Plant and Soil 266: 343354, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

343

Control of nutrient solutions for studies at high pH

Peter M. Kopittke1,2 & Neal W. Menzies1
1 Centre 2 Corresponding

for Mined Land Rehabilitation, University of Queensland, St. Lucia, Queensland, Australia, 4072. author

Received 12 March 2003. Accepted in revised form 11 March 2004

Key words: alkaline, high pH, hydroxyl ion, ion exchange resin, root growth, seedling nutrient requirement

Abstract Little is known about factors effecting plant growth at high pH, with research often limited by the inability to separate nutritional deciencies and HCO toxicity from the direct limitations imposed under high pH conditions. 3 Various methods of controlling dilute nutrient solutions for studies at high pH were investigated. For short-term studies, it was found that a solution without Cu, Fe, Mn and Zn and aerated with CO2 depleted air, greatly reduced nutrient precipitation at high pH, thus eliminating nutritional differences between treatments. Manual pH adjustment and the use of ion exchange resins as pH buffers were unsuitable methods of pH control. However, pH control by automated titration had little effect on solution composition while maintaining constant pH. The system described is suitable for studies in which the pH of the bulk nutrient solution must be maintained. The system was used to examine OH toxicity in mungbeans (Vigna radiata (L.) Wilczek cv. Emerald), with root length reduced at a bulk solution pH of 8.5 and greater. Abbreviations: DI deionised; EC electrical conductivity; ICPAES inductively coupled plasma atomic emission spectroscopy; LSD least signicant difference.

Introduction Although there has been much research into soil acidity, little work has investigated the effect of alkalinity on plant root growth (Tang et al., 1993b). The inability to separate the direct effects of alkalinity (hydroxyl ion (OH ) toxicities) from indirect effects (nutritional disorders and bicarbonate (HCO ) toxicity) in solution 3 culture experiments has restricted research. Plant growth at high pH is limited indirectly both by nutritional disorders and HCO toxicity. Iron de3 ciency (lime-induced chlorosis) is the most common nutritional disorder of plants growing on alkaline soils and is caused both by low Fe availability and by elevated concentrations of HCO (Marschner, 1995; 3 Russell and Wild, 1988). The availability of other nutrients such as Ca, Zn, P and Mn, is also low in alkaline conditions due mainly to adsorption and precipitation processes (Parker and Walker, 1986; Srivastava and
FAX No: +61-7-3365-3452. E-mail: p.kopittke@uq.edu.au

Sethi, 1981). Plant growth in many species is limited by excess HCO . Although the mechanisms are not 3 fully understood, it is believed that HCO adversely 3 effects the root plasma membrane resulting in root growth inhibition (Alhendawi et al., 1997). However, the addition of HCO in low concentrations has been 3 observed to increase growth (Bialczyk et al., 1994; Cramer and Lips, 1995). The direct effect of alkalinity, the high OH concentration, is thought to limit root growth, although the concentration at which this toxicity develops is unknown. Tang et al. (1993b) observed that the elongation of the roots of Lupinus angustifolius L. was particularly sensitive to increases in pH and that this decrease in root elongation was caused primarily by a decrease in cell elongation rather than cell division. The formation of lateral roots and root hairs is also delayed in high pH soils (Canmore-Neumann et al., 1997; Tang et al., 1993a).

344 Previous studies at high pH have often failed to separate the growth limitations caused by nutritional disorders and HCO from the direct effect of OH . 3 Fuller and Richardson (1986) concluded that the aluminate ion (Al(OH) ) was toxic to root growth even 4 though the precipitation of P and Mg from the nutrient solution resulted in aluminate treatment plants having shoots with tissue P and Mg concentrations signicantly lower than the control. Kinraide (1990), also investigating aluminate toxicity, buffered nutrient solutions with HCO at possibly toxic concentrations 3 (5.9 mM). The presence of HCO could also result 3 in the precipitation of nutrient cations as carbonates from the nutrient solution in the high pH treatments. Investigating the effects of high pH on root growth, Peiter et al. (2001) used a complete nutrient solution at pH values between 5.0 and 8.0. In the higher pH treatments, the availability of Fe, Cu, Mn and Zn (although not measured) would most likely have been lower than in the control. Therefore, the observed reduction in root length at these higher pH values could not, with certainty, be attributed to OH toxicity. Solution pH control was also poor, with the pH 8.0 treatment varying between pH 6.5 and 8.5 over the duration of the investigation. In the present study, we sought to develop a solution culture system to control plant growth conditions in the bulk nutrient solution, thus allowing the investigation of constraints to plant growth at high pH (such as aluminate toxicity) without nutritional or HCO 3 growth limitations. The use of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald) in short-term solution culture systems with incomplete nutrient solutions was examined. The pH and nutritional changes in unbuffered, dilute nutrient solutions at high pH were investigated, and the use of ion exchange resins and automated pH titration as methods of solution pH control evaluated. Experiment 1 Seedling nutrient requirement This experiment aimed to establish if mungbean seedlings require nutrients, other than Ca and B, during a short-term growth experiment. The experiment was conducted as a completely randomised design (CRD) with two treatments (complete and simple nutrient solutions) and ten replicates. Polypropylene containers (22 L; 265 mm diameter by 400 mm deep) were lled with 21 L of nutrient solution. The complete solution comprised (M) 470 N; 700 Ca; 256 Cl; 500 S; 133 K; 50 Mg; 5 Fe (Fe(III)-EDTA); 3 P; 0.2 Mn; 0.16 B; 0.10 Zn; 0.04 Cu; 0.01 Mo, while the simple solution contained (M) 700 Ca, 1400 Cl, and 0.16 B. To minimise light penetration into the root chambers (Schwarz and Schneider, 1985), all containers and lids were covered with three coats of metallic paint. Mungbean seeds (Vigna radiata (L.) Wilczek cv. Emerald) were imbibed in aerated 200 M CaSO4 solution for 2 h following seed surface sterilisation (40 mM sodium hypochlorite (NaOCl) for 15 s). Seeds were then placed on germination trays lined with moistened (200 M CaSO4 ) tissue paper and maintained at 25 C for 48 h. The aeration rate through the nutrient solutions was held constant at approximately 10.0 mL s1 per pot. The CO2 was rst removed by passing the air through two PVC bubbling columns (150 mm diameter by 1.75 m long), the rst containing 20 L 0.5 M NaOH and the second 20 L DI water. Uniform airstream dispersion was achieved through the use of eight aquarium air stones in each of the columns. Both the NaOH and DI water were replaced daily. Solutions were aerated for 24 h prior to planting. Four seedlings with radicle lengths of 10 1 mm were transferred to each of the containers with solution pH adjusted to pH 6.0 using saturated Ca(OH)2 (approximately 0.03 M) or 0.01 M HCl. Solutions were replaced every 3 d with replacement solutions also aerated for 24 h before use. Electrical conductivity (EC) was measured daily. Measurements of root length (to the nearest mm) were taken daily for 11 d. Solution pH was also monitored daily and adjusted to 6.0 (0.10), and unusual growth characteristics noted. After 11 d of growth, plants were harvested and shoot elemental concentrations determined. Tissue N concentrations were measured as detailed by Rayment and Higginson (1992) using a LECO CNS 2000, with all other elements determined by inductively coupled plasma

Materials and methods All experiments were conducted in a controlled environment glasshouse. High pressure sodium lamps were used to supplement natural sunlight providing 16 hours of light per day with temperatures maintained at 30 C during the light period and 25 C during the dark (Imrie and Lawn, 1990).

345 atomic emission spectroscopy (ICPAES) after acid digestion as described by Martinie and Schilt (1976). Root lengths were analysed in GenStat 6 (GenStat 2002) as a CRD using a repeated measures analysis. Comparisons between means were made using Fishers protected least signicant difference (LSD) test. The variance ratios and LSDs for the time and interaction terms were adjusted for the degree of autocorrelation between times by the Greenhouse-Geisser epsilon (Greenhouse and Geisser, 1959). Mungbean tissue nutrient concentrations were analysed using one-way analysis of variance (CRD) and treatment means compared using LSDs as above. Experiment 2 pH changes in unbuffered nutrient solutions at high pH At high pH, the introduction of CO2 to the nutrient solution will cause precipitation of CaCO3 and a pH reduction. This experiment evaluated the effects of aeration stream quality (CO2 concentration) and plant growth on nutrient solution pH. In order to remove a greater proportion of the CO2 from the air, the use of a molecular sieve (4, 8 12 mesh, Sigma) was investigated. Before use as an aeration stream, air was passed sequentially through 20 L of 0.5 M NaOH, 25 L of DI water, 5 kg silica gel (510 mesh) and nally 1.5 kg molecular sieve. The air was passed through the molecular sieve at a pressure of 35 kPa for maximum CO2 removal (as per manufacturers guidelines). A diaphragm regulator then reduced the air pressure such that the aeration rate remained 10.0 mL s1 per pot. Solutions were aerated for 24 h before being raised to pH 9.0 for 48 h. The experiment was run both with and without plants; each with solution pH adjusted every 12 h. The 0.5 M NaOH solution and DI water were replaced daily and the molecular sieve regenerated in a vacuum oven before beginning the trial. Samples were taken from the aeration stream, after passing through the columns, and analysed for CO2 using gas chromatography (Shimadzu GC-17A with 1.8 m 3 mm column using Porapak N 80100 mesh, He carrier gas at 30 mL min1 , an oven temperature of 65 C, and a thermal conductivity detector). Experiment 3 Ion exchange resins as buffers This experiment evaluated the effectiveness of two anion exchange resins as pH buffers in the pH 6 to 10 range. Samples of Dowex Type 1 (1X8, 100 200 mesh, Aldrich) and Bio-Rex 5 (100200 mesh, Bio-Rad) exchange resins (15 g) were packed into separate columns and rinsed with methanol until the efuent from each was clear. Five volumes of DI water were then passed through the resins. They were then converted to their OH form by passing 20 volumes of 1 M NaOH through the columns followed by ve volumes of DI water (or until the efuent pH < 9.0). A sub-sample of each resin (0.5 g oven dry equivalent) was then mixed with 15 mL 1.5 mM KCl and titrated with standardised 0.1018 M HCl. Solutions containing 0.5 g of the Dowex Type 1 resin in both the OH and Cl forms in ratios of 85:15 and 50:50 (OH :Cl ) were mixed and titrated. The Dowex Type 1 resin was converted back to the Cl form using 20 volumes of 1 M NaCl followed by ve volumes of DI water. Acetate, bicarbonate and phosphate forms of the resin were formed using 20 volumes of 0.5 M solutions of ammonium acetate (C2 H7 NO2 ), ammonium bicarbonate (NH4 HCO3 ), and ammonium dihydrogen orthophosphate (NH4 H2 PO4 ) respectively, each raised to pH 9.7 with NH4 OH. Each of these resin forms was then titrated as above. Samples of solutions from the phosphate form were taken periodically throughout the titration and solution phosphate concentration measured spectrophotometrically (Motomizu et al., 1983). Experiment 4 pH control by automated titration The experimental system as detailed in Experiment 1 was used to determine the suitability of automated pH titration units for maintaining nutrient solutions at high pH. Five 22-L polypropylene containers were lled with 21 L of complete nutrient solution. After an initial 24 h aeration, each container was connected to a separate pH titration unit (TPS, miniCHEM-pH) and peristaltic pump (Masterex 5 RPM with Masterex Tygon tubing, L/S 17) which was used to raise and maintain the pH at 9.0 by the addition of saturated Ca(OH)2 solution (approximately 0.03 M). The air intake of the Ca(OH)2 reservoir was tted with a soda lime trap to remove CO2 . The solutions were maintained at pH 9.0 without plants for a total of 8 d and were not renewed during this period. Samples were taken for analysis immediately after mixing of the solution, after the initial 24 h of aeration, and then daily. Samples were ltered using a 0.22 m lter (Millipore GSWP) and analysed for B, Mg, P, S, K, Ca, Mn, Fe, Cu, Zn and Mo by ICPAES (or by ICPMS if below the detection range). Solu-

346 tion EC was measured daily and each pH electrode calibrated every 3 d. The solutions were then replaced and aerated for 24 h before being raised to pH 9.0 by the titration units. Four seedlings (see Experiment 1) were then transferred to each container. The solutions were again maintained at pH 9.0 for 8 d without being renewed. All measurements and observations were performed as described above. PhreeqcI 2.6 (Parkhurst, 2002) (a low temperature, aqueous, thermodynamic geochemical model), using the Minteq database, was used to calculate the expected change in nutrient availability over time in complete solutions aerated by gas with a CO2 partial pressure of 0.35 Pa and atmospheric O2 . A series of gas additions were made (864 L each, representing one day of aeration) to the initial solution. Solution pH was xed through the use of Ca(OH)2 . The use of chelating agents (EDTA and DTPA) to prevent nutrient precipitation at high pH was also examined using GEOCHEM-PC 2.0 (Parker et al., 1995). Experiment 5 Effect of high pH on root growth An experimental system using unbuffered nutrient solutions at high pH was examined to determine if the indirect growth limitations imposed on plants at high pH can be separated from the direct effect of high pH (OH ). The experiment was conducted as per Experiment 4 in a CRD with eight treatments (pH 6.0 (control), 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0) and three replicates. Each container was lled with complete nutrient solution minus Cu, Fe, Mn and Zn. After the initial 24 h aeration, each of the solutions was adjusted to the correct pH by the pH titration unit, with saturated Ca(OH)2 solution or 0.01 M HCl, and four seedlings transferred to each container. The seedlings were allowed to grow for a total of 6 d, with solutions replaced after 3 d of growth. Samples and measurements were taken as per Experiment 4, with tissue N concentrations also measured as per Experiment 1. The HCO concentration in each of the solutions was 3 determined by titration with standardised 0.0046 M HCl (Greenberg et al., 1992). Data were analysed as a CRD using a repeated measures analysis as described previously. Results Experiment 1 Seedling nutrient requirement A signicant interaction was found between solution nutrient composition and time (P < 0.001), indicating signicantly different patterns of response across time by the nutrient solutions examined (Table 1). No signicant differences were found between root lengths of seedlings grown in the simple and complete nutrient solutions from days zero to seven. However, on day eight, root lengths were signicantly lower in the simple solutions (P < 0.001, LSD (5%) = 10.3), corresponding to the observation of Mg deciency in the simple solution on day nine. Leaves were observed to droop and curl downwards and were light green in colour with brown necrotic areas between the major veins. Symptoms of K deciency were also observed on day ten, with affected leaves being crimped and crinkled and shiner than normal leaves. These visual symptoms were conrmed by tissue analysis on day 11 with shoot concentrations of both Mg and K signicantly lower in the simple solution plants (P < 0.001) than in the complete nutrient solution plants (Table 2). Tissue concentrations of Ca and Zn (P = 0.002), and Fe and Mn (P < 0.001) were also signicantly lower in the simple solutions. Experiment 2 Unbuffered nutrient solutions at high pH The addition of plants to the solution resulted in a greater rate of decrease in average solution pH, with the average pH change over 12 h (without molecular sieve) being 0.60 with plants and 0.21 without. However, the use of a molecular sieve reduced this average rate of pH decrease, being 0.51 with plants and 0.09 without plants. The use of molecular sieve in addition to NaOH reduced CO2 concentrations further, with the partial pressure of CO2 being 15.0 Pa after passing through the NaOH, and below the detection limit (approximately 3 Pa) after passing through both NaOH and molecular sieve. Experiment 3 Ion exchange resins as buffers When in the OH form, the Dowex Type 1 resin provided greater buffering against HCl addition than Bio-Rex 5 (Figure 1). However, the pH at which the OH form Dowex Type 1 resin buffered, pH 11.6, could not be altered; the mixing of OH and Cl forms proportionally reducing the buffering capacity

347

Figure 1. Titration curves of the (a) Dowex Type 1 and (b) Bio-Rex 5 anion exchange resins in OH :Cl ratios of 100:0 ( ), 85:15 ( ), and 50:50 ( ).

Figure 2. (a) Titration curves of 1.5 mM KCl solution mixed with the Dowex Type 1 anion exchange resin in various forms (acetate ( ), bicarbonate ( ), phosphate ( )), and chloride resin ( ), and (b) phosphate concentration in solution while titrating Dowex Type 1 in the phosphate form.

rather than changing the buffering pH (Figure 1). The buffering of the acetate, bicarbonate and phosphate forms of the Dowex Type 1 resin were also examined with the bicarbonate form providing the greatest buffering (Figure 2). However, the use of these forms resulted in the release of comparatively high concentrations of the buffering ion into solution (Figure 2). Experiment 4 pH control by automated titration With the exception of Ca, the concentrations of all measured plant macronutrients remained constant over the entire nine day experimental period. The Ca concentration increased 10.7% due to the addition of Ca(OH)2 for pH control (approximately 10 to 20 moles L1 d1 ) (Figure 3). Of the micronutrients measured (B, Cu, Fe, Mn, Mo and Zn), only concentrations of the anionic nutrients (B and Mo)

remained constant. The metal cation concentrations all decreased with time, with Cu and Zn showing the most rapid decrease. The EC of solutions both with and without plants increased approximately 20% over the duration of the experiment (Figure 4). Experiment 5 Effect of high pH on root growth A signicant interaction was found between pH and sample time (P < 0.001), indicating signicantly different patterns of response across time by the pHs examined (Figure 5). No signicant differences were found between root lengths at pH 6.0, 7.0, 7.5 and 8.0 for the duration of the experiment. Root lengths on day six in solutions at pH 8.5, 9.0, 9.5 and 10.0 were signicantly lower than the control, and also signicantly different to each other (P < 0.001, LSD = 14.64), root length decreasing with increasing pH.

348

Figure 3. Measured concentrations of selected plant available nutrients in complete nutrient solutions at pH 9.0 aerated with CO2 depleted air in the presence ( ) and absence of plants ( ). (a) P, (b) S, (c) Ca, (d) Fe, (e) Cu, (f) Zn. The dotted line ( ) indicates the change in concentration predicted by PhreeqcI modelling of this system (assuming a CO2 partial pressure = 0.35 Pa) (means are the average of ve replicates).

No nutritional deciency symptoms were observed throughout the duration of the experiment. This was conrmed by plant tissue analysis, with concentrations of all nutrients in all treatments greater than those expected to cause deciency (Table 3). Solution concentrations of all nutrients included in the nutrient solution remained relatively constant in all treatments throughout the experiment, data for Ca presented in Figure 6 representative of the effects on the other nutrients. Average EC across all treatments rose 6.9% in the initial 3 d of growth and 5.3% in the second 3 d of growth after solution renewal (Figure 6). The greatest average EC increase was in the pH 10.0 treat-

ment; the EC rose 11.6% from day zero to three and 10.0% from day three to six. Bicarbonate concentrations increased over time but were generally low, with concentrations greatest in the pH 8.0 and 8.5 treatments (up to approximately 0.3 mM) and lowest in the control (approximately 0.1 mM) (Figure 6).

Discussion Experiment 1 Seedling nutrient requirement The initial growth of seedlings is dependent upon the nutrient stores contained within the seed until the root

349
Table 1. Mean daily mungbean (Vigna radiata (L.) Wilczek cv. Emerald) root lengths (mm) over 11 d growth at pH 6.0 in a complete nutrient solution, or in a simple solution containing only Ca, B and Cl (within a column, means with same letter are not signicantly different at the 5% level) (means are the average of ten replicates) Treatment 0 1 2 3 4 5 6 7 8 9 10 11

Complete 10a 29a 59a 105a 156a 201a 254a 302a 352a 397a 434a 475a Simple 9a 29a 63a 112a 162a 203a 255a 306a 341b 358b 379b 397b

Table 2. The concentration of selected nutrients in mungbean (Vigna radiata (L.) Wilczek cv. Emerald) plant tops following 11 d growth at pH 6.0 in a complete nutrient solution, or in a simple solution containing only Ca, B and Cl (within a column, means with same letter are not signicantly different at the 5% level) (means are the average of ten replicates) Treatment N K P Ca S Mg Fe Mn Zn Cu B (mg g1 ) (mg g1 ) (mg g1 ) (mg g1 ) (mg g1 ) (mg g1 ) (g g1 ) (g g1 ) (g g1 ) (g g1 ) (g g1 ) Complete 40.8a Simple 38.5a LSD (5%) NSD 29.6a 11.4b 2.6 2.1a 2.2a NSD 18.3a 16.4b 1.0 2.8a 2.6a NSD 2.9a 0.9b 0.15 302a 106b 33.6 75.8a 27.7b 4.2 98.0a 64.4b 18.0 1.3a 1.8a NSD 30.0a 38.9b 6.3

NSD No signicant difference

Figure 4. Change in EC of unbuffered complete nutrient solution at pH 9.0 over time with ( ) and without ( ) plants (means are the average of ve replicates).

Figure 5. Mungbean (Vigna radiata (L.) Wilczek cv. Emerald) root length over time at pH 6.0 ( ), 7.0 ( ), 7.5 ( ), 8.0 ( ), 8.5 ( ), 9.0 ( ), 9.5 ( ), and 10.0 () in nutrient solutions without supply of Zn, Cu, Mn and Fe (means are the average of three replicates).

is sufciently developed that it is able to exploit the soil mineral reserves (Hole and Scaife, 1993). Typically, the seed nutrient supply is not in the ratios required by the seedlings, with external supplies of some nutrients required almost immediately after germination (Fenner and Lee, 1989). To determine how long mungbeans could be grown in simple nutrient solutions, and which nutrients would be most limiting, mungbeans were grown in solutions containing only Ca and B. The seed nutrient reserves of mungbeans were able to meet the seedling requirements for all nutrients not supplied in the solution for the rst 7 d of growth,

with no signicant difference in root growth between treatments over this period. On day eight, root length was signicantly lower in solutions containing only Ca and B than in complete solutions (P < 0.001) (Table 1). This reduction in root growth corresponded with the observation of Mg deciency on day nine and K deciency on day ten. Tissue concentrations of Mg (0.9 mg g1 ) and K (11.4 mg g1 ) were signicantly lower in the simple solutions (P < 0.001) (Table 2) and were below the critical concentrations of deciency (1.53.5 mg Mg g1 and 10 mg K g1 (Bell et al., 1990; Marschner, 1995)).

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Figure 6. Changes in (a) concentrations of plant available Ca, (b) solution EC, (c) solution bicarbonate concentrations and (d) total alkalinity concentrations over time in nutrient solutions at pH 6.0 ( ), 7.0 ( ), 7.5 ( ), 8.0 ( ), 8.5 ( ), 9.0 ( ), 9.5 ( ), and 10.0 (), before and after solution renewal on day three (means are the average of three replicates).

Although Ca was supplied in both treatments, at harvest (day 11), tissue concentrations of Ca of plants grown in simple solutions was signicantly lower than that of plants grown in the complete solutions (P = 0.002) (Table 2). Although Ca concentrations in simple solutions were lower, no symptoms of Ca deciency were observed indicating that Ca was not limiting. Signicantly lower tissue concentrations of Fe, Mn and Zn were also found in the simple solution. Although these nutrients were not supplied with the simple solution, their concentrations were above the critical concentrations for deciency (72, 19 and 19 g g1 , respectively) (Bansal and Nayyar, 1989; Gupta and Mittal, 1981; Smith et al., 1984). This suggests that the seed reserves of these nutrients were sufcient to maintain growth over the 11 day duration of the experiment without limitation. These results are similar to those reported for other species, that seedlings deplete their mineral reserves in the general order: Ca, P, N, Mg, K, S, Fe (Fenner, 1986; Fenner and Lee, 1989; Krigel, 1967). Simple nutrient solutions containing only Ca and B are therefore suitable for short-term mungbean solution culture experiments over a maximum period of 7 d. The addition of further

nutrients, in particular Mg and K, would allow longer growth periods and would also produce a solution with a composition closer to that of the soil solution. Experiment 2 pH changes in unbuffered nutrient solutions at high pH The average pH decrease over 12 h in the absence of molecular sieve was greater with plants (0.60) than without (0.21). The observed increase in the rate of pH change in solutions with plants is attributed primarily to the release of CO2 and the formation of HCO as 3 a result of plant root respiration. It is also likely that the activity of the root plasma membrane H+ -ATPase contributed to the medium acidication (Morsomme and Boutry, 2000). Use of a molecular sieve to remove further CO2 from the air prior to aeration of solutions with plants reduced the average pH decrease from 0.60 to 0.51 pH units every 12 h. Although the removal of all detectable CO2 from the air prior to solution aeration limited the rate of pH decrease, the release of CO2 by plant roots into these unbuffered, dilute solutions resulted in a comparatively rapid pH decrease. The observed rate of pH

351 change with plants was too large for acceptable control to be achieved through manual pH adjustment. Experiment 3 Ion exchange resins as buffers Ion exchange resins have previously been investigated as a means of controlling the pH of nutrient solutions (Hageman et al., 1961; Harper and Nicholas, 1976; Nicholas and Harper, 1993). However, resins (typically weak cation exchangers) have only been used to buffer solutions in the neutral to acidic range. Strong and intermediate anion exchange resins were examined for their ability to buffer pH changes in alkaline conditions. The OH forms of both the Dowex Type 1 and Bio-Rex 5 resins provided good pH buffering. However, unlike previous studies in neutral and acidic conditions, the pH at which these resins buffered could not be altered by the mixing of OH and Cl forms (Figure 1). Using Amberlite IRC-50 (a weak cation exchange resin), Hageman et al. (1961) and Harper and Nicholas (1976) buffered solutions between pH 4 and 8 through the use of varying ratios of the H+ and Ca2+ forms. When converted to the acetate, bicarbonate and phosphate forms, the pH at which the Dowex Type 1 resin buffered could be changed by altering the pH (through the addition of NH3 ) of the solution used to convert the resin (Figure 2). Of these three forms, the bicarbonate form provided the greatest degree of buffering. However, the use of such forms as pH buffers in dilute nutrient solutions is limited. Titration of the phosphate form resin in solution resulted in the release of comparatively large quantities of phosphate, with the phosphate concentration (500600 M, Figure 2) more than two orders of magnitude greater than that desired in the nutrient solution. Experiment 4 pH control by automated titration Automated pH titration controllers have been widely used in owing solution culture units under acidic conditions (Dring et al., 1996). However, the effect of auto pH titration on solution composition in alkaline conditions is unknown. To test the suitability of pH controllers for maintaining alkaline solution cultures, the solution pH, EC and plant available nutrient concentrations of solutions held at pH 9.0 were measured over 9 d. By day three, the average EC of solutions with plants increased 12.5% compared to a 3.5% increase in those without plants (Figure 4). However, over the entire 9 d, the increase in average EC was greater in solutions without plants (21.1%) than in those with plants (17.4%). The EC of solutions without plants increases gradually over time due to the addition of titrant for pH control. However, changes in the EC of solutions with plants are dependant upon the relative balance between plant nutrient uptake, and the addition of titrant for pH control. During the initial stages of growth, when seedlings are reliant on seed nutrient reserves, there is little nutrient uptake from solution. Increases in solution EC are greater in pots with plants than in those without plants due to a greater rate of titrant addition resulting from the release of CO2 by root respiration and activity of the root plasma membrane H+ -ATPases. As growth continues and the nutrient uptake (largely NO and K+ ) from solution increases, 3 the overall rate of increase in EC was reduced. The plant available concentrations of all measured macronutrients, other than Ca (i.e., N, K, Mg, S and P), remained relatively constant over the 10 d. The Ca concentrations increased 10.7% over the duration of the experiment due to the addition of Ca(OH)2 by the titration units to maintain solution pH (Figure 3). Of the micronutrient concentrations measured, only B and Mo remained constant. Availability of Cu, Fe, Mn and Zn decreased over time due to the formation of Cu(OH)2, Fe(OH)3 , MnCO3 and MnSO4 , and Zn(OH)2 respectively (Parkhurst, 2002). As this decrease in micronutrient availability is due to precipitation, primarily as hydroxides, the loss of these micronutrients in any experimental system at high pH is unavoidable. Modelling using GEOCHEM-PC indicated that the addition of EDTA and DTPA chelating agents could not prevent the precipitation of these nutrients. To ensure that the treatment differences obtained in solution culture studies of the direct effects of high pH are not due to nutritional differences between pH treatments, the micronutrients (Cu, Fe, Mn and Zn) should be excluded from the solution. The availability of all of the remaining nutrients (Ca, K, Mg, N, S, P, B and Mo) will therefore be constant, even between treatments of different pH. The periodic replacement of solutions would also help limit increases in EC and reduce solution nutrient depletion due to plant uptake. Experiment 5 Effect of high pH on root growth Mungbean root length was not limited by OH up to a pH of 8.0, with no signicant differences in root length between treatments at pH 6.0 (control), 7.0, 7.5 and 8.0. Upon trial termination (day six), root

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Table 3. Concentration of selected nutrients in mungbean (Vigna radiata (L.) Wilczek cv. Emerald) plant tops following 6 d of growth in nutrient solutions of various pH values without supply of Zn, Cu, Mn and Fe (means are the average of three replicates) Solution pH N K P Ca S Mg Fe Mn Zn Cu B (mg g1 ) (mg g1 ) (mg g1 ) (mg g1 ) (mg g1 ) (mg g1 ) (g g1 ) (g g1 ) (g g1 ) (g g1 ) (g g1 ) 75.4 67.0 65.5 68.1 73.5 63.1 69.4 63.6 35.4 32.4 36.7 33.8 34.5 37.0 30.7 30.0 10a 11.8 9.8 11.0 10.9 10.3 10.5 8.4 7.5 1.13.0b 7.4 6.7 7.4 7.8 7.3 7.4 6.4 4.9 4.7 4.9 6.0 9.0 3.2 5.7 4.7 3.6 2a 2.6 2.4 2.5 2.6 2.6 2.7 2.5 2.3 1.53.5c 85.0 88.3 93.7 89.0 90.0 90.7 118 106 72d 48.8 47.5 48.2 47.0 45.8 45.6 46.0 42.2 19e 34.8 22.6 29.9 32.7 31.5 31.7 27.7 26.5 19f 6.5 6.6 6.5 6.9 6.5 6.6 7.1 6.3 4a 92.3 81.3 79.3 84.9 90.7 89.3 70.4 68.1 16a

6.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Critical value 38a

Critical value of deciency for black gram (Vigna mungo)

General critical value of deciency

a (Bell et al., 1990) b (Gaind and Gaur, 1991; Rashid and Bughio, 1994) c (Marschner, 1995) d (Smith et al., 1984) e (Bansal and Nayyar, 1989) f (Gupta and Mittal, 1981)

lengths at pHs 8.5, 9.0, 9.5 and 10.0 were signicantly lower than the control and were different to each other (P < 0.001), root length decreasing with increasing pH (Figure 5). Although pH titration units effectively maintain bulk nutrient solution pH, Nikolic and Rmheld (2003) concluded that while organic buffers are able to diffuse from the nutrient solution into the root apoplast, titration units have limited ability to control root apoplastic pH. Therefore, while root growth was reduced at a bulk solution pH of 8.5, it is likely that the root apoplastic pH was lower than that of the bulk solution due largely to the acidication by plasma membrane H+ -ATPase activity (Kurdjian and Guern, 1989). Under alkaline conditions, apoplastic pH has been observed to be up to two pH units lower than that of the bulk solution (Peters and Felle, 1999). To ensure root growth was not being limited indirectly, solution HCO and nutrient concentrations, and 3 plant tissue nutrient concentrations were measured. The HCO concentrations were maintained below 3 0.3 mM, one order of magnitude lower than the concentration reported to be toxic (Alcntara et al., 2000; Alhendawi et al., 1997; Chaanin et al., 1994; Peiter et al., 2001; Yang et al., 1994). Although total alkalinities were similar in all treatments above pH 8.0, HCO concentrations were greatest in the pH 8.0 3 and 8.5 treatments due to the formation of CO2 3 in the more alkaline treatments (Figure 6) (Lindsay,

1979). The formation of H2 CO3 (and hence CO2 and H2 O) resulted in lower total alkalinities in treatments between 6.0 and 7.5. Plant growth was also not limited by nutritional disorders, with no symptoms of deciency in any treatments and solution nutrient availability constant across all pH treatments (Figure 6). The critical values for deciency, although established under a variety of experimental conditions, also indicate growth was not limited in any treatment (Table 3). Solution EC increased an average of 6.9% from day zero to four and 5.3% from day three to six. A maximum increase of 11.6% was observed between days one to four in the pH 10.0 treatment. Although this increase is greater than that observed in the control (7.0%) over the same period of time, it is not sufcient to limit plant root growth. In conclusion, the results presented here demonstrate that nutrient solutions without Cu, Fe, Mn and Zn maintained by pH titration units and aerated in the absence of CO2 are suitable for studies at high pH. Using this experimental system, OH was found to limit the root growth of mungbeans at a bulk nutrient solution pH of 8.5 and greater.

353 Acknowledgements The author wishes to acknowledge Graham Kerven for his technical assistance and guidance, particularly with the ion exchange resins and pH titration units. The guidance of David Edwards and Pax Blamey is gratefully acknowledged. The statistical help and advice provided by Rosemary Kopittke is also much appreciated. The authors also wish to thank Ian Fulton for his support and for placing the research in the context of the alumina industry. This work was conducted as part of an environmental research program funded by Alcan Gove Pty Ltd.
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