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>

<ol>
	<li>Holden, P. A., Fierer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+processes+in+the+vadose+zone.">Microbial processes in the vadose zone.</a> Vadose Zone Journal. 4, 1-21 (2005).</li><li>Whitman, W. B., Coleman, D. C., Wiebe, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotes:+The+unseen+majority.">Prokaryotes: The unseen majority.</a> Proceedings of the National Academy of Sciences. 95, 6578-6583 (1998).</li><li>Kieft, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+abundance+and+activities+in+relation+to+water+potential+in+the+vadose+zones+of+arid+and+semiarid+sites.">Microbial abundance and activities in relation to water potential in the vadose zones of arid and semiarid sites.</a> Microbial ecology. 26, 59-78 (1993).</li><li>Wang, G., Or, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+films+limit+bacterial+cell+motility+and+colony+expansion+on+partially+saturated+rough+surfaces.">Aqueous films limit bacterial cell motility and colony expansion on partially saturated rough surfaces.</a> Environ. Microbiol. 12, 1363-1373 (2010).</li><li>Griffin, D. M., Parr, J. F., Gardner, W. R., Elliott, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Water Potential Relations in Soil Microbiology SSSA Special Publication. , 141-151 (1981).</li><li>Papendick, R. I., Camprell, G. S., Parr, J. F., Gardner, W. R., Elliott, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Water Potential Relations in Soil Microbiology SSSA Special Publication. , 1-22 (1981).</li><li>Boswell, G. P., Jacobs, H., Davidson, F. A., Gadd, G. M., Ritz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+consequences+of+nutrient+translocation+in+mycelial+fungi.">Functional consequences of nutrient translocation in mycelial fungi.</a> J. Theor. Biol. 217, 459-477 (2002).</li><li>Jennings, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+solutes+in+fungi.">Translocation of solutes in fungi.</a> Biol. Rev. Camb. Philos. Soc. 62, 215-243 (1987).</li><li>Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+root+exudates+in+rhizosphere+interactions+with+plants+and+other+organisms.">The role of root exudates in rhizosphere interactions with plants and other organisms.</a> Annu. Rev. Plant Biol. 57, 233-266 (2006).</li><li>Furuno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycelia+promote+active+transport+and+spatial+dispersion+of+polycyclic+aromatic+hydrocarbons.">Mycelia promote active transport and spatial dispersion of polycyclic aromatic hydrocarbons.</a> Environ. Sci. Technol. 46, 5463-5470 (2012).</li><li>Gao, Y., Cheng, Z., Ling, W., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbuscular+mycorrhizal+fungal+hyphae+contribute+to+the+uptake+of+polycyclic+aromatic+hydrocarbons+by+plant+roots.">Arbuscular mycorrhizal fungal hyphae contribute to the uptake of polycyclic aromatic hydrocarbons by plant roots.</a> Bioresour. Technol. 101, 6895-6901 (2010).</li><li>Semple, K. T., Morriss, A. W. J., Paton, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioavailability+of+hydrophobic+organic+contaminants+in+soils:+fundamental+concepts+and+techniques+for+analysis.">Bioavailability of hydrophobic organic contaminants in soils: fundamental concepts and techniques for analysis.</a> Eur. J. Soil. Sci. 54, 809-818 (2003).</li><li>Tecon, R., Binggeli, O., vander Meer, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-tagged+fluorescent+bacterial+bioreporter+for+the+study+of+polycyclic+aromatic+hydrocarbon+diffusion+and+bioavailability.">Double-tagged fluorescent bacterial bioreporter for the study of polycyclic aromatic hydrocarbon diffusion and bioavailability.</a> Environ. Microbiol. 11, 2271-2283 (2009).</li><li>Tecon, R., Wells, M., vander Meer, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+green+fluorescent+protein-based+bacterial+biosensor+for+analysing+phenanthrene+fluxes.">A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.</a> Environ. Microbiol. 8, 697-708 (2006).</li><li>Schamfuss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+mycelia+on+the+accessibility+of+fluorene+to+PAH-degrading+bacteria.">Impact of mycelia on the accessibility of fluorene to PAH-degrading bacteria.</a> Environ. Sci. Technol. 47, 6908-6915 (2013).</li><li>Smibert, R. M., Krieg, R. M., Gerhardt, P., Murray, R. G. E., Costilow, R. N., Nester, E. W., Wood, W. A., Krieg, N. R., Phillips, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of methods for general bacteriology. 19, 409-443 (1981).</li><li>Wu, C. H., Warren, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+autofluorescence+in+fungi+and+its+correlation+with+viability.">Natural autofluorescence in fungi and its correlation with viability.</a> Mycologia. 76, 1049-1058 (1984).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Pedersen, C., Sylvia, D., Mukerji, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+8+Concepts+in+Mycorrhizal+Research.">Ch. 8 Concepts in Mycorrhizal Research.</a> Handbook of Vegetation Science. Vol. 19, 195-222 (1996).</li><li>Furuno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+mycelia+allow+chemotactic+dispersal+of+polycyclic+aromatic+hydrocarbon-degrading+bacteria+in+water-unsaturated+systems.">Fungal mycelia allow chemotactic dispersal of polycyclic aromatic hydrocarbon-degrading bacteria in water-unsaturated systems.</a> Environ. Microbiol. 12, 1391-1398 (2010).</li></ol>

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 quantification of the total transport. However, some points have to be carefully considered prior to and while performing the experiment. An important point is to keep the microcosms free of any bacterial or chemical cross contaminations. This can be challenging, since many microcosms are run in parallel and thus, big incubation chambers (such as desiccators) are required. Hence, it is crucial to sterilize all parts carefully prior to the setup construction. Another critical point may be the application of plastic Petri dishes for the microcosms. Although PAH and bioreporter do not get in direct contact, some amount of PAH may be absorbed by the plastic material. However, we consider this not problematic for the described protocol, since this would result, if any, in an underestimation of mycelial PAH-translocation and/or bioavailability. In any case, if precise quantification is required, it should be considered exchanging the plastic parts with glass parts, although handling will be more inconvenient. Other points that should be considered involve the chosen bacterial and fungal strains (possible mutual inhibition, growth rate, mobility and possible autofluorescence), the chosen chemicals (toxic effects on microorganisms, volatility, detection limits) and practical aspects (bleaching, limitation of eGFP induction and calculation of eGFPrel). A summary of potential pitfalls and respective comments can be found in Table&#160;3.
We used this model microcosm setup to demonstrate mycelial FLU transport and subsequent bacterial degradation in an unsaturated system directly on a cell-mycelia interaction level. Our model set-up can be varied and adjusted to meet different needs with various bacterial and fungal strains, chemical compounds, etc. Thus, the system could be used for example (i) to evaluate the influence of mycelia on bacteria in the presence of an otherwise restricted chemical (e.g., toxic) source, (ii) to predict the influence of mycelia on the bioavailability of compounds for bacterial degradation, (iii) to study contaminant bioavailability depending on the distance of bacteria to the transport vector or (iv) to investigate the effect of different chemical sources (e.g., crystalline, in soil or dissolved). However, the adjustment of the system to alternate conditions will be delicate and should be conducted step by step in order to achieve clear results. So far, we have not tested the system for other bacterial or fungal strains and other compounds than PAH. However, since mycelial networks are known to transport also other contaminants than PAH like pesticides19 or soluble substances like salicylate20, we think that the system can be expanded to different chemical compounds given a compound-specific bacterial biosensor.
Please note that depending on the applied bioreporter strain a different calculation of eGFPrel might be preferable. Instead of equation (1) the two following options could be applied:
<img alt="Equation 2" src="/files/ftp_upload/52334/52334eq2.jpg" width="264px" /> (2)
<img alt="Equation 3" src="/files/ftp_upload/52334/52334eq3.jpg" width="300px" /> (3)
However, in the presented protocol equation (1) was chosen due to the following reasons: (i) eGFP intensity was reported to correlate to the PAH flux to the cell14 whereas (ii) mCherry was reported to show considerable variations for different cells13. Equation (2) and (3), in contrast, may be chosen to prevent false information from potential light artefacts in the sample. In any case, we compared all three calculation methods with each other, whereby no statistically significant differences could be found. Still, due to the reported variations in the fluorescence intensity of mCherry, we recommend using equation (1) or (2).
In the end, some limitations of the technique should be considered. Since the microcosms were designed to prevent gas-phase transport of the tested compound, mycelial uptake of the compound is reduced. This results in lower transport rates than would be expected with unhindered uptake. Hence, the sensitivity of the bioreporter strain must be high enough to detect small changes in bioavailable fractions. Further, the setup will probably not be suitable for fungal strains with a low growth rate. In this case, long term incubation might favor gas-phase transport of the contaminant thus leading to false-positive results. It might be possible to modify the setup in a way that the contaminant can be applied at a later time point. However, hyphae may then be already inactive or dead at the inoculation point. Finally, as stated above, the range of chemical compounds which are transported by mycelial networks is big. However, the availability of an appropriate bioreporter strain might be a limiting key factor.

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><tbody><tr><td>Confocal Microscope</td><td>Leica</td><td></td><td>TCS SP5X, LAS AF - Version 2.6.1; or equivalent CLSM</td></tr><tr><td>GC HP 7890 Series GC and Agilent 5975C MSD</td><td>Agilent</td><td></td><td>an equivalent GC/MS may be used</td></tr><tr><td>GC capillary column J&amp;amp;W 121-5522&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Agilent</td><td></td><td></td></tr><tr><td>Cork borer</td><td>Fisher Scientific</td><td>12863952</td><td>or any other</td></tr><tr><td>Cover slips</td><td>Marienfeld</td><td>107222</td><td>High performance, No.1.5H</td></tr><tr><td>GC/MS insterts</td><td>WICOM</td><td>WIC 47080</td><td></td></tr><tr><td>GC/MS vials 2 ml</td><td>WICOM</td><td>WIC 41150</td><td></td></tr><tr><td>Lids / septa for screw cap vials</td><td>DIONEX</td><td>49463 / 049464&amp;nbsp;</td><td></td></tr><tr><td>Lids for GC/MS vials</td><td>WICOM</td><td>WIC 43948/B</td><td></td></tr><tr><td>Objective Slides</td><td>Menzel</td><td></td><td>ordinary</td></tr><tr><td>PDMS coated glass fibers</td><td>Polymicro Technologies, Inc.</td><td></td><td>V (PDMS) = 13.55&amp;nbsp;&amp;plusmn;&amp;nbsp;0.02&amp;nbsp;&amp;micro;l&amp;nbsp;m&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Petri Dishes small / big</td><td>Greiner</td><td>633-102 / 628-102</td><td></td></tr><tr><td>Screw cap vials 40 ml</td><td>DIONEX</td><td>48783</td><td>other glass vials may be used</td></tr><tr><td>Screw cap vials 60 ml</td><td>DIONEX</td><td>48784</td><td>other glass vials may be used</td></tr><tr><td>Acenaphthylene d&lt;sub&gt;08&lt;/sub&gt;</td><td>Dr. Ehrenstorfer</td><td>C 20510100</td><td></td></tr><tr><td>Acetone</td><td>Carl Roth</td><td>9372.2</td><td></td></tr><tr><td>Activated carbon</td><td>Sigma-Aldrich</td><td>242276-1kg</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth</td><td>2267.4</td><td></td></tr><tr><td>Fluorene</td><td>Fluka</td><td>46880</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Carl Roth</td><td>T832.2</td><td>50 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>P7171</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Minimal Medium:&lt;/strong&gt; 100 ml &lt;strong&gt;Solution 1&lt;/strong&gt; + 25 ml &lt;strong&gt;Solution 2&lt;/strong&gt; + 5 ml &lt;strong&gt;Solution 3&lt;/strong&gt; ad. 1,000 ml aqua dest</td></tr><tr><td colspan="4">&lt;strong&gt;Solution 1&lt;/strong&gt;</td></tr><tr><td>Ammonium sulfate</td><td>Carl Roth</td><td>3746.1</td><td>5 g L&lt;sup&gt;-1&amp;nbsp;&lt;/sup&gt;</td></tr><tr><td>Magnesium chloride x 6&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>2189.1</td><td>1 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Calcium nitrate x 4&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>P740.1</td><td>0.5 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Solution 2&lt;/strong&gt;</td></tr><tr><td>Disodium phosphate</td><td>Carl Roth</td><td>P030.1</td><td>55.83 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Monopotassium phosphate</td><td>Carl Roth</td><td>3904.1</td><td>20 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Solution 3&lt;/strong&gt; (pH 6.0)</td></tr><tr><td>Disodium EDTA</td><td>MERCK</td><td>1084180250</td><td>0.8 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Iron(II) chloride x 4&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>MERCK</td><td>1038610250</td><td>0.3 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Cobalt(II) chloride x 6&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>T889.3</td><td>4 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Manganese(II) chloride x 1&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>4320.2</td><td>10 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Copper(II) sulfate</td><td>Carl Roth</td><td>P023.1</td><td>1 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Sodium molybdate x 2&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>0274.1</td><td>3 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Zinc chloride</td><td>MERCK</td><td>1088160250</td><td>2 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Lithium chloride</td><td>Carl Roth</td><td>P007.1</td><td>0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Tin(II) chloride x 2&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>4473.1</td><td>0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Boric acid</td><td>Riedel-de-Haen&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>11606</td><td>1 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Potassium bromide</td><td>Carl Roth</td><td>A137.1</td><td>2 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Potassium iodide</td><td>Carl Roth</td><td>6750.1</td><td>2 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Barium chloride</td><td>Carl Roth</td><td>4453.1</td><td>0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>MMA</td><td></td><td></td><td>Minimal medium + agarose 0.2%</td></tr><tr><td>Phenanthrene d&lt;sub&gt;10&lt;/sub&gt;</td><td>Dr. Ehrenstorfer</td><td>C 20920100</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Potato Dextrose Agar:&lt;/strong&gt; 24 g L&lt;sup&gt;-1&lt;/sup&gt; broth + bacto-agar 1.5%; pH 6.8</td></tr><tr><td>Potato Dextrose broth</td><td>Difco/ Beckton Dickinson</td><td>254920</td><td></td></tr><tr><td>Bacto-agar</td><td>Difco/ Beckton Dickinson</td><td>214040</td><td></td></tr><tr><td>Sodium acetate x 3 hydr.</td><td>Carl Roth</td><td>6779.1</td><td></td></tr><tr><td>Sodium sulfate</td><td>MERCK</td><td>1066495000</td><td></td></tr><tr><td>Toluene</td><td>MERCK</td><td>1083252500</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;mTY medium:&lt;/strong&gt; 3 g&amp;nbsp;L&lt;sup&gt;-1&lt;/sup&gt; yeast extract, 5&amp;nbsp;g&amp;nbsp;L&lt;sup&gt;-1&lt;/sup&gt; bacto tryptone and 50 mM NaCl</td></tr><tr><td>Yeast extract</td><td>Merck</td><td>1037530500</td><td></td></tr><tr><td>Tryptone</td><td>Serva</td><td>4864702</td><td></td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>ImageJ with logi tool plugin</td><td></td><td></td><td>http://rsb.info.nih.gov/ij/download.html and http://downloads.openmicroscopy.org/bio-formats/4.4.10</td></tr><tr><td>&lt;em&gt;Pythium ultimum&lt;/em&gt; strain 67-1</td><td></td><td></td><td>Obtained from the lab of Dr. Christoph Keel; Department of Fundamental Microbiology, University of Lausanne, Switzerland</td></tr><tr><td>&lt;em&gt;Burkholderia sartisoli&lt;/em&gt; RP037-mChe</td><td></td><td></td><td>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.

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

Nanyang Technological University

Research

JoVE Journal

Environment

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

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

Chemistry

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Bioengineering

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Biology

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

Characterization of Aquatic Biofilms with Flow Cytometry

Characterizing Electron Transport through Living Biofilms

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches