Figure 1. Left: Chamber during rest. Middle: Chamber during NEE measurements. Right: Chamber during ER measurement.

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Technical abstract ECO FluX chamber Introduction This ECO FluX gas flux measurement chamber sets new standards for measuring CO dynamics at the ecosystem level. Because the chamber can be transparent as well as darkened, both Net Ecosystem Exchange (NEE) and Ecosystem Respiration (ER) can be measured directly and Gross Ecosystem Photosynthesis (GEP) can be estimated by their difference (GEP = NEE ER), thus producing direct data of the processes behind NEE. The chamber covers a ground area of 3,100 cm and has a volume of 0 L (at standard height of 80 cm) and is therefore big enough to include plants communities in many natural lowstature ecosystem types as well as many agricultural crops. The chamber consists of two moveable flexible tubes and a moveable lid (Figure 1). The inner tube (inner diameter (ID) of 630 mm) is transparent and, together with the lid, it acts as the closed, airtight gas flux chamber. It operates both for NEE and ER measurements. The outer, darkened tube (ID of 75 mm) is active only during ER measurements (middle panel). The chamber height (standard of 80 cm) may be set according to the desired chamber volume and the vegetation height by adjusting the angle of closure. Figure 1. Left: Chamber during rest. Middle: Chamber during NEE measurements. Right: Chamber during ER measurement. Basics of the ECO flux chamber In contrast to most other chamber designs the ECO flux chamber closes from the ground and upwards to the desired height where it then meets the lid. This prevents plant damage during closure of the chamber. The movement of the arms of the lid and the chamber tubes takes place over one axis by only one motor/gear unit. When inactive, the chamber lid and tubes rests close to the ground and well protected against wind. The simple and sturdy design makes the chamber highly reliable even under very windy conditions. See a video of the chamber at https://youtu.be/ukfiio6s6dg. 1

Installation in the field To minimize shading effects from the lid during measurements, the entire construction the chamber is installed north/south direction with the chamber itself facing south. At latitudes with maximum solar angles at noon < 70 this ensures that the lid never shades the base area of the chamber (Figure, left panel). Therefore a transparent chamber lid is not needed. The non-transparent lid enables full darkening of the chamber during ER measurements. Figure. Left: In the field, the chamber is installed in north/south direction, so that the lid does not shade the chamber base area directly. Right: The chamber controller unit. Non-soil-intrusive double collar Another novelty of the chamber design is the innovative, non-soil-intrusive double collar, which avoids the root damage of classical soil penetrating collar designs upon field installation (Figure 3, left panel). When the rim between the two steel sockets is filled with soil after field installation gaseous fluxes through the soil near the soil collar are minimized potential biases caused by lateral diffusion (as chamber concentration changes during measurements) are kept minimal. The base is firmly fastened to the ground with three large plastic soil screws (Figure 3, right panel). Figure 3. Left: The chamber collar with inner, transparent tube. Right: The base is firmly fastened to the ground with three large plastic soil screws.

Auxillary measurements A number of important physical parameters are measured simultaneously with flux measurements. These are PAR (Photosynthetically Active Radiation) inside the chamber, chamber temperature, soil temperature and soil water content (SWC). Furthermore, a fan inside the chamber ensures good mixing of the air inside in order to prevent gradients in CO concentrations arising inside the chamber during measurements (Fig. 4, right panel). Auxiliary sensors are sold separately. Figure 4. Left: Chamber PAR sensor. Right: Chamber fan. Chamber temperature, soil temperature and soil water content may also be measured (not shown). Chamber application in the field The ECO flux chamber is developed to be controlled by the LI-8100/LI-8150 Multiplexer CO /H O infrared gas analyzer system from LI-COR Biosciences (please acquire quotation directly from LI-COR at www.licor.com). This also means that data may be analyzed using standard LI-COR software. Data may of course be exported from the LI-COR software and analyzed e.g. with other software tools, such as R (http://www.r-project.org/). Please contact us for further details. Flux calculations During field measurements, the first measurement with the transparent tube allows for both photosynthesis and respiration at the ecosystem level to take place. An example of such a measurement during high light intensity is shown in Figure 5, left panel in a situation where GEP > ER thus leading to decreasing CO concentrations during the measurement. During the second measurement when the chamber is darkened, photosynthesis has stopped and only ecosystem respiration continues. An example of such a measurement is shown in Figure 5, right panel. In between measurements, all gas tubes are flushed for -5 minutes to make sure gas concentrations in the tubes are at equilibrium with the atmosphere prior to measurements. Furthermore, a dead band is set to make sure that disturbance during the initial chamber closure period are not used for flux calculations. The dead band is longer for ecosystem respiration measurements because photosynthesis may continue up to 60 s after darkening the chamber. When both light and dark measurements have been performed, GEP is calculated as NEE ER as described above. 3

Figure 5. Left: Screen shots from LI-COR software during chamber test in the field. The graph shows declining chamber concentrations of CO after chamber closure indicating that GEP > ER during this measurement. Right: Chamber concentrations of CO after chamber closure with chamber darkened and only ER is taking place. ECOFluX example data from the field The chamber has been long term tested, e.g. producing more than 8.000 measurements while measuring intensively during a six week period (Figure 6). The high-frequency data obtained with the chamber enables both measurements and modelling of the ecosystem processes behind observed gas fluxes and their dependence on climatic drivers in much higher detail than previously possible. For example, sporadic events like rain and fast temperature changes, as indicated with arrows in the graph, and their effects on gas fluxes can now be followed (Figure 6). 1 10 Net Ecosystem Exchange Ecosystem Respiration Gross Ecosystem Photosynthesis 5 µmol CO m s Temperature drop Rain event 0-5 -10 08/09/15 15/09/15 /09/15 9/09/15 06/10/15 13/10/15 0/10/15 7/10/15 Date Figure 6. Example data of CO fluxes in a heathland ecosystem measured with the ECOfluX chamber. Negative values indicate CO uptake and positive values indicate CO release. Note how effects of sporadic events like rain and fast temperature changes on the gas fluxes can be followed. 4

CO (ppm) CH 4 (ppm) We have also performed an initial investigation to test if the chamber system works with an additional CRDS CH 4 /CO analyzer (Los Gatos Inc., USA) in parallel to the default LI-8100 infrared gas analyzer (LI-COR, USA). Addition of more analyzers is valuable as it increases the versatility of the chamber system. The test showed high degree of reproducibility between the CO concentrations measured with the two analyzers and indicated that we could detect uptake of atmospheric CH 4 (negative slope) (Figure 7). 460 450 Chamber transparent CO, LI-COR 8100 CO, Los Gatos UGGA CH 4, Los Gatos UGGA Chamber darkened.09.08 440 430.07.06.05 40.04 410.03 13:15 13:0 13:5 13:30 13:35 Time of day (/10/15) Time of day (/10/15) Figure 7. Test of combination of LI-COR 8100 with a Los Gatos UGGA (Ultraportable Greenhouse Gas Analyzer) and the ECO flux chamber showing high reproducibility of results from the two sensors used in parallel in the system as indicated by the overlap of the CO curves from the two sensors. For further information - please contact: Prenart Equipment ApS Buen 14 DK-000 Frederiksberg Tel.: +45 395 0900 Web: http://www.prenart.dk, E-mail: pj@prenart.dk 5