We present a dynamic twin-cuvette system for quantifying the trace-gas
exchange fluxes between plants and the atmosphere under controlled
temperature, light, and humidity conditions. Compared with a single-cuvette
system, the twin-cuvette system is insensitive to disturbing background
effects such as wall deposition. In combination with a climate chamber, we
can perform flux measurements under constant and controllable environmental
conditions. With an Automatic Temperature Regulated Air Humidification
System (ATRAHS), we are able to regulate the relative humidity inside both
cuvettes between 40 and 90 % with a high precision of 0.3 %. Thus,
we could demonstrate that for a cuvette system operated with a high flow
rate (> 20 L min
List of previous studies in the research field of O
The atmosphere–biosphere exchange of various trace gas species plays an important role for the climate and ecosystem interaction. The removal and emission of trace gases by the biosphere represents a significant factor, and its understanding is essential for atmospheric chemistry and the calculation of global trace gas budgets. While there is an increasing interest in the underlying mechanism of trace gas exchange of plants, various methods to determine the exchange flux of trace gases exist – in the field and under controlled laboratory conditions. For flux measurements on ecosystem level micrometeorological methods such as the eddy covariance or gradient method are used, causing only minimal disturbance (Horst and Weil, 1995). However, to understand the mechanism and processes in more detail, measurement on the plant and leaf scale under controlled environment conditions are often used. Therefore, enclosure techniques are mainly used to perform experiments under constant environmental conditions for investigation of the interaction between the plant and the atmosphere with higher resolution and reliability. For these experiments, it is important to minimize the disturbance of the environmental conditions such as radiation, humidity, temperature, and the trace gas concentration to ensure an optimum of plant physiological activity (Pape et al., 2009). Especially for flux measurements of trace gases whose exchange processes are predominantly controlled by the leaf stomata, such reproducible preconditions should be achieved.
A commonly used technique for the measurement of trace gas uptake and
release on plant and leaf scale is the dynamic cuvette technique (e.g.,
Breuninger et al., 2012). In the field, it can be ensured that the inner
trace gas concentration and other related quantities are constant and close
to the ambient conditions outside of the cuvette by the continuous renewal
of the air inside the cuvette. For laboratory measurements, the dynamic
cuvette leads to a temporally constant trace gas mixing ratio inside the
cuvette. Typically, systems employ one single dynamic cuvette, in which the
trace gas concentration is measured at the entrance position of the cuvette
and inside the cuvette to retrieve the trace gas flux. One disadvantage of
such cuvette systems is the potential adsorption and/or desorption effects on
the cuvette walls, which is critical, especially for reactive trace gases
(Kulmala et al., 1999; Pape et al., 2009). Furthermore, the influence of
humidity is an important factor for the surface deposition of various water
soluble trace gas species (e.g., NH
Laboratory studies which were designed to compare the PAN deposition with
O
Flow chart of the dual cuvette system. 1 – plant cabinet; 2 – water
storage tank (see Sect. 2.1.3); 3 – ATRAHS (see Sect. 2.1.3); 4 – Temperature and relative humidity sensor
for humidity regulation (see Sect. 2.1.3); 5 – mass
flow controller (MFC); 6 – dynamic cuvette (reference); 7 – dynamic cuvette
(sample); 8 – temperature and relative humidity sensor for monitoring;
9 – Teflon-valve block; 10 – MFC for gas addition; 11 – O
Layout of the dynamic cuvette as used in this study. 1 – PVC frame; 2 – acrylic glass cap; 3 – fan coated with Teflon; 4 – FEP foil; 5 – inlet PFA-tube with additional ring; 6 – sample tubes; 7 – clamps; – 8 silicon strip.
The experimental setup consisted of two dynamic cuvettes (Kesselmeier et
al., 1996; Chaparro-Suarez et al., 2011; Breuninger et al., 2012) (see
Figs. 1 and 2). The
entire plant sample above the soil was introduced into the sample cuvette.
The leaf temperature was measured by thermocouples (Type E, OMEGA
Engineering. Inc., USA) at four different positions of the plant. Pressurized
air flow was provided by a compressor and was purified by different filter
cartridges filled with glass wool (Merck, Germany), silica gel (2–5 mm Merck,
Germany), Purafil® (KMnO
The wall material of the cuvettes was made of FEP foil (Saint Gobain
Performance Plastics Corporation, USA) to minimize wall effects for the
measured trace gases. All tubing and tubing connections that were in
contact with the air flow consisted of PFA-Teflon®
(Swagelok, USA). A fan (APC Propellers, USA), which had been coated with
Teflon® by the MPIC mechanical workshop, was
installed inside both cuvettes to assure well-mixed conditions in order to
reduce the aerodynamic resistance for the trace gas fluxes (Gut et al.,
2002). The purified air stream supplied with a certain mixing ratio of
O
The choice of the appropriate residence time in the cuvette is a key factor
for the operation of a dynamic cuvette system. On the one hand, it should be
short enough to exclude chemical reactions in the cuvette and to follow
potential fast changes in the environment. In the case of PAN, the thermal
decomposition under higher temperature made it necessary to keep the
residence time inside both cuvettes as short as possible. As the lifetime of
PAN was about 5 hours at the cuvette temperature of 25
Flow chart of the automatic temperature regulated air humidification system (ATRAHS). 1 – deionized water supply; 2 – water storage tank; 3 – humidifier tank; 4 – float valve; 5 – V25 control device; 6 – T/RH sensor (initial regulation); 7 – T/RH sensor (monitoring); 8 – dynamic cuvette; 9 – heat element.
We present an automatic temperature regulated air humidification system
(ATRAHS) to control the relative humidity inside the cuvettes. Due to the
high operating main flow rate of 20 L min
Draft of the leaf wetness sensor. 1 – data cable; 2 – sensor; 3 – electrode clams; 4 – plant leaf. Modified from Burkhardt and Gerchau (1994).
The leaf wetness sensors were used to identify and characterize the
formation of liquid water films on the leaf surface. The technique is based
on the measurement of the electrical resistance of the surface between two
electrodes, which are fixed on the leaf with metal clamps (see
Fig. 4). The sensor design is based on that
published by Burkhardt and Eiden (1994), and has been updated according to
suggestions of J. Gerchau (2011, personal communication). Briefly, an
oscillator generates an alternating voltage (2 VAC) with an adjustable
frequency between 0.5 and 2 kHz. This voltage is applied to the leaf
surface, which acts as a resistor in a voltage divider circuit. The
resistance of the leaf surface, which is derived directly from the resistive
voltage drop, depends on the leaf surface wetness. A major drawback of the
old sensor design was the temperature dependence of the signal (e.g., Altimir
et al., 2006) caused by changing resistance of the cable between the leaf
clamp and the measuring device. This has now been solved by using a
miniaturized sensor board measuring the leaf resistance with a voltage
divider circuit including a capacitor and a pre-amplifier directly at the
clamps of the sensor (see Fig. 4). Additionally,
the oscillator can be tuned to four different frequencies from 0.5 to 2 kHz,
which allows conductivity measurements also at high ionic strengths
(activities), i.e., at low ion mobility. Furthermore, all sensors are now
evaluated independently with individual sensor boards as well as individual
amplifier and rectifier circuits before A/D conversion in order to avoid
interferences between different leaf measurements. In this study, we present
measurements of the optimized version of the leaf wetness sensor. For the
sample cuvette, three sensors were mounted on plant leaves at different
heights to obtain an average value, which was regarded as representative for
the entire plant. The background signals were measured by sensors without
leaves in the reference cuvette. The net-signals were used to calculate the
electrical surface conductance
A long-term flux measurement was performed over a period of 14 days to demonstrate the quality of the twin-cuvette setup.
The experiments were performed with 3-year-old tree individuals of
The diurnal cycle of the plant was simulated by the plant cabinet system
with 13 h of light and 11 h of dark period. The light intensity
(450–650 nm) was kept constant at 600
For a non-destructive determination of the leaf area the shape of every single leaf of the sample plant was drafted by hand and digitalized via a photo scanner (Epson Perfection 3170 Photo). The scans were evaluated by the program “Compu eye, Leaf & Symptom Area” (Bakr, 2005) to obtain the overall leaf area.
Abscisic acid (ABA) is a plant hormone, which affects the closure of the
leaf stomata. For the experiments, a branch was cut from the plant under
water to prevent embolism. The stock solution of ABA was created by
dissolving solid ABA (CAS 14375-45-2, Sigma Aldrich, USA) in 5 mL ethanol
and then filling up to 100 mL with deionized water. The nutrient solution
was concentrated with 250
As we could assume constant conditions in the cuvettes over the time of 10
min switching intervals, the mixing ratios of all four measured positions
(inlet and outlet of both cuvettes) could be used to derive the deposition
fluxes of O
The deposition velocity
The stomatal conductance is an important parameter to evaluate the gaseous
uptake from the ambient air into the plant leaves. The stomatal conductance
of water vapor
The saturation water vapor pressure SVP (hPa) was calculated with the
Goff–Gratch equation (Goff and Gratch, 1946), which is dependent on the leaf
temperature
The diffusion coefficients of O
The internal leaf O
As mentioned above, the advantage of a twin-cuvette setup was to account for
disturbing background effects such as wall deposition, assuming that these
effects were equal for both cuvettes. In order to check the accuracy and
stability, the mixing ratio measurements of O
Given that the inlet mixing ratios between both cuvettes are identical
(vmr
The described evaluation experiment was conducted over 8 hours at which
the inlet O
In addition to accounting for effects in the cuvettes, the O
Calibration of the O
The precision of the O
The random errors of
The significance of the mixing ratio difference between each sampling
position of the reference and sample cuvette was proved with an independent
two-sample-t-test, using a
Comparison of the all key parameters between both empty cuvettes to
indicate their identity.
The comparison of both empty cuvettes (see Sect. 2.3.1) showed only insignificant differences (i.e.,
Minimal resoluble mixing ratio difference of O
To include the potential influence of cuvette system on the LOD and the
precision of the O
Detection limit (LOD) and precision of the twin-cuvette system at
different sampling positions. The determination of the LOD is based on
3
The initial humidity (RH
Relative humidity controlled by ATRAHS at different levels from
50 to 100 %. Straight red line: RH
Multipoint calibration curve of O
Systematical determination of the O
For both, O
Wall deposition rate of O
Long-term flux measurement of
The twin-cuvette system could be successfully adapted to the needs of the
plants and the experimental conditions needed. Measurements were performed
under a light/dark cycle with a maximum of
480
Comparison of the deposition velocity ratio of O
Behavior of O
The relative humidity in
the sample cuvette was significantly enhanced by the transpiration of the
plant. During the dark periods the relative humidity of the sample cuvette
was 38 %, a similar value as compared to the reference cuvette when the
leaf stomata were closed and plant transpiration was inhibited. During the
light periods, the humidity level inside the sample cuvette reached 47 %.
The water transpiration and CO
A clear linear relationship was found between the O
The ABA nutrient solution (
O
PAN mixing ratio
Of special interest is the question whether plant surfaces may act as a sink
for trace gases without taking into account the stomatal deposition. Water
soluble compounds can be affected significantly by water films on such
surfaces (Burkhardt and Eiden, 1994). Our twin-cuvette system with the
automatic temperature regulated humidification system (ATRAHS) can support
experimental conditions to investigate such questions (see Fig. 6). As
demonstrated, the electrical surface conductance
Relationship between the measured stomatal conductance
Relationship between relative humidity and electrical leaf surface conductance.
With the presented twin-cuvette system it is possible to measure stable
O
Comparison of O
To investigate the uptake of trace gases by plant leaves under controlled
laboratory conditions, the simulation of environmental parameters such as
light, temperature, and relative humidity inside the cuvettes is essential
(Niinemets et al., 2011). For the non-stomatal deposition of O
During the long-term experiment a decreasing trend of the water flux was
observed over the measurement period, which became constant after 10 days.
This effect might be due to the acclimatization process of the plant by
changing the location from the greenhouse to the plant cabinet. From previous
studies it is known that plant samples need a significant amount of days to
adapt for changes in temperature, light intensity and relative humidity
(Kesselmeier et al., 1998). Also, as the measurement was performed in
October, the onset of winter stress could have played a role in the reduction
of the water flux with time as it also effects the transpiration rate (Oh and
Koh, 2014). A similar reducing trend could also be observed for the
deposition fluxes of O
By using a comparative measurement technique with a twin-cuvette system, we
are able to perform trace gas exchange measurements with high precision and
resolution of the measured trace gas mixing ratios. In our experimental
setup, it is possible to control multiple environmental parameters such as
light, temperature, trace gas mixing ratio and relative humidity inside the
dynamic cuvettes. The comparison of fluxes between a single and the twin –
cuvette system revealed an overestimation of fluxes by the single-cuvette
system for both O
The authors gratefully acknowledge financial support by the Max Planck
Society and by the German Science Foundation (DFG project HE 5214/4-1). We
thank Ivonne Trebs for initiating the research on O