Journal of Bioscience and BioengineeringVOL. 109 No. 1, 47–50, 2010

www.elsevier.com/locate/jbiosc

Enhanced heavy metal phytoextraction by Echinochloa crus-galli using root exudates

Sunghyun Kim,1 Hyewon Lim,2 and Insook Lee1,⁎

⁎ CorrespondE-mail add

1389-1723/$doi:10.1016/j

Division of EcoScience, Ewha Womans University, 11-1 Daehyundong, Seodaemungu Seoul 120-750, South Korea1 and Complementary Medicine,Peninsula Medical School, University of Exeter, Devon EX2 4NT, UK2

Received 28 April 2009; accepted 20 June 2009

Available online 29 July 2009

Heavy metal uptake and growth by Echinochloa crus-galli were investigated to determine if the use of root exudatesenhanced phytoextraction. E. crus-galli were planted in soils contaminated with 600 mg kg−1 Pb, 40 mg kg−1 Cd and100 mg kg−1 Cu. E. crus-galli were then cultivated with and without root exudates from Belamcanda chinensis for 4 weeks.The growth of E. crus-galli in metal-contaminated soils that contained root exudates showed increased roots and shoots whencompared to E. crus-galli grown without root exudates (p<0.05). In addition, the accumulation of metal in E. crus-galli thatwas cultivated with the root exudates was two- to fourfold higher than in plants that were cultivated without the rootexudates. The exchangeable soil fraction in the rhizosphere of E. crus-galli grown with root exudates was greater than when E.crus-galli was grown without root exudates. Finally, the BCF and TF values of Cd, Cu and Pb were greater when the rootexudates were added (p<0.05). Taken together, these results indicate that root exudates can be used as a natural chelatingagent to enhance phytoextraction.

© 2009, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Echinochloa crus-galli; Heavy metal; Natural chelate; Phytoextraction; Root exudates]

Heavy metal contamination is a major environmental problem inagricultural lands. Among the methods used to remediate heavymetal-contaminated soils, traditional physicochemical techniquesinclude turning and deep plowing of soil, the addition of fresh soil,ridging precipitation, sorption, leaching, and washing. However, theseremediation methods require high energy input and expensivemachinery that causes secondary pollution (1). Another remediationmethod in which plants are used to clean soils, phytoextraction, hasthe potential to remediate metal contamination in soils (2). Whencompared with traditional methods, phytoextraction is a cost-effective and environmentally friendly alternative (3). Early studiesof phytoextraction focused on hyperaccumulating plants that wereknown to be able to accumulate and tolerate high levels of heavymetals. However, such hyperaccumulating plants generally have a lowefficiency due to their slow growth and small biomass (4).

To overcome these problems, synthetic chelating agents have beenused to increase the uptake and translocation of metals and to achievea high removal rate (5). Synthetic chelating agents such as EDTA caneffectively increase the solubility of metal contaminants in soils (6).However, the use of synthetic chelating agents often leads to poorbiodegradability and decreased plant growth and biomass production(7, 8).

Recently, the use of natural low molecular weight organic acids(NLMWOA) as an alternative to synthetic chelating agents was foundto be useful for the remediation of heavy metals. It is well known thatexudation of NLMWOA by roots plays a significant role in heavy metal

ing author. Tel.: + 82 2 3277 2375; fax: +82 2 3277 2385.ress: islee@ewha.ac.kr (I. Lee).

- see front matter © 2009, The Society for Biotechnology, Japan. All.jbiosc.2009.06.018

solubility (9, 10) and increased root growth (11). Some studies haveshown that the application of NLMWOA has positive effects on thephytoextraction of heavy metals from soil (9, 10). However, higherconcentrations of NLMWOA lead to decreased biomass, while lowerconcentrations of NLMWOA result in poor phytoextraction (12). Inaddition, the effectiveness of NLMWOA is dependent on the species ofplant being used (12).

Echinochloa crus-galli were found to have a high potential for thephytoextraction of heavy metal-contaminated soil in a previous study(13). Therefore, this study was conducted to investigate the ability ofroot exudates to enhance the phytoextraction of Cd-, Cu-, and Pb-contaminated soil. Specifically, soil pot experiments were used toevaluate the effects of applying root exudates to soil on the uptake ofCd, Cu, and Pb by E. crus-galli. In addition, the effects of root exudateson the growth of E. crus-galli were evaluated.

MATERIALS AND METHODS

Soil preparation and experimental set up Natural soil was sampled from thecampus of Ewha Womans University, Seoul, Korea. The physicochemical properties ofthe soil were as follows: soil texture — loamy sand (54.1% sand, 30.9% silt, and 15.0%clay); total organic matter 4%; total moisture content 3%; cation exchange capacity(CEC) 10.2 mmol kg−1; dehydrogenase activity 27 μg−1; pH 5.5. The soil was passedthrough a 2 mm sieve and then air-dried. For each experiment, 27 kg soil aliquots wereartificially contaminated with 600 mg kg−1 Pb, 40 mg kg−1 Cd and 100 mg kg−1 Cu. Pb,Cd, or Cu was added as an aqueous solution of Pb SO4, Cd SO4, and CuSO4 in water,respectively. The soils were then thoroughly mixed to ensure uniformity and then aged1 week to stabilization. Twenty-four hours prior to the start of each test, contaminatedsoils were measured. Soils were divided into three groups, a total of 27 pots, each ofwhich included nine 1.5 l cylindrical plastic pots, diameter 10 cm. The seeds of E. crus-galliwere sown directly in the 1 kg test pots, after which the seedlings were thinned to adensity of three per pot. The pots were then placed in a growth room that was

rights reserved.

TABLE 1. The concentration of organic acids in root exudates.

Succinic ug g−1 Acetic

Malic Maleic Oxalic Citric

1.6 n.d. n.d. 37.6 40.3 0.2

n.d. = none detected.

FIG. 1. Effects of root exudates on (A) Cd, (B) Cu, and (C) Pb accumulation in Echinochloacrus-galli. a Values shown are the means±SD (n=27). Values within a row or within acolumn followed by the same letter do not differ significantly (<0.05).

48 KIM ET AL. J. BIOSCI. BIOENG.,

controlled at 25 °C and subjected to a 16 h light/8 h dark cycle. In some treatments,50 ml of root exudates from Belamcanda chinensiswas applied. After 4 weeks, the plantswere carefully harvested.

Collection of root exudates and analysis of organic acids B. chinensis wasgrown for 150 days in the herb garden at Ewha Women's University, Korea. This areahas a continental climate, making the weather very warm and rainy in the summer andcold and dry in the winter. The mean temperature and precipitation were 12.3 °C and1235.2 mm, respectively (Korea Meteorological Administration 2008). B. chinensis wassown and grown in plots (each 2×2 m). Plants were harvested by gently removingthem from the soil. Prior to analysis, the plants were washed with water to remove thesoil deposits.

Plant sampling and the preparation of extracts were performed as described by Yunand Kil (14). At harvest, the fresh plants were separated into leaves, stems, and roots.Fresh roots (100 g) were immersed in 1 l of distilled water for 48 h, after which thesolutions were filtered through a 0.45 μm syringe filter and used undiluted.

Organic acids were analyzed by ion chromatography (Dionex, DX-500with an ED40Electro-chemical Detector, IonPac AS11 (4×250 mm) column and an ASRS-Ultra IIAnion self-regenerating suppressor). The total concentrations of the root organic acidswere calculated as the sum of the concentrations of individual organic acids.

Growth and metal uptake Plants were harvested from the soil. Prior toanalysis, the plants were washed with water to remove the soil deposits. The shoot androot lengths were then measured. To determine the amount of heavy metals in theplant, the roots and shoots were separated and oven dried at 70 °C for 48 h. Samples ofthe plants were then digested in concentrated HNO3 in a microwave digester (MDS-2000, CEM). The heavy metal contents were then determined using an atomicabsorption spectrophotometer (AAS analysis 100, Perkin Elmer), which was calibratedusing certified reference materials (No. 10-c Rice Flour; National Institute forEnvironmental Studies in Japan).

Analysis of the soil heavy metal fraction The concentrations of total,exchangeable and soluble heavy metals in the soil samples were measured. Soilsamples were dried at room temperature and then analyzed for water-soluble heavymetals by equilibrating 1 g of soil with 20 ml of 0.01 M KNO3 for 2 h (15). Theexchangeable heavy metals were then analyzed by extracting 1 g of the soil with 20 mlof 1 N NH4COOH for 1 h (16). To determine the total heavy metal concentration, 0.5 g ofsoil was extracted with 2.4 ml of aqua regia (35% HCl 1.8 ml+65% HNO3 0.6 ml) in anautomatic microwave digester (MDS-2000, CEM). The extracted heavy metals werethen analyzed using an atomic absorption spectrophotometer (AAS analysis 100, PerkinElmer) that had been calibrated using certified reference materials (MESS-2 MarineSediment; National Research Council of Canada).

Statistical analysis Data were analyzed by Tukey's test following one-wayANOVA using the SPSS 9.0 software. One-way ANOVA was used to determine thesignificance of differences in metal accumulation and growth among treatments.

RESULTS

Organic acids of the root exudates Table 1 shows the organicacid concentrations of the root exudates of B. chinensis, which affectedthe growth rate and heavy metal uptake of E. crus-galli. The primaryorganic acids in the exudates of B. chinensis were citric and oxalicacids. In addition, therewere small amounts of succinic and acetic acidin root exudates. However, malic and maleic acid were not detected.

Growth rate of E. crus-galli The root and shoot growth ratesare shown in Table 2. The application of root exudates had no toxic

TABLE 2. Effects of root exudates on shoot and root growth of Echinochloa crus-galli.

Metal Treatment Shoot (cm) Root (cm)

No metal Without exudates 31.8±4.7 18.8±4.6With exudates 32.9±1.4 19.5±2.1

Cd Without exudates 10.3±1.4 10.7±2.4With exudates 11.4±1.1⁎ 15.3±2.7⁎⁎⁎

Cu Without exudates 19.3±2.0 13.0±2.4With exudates 25.8±2.6⁎⁎⁎ 23.1±1.0⁎⁎⁎

Pb Without exudates 27.0±1.9 18.0±1.6With exudates 39.6±3.7⁎⁎⁎ 20.8±2.4⁎⁎⁎

Values are the means±SD (n=27). Significant t-test at ⁎p<0.05, ⁎⁎p<0.01,⁎⁎⁎p<0.001.

effects on the growth of E. crus-galli. Rather, the application ofexudates to Cu- and Pb-contaminated soil induced a significantincrease in the growth of shoots when compared to samples that didnot contain exudates (p<0.05). These findings indicate that exudatesfrom B. chinensis may affect the growth of E. crus-galli.

Accumulation and translocation of heavy metals As shownFig. 1, the accumulation of Cd, Cu and Pb in the roots and shootsincreased in response to the addition of exudates. Specifically, the Cd,Cu and Pb accumulation was 2-fold greater in the roots of plants thatwere treated with the exudates, while the accumulation of thesemetals was 3- to 4-fold higher in the shoots. Table 3 shows thebioconcentration factor (BCF) and translocation factor (TF) values forthe E. crus-galliwith or without exudates. The BCF values of Cd, Cu andPb treated soils increased in response to the addition of the exudates

TABLE 3. The bioconcentration factor (BCF) and the translocation factor (TF) of E. crus-gali cultured with and without exudates.

Treatment BCF TF

Cd Cu Pb Cd Cu Pb

Plant 0.41±0.09a 0.35±0.05a 0.3±0.01a 0.54±0.06a 0.28±0.09a 0.43±0.06a

Plant+exudate 1.8±0.5b 0.63±0.07b 0.8±0.03b 0.89±0.17b 0.83±0.14b 0.62±0.14b

BCF=Conroots/Consoil; TF=Conshoots/Conroots. Values shown are the means±SD (n=27). Values within a row or a column followed by the same letter do not differ significantly(<0.05).

ENHANCED HEAVY METAL PHYTOEXTRACTION 49VOL. 109, 2010

(p<0.05). The BCF values of E. crus-galli that were treated withexudates were highest for the Cd-contaminated soils. The TF valuesindicate the rate of translocation of the metal from the roots to theshoots. The TF values were greater for E. crus-galli cultivated with theexudates than for those that were cultivated without the exudates(p<0.05).

Changes in the soil heavy metal fraction To determine ifnatural chelation of E. crus-galli by treatment with the root exudatesof B. chinensis enhanced the rate at which Cd, Cu, and Pb wereremoved from the soil, we measured the total metal content in therhizosphere and bulk soil after four weeks of growth in contaminatedsoil. The Rhizosphere is the 1mm zone surrounding the roots of plantsin which complex relations exist among the plant and the soil itself.The changes in Cd, Cu, and Pb concentrations in each contaminatedsoil over a 4 week period are shown in Table 4. Soil containing plantsand the exudates showed a reduction in total metals when comparedto soil that did not contain the exudates. Notably, when plants weregrown in the presence of exudates there was a 52% and 14% reductionin the levels of Cd and Pb in the soil, respectively (p<0.05). Treatmentof the soil with root exudates also reduced Cu levels 21%; however,this difference was not significant. Additionally, the exchangeable Cd,Cu and Pb levels were slightly increased in soils that containedexudates when compared to those that did not. Again, this differencewas not significant. There was no soluble Cd or Cu detected in soilsthat did or did not contain the exudates.

DISCUSSION

The primary organic acids in the B. chinensis were citric and oxalicacids. The application of root exudates had positive effects on growthand the accumulation of metals in E. crus-galli. These findings aresimilar to those of previous studies that have suggested that NLMWOAincreases root growth and the accumulation of metals.

The results of this study demonstrate that citric acid and oxalic acidare capable of enhancing phytoextraction. This finding is consistentwith the results of several previously conducted studies. Duarte et al.(17) reported that the application of Cd with 25 μM citric acid resultedin an increase in Cd uptake by Halimione portulacoides of 27% to 61%.Nigam et al. (10) found that the accumulation of Cd in corn was

TABLE 4. The changes in total, exchangeable, and soluble metal contents in soil treatedwith and without exudates.

Metal Treatment Total Exchangeable(mg Pot−1)

Soluble

Cd No plant 43.7±4.1a 5.4±0.4a 1.3±0.1a

Plant 34.7±7.9a 10.8±0.8b n.d.b

Plant +exudates 16.5±5.5b 12.6±0.8b n.d.b

Cu No plant 93.8±6.1a 3.1±0.1a 0.5±0.2a

Plant 76.3±8.7b 11.7±1.1b n.d.b

Plant+exudates 59.9±7.2b 12.9±1.0b n.d.b

Pb No plant 581.1±15.3a 95.4±6.0a n.d.a

Plant 557.3±22.5a 134.7±14.1b 1.5±0.7b

Plant+exudates 481.1±19.1b 161.5±18b 0.6±0.2b

n.d. = none detected.Values are the means±SD (n=27); values within a row or a column followed by thesame letter do not differ significantly at the 0.05 level according to Tukey's test.

enhanced by the addition of citric andmalic acid to contaminated soil.Moreover, Chen et al. (18) reported that treatment with citric acidpromoted the translocation of heavy metals, especially Pb, from roots.Several previous studies have reported that treatment with oxalic acidincreased the concentration of Cu in the shoots of hyperaccumulatingplants (19, 20). Finally, application of 62.5 mM oxalic acid increasedthe Cu concentration in the shoots of tobacco (19). Root exudates havealso been found to increase the root and shoot growth of E. crus-gallieven though heavy metals have a toxic effect on the plant. The resultsof the present study provide evidence of the participation of organicacids in the development of heavymetals tolerance. These findings aresimilar to those of Duarte et al. (17), who reported that application ofNLMWOA at a concentration of 62.5mM induced no adverse effects onthe shoots of tobacco, but instead led to a slight increase in the growthof shoots. These results suggest that root exudates, especially citricand oxalic acid, lead to the release of damaged cells (21). In addition, ithas been suggested that NLMWOA has the ability to detoxifyintracellular heavy metals via binding of the metal (21). The highstability constants for metal-citrate and metal-malate complexes alsosuggest that these non-toxic complexes will preferentially form uponrelease into the rhizosphere (22). However, detoxification mechan-isms have only been shown to operate in hydroponic cultureconditions and not in soil (23).

The results of this study demonstrated that exchangeable metalcontents increase in soil that contains exudate. These findings indicatethat heavy metals are more easily transported in soil due to exudate,in addition to being more easily absorbed (24). Exchangeable metalsin the soil are considered to be easily available for plant uptake (25).Rovira (26) reported that LMWOA from plant roots are composed ofcitric acid, oxalic acid, tartaric acid, and acetic acid, which can combinewith metal ions. Taken together, these results indicate that the rootexudates of B. chinensis can be as efficient as natural chelates for use inphytoextraction by E. crus-galli. In addition, the application of rootexudates from B. chinensis is unique in that it enhanced phytoextrac-tion and stimulated growth of E. crus-galli. In this study, the impor-tance of the influence of the root exudates in rhizosphere metaldegradation is apparent.

ACKNOWLEDGMENTS

This study was conducted with the support of the 21st CenturyFrontier R&D program (R0120050001026802007) and the 2008KOSEF-DAAD Summer Institute Program provided by the KoreaScience and Engineering Foundation.

References

1. Cunningham, S. D. and Ow, D. W.: Promises of phytoremediation, Plant Physiol.,110, 715–719 (1996).

2. Susarla, S., Medina, V. F., and McCutcheon, S. C.: Phytoremediation: anecological solution to organic chemical contamination, Ecol. Eng., 19, 647–668(2002).

3. Salt, D. E., Smith, R. D., and Raskin, I.: Phytoremediation, Annu. Rev. Plant Physiol.Plant Mol. Biol., 49, 643–668 (1998).

4. Baker, A. J. M. and Brooks, R. R.: Terrestrial higher plants with hyperaccumulatemetallic elements — a review of their distribution, ecology and phytochemistry,Biorecovery, 1, 81–126 (1989).

50 KIM ET AL. J. BIOSCI. BIOENG.,

5. Lasat, M. M.: Phytoextraction of metals from contaminated soil: a review of plant/soil/metal interaction and assessment of pertinent agronomic issues, J. Hazard.Subst. Res., 2, 1–25 (2000).

6. Ebbs, S. E., Norvell, W. A., and Kochian, L. V.: The effect of acidification andchelating agents on the solubilization of uranium from contaminated soil,J. Environ. Qual., 27, 1486–1494 (1998).

7. Grcman, H. S., Velikonja-Bolta, D., Vodnik, B., and Kos, D. L.: EDTA enhancedheavy metal phytoextraction: metal accumulation, leaching and toxicity, Plant Soil,235, 105–114 (2001).

8. Blaylock, M., Salt, D. E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik,Y., Ensley, B. d., and Raskin, E.: Enhanced accumulation of Pb in Indianmustard by soil-applied chelating agents, Environ. Sci. Technol., 31, 860–865(1997).

9. Krishnamurti, G. S., Cielinski, R. G., Huang, P. M., and vanRees, K. C. J.:Kinetics of cadmium release from soils as influenced by organic acids:implementation in cadmium availability, J. Environ. Qual., 26, 271–277(1998).

10. Nigam, R., Srivastava, S., Prakash, S., and Srivastava, M. M.: Cadmiummobilization and plant availability — the impact of organic acids commonlyexuded from roots, Plant Soil, 230, 107–113 (2001).

11. Uren, N. C. and Reisenaner, H. M.: The role of root exudates in nutrient acquisition,in: P. B. Tinker, A. Lanchli (Eds.), Advances in Plant Nutrition, Praeger, New York,1998, pp. 79–114 (1998).

12. Long, X. X., Ni, W. Z., and Ye, Z. Q.: Effect of organic acids application on Zn uptakeand accumulation by two ecotypes of Sedum alfredii Hance, J. Plant Nutr. Fertil., 8,467–472 (2002).

13. Kim, S. H, Park, J. S., and Lee, I. S.: Characterization of Cadmium binding ligandsfrom roots of Echinochloa crusgalli var. frumentacea, J. Plant Biol., 52, 167–170(2009).

14. Yun, K. W. and Kil, B. S.: Assessment of allelopathic potential in Artemisia princesvar. orientalis residues, J. Chem. Ecol., 18, 1933–1940 (1992).

15. Sposito, G., Lund, L. J., and Chang, A. C.: Trace metal chemistry in arid-zone fieldsoils amended with sewage sludge. I — Fractionation of Ni, Cu, Zn, Cd, and Pb insolid phases, Soil Sci. Soc. Am., 46, 260–264 (1982).

16. Miller, W. O., Martens, D. C., and Zelanzny, L. W.: Effect of sequence in extractionof trace metals from soils, Soil Sci. Soc Am., 50, 598–601 (1985).

17. Duarte, B., Delgado, M., and Cacador, I.: The role of citric acid in cadmium andnickel uptake and translocation, in Halimione portulacoides, Chemosphere, 69,836–840 (2007).

18. Chen, Y. X., Lin, Q., and Luo, Y. M.: The role of citric acid on the phytoremediationof heavy metal contaminated soil, Chemosphere, 50, 807–811 (2003).

19. Evangelou, M. W. H., Ebel, M., and Schaeffer, A.: Evaluation of the effect of smallorganic acids on phytoextraction of Cu and Pb from soil with tobacco Nicotianatabacum, Chemosphere, 63, 996–1004 (2006).

20. Nascimento, C. W. A., Amarasiriwardena, D., and Xing, B.: Comparison of naturalorganic acids and synthetic chelates at enhancing phytoextraction of metals from amulti-metal contaminated soil, Environ. Pollut., 140, 114–123 (2006).

21. Lee, J., Reeves, R. D., Brooks, R. P., and Jaffre, T.: Isolation and identification of acitrate-complex of nickel from nickel-accumulating plants, Phytochemistry, 16,1502–1505 (1977).

22. Jones, D. L. and Kochian, L. V.: Aluminum-organic acid interactions in acidsoils. 1. Effect of root-derived organic-acid on the kinetics of Al dissolution,Plant Soil, 182, 221–228 (1996).

23. Parker, D. R. and Pedler, J. F.: Probing the malate hypothesis of differentialaluminum tolerance in wheat by using other rhizotoxic ions as proxies for Al,Planta, 205, 389–396 (1998).

24. White, M. C., Decker, A. M., and Chaney, R. L.:Metal complexation in xylem fluid. I:Chemical composition of tomato and soybean stem exudates, Plant Physiol., 67,292–300 (1991).

25. Kabata-Pendias, A.: Behavioural properties of tracemetals in soils, Appl. Ecochem.,2, 3–9 (1993).

26. Rovira, A. D.: Plant root exudates, Bot. Rev., 35, 35–57 (1969).