overgrown with P. ultimum and place one patch (PDA 2 in Figure 1) bottom down onto the PDA ring so that the mycelial mat is facing the FLU crystals.
	<ol>
		<li>Repeat the previous step without adding FLU crystals. 
		NOTE: This is the negative control setup (CONNEG) to determine background fluorescence of bioreporter cells.</li>
	</ol>
	</li>
	<li>Use a 1&#160;cm cork borer to punch out circular patches from the agar containing activated carbon. Place four patches in each microcosm setup to further decrease the gaseous FLU concentration (Figure 1).</li>
	<li>Prepare a positive control (CONPOS). Add some FLU crystals to 200&#160;&#181;l of MMA cell suspension and put it in one cavity of an empty slide.</li>
	<li>Place a Petri dish (d&#160;=&#160;10&#160;cm) with some powder of activated carbon on the bottom of the incubation chamber to minimize gaseous FLU concentration in the chamber. Also, place four to five 50 ml beakers with sterile water on the bottom to maintain high humidity in the chamber.
	<ol>
		<li>Transfer microcosm setups into incubation chambers and incubate at 25&#160;&#176;C for 96&#160;hr. Place samples randomly in the different growth chambers to exclude location effects. 
		NOTE: This incubation time applies to the oomycete P. ultimum. It might be different for other organisms.</li>
	</ol>
	</li>
</ol>
4. Confocal Laser Scanning Microscopy
<ol>
	<li>Ideally use a CLSM setup with upright microscope. 
	NOTE: It may be equipped with conventional lasers offering e.g., 488, 561 and 633 nm lines for excitation or with a tunable white laser source.</li>
	<li>Use the following settings for collection of images: Excitation: 490 nm (eGFP) and 585 nm (mCherry) at the appropriate laser intensity (simultaneous excitation); emission ranges: 500 to 550 nm (eGFP) and 605 to 650 nm (mCherry); objective lens: 63X NA 0.9 water immersible (due to its long working distance); step size for z-stacks: 0.5&#160;&#181;m.</li>
	<li>Open microcosm setup and use a scalpel to remove PDA 1, 2, 3 and the PDA barrier. Put a glass cover slip on top of the MMA and press it gently to remove air bubbles. Mount slide on microscope stage. Add a droplet of water on top of the cover slip and use mCherry settings to find bioreporter cells in the sample.</li>
	<li>Get a quick overview of the sample and optimize signal to noise ratio using a glow-over-under lookup table for red (mCherry) and green (eGFP) channel. Choose a random position on the sample to analyze bioreporter fluorescence. 
	NOTE: hyphae may show autofluorescence in the range of bioreporter emission (e.g.,&#160;green autofluorescence)17. Be sure to select areas without hyphae if this is the case.</li>
	<li>Define and record z-stacks for each position in the green (eGFP) and the red (mCherry) channel. Avoid bleaching of the sample by long exposure to epifluorescence light or laser light.
	<ol>
		<li>Repeat previous step for ten random, evenly distributed positions.</li>
	</ol>
	</li>
	<li>Repeat all steps using the same settings for all samples of the test track (SAM), CONAIR , CONNEG and CONPOS.</li>
</ol>
5. Image Analysis
NOTE: To analyze red (mCherry) and green (eGFP) fluorescence in the z-stacks recorded, amongst other options the free software ImageJ18 (http://rsb.info.nih.gov/ij/download.html) can be used.
<ol>
	<li>Make sure to install the logi_tool plugin (http://downloads.openmicroscopy.org/bio-formats/4.4.10/).</li>
	<li>Open file in ImageJ. Select &#8220;split channels&#8221; and close green channel. Use the macro &#8216;mCherry area&#8217; provided as supplementary code file to quantify area of red fluorescence in z-stack. Adjust preferred settings in the macro (indicated in bold).
	<ol>
		<li>Output is a table with the measured pixels above threshold (and above the defined minimum size) in z- stack. 
		NOTE: The sum of all values is the total number of red pixels (area) in the stack.</li>
	</ol>
	</li>
	<li>Open file in ImageJ. Select &#8220;split channels&#8221; and close red channel. Use the macro &#8216;eGFP int&#8217; provided as supplementary code file to quantify intensity of green fluorescence in z- stack. Adjust preferred settings in the macro (indicated in bold).
	<ol>
		<li>Output is a table with the mean intensities and area of all green objects above threshold (and above the defined minimum size) in z-stack. To calculate the total intensity of green pixels in the stack, multiply each mean intensity by the corresponding area.</li>
	</ol>
	</li>
	<li>Calculate the relative eGFP induction (eGFPrel): 
	<img alt="Equation 1" src="/files/ftp_upload/52334/52334eq1.jpg" width="300px" /> (1) 
	NOTE: Since mCherry is expressed constitutively, the resultant value of eGFPrel is a relative measure for the amount of eGFP expressed by a certain amount of cells. Please also refer to Table 3 for further information on calculation of eGFPrel.</li>
</ol>
6. Chemical Quantification
NOTE: Microcosm setups may also be used to perform chemical quantification of translocated FLU amounts with or without an abiotic contaminant sink.
<ol>
	<li>Wrap 15&#160;ml glass vials, 1&#160;ml GC/MS vials and inserts in aluminum foil. Fill evaporating dishes with Na2SO4; place all in muffle furnace for 5&#160;hr at 450&#160;&#176;C to remove possible contaminations. Store the Na2SO4 inside of an activated desiccator.</li>
	<li>Cut PDMS coated glass fibers into 0.5&#160;cm length. Wash fibers by shaking four times 2&#160;hr in fresh methanol. Repeat washing steps another four times with ultrapure water and store clean fibers in ultrapure water in a sealed glass vial.</li>
	<li>Prepare 1.5&#160;L of toluene/acetone (3:1, v/v) containing 10&#160;&#181;g L-1 phenanthrene-d10.</li>
	<li>Perform steps 1, 2 and 3 of the protocol. Prepare microcosms without bioreporter cells to study FLU transport (SAM(-) and CONAIR(-)) and with bioreporter cells (SAM) to verify FLU degradation.
	<ol>
		<li>Optional (without bioreporter cells): Place three PDMS coated glass fibers (usually used for SPME experiments) as artificial and quantifiable contaminant sink inside MMA of SAM(-) using fine forceps.</li>
		<li>Remove glass fibers using fine forceps and transfer each one into a GC/MS vial with insert.</li>
		<li>Add 200&#160;&#181;l of toluene and shake vertically O/N and analyze via GC/MS. Use the settings provided in Table 2.</li>
	</ol>
	</li>
	<li>Transfer MMA from the middle cavity into a 15&#160;ml glass vial and add about three spatulas of Na2SO4. Mix thoroughly using a spatula and add 10 ml of solvent. Vortex the mixture and shake horizontally O/N in the dark.
	<ol>
		<li>Transfer 100&#160;&#181;l of sample into GC/MS vial with insert. Add 10&#160;&#181;l of acenaphthylene-d08 (10&#160;mg L-1) and analyze via GC/MS using the same program as described in 6.4.3.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interest.

The presented microcosm setup proved suitable to study bioavailability of spatially separated chemicals to degrading organisms after uptake and transport by mycelia. Potential gas-phase transport of partially volatile compounds is prevented and bacterial bioreporter cells can be visualized without elaborate sample preparation and thus with minimal disturbance of the sensitive system. At the same time, chemical analysis of the sample can be easily conducted allowing for a good control of the gained results and for quantif...

Soil is densely populated by a wide range of microorganisms1,2 such as bacteria. However, conditions in this habitat are challenging, especially in terms of water availability3. Bacteria permanently need to search for optimal conditions in heterogeneous environments4, but the absence of continuous water films is resulting in restricted mobility5 hindering them to spread freely. Also, diffusion rates of solutes (e.g., nutrients) are lowered under unsaturated conditions6. Thus, bacteria and nutrients are often physically separated and nutrient accessibility is limited3. As a consequence, a transport vector for chemical compounds which does not require a continuous water-phase could help to overcome these limitations. In fact, many microorganisms such as fungi and oomycetes have developed a filamentous growth form enabling them to grow through air-filled pore spaces thereby reaching and mobilizing also physical separated nutrients7 and carbonaceous8 substances over long distances. They may even act as biological transport vectors which deliver sugars and other energy sources to bacteria9. Uptake and transport in mycelial organisms has also been shown for hydrophobic organic pollutants such as polycyclic aromatic hydrocarbons (PAH) in Pythium ultimum10 or in arbuscular mycorrhizal fungi11. Since PAH are ubiquitous and poorly water soluble contaminants12 in soil, mycelia-mediated transport might help to increase contaminant bioavailability for potential bacterial degraders. Whereas the total amount of contaminant transport can be quantified directly by chemical means10, bioavailability of contaminants transported by mycelia to degrading bacteria and other organisms cannot be assessed easily.
The following protocol presents a method to evaluate the impact of mycelia on contaminant bioavailability to bacterial degraders in a direct manner; it allows gathering information about the spatiotemporal impact of contaminants on microbial ecosystems. We describe how to set up an elaborate unsaturated microcosm system mimicking air-water interfaces in soil by linking a physically separated PAH point source with PAH-degrading bioreporter bacteria via mycelial transport vectors. Because airborne transport is excluded, the effect of mycelial-based transport on PAH bioavailability for bacteria can be studied in an isolated way. In more detail, three-ring PAH fluorene, the mycelial organism Pythium ultimum and the bioreporter bacterium Burkholderia sartisoli RP037-mChe13 were applied in the described microcosm setups. The bacterium B. sartisoli RP037-mChe was originally constructed to study phenanthrene fluxes to the cell14 and expresses enhanced green fluorescent protein (eGFP) as a result of the PAH flux to the cell, whereas the red fluorescing mCherry is expressed constitutively. Detailed information on the reporter construction is given by Tecon et al.13 In preliminary tests, the bacterium revealed no swimming and only very slow swarming ability. It was able to migrate slowly on hyphae of Pythium ultimum when applied as a dense suspension on top of the hyphae. Since bacteria were embedded in agarose in the following protocol, migration on hyphae did not occur.
Using confocal laser scanning microscopy (CLSM), the bioreporter bacteria can be visualized directly in the microcosms and expression of eGFP can be quantified in relation to the amount of cells (proportional to the mCherry signal) with the help of the software ImageJ. This allows comparing bioavailability qualitatively in different scenarios (i.e., higher or lower). FLU was found to be bioavailable after mycelial transport by P. ultimum (i.e., it was higher than in a negative control). Furthermore, the protocol describes how to quantify the total amount of mycelia-mediated transport via chemical means and to verify contaminant bioavailability using silicon-coated glass fibers (SPME fibers) in identical microcosms. Results using this microcosm setup have been published and discussed for the combination of P. ultimum, fluorene and B. sartisoli RP037-mChe15. Here, the focus lies on a detailed method description and the identification of potential pitfalls of the protocol to provide this knowledge for potential further applications. Further applications may involve various fungal, bacterial species (e.g., from contaminated sites), and other contaminants (e.g., pesticides) or contaminant-supply (e.g., aged soils).

<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.

The results presented here have already been published earlier15. Please refer to the article for detailed mechanistic and environmental discussion.
After image recording via CLSM, a maximum intensity projection can be conducted using the respective microscope software or ImageJ to gain a first visual impression of the sample and the controls (Figure 2). Later, the data sets may be projected differently in order to show meaningful features by specific visualization ...

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

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

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

a-whole-cell-bioreporter-approach-to-assess-transport-bioavailability

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10.3K Views.  Helmholtz Centre for Environmental Research - UFZ. A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

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.

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

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.

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 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.

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.

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.

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.

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.

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.

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.

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. ...