PITFALLS	COMMENT
1	Preliminary growth and vitality tests	Study the growth of mycelial organism of choice. How long does it take to reach the MMA patch? Is it inhibited by the cycloheximide barrier? Also check whether bioreporter cells stay vital in MMA the whole time via plaiting assays. If not, one also may add a carbon source to the MMA which does not result in bioreporter induction. Exclude possible mutual inhibition of bacteria and mycelia.
2	Mycelial growth rate	The growth rate of the mycelial organism should not be too slow, because otherwise gas-phase transport of PAH increases with prolonged incubation time. The protocol may be adapted to add PAH at a later time point, but then again, mycelia at the inoculation point may not be active anymore.
3	Bacterial mobility	If a point scanning laser microscope is used and the bacterial strain is motile, the recording and quantification of z-stacks may be impossible due to movement inside of MMA. Thus, bacterial movement should be prevented, e.g., by increasing the solidity of MMA, by using CLSM with a fast scanner or a spinning disk laser microscope.
4	Vapor-phase transport	Vapor-phase transport of the chemical (e.g., PAH) towards the bioreporter cells must be excluded sufficiently since bioreporter cells are extremely sensitive to chemicals in the vapor-phase. This is the crucial point of the whole protocol in order to prevent false-positive results caused by gaseous transport. The gas phase concentration can be estimated via chemical analysis of CONAIR(-) and vapor-phase concentrations may be adjusted for example by adding more or less agarose patches with activated carbon.
5	Toxicity	Keep in mind, that high concentrations of the tested chemical might have toxic effects on the mycelial organism and/or bacteria.
6	Autofluorescence	Check if the chosen mycelial organism shows some autofluorescence in the range of the expected bioreporter signal. Be sure to check at different growth stages since autofluorescence may vary17. If autofluorescence is detected, be sure to record z-stacks in mycelia-free areas.
7	Bleaching	mCherry fluorescence is usually stable and not sensitive to bleaching in the applied bioreporter cells. In contrast, eGFP bleaches rather quickly. Therefore, don’t use the eGFP channel to visualize the sample prior to z-stack recording.
8	eGFP induction	We noticed a low eGFP induction in SAM compared to CONPOS. This is accounted for by the strong spatial restriction and low accessibility of the FLU source to maintain very low vapor-phase concentration of FLU (cf. last results paragraph). Depending on the chosen bioreporter strain and chemical, the uptake area at PDA 1 may be varied to increase the transport rate (Figure 5B). However, it has to be considered that this also increases vapor-phase transport towards the bioreporter cells.
9	Calculation of eGFPrel	The presented calculation method for eGFPrel where the intensity of green pixels is compared to the area of red pixels is only one possibility. Please refer to the discussion for further information.
10	Pixel size	Please be aware that the magnification of the chosen lens and a potentially applied zoom factor affect the pixel size of the image. This must be taken into consideration prior to image analysis.
11	Bioavailable fractions	Keep in mind that the bioavailability of the transported compound is assessed qualitatively (not quantitatively) via eGFP induction. No correlation of the calculated relative eGFP-induction and the bioavailable fraction was attempted. However, this may be addressed in future applications.
12	Detection limits	Chemical detection. For chemical quantification of organic compounds, a wide range of concentrations can be detected reliably. We detected amounts in the range 0ng and 1,000ng. eGFP quantification. We compared relative eGFP induction in samples without contaminant transport (i.e., 0ng transported), with mycelia-mediated transport (i.e., between 20 and 40ng transported; cf. Figures 4 and 5) and with direct contact to the contaminant source (i.e., maximum available amount). These three cases proved to be well distinguishable with the described method. However, we cannot make a more detailed statement on the resolution of the relative eGFP induction. This issue may be addressed in the future by linking different contaminant amounts with the correlating eGFP induction in the microcosm setups.
13	Petri dish material	Plastic Petri dishes may result in an underestimation of mycelial PAH translocation and/or bioavailability. This should not be problematic, if qualitative statements are aspired. In case precise quantitative measurements are required, application of glass Petri dishes should be considered despite preparation and handling will be more inconvenient.