Separation and Purication Technology 102 (2013) 3442

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Separation and Purication Technology

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Application of EAPR system on the removal of lead from sandy soil and uptake by Kentucky bluegrass (Poa pratensis L.)
Rudy Syah Putra a,b,, Yasuhisa Ohkawa a, Shunitz Tanaka a
a b

Division of Environmental Science Development, Graduate School of Environmental Science, Hokkaido University, Kita-ku, Kita 10 Nishi 5, Sapporo 060-0808, Japan Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Islam Indonesia, Jl. Kaliurang km. 14, Yogyakarta 55584, Indonesia

a r t i c l e

i n f o

a b s t r a c t
The combined use of electrokinetic remediation and phytoremediation (i.e. electro-assisted phytoremediation, EAPR) to remove lead from contaminated sandy soil and uptake by Kentucky bluegrass (Poa pratensis L.) has been demonstrated in a laboratory-scale experiment. The effectiveness of two dimensions (2D) of electrode conguration used in the EAPR system was also evaluated through the agar media for rapid process (48 h) and then compared with the experiment in the soil matrix for 15 d. The results obtained from EAPR system were compared with plants exposed to lead in the contaminated soil by using phytoremediation for 30 d process. Here we report a basic difference in plant responses to the lead, depending on the growth condition with respect to the uptake parameters, e.g. bioaccumulation coefcient (BC value), translocation factor (TF value), distribution concentration of lead in roots and shoots system and soil pHs. Several survival parameters for plant were also monitored including water content (dry weight to fresh weight DW/FW ratio), biomass accumulation (dry matter), and plant chlorophyll (total content and chlorophyll a/b ratio). The effectiveness of common agrochemical-urea, used to facilitate the healthy growth of plants during the stress period was also discussed. Our nding showed that when the plant was grown in the soil by using EAPR system, a high amount of lead was founded in the root and shoot system relative to phytoremediation process. Generally for each method, the BC and TF value of plant grown in EAPR system was high, showing high absorption of lead concentration in the root and then deposited it into the shoots system. Upon the addition of urea, the water content, biomass accumulation and chlorophyll content have shown positive responses for plants in overcoming the stresses of lead in the soil. 2012 Elsevier B.V. All rights reserved.

Article history: Received 1 June 2012 Received in revised form 22 September 2012 Accepted 25 September 2012 Available online 3 October 2012 Keywords: Phytoremediation EAPR Lead Urea Kentucky bluegrass (Poa pratensis L.)

1. Introduction Phytoremediation is the use of plants and plant processes to remove, degrade, or render harmless hazardous materials present in the soil or groundwater [1,2]. This emerging technology may offer an attraction for cleanup of soil because it is cost-effective, nonintrusive and requires little maintenance. However, its application is limited to surface contamination only, because the cleanup depth is strictly determined by the length of the plant roots. It is a passive technology in terms of contaminant transport, and the movement of contaminants in the soil is induced exclusively by the slow plant root suction and thus the efciency of removal of contaminants depends on the extension of the plant roots in the soil surface [3]. Plants commonly employed for phytoremediation

Corresponding author at: Department of Chemistry, FMIPA Islamic University of Indonesia, Jl. Kaliurang km. 14, Yogyakarta 55584, Indonesia. Tel.: +62 274 895920; fax: +62 896437. E-mail address: rudy.syahputra@uii.ac.id (R.S. Putra).
1383-5866/$ - see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2012.09.025

have to unite a high biomass plant, rapid growth, and high hyperaccumulation, such as Vetiver grass (Vetiveria zizaniodes) and Indian mustard (Brassica juncea) [4,5]. Lead is a common and serious pollutant because of its toxicity. It has limited soluble forms in the soil matrix and availability for plant uptake due to complexation with organic matter, sorption on oxides and clays, and precipitation as carbonates, hydroxides and phosphates [6]. Because of its limited bioavailability, an approach to increase its bioavailability is essential to the success of phytoremediation. The combined use of electrokinetic with phytoremediation, which is referred later on as electro-assisted phytoremediation (EAPR) is one of preference to enhance the phytoremediation process. The combined technology will overcome the problem encountered by the individual technologies such as the phytoremediation problem of applying phytoextraction to deep subsurface contaminant plumes and the electrokinetic remediation problem of slow and long term accumulation of contaminants in distant electrode wells [3]. In the EAPR system, the key element is electrokinetic mobilization of metal ions in the soil from a

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Fig. 1. Schematic diagram for the different system of phytoremediation process (a) and EAPR system (b) for removal of lead from contaminated soil.

contaminated zone which is located deeper in the soil, than the root zone which increases metal ion availability to the plant root by electro-migration process and thus plant metal uptake and subsequent extraction by phytoremediation [7]. The method also makes possible to use plants whose root is not long and consequently to enhance the variety species of plants for phytoremediation process. For hyper-accumulator plants, high translocation of the heavy metal from root system would occur to the aerial part of plants, such as shoots and leaves [8]. In addition, the method utilizes the electrokinetic phenomena to deliver water and nutrients elements needed for plant growth [3]. Consequently, there might be higher nutrient element levels available to the plant roots growing under electrokinetic treatment. Fig. 1 shows the different system of phytoremediation process and the enhancement process

by using EAPR system on the removal of lead from contaminated soil. Several studies had been reported on the improvement of soil phytormediation by using the combination process with electrokinetic remediation. The electrical eld can either be applied together with phytoremediation [7,9], or the phytoremediation can be applied after the electrokinetic treatment [10]. Various plants and heavy metals were used as a model for studies, such as perennial ryegrass (Lolium perenne) used to treat Cu/Cd/As [7] and Pb uptake from soil by Indian mustard (Brassica juncea) through the enhancement of EDTA [5]. Other studies were also reported by using similar plant for simultaneous removal of multi-element (Cd, Cu, Zn and Pb) from soil [11]. Meanwhile, Aboughalma et al. [12] and Bi et al. [13] applied the EAPR system to remove Zn, Pb,

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Cu and Cd by using potato plant, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum) with special emphasis on treated soil by direct current electric eld (DC) compared with alternative current (AC). Zhou et al. [14] evaluates the application of vertical DC electrical eld on the removal of Cu/Zn by using ryegrass when EDTA/ EDDS was applied to increase the bioavailability of heavy metal in the soil. Although from previous results have veried the enhancement of heavy metal uptake by plants after the application of electrical eld, the pH reduction had been an un-favorable parameter limiting the plant growth [7] and a reduced growth of potato and tobacco plants were found under the DC eld application [12,13]. Thus several aspects of EAPR system still need to be adjusted. Furthermore, these studies had not discussed the inuence of electrode conguration on the removal efciency since these studies had used horizontally one-dimensional (1D) electrode conguration. For 1D electrode conguration, the pollutant was transported in one direction only from anode toward cathode compartment. The inuence of positioning of electrodes in terms of geometrical arrangements for EAPR system had been discussed by Hodko et al. [3]. The study had been applied on two-dimensional (2D) or axisymmetrical electrode conguration on the removal of lead from articially contaminated soil through the enhancement with EDTA as a lead-complexing agent for the extracted lead from soil matrix. In that conguration, the cathode was placed at the center and the anodes were placed on the perimeter to maximize the spread of the acidic environment generated by the anodes and to minimize the extent of the basic environment generated by the cathode [15]. These results have demonstrated that the application of an electric eld has produced plants with much higher concentration of metals compared to the plants which were grown in the phytoremediation process. However, the enhancement of electro-migration process of lead ion from soil matrix through electro-assisted by using 2D electrode conguration was not shown in that report and unclear to discuss, since the EK cell was lled only with contaminated soil. Therefore, this effect requires attention in the present study. Kentucky bluegrass (Poa pratensis L.) was selected as the test plant in this study as it is frequently used in the northern region of Japan to provide an amenity of grass cover. It has vigorous growth to produce an adequate biomass for harvest measurements and subsequent elemental analysis, and also it has been known that high demand for macronutrients would potentially exacerbate any imbalance in nutrient supply that will arise during the experiment [16]. Furthermore, this species is environmentally tolerant to grow well on heavier (clay like) soils at pH 5.88.2 and a tolerable for heavy metal concentration such as Pb and Cd [16,17]. In this study, two major issues are addressed. First, the evaluation of 2D electrode conguration on the enhancement of electro-migration process of lead ion through electro-assisted would be studied by using agar matrix representing liquid media and soil matrix (i.e. sandy soil). The aim was to examine whether the result obtained from liquid media would be similar as that from soil matrix. It was of interest to determine what extent the results from such experiment can serve as a reliable basis for concluding about the suitability of electrode-assisted in the EAPR system. Second, the survival strategy of plants growing on lead contaminated soil was assessed by: (i) evaluation of the survival indicator to determine the level of lead tolerance; (ii) determination of Pb accumulation and the efciency of root-to-shoot translocation; and (iii) the effect of chaotropic agent-urea on the enhancement process of the EAPR system. 2. Experimental 2.1. Chemicals, plant materials and soils The highest grade commercially available reagents were purchased from Wako Pure Chemicals, Co. (Tokyo, Japan), except as

stated otherwise. Deionized water was used throughout to obtain the desired concentrations in the solution. Kentucky bluegrass (Poa pratensis L.) was supplied from G-GAIA Co., Ltd. (Sapporo, Hokkaido, Japan) with 2 cm thickness of massive root. For all experiment units, the plant was grown in the growth chamber with the dimension of 180 (L) 60 (W) 180 (H) cm. The light source was provided from uorescent tubes (i.e. NEC Biolux, FL 40 SBR) with light intensity for about 46 lmol m2 s1 and it was run with an intra-matrix timer to provide 16/8 h light/dark cycle. The laboratory ambient temperature was maintained by an air conditioner at 25 C. This system brought the chamber temperature to 2428 C in light/1820 C in dark condition and 4045% humidity. Upon purchase, plants were carefully selected to provide the uniform distribution of shoot and root biomass and then washed to remove soil before being placed in the trays containing halfstrength of Hoagland solution. After that the plants were transferred to the growth chamber to acclimatize for 2 weeks before being used for pot experiments under the EAPR system and phytoremediation process. The modied half-strength Hoagland solution was prepared according to the references [1820] by mixing 0.0676 g of KH2PO4, 0.253 g of KNO3, 0.59 g of Ca(NO3)24H2O, and 0.20 g of MgCl26H2O in 1 L of solution. The solution pH was adjusted to 6.75 by adding a few drops of 1 M NaOH. A commercially-produced top soil used in this study was purchased from an agriculture shop and the soil properties are summarized in Table 1. The soils were articially amended with 1000 mg/kg lead concentration (i.e. Pb(NO3)2 salts) and then treated with six cycles of saturation process by using de-ionized water and air-dried before being aged for one year. High amended concentration of lead in the soil was representing the soil from industrial site as reported in other study [12,13,21]. The basal fertilizer was applied to the soil according to the manufacturers recommended rate application of 8 g/L (e.g. 112 g for 14 L soil in a container, 8:8:8:2.8 NPK and Mg) before being used for all experiment units. 2.2. Evaluation of 2 D electrode conguration of EAPR cell The aim of study was to evaluate whether the construction of 2D electrode conguration could be used efciently to enhance the electro-migration of lead ion from the deep contaminated plume during the electro-assisted process or not. Therefore, the contaminated part was set-up in the bottom tray; meanwhile

Table 1 Chemical and physical properties of the soil used in the EAPR and phytoremediation studies. Parameter Particle size distribution Sand Silt Clay Soil texture Organic C Total N Cation exchange capacity pHa Redox potentiala Electrical conductivityb Total heavy metalc Pb Cd
a b

Unit % % % % % meq/100 g mV mS/cm mg/g mg/g

Value 93.6 0.0 2.7 Sandy 4.82 0.27 26.5 7.48 0.015 44 2.65 2.90 0.46 0.0078 2.31.E-04 0.0011 5.77.E-05

Determined by using 1 g: 2.5 mL of soil: water ratio (n = 3). Determined by using 1 g: 5 mL of soil: water ratio (n = 3). c Total digestion of soil using 1: 3 of HNO3: HClO4 ratio. Heavy metal concentration was determined by using ame AAS (n = 3).

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another part in the tray was lled with clean agar as well as clean soil. Fig. 2 shows the modied 2D electrode conguration of EAPR cell system that was used in this study. The rst test was run in the agar matrix representing as a liquid media, where the rapid assessment of the lead ions movement in a short time (i.e. 72 h) could be evaluated. Each layer of agar matrix was prepared by pouring of 15 g agar in 1 L of 0.0025 M KNO3 solution for total 3.8 L volume. The contaminated agar matrix was prepared by addition of 100 mg/L of lead concentration. The results from this step were further used to design the effectiveness of electrode conguration in the soil matrix for 15 d of EK process. The studies in soil matrix were carried out in 3.5 kg of total soil. 100 mg/kg of lead was added in 1 kg soil and then used as a contaminated soil. Each layer of soil in the EK cell was separated by lter paper. 0.1 M of acetate buffer in pH 3.75 was rinsed in the anode well as a purging solution during the electro-assisted process. All studies were run in 50 mA of constant current and repeated for twice.

prepared as a control plant and also one tray in the absence of plant was prepared to study a potential lead adsorption by tray walls. Periodically, the soil pH near the cathode area was measured during the growth period under the EAPR system and a constant amount of soils was withdrawn from each tray at the end of the experiment for further measurement of remaining lead concentration in the soil. 2.4. Plant analysis Directly after the completion of the experiment, plants were harvested, weighed and separated into shoot and root. The root part was then washed thoroughly with running tap water. The shoot and root were cut into small pieces, dried for 2 days at 80 C. The air-dried root and shoot samples were ground to a ne powder with a mortar. Approximately 200 mg of samples were digested in a mixture of concentrate HNO3 and HClO4 (10:5 v/v) for overnight and then adjusted to a volume of 50 mL of deionized water. The lead content in the acid extract was determined by a ame atomic absorption spectrophotometer (Hitachi A-2000, Japan). The concentrations of element in this study were reported on a dry matter basis. The translocation factor (TF), dened as the ratio of metal concentration in plant shoot to those in roots was calculated to check the effectiveness of plants in translocation of metal ions to their aerial parts [24]. Second parameter is a bioaccumulation coefcient, dened as the ratio of metal concentration in root tissue to the initial metal concentration in soil was calculated to check the absorption of lead from soil to the root system [17]. 2.5. Plant growth and physiological parameters Plants were observed and monitored every day to take note of changes in appearance. Chlorophyll content of plants was measured according to Moran and Porath [25]. A sample of 200 mg plant shoots were cut into 0.5 cm segments and incubated in

2.3. Batch soil experiment on EAPR system and phytoremediation process The EAPR and phytoremediation study consisted of the following treatment: (i) 1000 mg/L of initial lead concentration in the soil; and (ii) two concentrations of chaotropic agent-urea (0.01% and 0.1% w/v). The urea concentrations (up to 0.1% w/v) were chosen to mimic typical urea-N application rate for major agronomic crops (100 kg urea/ha, [22]). The 0.1% urea treatment is signicantly lower than the optimum urea concentration (i.e. 2 M) used in laboratory experiments to maximize chaotropic effect on a large quantity of dissolved hydrophobic organics [23]. After acclimatization in nutrient solution, a bundle of plant was directly placed in the trays with lead contaminated soil. The soil moisture content of 70% was maintained by adding 0.0025 M KNO3 solution every 2 days. A 50 mA constant current was applied in EAPR system for one-month operation. Other tray with the absence of lead concentration, but presence of Kentucky bluegrass (Poa pratensis L.) was

Fig. 2. Schematic diagram of EAPR cell and image photo of cell: (a) electrode conguration, (b) cell with 3 kg soil and (c) EAPR ready cell runs with Kentucky bluegrass as an accumulator plant, (d) dimensional size of cell, and (e) lateral image of 2D electrode conguration, respectively.

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acetone for 24 h at 4 C in the dark room. The absorbance of the solutions was measured with spectrophotometer (JASCO JS460, Japan) at k645 and k663 nm. Chlorophyll concentrations (mg/ml) were calculated by using the following equations:

Chl a 12:7 Absorbance at k663 nm 2:69 Absorbance at k645 nm Chl b 22:9 Absorbance at k645 nm 4:68 Absorbance at k663 nm Total Chl 8:02 Absorbance at k663 nm 20:2 Absorbance at k645 nm 3 2 1

3. Results and discussions 3.1. Lead migration in 2D electrode conguration of EAPR cell 3.1.1. Evaluation in agar matrix The measurement of lead concentration in the agar matrix was determined by nine sampling points. For each column was sectioned into four layers as shown in the Fig. 3a and b. All samples were homogenized by re-dissolving into agar solution by addition a few drop of concentrate HNO3. Lead concentration was measured by using ame-AAS. Fig. 3c shows that the lead ions were transported from contaminated layer in the bottom of the tray through the clean agar matrix toward to the cathode unit at the top part of tray and then highly precipitated in the center of cathode. These results would presumably be the effectiveness of the electromigration pattern of the lead ions in the liquid media. However, in the EAPR system (i.e. soil matrix), the upward, counter gravitational transport of lead ions was enhanced by electro-osmosis which occurs in the direction from the anode toward the cathode. Once the contaminants reach the accumulative of root zone, the lead ions might be extracted from the soil by plant roots.

3.1.2. Evaluation in soil matrix After the electro-assisted process, the soil was sectioned into nine sampling points and then samples were taken from every layer in each column. The soil was dried, grinded and then 5 g of samples were digested by 1 M HCl for overnight. The nal samples were adjusted with 50 mL of deionized water (1:10 w/v). The lead content was then determined by ame AAS. Fig. 4c shows the lead distribution for every column in each layer after the electro-migration from the contaminated soil (see Fig. 4a and b). The deposition of lead in the rst, second and third soils layer were quiet low subsequently from contaminated soil. For example, in the rst layer, it was 2.403.92 mg/kg meanwhile in the second layer was 2.48 3.68 mg/kg. This results suggesting that the accumulative rate concentration in the soil matrix at the center part of EAPR cell was quiet similar. The different electro-migration pattern of the lead ions in the soil matrix compared with the liquid media presumably as a result of slow electric current density from anode with the distance toward the cathode. However, based on these results, the design of 2D electrode conguration in this study could be used effectively to enhance the electro-migration of lead ion from contaminated soil toward the accumulative zone of plant (i.e. cathode area). 3.2. pH proles on the EAPR and phytoremediation system The decreasing soil pH would increase the solubility of metals, although the acidic condition and high amount of soluble metals could be the limiting factors for plant growth [3,26]. Therefore in this study, the soil pH near the cathode area (i.e. massive root zone) under the EAPR system was monitored and the results were compared with the phytoremediation process. Fig. 5a shows that the proles of soil pH tend to be mild acidic compared to that of the treatments without the presence of lead in the soil with the plant (i.e. control pH). A similar result was also shown when urea was added in the soil (see Fig. 5b). These results suggested that the soil system has a strong buffer capacity. Fortunately, the growth of Kentucky bluegrass (Poa pratensis L.) was not inuenced by the increase or decrease of pH through the addition of urea, because this

Fig. 3. Lead migration from contaminated agar toward clean agar matrix. Image photos of sampling points (a), sectional agar matrix in the chamber (b) and lead distribution in agar matrix after EK process (c). The experiment was run for 72 h in 50 mA of constant current. Agar matrix was prepared by mixture of 15 g agar in each layer for total 3.8 L solution. The contaminated agar was prepared by addition of of 100 mg/L of lead concentration and 0.0025 M KNO3 as a background electrolyte.

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Fig. 4. Lead migration from contaminated soil toward clean soil. Sampling points of soil (a), sectional of soil in the EK cell (b) and (c) lead distribution after EK process. The experiment was run in 3.5 kg total soil which 1 kg of soil was contaminated with 100 mg/kg of lead concentration. The study was run for 15 days in 50 mA of constant current. 0.1 M acetate buffer at pH 3.80 has been used as a purging solution. The sampling point at numbers S1, S3, S7 and S9 were the soils in the anode position.

Fig. 5. pH proles for soil remediation in each method. A daily pH was measured after watering by using 0.0025 M KNO3 solution. Control pH was dened here as the plant grown in the soil without lead contaminated soil. The soil pH was measured near the cathode for EAPR system, meanwhile in the control plants were measured near the root area.

plant has been documented to be able to grow well under mild acidic and alkaline conditions [27]. 3.3. Pb concentration in plants on the EAPR and phytoremediation system To evaluate the accumulation capacity of lead in the studied plants, the concentration of lead was measured in roots and shoots of plant grown in the EAPR system and phytoremediation process. Fig. 6a and b show that the high translocation of lead ion into the shoot system (i.e. high TF value) in each method either in amended

urea treatment or the absence as well. In addition, high storage capacity of Pb metal in roots was indicated with a higher BC value for EAPR system than that in the phytoremediation process. These results have suggesting the electro-assisted enhancement was quiet effective to increase the transport rate of lead ion from pore uid to the massive root zone. A critical point to the success of the EAPR system is not only on the right timing between the transportation rates of pollutant to the accumulative root zone of the plant [3], but also a selection of appropriate plant species with highly accumulation capacity for lead. For example, Vetiver grass (Vetiveria zizaniodes (L) Nash)

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Fig. 6. The concentration of lead uptake by Kentucky bluegrass for each remediation method (n = 3) without urea (a) and amended urea (b). The soil used in the phytoremediation and EAPR system were prepared with 1000 mg/kg of 3 kg lead contaminated soil. A daily watering was using 0.0025 M KNO3 solution and 50 mA constant current was applied in EAPR system for one month operation. Bioaccumulation coefcient (BC) = lead concentration in root (mg/kg)/500 mg/L of lead concentration in solution or 1000 mg/kg of lead concentration in soil; translocation factor (TF) = lead concentration in shoot (mg/kg)/lead concentration in root (mg/kg).

Table 2 Accumulative biomass and water content (DW/FW ratio) in the tissues of plant collected for each method in the EAPR and phytoremediation studies. Test No. Experiment Biomass (g dry wt.) Shoot (1) 1 2 3 4 5 6 EAPR Phyto EAPR 0.1% Urea Phyto 0.1% Urea Phyto 0.01% Urea Control 2.40 1.93 2.50 1.33 1.70 1.50 Root (2) 39.1 42.4 39.0 36.6 40.8 33.8 Ratio (DW/FW)tot (3) 0.19 0.26 0.19 0.23 0.22 0.20 (DW/FW)shoot (4) 0.35 0.26 0.30 0.26 0.23 0.18 (DW/FW)root (5) 0.19 0.27 0.19 0.23 0.22 0.20

Water content indicator in the tissue was determined by dry weight (DW) to fresh weight (FW) ratio of plant tissue. The following equations are formulated to calculate the (DW/FW) ratio in the total, shoot and root. [1] (DW/FW)total = Value in column (1) + Value in column (2) / total fresh weight of plant tissues (g wet wt.); [2] (DW/FW)shoot = Value in column (1) / fresh weight of shoot (g wet wt.); [3] (DW/FW)root = Value in column (2) / fresh weight of root (g wet wt.).

has ability to accumulate up to 17-fold and 3-fold mg Pb/kg dry weight of root and shoot tissue when exposed to 1200 mg/L of lead concentration [28]. In this study, the designed cell of EAPR system was quiet enough to accommodate the plant for consecutive accumulation of lead ion in one-month operation. In addition for the EAPR system, the presence of urea in the soil was not signicantly implied to the increasing of lead deposition in the plant. 3.4. Pb tolerance under EAPR and phytoremediation system To assess the tolerance of plants for lead, several parameters of the plants were monitored during the EAPR process as well as phytoremediation, including water content (e.g. it was indicated with DW/FW ratio) and biomass productivity (dry matter) shown in the Table 2. Dry weight to the fresh weight (DW/FW) ratio of plants was determined as water content indicator in the tissue under the stress conditions [29]. Plant appearance was also monitored by the measurement of chlorophyll content (see Fig. 7a and b). Chlorophyll contents and the Chl a/b ratio are the parameters for

photosynthetic activity and also often used as the indicators of stress in plants. These parameters have been used for detecting and assessing the exposure of plants to environmental contaminants [3032]. Table 2 shows that a substantial increase the (DW/FW) ratio in shoot and biomass production for treated plant with EAPR system were a higher than that in phytoremediation process. In addition, stimulatory responses (hormesis) on high value of total chlorophyll content were also observed on the treated plant with EAPR system (see Fig. 7a), even though the plant was cultivated in the high lead concentration. The low content of chlorophyll a/b ratio has indicated that the plants were being exposed for less toxic chemical stressing [29] as well. However, the low ratio of DW/FW in the root for treated plant with EAPR system resulting from advective movement of the pore uid from the anode toward the cathode as it was a nature condition in the electrokinetic system. Based on these results, it was concluded that the enhancement of phytoremediation by electro-assisted system did not imply to disrupt the circulation of nutrient ion in the plant tissues.

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Fig. 7. Total chlorophyll content and chlorophyll a/b ratio in the plant for each remediation method (n = 3) without urea (a) and amended urea (b). Chlorophyll in control plant was dened here as the plant grown in the soil without lead contaminated soil.

The visual characteristics of Kentucky bluegrass (Poa pratensis L.) were compared between plant in lead-spiked systems and the absence of lead. The plants grown under the EAPR system or phytoremediation process in this study did not show any phytotoxic symptoms (e.g. discoloration, pigmentation, yellowing, and withering), suggesting higher tolerance of plant to the lead concentration. Therefore, the plants, even though under that stress, were still able to maintain relatively normal level of photosynthetic pigments. The presence of chaotropic agent-urea in the contaminated soil of EAPR system has promoted plant survival and growth, thereby increasing effectiveness of remediation process. Furthermore, the presence of urea especially at low concentration of urea (i.e. 0.01% w/v) in the treated plant with EAPR system has promoted the healthy growth of plants and survival, therefore increasing the effectiveness of remediation process. 4. Conclusions The results from our study has considerable potential towards achieving our long-term goal, i.e., to develop a low-cost, environment friendly combined electrokinetic and phytoremediation system capable of remediating lead contaminated soils. Several major points emerged from this evaluation as treatment options for lead contaminated soils. They are as follows: 1. In this study, for 2D electrode conguration of EAPR system, the application of electric across the contaminated plume with lead in the agar media or soil matrix, causes Pb2+ to migrate from anode to cathode, accompanied by marked changes in the lead concentration consecutively from the bottom of tray toward to the top of surface level (i.e. close to the cathode part which the accumulative root zone was present). Therefore, the design of electrode conguration in this study could be used to enhance the electro-assisted process for the electromigration of lead in the contaminated matrix. 2. The characteristic features of Kentucky bluegrass (Poa pratensis L.) which was discovered in the EAPR system was clearly demonstrating that with respect to all studied parameters; the

response of plant to remove the lead ion from contaminated soil was high. The translocation rate (i.e. TF value) of lead from roots to shoots system in the EAPR system was higher than that in the phytoremediation process. Therefore, total deposition of the concentration of lead in the Kentucky bluegrass (Poa pratensis L.) was high in the EAPR system. The similar results were also shown for the absorption in roots system (i.e. high BC values), whereas the low BC value was shown when plants were cultivated by phytoremediation process. 3. The contaminant tolerance and growth potential of a plant species are directly related to its biochemistry and physiology. Kentucky bluegrass (Poa pratensis L.) was able to increase or maintain water content in photosynthetic tissue, and to increase root growth development, by transporting more photosynthetic material through the root system during stressful conditions. The presence of chaotropic agent-urea in the contaminated soil of EAPR system has promoted plant survival and growth, thereby increasing effectiveness of remediation process. This indication was shown by the characteristic of increasing water content in shoots, increasing of biomass accumulation in roots, and maintaining the content of chlorophyll as a positive response for plants in overcoming the stresses of lead in the soil.

Acknowledgements The authors would like to thank Japan Rail road (JR) Hokkaido, Japan for their nancial support in part of this study and also greatly appreciate to G-GAIA Co., Ltd. (Sapporo, Hokkaido, Japan) for providing the Kentucky bluegrass. A scholarship from Ministry of Education of the Republic of Indonesia to R.S.P. for Doctoral program is also greatly acknowledged. References
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