High temporal resolution measurement of nitrate uptake from flowing solutions

Bioresource Technology 53 (1995) 113-123

0960-8524(95)00062-3

ELSEVIER

© 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/95/$9.50

HIGH TEMPORAL RESOLUTION M E A S U R E M E N T OF NITRATE UPTAKE FROM FLOWING SOLUTIONS D. Raj Raman The University of Tennessee, Department of Agricultural Engineering, PO Box 1071, Knoxville, TN 37901-1071, USA

Roger M. Spanswick Cornell University, Division of Biological Sciences, Section of Plant Biology, Plant Science Building, Ithaca, NY 14853-5908, USA

&

Larry P. Walker Cornell University, Department of Agricultural and Biological Engineering, Riley-Robb Hall, Ithaca, NY 14853-1701, USA (Received 6 December 1994; revised version received 24 April 1995; accepted 25 April 1995)

Abstract The nitrate (NO j-) uptake rates of intact, 23 day old rice plants were measured to determine the relationship between the plant's NOA- nutrition history and the NO j- uptake rate. A system for measuring NO juptake was designed, built and tested. Specific design goals, which were met, include: low handling shock to the plants, high measurement accuracy (4%), high temporal resolution (10 min) and minimal mass-transfer limitations to uptake. Important design factors were identtfied and the overall uncertainties in the reported measurements were computed. The observed uptake rates were dependent on the NOA- concentration ([NO j-]) to which the plants were exposed for the 24 h prior to testing; plants pretreated at higher [NO j-] had lower uptake rates from 200 p u NO j- solutions than plants pretreated at lower [NO j-].

understanding of the biological and physiochemical mechanisms underlying the treatment processes. Without this understanding, design guidelines have been vague, as is evident in the large scatter of hydraulic- and BOD-loading rates of existing wetland treatment systems (Reed & Brown, 1992). A rational design method is needed to make constructed wetland treatment systems more cost effective and reliable. Such a method would express pollutant removal rates as a function of environmental conditions and waste strengths and would require a knowledge of the process kinetics. Since Rogers et al. (1991) have demonstrated that, under certain circumstances, over 90% of the N removal can be accounted for by plant uptake mechanisms, a knowledge of the kinetics of N uptake will probably be one part of a rational design method. In wastewater, N occurs primarily in the mineral forms of ammonium (NH4+) and nitrate (NO3). Of this pair, the uptake kinetics of NO3 are of particular interest because NO3 is a pollutant of ground water and because microbiological processes on plant roots may convert NH2- to NO3 (Hammer & Bastian, 1989). Numerous methods for measuring NO3 uptake have been reported (Blom-Zandstra & Jupijn, 1987; Bloom & Chapin, 1981; Clement et al., 1974; Doddema & Telkamp, 1979; Glass et al., 1987; Goyal & Huffaker, 1986; Henriksen et al., 1990; Ingemarsson et al., 1987; Ingestad & L u n d , 1979; Lee & Drew, 1986; Mattsson et al., 1991; review by Wild et aL, 1987; Youngdahl et aL, 1982). They vary in their accuracy, temporal resolution, spatial resolution and invasiveness, the latter being of interest because mechanical disturbance of plants has been shown to

Key words: Nitrate, ion-uptake, measurement error, pretreatment, recirculating system. INTRODUCTION

Constructed wetlands are gaining popularity as a wastewater treatment technique, due to their low cost, low energy requirements and simplicity. In these systems, removal of organic carbon (typically measured as biochemical oxygen demand, or BOD) occurs through sedimentation and through the activity of aerobic and anaerobic microorganisms affiliated with plant roots, while nitrogen (N) removal occurs through microbial nitrification-denitrification, through plant uptake of N and through volatilization of ammonia (Brix, 1993). The design of these systems has been hampered by a lack of 113

114

D. Raj Raman, R. M. Spanswick, L. P. Walker

inhibit uptake for several hours (Bloom & Sukrapana, 1990). Although NO3 uptake can be determined by assaying N accumulation in plants over time (e.g. Lee & Drew, 1986), the bulk of investigations have used a depletion technique, in which the concentration drop of a solution in contact with plant roots is measured to determine the net NO3 uptake. Depletion techniques can be applied at the microscopic level, as in the microelectrode flux estimation technique (MFET), wherein the uptake rate is determined by measuring the concentration gradients in the unstirred layer around roots (Henriksen et al., 1992; Henriksen et al., 1990; Newman et al., 1987). The MFET is capable of making multiple, non-invasive uptake rate measurements over short time periods ( - 5 min resolution) and over small root segments (Henriksen et al., 1992). However, extrapolation of the locally measured uptake rate to the entire plant is problematic, due to the demonstrated variations in uptake rate along the root axis (Henriksen et al., 1992). Alternatively, depletion techniques can be applied at the gross level, either in a batch mode or in a flow-through manner, where the ion concentration is continuously measured by a variety of techniques (Bloom & Chapin, 1981; Clement et al., 1974; Doddema & Telkamp, 1979; Glass et al., 1987; Goyal & Huffaker, 1986; Henriksen et al., 1990; Ingemarsson et al., 1987; Ingestad & Lund, 1979; Mattsson et al., 1991; Youngdahl et al., 1982). A NO3 ion selective electrode (ISE) was used in this investigation for the following reasons: an ISE is relatively simple and of low cost; it has a logarithmic response, which allows accurate determinations of concentration in the range of 10--104#M;and it is capable of continuously sensing concentrations in flowing solutions, allowing the construction of a flow-through measurement system which minimizes handling shock. Regardless of the sensing technique used, a NO3 uptake measurement system must have high temporal resolution, because plants feedback-regulate NO~- uptake (Deane-Drummond, 1982; Doddema & Otten, 1979; Lee & Rudge, 1986; Mattsson et al., 1991; Siddiqi et al., 1989); i.e. NO~- uptake mechanisms appear to compensate for changes in the external NO£ supply. Feedback regulation of NO3 uptake implies that a fixed pair of kinetic parameters will probably not be applicable to wetland wastewater treatment systems, as the plant uptake systems will adjust to meet a nitrogen-demand which will presumably be dependent on growth rate, light availability and other environmental factors. Feedback regulation of NO3 uptake also implies that kinetic experiments should be conducted after a well-defined pretreatment period to minimize variations in plant NO£ content. Furthermore, since plants apparently adjust their uptake rates with time-scales of the order of 2-3 h (Doddema & Otten, 1979), feedback regulation of NO3 uptake

implies that an experimental determination of kinetic parameters should be done over a short time-scale, to avoid variations in the uptake system status while making a set of uptake rate measurements for estimating the parameters; this suggests maximizing the temporal resolution of the uptake measurement system. Even in a system with high temporal resolution, there remains the problem of mass transfer limitations to the root, which has been addressed by agitating the solution around the roots with a magnetic stirring device (Bloom & Chapin, 1981; Ingemarsson et al., 1987; Mattsson et al., 1991), through high flow-rates (Clement et al., 1974), or through aeration (Blom-Zandstra & Jupijn, 1987; Glass et al., 1987; Mattsson et al., 1991). Mass transfer limitations can cause the root surface [NO~-] to be significantly lower than [NO~-] in the bulk solution, leading to errors in the estimation of kinetic parameters. Stirring devices within the root chamber are problematic because they require a large fluid volume relative to the root mass, or they risk damaging the plant roots. High flow-rate systems can minimize mass transfer limitations, but measurement sensitivity is reduced because the solution passing through the roots is only slightly depleted. However, a recirculating, high flow-rate system reduces mass transfer limitations, while allowing significant depletion of the bulk solution, and was chosen for the current work. It is always important to estimate the uncertainty in the measurements made by an experimental apparatus. For example, if there is a high degree of uncertainty in the uptake rate measurement, it may be impossible to determine whether variations between replications reflect genuine differences between treatments, or simply imprecision in the measurements; if the uncertainty is low, then minor variations between treatments may be detected. In the case of depletion uptake measurement systems, determination of the uncertainty in the measurements is difficult for two reasons: first of all, as Henriksen et al. (1992) pointed out for the MFET, there is no device available with a constant NO~uptake rate which could be used to calibrate the measurement system by means of a standard; secondly, since the NO3 uptake rate depends upon the previous nutritional history of a plant, repetitive measurements of the uptake rate at a particular concentration are questionable, since exposing the plant to a particular concentration alters its nutritional history. These problems motivated the use of an error propagation calculation to determine the uncertainty in the experimental measurements. This paper reports on the development of a measurement system to quantify the NO3 uptake rate of intact rice plants in flowing solutions, with the ultimate goal of studying the kinetics of NO3 uptake by rice. The measurement system minimizes mass transfer limitations to NO3 uptake, has a high

Measuring nitrate uptakefrom flowing solutions temporal resolution to allow examination of the time-course of NO3 uptake and is capable of testing 16, 3-week-old rice plants at a time. The flow pattern in the plant chamber was analyzed, an estimate of the NO3 depletion between the bulk solution and the root surface was computed, the temporal response of NO3 uptake over several hours was observed to verify that the experimental technique did not excessively disturb the plants and preliminary experiments were performed to examine the effect of pretreatment [NO3] on steady-state NO3 uptake.

115

15 x 0.84 n u n dia. holes

METHODS Plant containers In order for a depletion uptake measurement system to have high temporal resolution, with low noise in the measurement, the ratio of root mass to total solution volume should be large (see Appendix). This goal suggests that the roots fit snugly into the test container. However, tightly packing roots into an experimental apparatus could easily damage them and would constitute a large handling shock; this problem was circumvented by using the same container for the growth, pretreatment and uptake measurement phases of an experiment. Each plant container had a nominal volume of 76 ml and was made of Plexiglas and stainless steel, to avoid phytotoxicity. The container consisted of two Plexiglas pieces: a shallow (1 cm) pentagonal basin with an opaque black bottom and clear sides (Fig. 1) and an opaque black top (Fig. 2). The clear sides enabled inspection of the container ports during cleaning and assembly; electrical tape was used to cover the sides to prevent light penetration. The top was secured to the basin after a thin layer of silicone grease was applied between the two pieces to form a watertight seal. Solution delivery was provided by a 15 × 0.84 mm hole manifold. Pieces of stainless steel tubing (0.64 cm OD), 2 cm in length, were epoxied into the inlet and outlet ports of the pentagonal basin to enable tubing attachment. The plants were mechanically supported by urethane foam cylinders inserted into 16 1.5-cm holes in the container top. Germination and growth Rice seeds (Oryza sativa L. cv IR-36, USDA Rice Experiment Station, Beaumont, TX) were germinated on paper soaked with 500 ~M CaSO4 for 2"5 days at 32°C; after germination, the 2.5 day old seedlings were folded in a piece of Teflon tape and placed in the center of a 1'8 cm dia × 1-3 cm cylinder of gray urethane foam, by means of a radial slit along the length of the cylinder. A seedling-containing foam cylinder was inserted into each of the 16 holes in the plant container and the loaded container was transported to the growth chamber in an insulated container.

Fig. 1. Plant container bottom. Fluid flowed into the manifold (top left corner), through the container and out of the port (bottom center). All dimensions are in cm.

16 x 1.5 cm dia. holes for ureathane foam cylinders that support one rice plant each ,.

I.ooo -II 0000 OOOO

Fig. 2. Plant container top. The top was made entirely of opaque, black, Plexiglas. The six small holes along the edge were for screws which secured the top to the bottom. All dimensions are in cm.

The growth chamber was maintained at 25°C and 16 h day-length and the photosynthetically active (400-700 nm) photon irradiance in the chamber varied from 150 /Jmol m -2 s -~ at the plant container surface, to 275 /~mol m -2 s ~ at the shoot tops of

116

D. Ray Raman, R. M. Spanswick, L. P. Walker

Table 1. Ion concentration in growth and experimental solutions. The high concentrations of SO~- and Na + in the experimental solutions reflect the use of Na2SO4 for ionic strength adjustment

Chemical species

Na +

NO; K÷ Ca 2 +

NH~HPO 2- and HEP04 Mg2+

so CIH3BO3 FeHEDTA Mn2+ and Zn 2÷ Cu2÷ and Mo species

Concentration in growth solution

(m)

Concentration in pretreatment and experimental solutions

0 1400 605 400 200 200 100 100.5 5 2.5 2 0.2 0-05

2000 x(variable) 200 50 + 0-5x 0 200 100 1150.5 5 2.5 2 0.2 0.05

22 day old plants. Twenty-eight liters of growth solution (1/10th strength modified Johnson solution, Table 1) was delivered to the plants at a rate of 0.1-0.2 1 min-1 chamber-1, by a dual reservoir solution delivery system inside the growth chamber. Fluid flowed out of the upper reservoir, through a PVC manifold, through black latex tubing, into the plant containers, then drained through black latex tubing into a PVC collection manifold and into the lower reservoir. When 4 1 of solution had flowed into the lower reservoir, a liquid-level sensing submersible centrifugal pump turned on for approximately 4 s, rapidly returning the solution to the upper reservoir. In this way the liquid level in the upper reservoir was maintained at 12-16 cm above the plant container(s) so that the supply pressure was held between 1.2 and 1.6 kPa. The growth solution was replaced weekly. Pretreatment

After 19.5 days in the growth chamber, the 22 day old plants were moved to the lab and connected to the pretreatment solution delivery system (the plants were deprived of recirculating solution and light for less than 10 min). Here, the plants received a complete nutrient solution, with N O 3 as the sole form of nitrogen (Table 1), for 22-24 h. The pretreatment [NO3] was 50, 200 or 800 p i . The large volume (38 1) of recirculating solution ensured that concentration changes during the 24 h pretreatment were insignificant. A constant flow-rate (2.1 ml s -1) gear pump circulated the fluid through the plant container. The fluid was not temperature controlled, but had come to equilibrium with the lab temperature, which varied from 20 to 27°C. Illumination was provided by a single 90 W halogen flood lamp located 40 cm above the plant container, filtered by 5 cm of water to reduce the heat load on the plants. The photosynthetically active (400-700 nm) photon irradiance varied from 50 pmol m -2 s -1 at the surface

(m)

of the plant container, to between 100 and 450 pmol m -2 s -1 at the shoot tops. A timer maintained the same photoperiod as in the growth chamber (05:00-21:00 h). Uptake measurement

Just prior to the start of uptake measurement, the plants were switched out of the pretreatment recirculating loop and placed in the measurement system (Fig. 3). The plant chamber was not drained or moved during this procedure. To minimize the shock to the plants the change was made as quickly as possible (ca. 2 min). Figure 3 illustrates the fluid flow pattern in the measurement system; elements shown in gray received 25°C water from a constant temperature recirculating water-bath to help maintain constant temperature conditions. The system had two operating states, which were characterized by different fluid flow paths: a 6"5 min depletion mode, during which solution recirculated through the system; and a 3.5 min flushing (or injection) mode, during which fresh solution entered the system while depleted solution drained from the system. Transitions between the two modes were made by simultaneously energizing solenoid valves V1 and V2. Solutions that were about to be injected were brought to 25°C by the preconditioner, a 900 ml PVC cylindrical vessel containing a stainless-steel heat-exchange coil and 5 cm long magnetic stir bar revolving at 1000 rpm. The [NO3] was monitored with a N O 3 ISE and reference electrode held in contact with the recirculating solution by means of a stainless-steel flow-through measurement cell. The ISE and reference electrode were connected to an instrumentation amplifier (WPI model 750, W-P Instruments, New Haven, CT) and the output of the amplifier was periodically recorded by a high resolution (3-1 /~V) data-acquisition and control (DAC) board (Data Translation Model DT2805/5716A,

Measuring nitrate uptake from flowing solutions

I I I

P

V

To drain

Fig. 3. System overview. Gray devices had provisions for temperature control and the dotted paths conducted fluid during the injection mode only. During the injection mode, V2 was open and V1 allowed fluid to flow from the preconditioner, through the condenser, plant container, ISE cell, pump and into the drain; this flushed the system with fresh solution. During the recirculating mode, V2 was dosed and V1 allowed fluid to flow from the pump, into the condenser and around the loop. Data Translation Inc., Marlboro, MA) in a personal computer (IBM-PC XT). To reduce electrical noise, a single-pole filter was placed at the input of the DAC board, effectively eliminating all frequencies above 20 Hz. The amplifier output was sampled at 500 Hz and groups of 100 samples were combined to compute an average value and a root mean square (rms) noise value for the signal. Every 15 s the EMF and rms noise values were stored to a file, along with the temperature of the recirculating fluid, which was sensed by means of a thermistor and bridge circuit. During the injection mode, the flow-rate of fresh solution into the system was dependent on the pressure at the preconditioner outlet. In order to match the solution outflow rate to the gear pump rate, the preconditioner could be pressurized up to 10 kPa. The pressure was controlled by varying the bleed rate from a pressure reservoir and the pressure was monitored with a water manometer. The flow-rate could be controlled precisely by observing the liquid level in the plant chamber and adjusting the pressure accordingly. The N O z uptake rate from 200 /~M NO3 solutions was measured at 10 min intervals for 6 h. At the end of an experiment, the volume of liquid in the plant chamber was determined and added to the known constant volume of the tubing, valves and condenser, to determine the system volume (V~ys). Shoots and roots were harvested and weighed (the root mat was dried by blotting with paper towels), then the biomass was dried at 70°C for at least 4 days and the dry weights were determined. The uptake experiments served two purposes: they estab-

117

lished the time course of uptake immediately after measurements began, to determine whether significant variations occurred due to the switch from pretreatment to experimental systems; and they determined whether a 24 h exposure to 50, 200 or 800 pM NO3 solutions affected the NOz uptake rate.

Data analysis The slope (m), relating the EMF across the electrodes to the logarithm of the concentration, was determined from a three-point calibration (50, 100 and 200 #M) at the end of an uptake experiment. The EMF measured just after an injection of fresh solution was used to compute the electrode intercept (Eo) for each injection. Once m and Eo were known, the raw EMF data were converted to concentrations by application of the Nernst equation:

C=colO (e-e°)/m

(1)

The rms noise in the EMF was converted to a variance in each concentration estimate and the depletion rate (dc/dt) was estimated by a weighted least-squares linear regression (Beck & Arnold, 1977), with the inverse of the variance in the concentration estimate (1/ac2) as the weighting factor (wi). This procedure minimized the effect of EMF measurements with high variance. However, because the ISE cell was immediately downstream of the plant container (due to hydraulic considerations; see Raman, 1994), there was a bias in the intercept determination due to the depletion of the solution and therefore a bias in the estimated value of dc/dt. This bias was eliminated by applying the following formula (Raman, 1994):

(dc/dt)*

(dr)

1+

(dc/dt)*

where (dc/dt)* denotes the depletion rate estimated from the raw data using the incorrect value of the intercept, dc/dt is the true depletion rate, Q is the volumetric flow-rate of solution through the plant container and co is the concentration of the injected solution. After applying eqn (2) to get the best estimate of the depletion rate, the uptake rate per unit dry weight (V) was computed according to the following formula:

F - ( dc/dt ) Wsys W

(3)

where W is the root dry weight. RESULTS AND DISCUSSION

Flow patterns in plant container The nature of the flow through the plant roots can be described by characterizing the plant container as

D. Raj Raman, R. M. Spanswick, L. P. Walker

118

one of three types of flow-through chemical reactor (Metcalf & Eddy, 1991). Reactors in which complete mixing occurs are called continuously stirred tank reactors (CSTRs), while reactors in which no mixing occurs are called plug-flow reactors (PFRs). Between these two extremes are arbitrary-flow reactors (AFRs), in which partial mixing occurs. The flow pattern in a plant container holding 23 day old rice plants was determined by measuring the effluent concentration from the reactor when a step change (from 500 to 1000 pM N O 3 ) was applied to the influent. When this was done, the effluent from the plant container changed exponentially (Fig. 4), as predicted for a CSTR (Weber, 1972). Furthermore, the time constant (z) of the exponential decay was within 2% of the hydraulic residence time of the container (0), as predicted for a CSTR (Weber, 1972). The CSTR-like behavior of the effluent concentration indicated a high degree of mixing in the container and suggests that all the roots receive essentially the same concentration of nutrient solution.

Error propagation Uncertainties in the EMF, slope, intercept, system volume and plant weight must all be taken into account when computing the uncertainty in the uptake rate. We have chosen to use the standard deviation (o-) to describe the uncertainty; the notation o-x(y) will refer to the standard deviation of x due to the uncertainty in y. When it was not possible to determine the standard deviation in a measurement by repeated measurements and standard statistical methods, the standard deviation was estimated by dividing the estimated maximum error by three. This decision was based on the fact that 99.7% of all measurements are predicted to fall within 3o- of the mean. The standard deviations in the measured quantities are listed, with explanations, in Table 2. Once the standard deviations in the raw data were known, the standard deviations in the computed quantities were found by applying the following equation (Kline & McClintock, 1953):

O-N=/i~=I(

ON~ 2

(4)

o-ui ~U i }

where N is a function of Ul, u2 .... u,,. The following equation was used to compute the standard deviation in the regression slope estimate (Beck & Arnold, 1977): O.slope = [ X W i X 2 __~ XWiXi] -- 1/2

(5)

Because all the concentrations in a particular depletion run were calculated with the same estimated slope and intercept, the errors in the concentration were correlated, not uncorrelated. Simply using eqn (5) to assign an uncertainty to each individual concentration estimate ignores this fact. The proper way to include slope and intercept uncertainties in the

1000

9oo

~6oo 5OO 400

I

0

I

I

1

I

2 Time (min.)

I

I

3

i

I

4

4. Effluent concentration from plant chamber. The concentration changes as a rising exponential, as predicted for a CSTR. Fig.

error propagation model is to determine the effect of slope and intercept on the entire dc/dt estimate. The following equations were used to include the effects of aeo and o-m in the total uncertainty (for derivation, see Raman, 1994): o- dc/dt(m )--

O'dc/dt(Eo) -- -

m

-

m

(6)

(7)

The standard deviation in the electrode EMF (aE) ranged from 0.1 to 0.2 inV. The standard deviation in the injection solution concentration was determined to be 0.014 times the concentration of injected solution (i.e. g=0.014). By examining the raw EMF data, aeo was estimated as 0.33 inV. On the basis of the scatter in m values observed between experiments, o-m was estimated as 1 mV/ decade. The maximum possible volume error was estimated as + 5 ml, making O-vs =1.7 ml. Taking these factors into account, the total standard deviation of the uptake rate estimate was approximately 3.5% of the mean.

Uptake rate measurements and evidence for regulation of uptake Sample data from the 2 h of uptake measurements after the switch from pretreatment to experimental configurations are given in Fig. 5. In this case, plants which had been pretreated for 22 h in 50 pM N O r were provided with 50 pM N O r for the first three injections and 200/AM N O r thereafter. The fourfold increase in [NO f ] resulted in a 2.5-fold increase in the N O f uptake rate (see Fig. 5), indicating a nonlinearity in the relationship between N O r uptake rate and [NO f ] in the range studied. The increase occurred immediately and the N O r uptake rate stayed relatively constant during the 40 min imme-

Measuring nitrate uptake from flowing solutions

119

Table 2. Standard deviations in measured quantities used in computing kinetic parameters

Measured quantity

Symbol

ISE/Reference EMF (E)

~rE (mV)

Injection solution concentration (Co)

O'CI =O~¢ 0 (IIM)

Intercept EMF for fresh solution (Eo)

aL-,, (mV)

System volume (Vsy~) Root dry weight (W) Slope (m)

0-V,ys (ml) aw (g) ffm

1.40 •

ll l l i

1.20 •

Comments Determined by taking 100 samples over 200 ms and computing rms noise Found to be essentially proportional to Co, value determined from knowledge of pipette and volume uncertainties Based on maximum error apparent from graphs of raw EMF vs t data Estimate maximum possible error and divide by 3 Estimated balance accuracy; ~rw considered negligible Estimated from scatter in slope determinations between experiments

i|lll

1.00 &

0.80 0.60

i

0.40

= 0.20

"L

0.00 10:00

from S0to20e~M . 10:30

~ , 11:00

. 11:30

12:00

15 ~ i - ~ ' ~ ' ~ ' ~ ' ~ ' ~ ' ~ 0 200 400 600

800

1000

Pretreatment nitrate ¢oncuaU-ation

Time 0~:mm a,m.)

Fig. 5. NO3 uptake for first 2 h after switching from pretreatment to experimental set-up. Plants were pretreated in 50/~M NO3 and were tested in 50/~M NO3 for the first three uptake rate determinations, after which time they were tested at 200 #M NO3. The uptake rate was virtually constant immediately after the switch from 50 to 200/ZM.

diately after the switch to 200 p u N O 3 ; apparently the transition had not caused a temporary depression or acceleration of uptake. The experiments allowed observation of the N O 3 uptake rate from solutions containing 200 # u N O 3 . In some experiments, the uptake measurements began with the plants exposed to the pretreatment concentration (as shown in Fig. 5). However, in all the steady-state experiments, plants were exposed to 200 #M N O 3 within 1 h of the start of the uptake rate measurements. It was therefore possible to compare the N O 3 uptake rates upon first exposure to 200 #M NO~-, to examine the effect of the pretreatment [NO3] on net N O 3 uptake. Figure 6 shows the initial uptake rate in 200 #M solutions as a function of pretreatment N O 3 concentration. The data clearly show a relationship between the pretreatment N O 3 concentration and the N O 3 uptake rate at 200 /ZM. Specifically, the uptake rate from 200 pM solutions appears to be inversely related to the pretreatment concentration, as would be expected if net N O 3 uptake is feedback regulated.

Fig. 6. Effect of pretreatment IN03] on the initial NO3 uptake rate from 200/~u solutions. Data shown is from 13 steady-state experiments, with pretreatment [NO3] of 50, 200 and 800 #M; re=0-67.

These experiments also showed that uptake rates under steady-state conditions varied 20-40% over the course of 6 h. However, the variation during 1 h was typically much lower (ca. 10%). The changes in uptake rate suggest that the plant status changes over the course of several hours, while the lack of variation over 1 h suggests that N O 3 uptake kinetics might be measured on a 1 h time-scale. The large amount of scatter in Fig. 6 reflects biological variations between sets of plants and problems in some of the experimental protocols, such as the limitedand spatially-variable-illumination; improvements in the lighting system are recommended. However, the results suggest that the measurement system is capable of rapid, accurate, measurements of N O 3 uptake, with minimal disturbance to the plants. Concentration differences between the bulk solution a n d t h e root s u r f a c e

To validate this system for uptake kinetic measurements, it is necessary to demonstrate that mass transfer to the root does not significantly limit N O 3 uptake, i.e. that the difference between the bulk solution concentration (Cb) and the solution at the root surface (cs) is not excessive. This concentration

D. Raj Roman, R. M. Spanswick, L. P. Walker

120

Table 3. Parameter values for analyzing the errors due to depletion near the root surface

Parameter

Value (unit)

Jm~, Km

Source

e

0"7 pmol m - 2 50 pM 0"75 X 10 -3 m 0.92

V Di

10 - 6 m E s -1 1.9 × 10 - 9 m E s -1

v'

2"5 xl0 -z m s -1

dp

Estimated from Vm~xand root diameter From kinetic experiments - - see text Estimated from root diameter - - see text Estimated from root mass Perry and Chilton (1973) Perry and Chilton (1973) Estimated - - see text

S -1

difference (Ac) cannot be measured directly, but can be estimated by assuming that the root container behaves like a packed-bed reactor, for which the relationship between Ac and several important variables have been described (e.g. Geankoplis, 1983). This approach was used, but the results must be interpreted with caution, because unlike a typical packed bed, the roots constitute a relatively small part of the container volume (ca. 8%) and because other phenomena, such as the high injection velocity into the container, greatly affect the flow pattern in the container. The following analysis uses the packed-bed equations, but suggests that one variable in particular, the superficial bed velocity, be calculated in a manner different from standard practice. In a packed-bed reactor, Ac can be related to the mass transfer coefficient (k') and the flux of ions to the root surface (Ji) as follows (Geankoplis, 1983):

Ji=k~Ac

JmaxC~

(9)

where Jm~x is the maximum flux rate and Km is the half-velocity concentration. Combining eqns (8) and (9) and solving for Ac yields: Ac-

Jmax

Cs

k'~(Km +Cs)

(10)

Dividing the preceding equation by cs and multiplying by 100 gives the percent concentration increase from the root surface to the bulk solution, which may be thought of as an error term: % error-

100Jmax

k" (Km + Cs)

~v'

= 2.5

mm/s " " v'=25

t

,0

7 ho

3 0 ' ~

mm/s

0

I00

2OO

3OO

cmcmtratim Fig. 7. Percent difference between root surface concentration and bulk solution concentration, as a function of root surface concentration, from eqns (11) and (12) with parameter values in Table 3. Middle curve (dashed line) corresponds to most realistic case.

(8)

At steady-state, the flux of ions towards the root surface is equal to the ion flux into the root. Assuming that the uptake kinetics can be described by a Michaelis-Menten model, Ji can be written as:

Ji--Km+Cs

50'

(11)

An expression for k" can be found by assuming the plant root container to be a packed-bed reactor, with the roots constituting the packing and combining several applicable equations from Geankoplis (1983):

k'=

1.09

v'Sc -2/3Re -2/3

(12)

/3

where v' represents the superficial bed velocity (m s - l ) , /3 represents the void fraction (dimensionless), Sc represents the Schmidt number (dimensionless) and Re represents the packed-bed Reynolds number (dimensionless). The Schmidt number is defined as follows: Sc = vD;-1, where o is the kinematic viscosity (m 2 s -1) of the solution and Di is the diffusion coefficient of the molecule in question (m 2 s - l ) , while Re=dpv'O -1, where dp is the effective particle diameter of the bed packing (m). By using the parameters listed in Table 3, eqn (11) was evaluated over a range of cs values to generate the curves shown in Fig. 7. The three curves correspond to low, medium and high estimates of v'. The low estimate of v' (2.5 x 10 -3 m s -1) is found by assuming plug flow through the root container, in which case v' equals the volumetric flow-rate divided by the cross-sectional area of the container. This is the strictly correct way of estimating v'. The high estimate of v' (250 x 10 -3 m s -1) is the velocity of the liquid as it comes out of the orifices in the injection manifold, a value which represents an upper bound on the actual average velocity. Since

Measuring nitrate uptake from flowing solutions testing demonstrated that the solution in the plant container was not plug flow in nature, but was well mixed, a realistic estimate of v' might be 25 x 10 -3 m s -a, an order of magnitude greater than the minimum possible velocity and an order of magnitude less than the maximum. The values of Jmax were based on the uptake rates observed for rice plants during this investigation, along with an estimate of the average root diameter (0.5 mm) and the assumption that ion uptake occurred equally over all parts of the root system, while the value of Km was taken from observations of uptake kinetics, which are reported in detail elsewhere (Raman et aL, 1994). Since dp is defined as the diameter of a spherical particle which has the same surface area to volume ratio as the bed-packing material, dp=l.5 droot for long cylindrical roots. From Fig. 7, it is evident that the depletion error at low concentrations approaches 20%. At 200 /~M, the error is already less than 5%. These conservative estimates show that the depletion caused by mass transfer limitations is probably less than 20%, even at low concentrations. CONCLUSIONS Pretreatment [NO3] affects subsequent N O 3 uptake in a manner consistent with a feedback regulation process, yet the effects of this regulation on the kinetics of N O 3 uptake are not evident. Analysis of the measurement system suggests that it possesses the desirable qualities of high temporal resolution, high accuracy, minimal handling shock and low mass transfer resistance necessary to perform kinetic investigations. An accompanying paper describes such experiments, as well as experiments exploring the effects of light deprivation on N O 3 uptake. ACKNOWLEDGEMENTS We would like to thank Doug Caveney for his help in fabricating the plant containers, ISE-cell and preconditioner, and Gordon Henriksen for his help with the plant growth protocols. Partial funding for this project came from U S D A grant 88-38420-3836.

REFERENCES Asher, C. J., Ozanne, P. G. & Loneragan, J. F. (1965). A method for controlling the ionic environment of plant roots. Soil Science, 100, 149-56. Beck, J. V. & Arnold, K. J. (1977). Parameter Estimation in Engineering and Science. John Wiley & Sons, New York. Ben-Yaakov, S. & Ben-Asher, J. (1982). System design and analysis of a continuous monitoring of the environment in nutrient solution culture. J. Plant Nutrition, 5, 45-55. Blom-Zandstra, M. & Jupijn, G. L. (1987). A computercontrolled multi-titration system to study transpiration, O H - efflux and nitrate uptake by intact lettuce plants

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(Lactuca sativa L.) under different environmental conditions. Plant, Cell Environ., 10, 545-50. Bloom, A. J. (1989). Continuous and steady-state nutrient absorption by intact plants. In Application of Continuous and Steady-state Methods to Root Biology (ed. J. G. Torrey & L. J. Winship). Kluwer Academic Publishers, Dordrecht, pp. 147-63. Bloom, A. J. & Chapin III, F. S. (1981). Differences in steady-state net ammonium and nitrate influx by coldand warm-adapted barley varieties. Plant Physiol., 68, 1064-7. Bloom, A. J. & Sukrapana, S. S. (1990). Effects of exposure to ammonium and transplant shock upon the induction of nitrate absorption. Plant Physiol., 94, 85-90. Brix, H. (1993). Wastewater treatment in constructed wetlands: system design, removal processes and treatment performance. In Constructed Wetlands for Water Quality Improvement (ed. G. A. Moshiri). Lewis Publishers, Boca Raton, Florida, pp. 9-22. Caldwell, C. D., Le Fevre, P. E. & Aikman, D. P. (1978). An open circuit apparatus for continuous determination of net ion uptake by seedlings grown hydroponically. Canadian J. Botany, 56, 2767-72. Clement, C. R., Hopper, M. J., Canaway, R. J. & Jones, L. H. P. (1974). A system for measuring the uptake of ions by plants from flowing solutions of controlled composition. J. Experimental Botany, 25, 81-99. Deane-Drummond, C. E. (1982). Mechanisms for nitrate uptake into barley (Hordeum vulgare L. cv. Fergus) seedlings grown at controlled nitrate concentrations in the nutrient medium. Plant Sci. Lett., 24, 79-89. Doddema, H. & Otten, H. (1979). Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate: III. Regulation. Physiologia Plantarum, 45, 339-46. Doddema, H. & Telkamp, G. P. (1979). Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate: II. Kinetics. Physiologia Plantarum, 45, 332-8. Geankoplis, C. J. (1983). Transport Processes and Unit Operations, 2nd Edn. Allyn and Bacon, Newton, MA, USA. Glass, A. D. M., Saccomani, M., Crookall, G., & Siddiqi, M.Y. (1987). A microcomputer-controlled system for the automatic measurement and maintenance of ion activities in nutrient solutions during their absorption by intact plants in hydroponic facilities. Plant, Cell Environ., 10, 357-81. Goyal, S. S. & Huffaker, R. C. (1986). The uptake of nitrate, nitrite and ammonium by intact wheat (Triticum aestivum) seedlings: I. Induction and kinetics of transport systems. Plant Physiol., 82, 1051-6. Hammer, D. A. & Bastian, R. K. (1989). Wetlands ecosystems: natural water purifiers? In Constructed Wetlands for Wastewater Treatment." Municipal, Industrial and Agricultural (ed. D.A. Hammer). Lewis Publishers, Chelsea, Michigan, pp. 5-19. Henriksen, G. H., Bloom, A. J. & Spanswick, R. M. (1990). Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. Plant Physiol., 93, 271-80. Henriksen, G. H., Raman, D. R., Walker, L. P. & Spanswick, R. M. (1992). Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes: II. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique. Plant Physiol., 99, 734-47. Ingemarsson, B., Oscarson, P., Ugglas, M. & Larsson, C. M. (1987). Nitrogen utilization in Lemna: II. Studies of nitrate uptake using 13NO3. Plant Physiol., 85, 860-4.

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D. Raj Raman, R. M. Spanswick, L. P. Walker

Ingestad, T. & Lurid, A.-B. (1979). Nitrogen stress in birch seedlings: I. Growth technique and growth. Physiologia Plantarum, 45, 137-48. Kline, S. J. & McClintock, F. A. (1953). Describing the uncertainties in single-sample experiments. Mech. Engng, 75, 3-8. Lee, R. B. & Drew, M. C. (1986). Nitrogen-13 studies of nitrate fluxes in barley roots. J. Experimental Botany, 37, 471-86. Lee, R. B. & Rudge, K. A. (1"986). Effects of nitrogen deficiency on the absorption of nitrate and ammonium by barley plants. Annals of Botany, 57, 471-86. Mattsson, M., Johansson, E., Lundborg, T., Larsson, M. & Larsson, C. M. (1991). Nitrogen utilization in N-limited barley during vegetative and generative growth: I. Growth and nitrate uptake kinetics in vegetative cultures grown at different relative addition rates of nitrate-N. J. Experimental Botany, 42, 197-205. Metcalf & Eddy, Inc. (1991). Wastewater Engineering: Treatment, Disposal and Reuse, 3rd Edn. McGraw-Hill, New York. Newman, I. A., Kochian, L. V., Grusak, M. A. & Lucas, W. J. (1987). Fluxes of H + and K + in corn roots: characterization and stoichiometries using ion-selective microelectrodes. Plant Physiol., 84, 1177-84. Perry, R. H. & Chilton, C. H. (1973). Chemical Engineers' Handbook, 5th Edn. McGraw-Hill, New York. Raman, D. R. (1994). The kinetics of nitrate uptake from flowing solutions by rice: influences of pretreatment nitrate concentration and irradiance. Ph.D. thesis, Faculty of the Graduate School, Cornell University. Raman, D. R., Spanswick, R. M. & Walker, L. P. (1995). The kinetics of nitrate uptake from flowing solutions by rice: influence of pretreatment and light. Biores. Technol., 53, 125-32. Reed, S. C. & Brown, D. S. (1992). Constructed wetland design - - the first generation. Water Environ. Res., 64, 776-81. Rogers, K. H., Breen, P. F. & Chick, A. J. (1991). Nitrogen removal in experimental wetland treatment systems: evidence for the role of aquatic plants. Res. J. Water Pollut. Control Fed., 63, 934-41. Siddiqi, M. Y., Glass, A. D. M., Ruth, T. J. & Fernando, M. (1989). Studies of the regulation of nitrate influx by barley seedlings using laNO 3 . Plant Physiol., 90, 806-13. Weber Jr, W. J. (1972). Physiochemical Processes for Water Quality Control John Wiley & Sons, New York. Wild, A., Jones, L. H. P. & Macduff, J. H. (1987). Uptake of mineral nutrients and crop growth: the use of flowing nutrient solutions. Adv. Agronomy, 41, 171-219. Youngdahl, L. J., Pacheco, R., Street, J. J. & Vlek, P. L. G. (1982). The kinetics of ammonium and nitrate uptake by young rice plants. Plant Soil, 69, 225-32.

APPENDIX On the relationship between system volume and signal to noise ratio The accuracy of a depletion measurement system depends primarily on two factors: the sensitivity of the measurement device to changes in concentration and the amount of concentration change caused by the plants in the system. For such systems, the signal may be defined as (dE/dt)o, the rate of change of the EMF across the ISE and reference electrode caused by the total net uptake (¢). The noise in

such systems is ascribable to drift in the electrode response, which may be designated (dE/dt)drift. The signal to noise (S/N) ratio for such systems can then be written as follows:

S

(dE/dt)o

N

(dE/dt)drift

(A1)

To relate (dE/dt)o to physical quantities, the chain rule is applied: (d~_t)¢ = dEdc dCdt

(A2)

where c represents the concentration of N O ; in solution and dc/dt represents the depletion rate. The depletion rate is a function of the total uptake rate and the system volume (Vsys), per the following equation: dc

¢

dt

Vsys

(A3)

while dE/dc may be found by differentiating the Nernst equation [eqn (A4)] with respect to c: E =Eo + 2"3m In c dE

2.3m

dc

c

(A4)

(AS)

where m is the electrode slope and Eo is the intercept voltage. Combining the previous four equations yields an expression for S/N (a negative sign has been dropped since only the magnitude of S/N is of interest): S

2.3m~b

N

cVsys(dE/dt)drift

(A6)

For the system described in this work, typical values of the variables in eqn (A6) were: m = 5 5 mV/decade, ¢ = 1 pmole/min, c=200 #M, Vsys=0"12 1 and (dE/dt)drift=0.15 mV/min, leading to a S/N of 35. In general, eqn (A6) reveals that S/N decreases at high concentrations, at large volumes and at high drift rates, while S/N increases with increases in the total uptake rate or electrode slope (sensitivity). As concentration decreases and approaches the ISE's limit of detection, the decrease in slope tends to decrease S/N, but this effect is counteracted by the inverse relationship between concentration and S/N. These counteracting effects make it impossible to draw general conclusions regarding the variation of S/N with concentration, in this range. Most important, from the point of view of designing an experimental system, is that a decrease in the system volume increases the signal to noise ratio. However, two components comprise the system volume, the vol-

Measuring nitrate uptakefrom flowing solutions ume associated with the plant container (Vp) and the volume associated with the other components of the measurement system (Vm) - - the volume of valves, tubing, condenser and pump. Because Vm is essen-

123

tially fixed, attempts to increase S/N by only minimizing Fp will not work. Instead, the ratio of ~b (which presumably increases with plant root biomass) t o Vsys should be maximized.