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O2, CO2 and pH Dynamics in the Capillary Fringe
VisiSens combined with an automated linear positioning system provides complete analyte maps
M. Wagner, N. Hack, G. Abbt-Braun, and H. Horn
Chair of Water Chemistry and Water Technology, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Germany
We investigated the biochemical transformation potential in the capillary fringe (CF) by measuring the distribution of dissolved gases and the pH value. Therefore, we combined the VisiSens A1, A2, & A3 with a linear positioning system to move the detector units along the CF in a lab-scale experiment. With this set-up we obtained complete pictures of analyte distributions in the CF and the images confirmed that the CF offers optimal conditions for high biological activity and biochemical transformation of organic compounds.
The capillary fringe (CF) is a highly active and dynamic zone in soil at the transition of the vadose zone and the aquifer. The height of the CF varies according to the grain size, the soil type and the groundwater level. These dynamics affect the biochemical transformation of organic compounds in the CF by microorganisms. Compared to fully water-saturated or completely dry zones, the biochemical transformation potential in the CF is elevated. Hence, it is of interest to investigate the distribution of dissolved gases, soluble compounds, as well as the pH value. The VisiSens imaging systems are in general well suited for this. Nevertheless, the field-of-view of a single camera is small in relation to the CF. To overcome this limitation, an automated linear positioning system was developed allowing to move the VisiSens cameras to any position along the CF in the lab-scale experimental set-up. This aims on drawing an almost complete picture of the O2, CO2 and pH value distributions in the CF. Further, it should allow correlating O2 and CO2 levels along with the pH value to the biochemical transformation capabilities of the CF.
Materials & Methods
In the lab a so-called Hele-Shaw cell was set up. It was filled with sand (height = 20 cm) mimicking a soil system close to reality with dimensions of 80 x 30 x 5 cm3 (Fig. 1A). Item aluminum profiles (item Industrietechnik GmbH, Solingen, Germany) were used to integrate the cell into the set-up. The linear positioning system (igus GmbH, Cologne, Germany) allowed a controlled moving of a camera carriage along each sensor stripe attached to the inner glass interface of the Hele-Shaw cell. The igus components were mounted to the item profiles. Five zones (A - E, Fig.1A) were defined containing a sensor stripe of 20 x 2 cm2 for O2, CO2 and pH (PreSens GmbH, Germany). Salicylic acid was used as organic model compound to be degraded in the CF. The cultivation medium together with nutrients was pumped into the cell at a height of 10 cm. The system was inoculated using a stock culture of Pseudomonas fluorescence (7 x 107 cells/mL). Thereby, the CF developed at different heights along the length of the Hele-Shaw cell. The igus system uses stepper motors for movement. Their control was realized using an in-house made stepper driver shield (populated with Pololu DRV8825 stepper motor drivers) attached to an Arduino Uno R3. The Arduino was flashed with the grbl firmware (http://bengler.no/gbrl, version 0.8 with an adapted homing code working with two axes). Briefly, grbl interprets g-code and converts these commands into movement of the stepper motors. A new version of the VisiSens software (VisiSens AnalytiCal 4) was developed by PreSens that allowed imaging of three different analytes simultaneously (O2, CO2, and pH) as well as was triggered by external software and vice versa. In particular a Python script (www.python.org) was developed which controlled the camera carriage movement by sending g-code to the Arduino. Once the position was reached and confirmed, VisiSens was triggered to acquire an image of the selected analyte. Each individual image of an analyte covered about 1.5 x 2 cm2 on a sensor stripe. To visualize the distribution of all three analytes over the entire height of the CF, the camera carriage was moved to overlap adjacent images by 20 % (Fig. 1B). Image acquisition started in the lower left corner in zone A in the order of pH value -> O2 -> CO2. Then the camera carriage was moved up acquiring the next set of three images. In total 130 images per analyte were captured over the Hele-Shaw cell (zones A - E). Acquisition time was about 20 min per run and the system was running reliably for 160 h. Image acquisition was performed by the newly developed VisiSens software and image analysis was realized using Matlab (MathWorks Inc., Nattick, USA). Matlab scripts were developed interpolating the distribution of O2, CO2 and pH value over the entire Hele-Shaw cell based on the acquired images.
O2, CO2 and pH Maps of the CF
As mentioned salicylic acid was used as model compound to study the aerobic degradation of organic substances in the CF. In Fig. 2 the O2 distribution over the entire Hele-Shaw cell is plotted. The top part (20 - 30 cm) is air-saturated and thus appears in dark red. The CF itself developed within a height of 5 - 20 cm. Within this region the O2 concentration was approx. 70 - 80 % air saturation. Due to the aerobic biochemical degradation of salicylic acid the O2 concentration dropped within the water- saturated zone. Dark blue regions indicate zones where the oxygen was almost consumed completely (approx. 20 % air sat.). By using the VisiSens system a boundary between the water-saturated zone and the CF has been visualized (green and yellow region in Fig. 2). Here a fair balance between the water content, O2 availability, and O2 consumption can be assumed. In addition, the presented system allowed extracting complete O2 profiles over the height of the CF in zones A - E. As shown in Fig. 2 the profiles reveal the development of oxygen consuming regions within the artificial soil system. It can further be understood as an image of salicylic acid distribution, which is driven by the inflow characteristics in zones A - B, and affected by gravity in zones C - E. A degradation product of salicylic acid is CO2. In Fig. 3 it is obvious that CO2 is produced and is accumulated in the CF above the water-saturated zone at a height of 8 cm along zones A - E (areas in cyan, green and yellow). Since the pH map (data not shown) did not show artifacts such as gas bubbles, it is assumable that the CO2 was dissolved in the liquid phase. The CO2 concentration reached its maximum of 5 % in zone A. Here, the biological activity and growth should mostly be influenced by the O2 and salicylic acid rich cultivation medium constantly pumped into the Hele-Shaw cell at a height of 10 cm.
The CF is a highly dynamic system providing good degradation capabilities for organic compounds. This is for example interesting in bio-remediation of contaminated sites. A Hele-Shaw cell creates a quite realistic lab-scale set-up to investigate the interaction of organic compounds with microorganisms in the CF. The greatest challenge is to capture the activity within the CF at four dimensions. These four dimensions (x, y, z, time) were assessed by developing an automated linear positioning system in conjunction with an optimized VisiSens software. The system provided highly resolved local concentration measurements for O2, CO2, and pH value, which further allowed the approximation of analyte maps for the entire Hele-Shaw cell. Finally it could be validated that the CF offers optimal conditions for high biological activity within a wide range of O2 availability and O2 consumption. Furthermore, the integration of O2 and CO2 sensor stripes may allow for the calculation of carbon mass balances.
The financial support by the German Research Foundation is gratefully acknowledged (FR536/39-2; Der dynamische Kapillarsaum - ein multidisziplinärer Denkansatz, Teilprojekt 5, DyCap II).