Plants and soil
Three plant species, Trifolium pratense, Dactylis glomerata and Ranunculus acris were chosen to represent a legume, a grass and a forb plant species, respectively. All plant species occurred in the mountain grassland study by Bamberger et al. (2011), during which a prolonged period of monoterpene deposition was observed to a grassland ecosystem associated with elevated ambient isoprenoid concentrations. The vegetation of this grassland ecosystem consisted of about 28 % grasses, 11 % Trifolium pratense and Trifolium repens and 4 % Ranunculus acris.
The plants for our experiment were grown for three months from seeds in pots with a surface area of 64 cm2 inside a greenhouse in Innsbruck, Austria. The soil consisted of steamed (for sterilisation) leaf mold, steamed basic soil, lava, coconut fibre, sand and rock flour (31 %, 15 %, 12 %, 15 %, 15 % and 12 % respectively), which was used by the Botanical Garden of the University of Innsbruck to simulate the soil of the study site by Bamberger et al. (2011). After their transport to the greenhouse facility of the EUS in Munich, the plants were subject to 14 h of a combination of natural and, when needed, artificial light with a close-to-natural wavelength composition (Powerstar HQI-TS, 400 W/D, OSRAM, Germany) to keep the incoming radiation stable. Day and night temperatures were set constant at 25° and 10 °C, respectively. All experiments were conducted during daytime and started when the photosynthesis and respiration rates of the plants and soils in their chambers were stable.
Experimental setup
For analysis the plants (n = 4 per species) were enclosed in PAR-transparent Perspex glass chambers (V = 5 L) (Fig. 1) to measure the bi-directional exchange of the BVOCs of interest (Ghirardo et al. 2012). Two of these chambers were used at a time to maximise the number of replicates during the experiment. Each of the cuvettes was connected via an inlet and an outlet to one infrared gas analyser (IRGA, GFS-3000, SN: KETA0103 and KETA0147, Heinz Walz, Effeltrich, Germany). These and all other connections were done using Teflon® PFA tubing, thermally insulated and heated wherever necessary to avoid condensation.
A constant flow of air containing 10,000 ppmv H2O and 380 ppmv CO2 was supplied to the inlet air to keep the rate of evapotranspiration and photosynthesis stable, starting 20–30 min before the first measurement. Two air pumps (Neuberger diaphragm vacuum pump, KNF, Freiburg, Germany), in combination with several overflow vents, were used to keep a steady flow of air (1280 sccm) throughout the cuvette. To minimize background contamination by ambient BVOCs, two-catalysers (Platinum (Pt) - Palladium (Pd), 390 °C; self-build, (Graus et al. 2010)) were used to purify the inlet air of both cuvettes. The ambient BVOC concentrations in the cuvettes (0, 3, 5 and 10 ppbv) were chosen to simulate similar conditions to the study of Bamberger et al. (2011), who measured monoterpene concentrations from below 1 ppbv to up to 7.5 ppbv. A certified gas standard (Apel-Riemer Environmental Inc., USA) containing isoprene as well as α-pinene was used to increase the concentration of both VOCs in the cuvettes simultaneously.
In order to gradually step through ambient concentrations in the cuvettes two mass flow controllers of 20 sccm (EL-FLOW Select Series Mass Flow Controller; BRONKHORST HIGH-TECH B.V., Ruurlo, Netherlands) were used to mix catalysed VOC-free air with the gas standard. The same mixing ratios (0–10 ppbv) were used for calibration with 10,000 ppm H2O, corresponding to chamber inlet conditions, prior to each measurement cycle. A calibration with 30,000 ppm H2O, reflecting typical conditions at the outlet of the chamber, was conducted once for each chamber after the experiment. The corresponding calibration factor was within the range of the calibration factors (n = 24) determined with 10,000 ppm H2O (6.9 ± 0.2 for isoprene and 3.4 ± 0.3 for α-pinene). The conversion from ncps (norm counts per second) as measured by the PTR-ToF-MS to the corresponding ambient concentration in ppbv was thus done based on the calibration factor determined prior to each experiment. The limit of detection for isoprene was on average 0.0078× + 0.03 and 0.0067× + 0.0187 for α-pinene, where x denotes the respective ambient concentration (ppbv) of the BVOC of interest.
Mass analysis was done with the original PTR-ToF-MS developed at the University of Innsbruck. Since PTR-ToF-MS is an already well established method, only a brief description of the fundamental operation principles is given here. A detailed description can be found elsewhere (Graus et al. 2010; Jordan et al. 2009).
PTR-ToF is a chemical ionization mass spectrometer developed at the Institute of Ion Physics at the University of Innsbruck. The instrument can be operated real time to record in situ concentrations. With its high mass resolving power up to m/dm = 5000 and a mass accuracy of less than 10 ppm (parts per million), isobars can be distinguished and empirical formulas can be identified (Graus et al. 2010; Jordan et al. 2009). A mass range of 0–300 m/z was recorded in a one second time resolution. The reaction chamber of the PTR-ToF-MS was operated at standard conditions (60 °C, 2.3 mbar, 580 V). For conversion of the raw data to ncps, the “PTR-TOF Data Analyzer” software in version 2.44 was used (Müller et al. 2013; Titzmann et al. 2010).
Four solenoid valves (M Series Miniature Solenoid Valve, Teqcom Industries Inc., Newport, USA) were used to switch the PTR-TOF’s connection between inlet and outlet air of the two cuvettes (Fig. 1). A permanent airflow (1280 sccm) was established from and to the IRGAs through the cuvettes to measure the rate of evapotranspiration and photosynthesis continuously. A temperature difference between the cuvettes (29 °C)and the surrounding air (21.5 °C) made it necessary to heat the outlet lines (37 °C/50 °C) to prevent condensation in the tubes (see dashed line in Fig. 1). The temperatures in the cuvettes were measured using two temperature and humidity sensors (Humi-pick: M21816 & M21817, Fa. Spirig, Rapperswil, Switzerland).
In a first measurement cycle (4 h), pots containing soil and above-ground biomass of one of the three plant species mentioned above were placed in the cuvettes, allowed to stabilize the gas exchange, and then exposed to a step-wise increase of VOC-ambient concentration (0,3,5 and 10 ppbv). In order to disentangle the exchange between the bare soil and the above-ground plant biomass, the above-ground parts of the plants were cut after this cycle. After 12 h, the remaining plant material (i.e. main stem) was sealed using a PTFE-paste (to prevent the volatilisation of wounding-induced VOCs (i.e. volatile breakdown products of unsaturated fatty acids: hexenylacetate, Z-hexanol, etc.) as reported by Brilli et al. (2011)). The bare soil was exposed to the same step-wise increase in VOC concentration levels. In between these two principal measurement cycles, which were conducted once per plant and soil, empty cuvettes were measured in the same fashion in order to characterize any residual exchange in the empty cuvettes. These background values were subtracted from the VOC concentrations at the outlet of the cuvettes. Although the inlet VOC concentrations were the same for both pots with vegetation and bare soils, the outlet concentrations differed because of the biological activity inside the cuvettes. Hence the difference between the VOC fluxes from the pots with vegetation and the soil measurements couldn’t be calculated by a simple subtraction and we thus do not explicitly infer the plant contribution by difference, but rather present results separately for pots with bare soils only and pots with vegetation cover and discuss the corresponding differences on a soil surface area basis.
The water vapor, CO2 and VOC exchanges (E) in the cuvettes were calculated using the formula of Caemmerer and Farquhar (1981).
$$ {E}_x=\frac{\left({X}_o-{X}_e\right)f}{A\left(1-{W}_o\right)} $$
(1)
Where f represents the flow rate (mol s−1), Χo and Χe the mole fractions (mmol mol−1 or μmol mol−1 or nmol mol−1) of the air leaving and entering the cuvettes, Wo the water vapor mole fraction (mol mol−1) exiting the cuvette and A represents the exposed soil surface area (0.0064 m2).
The CO2 and water vapor exchange was measured to ensure that plants were photosynthetically active and their gas exchange under steady-state conditions. During the experiments, the measured CO2 mole fraction, air temperature and relative humidity inside the cuvettes did exhibit only slight changes during the course of each measurement cycle (<28 ppm, <5 ° and <23 %, respectively) and was thus regarded as being in steady-state (data not shown). To ensure that no ambient air would enter the cuvettes and tubes, leak-tests were conducted at the beginning of every measurement.
After the gas exchange measurements, the soils were frozen for further analysis at −18 °C. The soil pH was then measured using a pH-meter (Lab pH meter inoLab® pH 110, WTW, Weilheim in Oberbayern, Germany) in a solution of 10 g sieved (2 mm) soil and 25 ml 0.01 M CaCl2. The soils used in this study were slightly acidic with a mean pH of 6.0 ± 0.2.
Statistical analyses
The linear regression model and the statistical group comparison, using a linear regression analysis and a paired t-test, respectively, were conducted using the Matlab statistics toolbox (Matlab Release R2014b, The MathWorks, Inc., Natick, Massachusetts, United States.), after confirming the assumption of normality and heteroscedasticity through QQ-plots and residual plots.