Ecological Engineering 37 (2011) 19421946

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Ecological Engineering
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Short communication

Phytoaccumulation of cadmium through Azolla from aqueous solution

Cai-yun Tan a , Xiao-quan Shan b , Guo-zhong Xu c , Yu-Man Lin a , Zu-liang Chen a,
a

School of Chemistry and Material Sciences, Fujian Normal University, Fuzhou 350007, China Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871, Beijing 100085, China c Azolla Research Center at Fujian Academy of Agricultural Science, Fuzhou 350013, China
b

a r t i c l e

i n f o

a b s t r a c t
Azolla, which is an aquatic fern, has proved to be effective in the uptake and accumulation of metals from polluted waters. Azolla spp., namely A. microphylla cv. MH3 and A. caroliniana Willd, were chosen as model plants so that Cd(II) could be accumulated from aqueous solution. An increase in uptake time and the concentration of Cd(II) in aqueous solution resulted in more Cd(II) accumulation in both species. Modied MichaelisMenten equation was employed to describe the concentration-dependent kinetics of Cd(II) uptake through the roots of A. microphylla cv. MH3, and the values of Km and Vmax were found to be 0.23 mg/L and 16.49 g/(g.f.wt.h), respectively. Cd(II) uptake by A. microphylla cv. MH3 occurs partly through Ca(II) channels and has the potential to be mediated by Zn(II) transporters. 2011 Elsevier B.V. All rights reserved.

Article history: Received 16 July 2010 Received in revised form 12 January 2011 Accepted 23 January 2011

Keywords: Phytoaccumulation Cadmium Ca(II) channel Zinc transporter

1. Introduction Phytoremediation, such as constructed treatment wetlands, is a technology that has the potential to solve pollution caused by heavy metals (Williams, 2002; Brix and Arias, 2005; Maine et al., 2006; Upadhyaya et al., 2007). However, choosing a plant species that can remove metal ions from polluted water depends on three variables in the environment: plant growth, biomass, and metal levels (Williams, 2002). Azolla, a oating aquatic fern, is found worldwide and it grows in all kinds of freshwater and wastewater. Azolla has high biomass productivity and a remarkable capacity to concentrate elements, including toxic heavy metals (Bennicelli et al., 2004). Azolla is easy to harvest and desiccate compared to other aquatic plants. These traits make it an ideal candidate for phytoremediation of Cd-contaminated wastewater (Arora et al., 2006). It has recently received attention as a potential plant for phytoremediation of heavy meals from polluted water. A. caroliniana has the capacity for taking up Hg(II) and Cr(III) (Bennicelli et al., 2004). A mechanistic study indicated that both apoplastic adsorption and symplastic storage were responsible for the overall accumulation of Pb(II) in the leaf of A. liculiodes (Benaroya et al., 2004). Living A. liculoides can be used to remove Pb(II), Cd(II), Ni(II) and Zn(II); the accumulation t the rst-order kinetic model well (Rakhshaee et al., 2006). A. liculoides grown in a high concentration of Cd(II)

was able to accumulate Cd(II) (Sela et al., 1989). However, it is necessary to understand the metal accumulation mechanism in order to assess the potential phytoaccumulation of Cd(II) from contaminated waters using Azolla species. The objective of this study was to examine whether Azolla is able to accumulate Cd(II) from aqueous solution. Hence, a hydroponic culture method was used to study the accumulation of Cd(II) by A. microphylla cv. MH3 and A. caroliniana Willd. The roles of Ca(II) channel, and Zn(II) transporters in the uptake of Cd(II) using A. microphylla cv. MH3 were studied.

2. Materials and methods 2.1. Azolla species collection, cultivation and selection A. microphylla cv. MH3 and A. caroliniana Willd were obtained from the Azolla Research Center at the Fujian Academy of Agricultural Sciences (Fuzhou, China) and cultured in a nutrient solution: 1.0 mM CaSO4 , 1.6 mM MgSO4 , 0.7 mM NaNO3 , 0.3 mM KH2 PO4 , 0.3 mM KCl, and 10.0 M EDTANa, 10.0 M FeSO4 , 24.3 M H3 BO3 , 7.7 M Na2 MoO4 , and 0.2 M ZnSO4 for 3 days. The nutrient solution was buffered to pH 6.0 with 2 mM MesTris. Two species were grown in a plant growth chamber with operating conditions consisting of 12 h light/12 h darkness and a temperature of 30/22 C. Two species were cultured in the nutrient solution for 3 days, then transferred to a 1.0 mg/L Cd(II) uptake solution for 1 day, and harvested, rinsed with ice-cold distilled water thoroughly. Then they were desorbed with 5 mM ice-cold CaCl2 solution for

Corresponding author. Fax: +86 591 83465689. E-mail address: zlchen@fjnu.edu.cn (Z.-l. Chen). 0925-8574/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2011.01.010

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Dry biomass (g)

40 min. Preliminary experiments indicated that desorption in 5 mM ice-cold CaCl2 solution for 40 min was enough to desorb Cd(II) adsorbed onto the cell wall of fern roots. The results are illustrated in Fig. 2(a). After desorption, two species were rinsed with icecold distilled water thoroughly again, excised into roots and leaves, dried at 80 C for 2 days, then the dried plant material and digested were weighed with HNO3 HClO4 for nal determination of Cd(II). A usable Azolla species was employed in the subsequent experiments and all tested experiments were carried out in triplicate. 2.2. Time course and kinetics of Cd(II) uptake by A. microphylla cv. MH3 A. microphylla cv. MH3 grown in the nutrient solutions for 3 days was transferred to 1.0 mg/L Cd(II) uptake solution. At different time intervals of 5, 10, 15, 20, 25, 30, 40, 60 min, and 2, 4, 8 h, A. microphylla cv. MH3 was harvested. The digestion and extraction of Cd (II) have been stated previously. This experiment was performed to test A. microphylla cv. MH3 uptake of Cd(II) at different Cd(II) concentrations. A. microphylla cv. MH3 was incubated with fresh nutrient solution containing various concentrations of Cd(II) (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, and 5.0 mg/L). After 20 min of uptake, they were harvested, desorbed in 5 mM ice-cold CaCl2 for 40 min, excised into roots and leaves, dried with paper tissue and nally digested to determine Cd(II). 2.3. Effect of Ca(II) channel blocker and Zn(II) deciency on Cd(II) inux into A. microphylla cv. MH3 The experiments were performed to test the potential contribution of Ca(II) channels and Zn(II) transporters to Cd(II) inux into A. microphylla cv. MH3. A Ca(II) channel blocker, 0.2 mM La(NO3 )3 , was added to the Cd(II) uptake solution (White, 1997). Zn(II) deciency was induced in A. microphylla cv. MH3 by replacing a full nutrient solution with a nutrient solution without Zn(II) for 3 days. Following this, A. microphylla cv. MH3 was incubated in a 1.0 mg/L Cd uptake solution without Zn(II). A. microphylla cv. MH3 grown in full nutrient solution and later incubated in 1.0 mg/L Cd(II) uptake solution with Zn(II) while a solution without La(NO3 )3 was used as control. After 4 h uptake, A. microphylla cv. MH3 was harvested and desorbed with 5 mM ice-cold CaCl2 for 40 min, excised into roots and leaves, and digested prior to Cd(II) determination. Cadmium contents in the ferns were established using ame atomic absorption spectrometry (FAAS) (Varian AA240, Shanghai, China) after digestion of samples with HNO3 HClO4 (3:1, v:v) on a hotplate. This continued until a clear solution or achromatous residue was obtained. After cooling, the residue was dissolved in 1% HNO3 prior to determining the amount of Cd(II). Cd contents in Azolla were calculated from rstly, the concentration and volume of the digested solution, and secondly, the plants dry weight. 3. Results and discussion 3.1. Accumulation of Cd(II) by two Azolla species Two species were used to compare the biomass and Cd(II) accumulation capability after culturing in the uptake solution for 1 day, and the results are shown in Fig. 1. In Fig. 1(a) there is no obvious difference in their dry biomass of the intact Azolla and leaves. Compared to A. caroliniana Willd (Fig. 1(c)), A. microphylla cv. MH3 roots have almost double the amount of biomass. As shown in Fig. 1(b), a much higher concentration of Cd(II) was found in the roots than in the leaves and intact ferns. For example, the Cd(II) contents in the roots of A. caroliniana Willd and A. microphylla cv. MH3 were

0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

Intact Azolla

Leaves

Roots

1400

Cd in Azolla ( g/g.d.wt)

1200 1000 800 600 400 200 0 Intact Azolla Leaves Roots

1400 1200 1000 800 600 400 200 0

Cd in Azolla (g/g.d.wt )

Intact Azolla

Leaves

Roots

Fig. 1. A comparison of dry biomass of the intact Azolla, roots and leaves after culturing in the uptake solution for 1 day (a), Cd(II) contents in the intact Azolla, roots and leaves after Cd(II) uptake for 1 day without desorption (b), and after desorption with 5 mM ice-cold CaCl2 for 40 min (c). Black pillar for A. microphylla cv. MH3, and white pillar for A. caroliniana Willd.

1364 and 1206 g/g, respectively, while only 60 and 72 g/g were detected in the leaves of A. caroliniana Willd and A. microphylla cv. MH3, respectively. Those results suggested that neither species is a hyperaccumulator of Cd(II). This nding was supported by Kirkham (2006). Furthermore, after desorption in 5 mM ice-cold CaCl2 for 40 min, Cd(II) contents in the roots fell from 1364 to 600 g/g and from 1206 to 522 g/g for A. caroliniana Willd and A. microphylla cv. MH3, respectively. In contrast, the Cd(II) contents in the leaves remained virtually unchanged after desorption, implying that Cd(II) in the leaves was translocated from the roots, but not directly taken up by leaf surface cell walls (Fig. 2(a)). There is less

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a
Cd remaining in Azolla (g g.d.wt)

900
800 700 600 500 400 300 200 100 0 0 10 20 30 40 50 60

Desorption time (min)

450 400 350

non-selective with the transition metals (Cutler and Rains, 1974). Compared to this, the Cd(II) in the leaves was almost constant after desorption, implying that Cd(II) in the leaves of both species was not directly taken up by leaves, while the leaf Cd(II) was probably transported from the roots and then stored symplastically. The uptake time is one of the most important factors affecting the concentration of heavy metals in plants. As shown in Fig. 2(b), a similar uptake pattern was observed for intact A. caroliniana Willd, its roots and leaves. Cd(II) uptake by A. microphylla cv. MH3 roots was biphasic, exhibiting an initial rapid phase and a later slower linear phase. The uptake of Cd(II) increased rapidly with more time up to 60 min, then uptake of Cd(II) rose slowly with a further increase in time to 8 h. The initial rapid phase (rst 60 min of uptake) presumably is the result of diffusion of Cd(II) into the free space and binding to root-cell walls. The slower phase of uptake of Cd(II) over the subsequent uptake time is believed to represent transport across the plasma membrane (Han et al., 2006). However, the uptake of Cd(II) did not reach saturation because the fern was still growing. Similar results were observed in one study concerning La(III) accumulation in wheat roots (Wang et al., 2004). 3.3. Concentration-dependent kinetics of Cd(II) uptake by A. microphylla cv. MH3 roots To investigate the concentration-dependent uptake kinetics of Cd(II) and to minimize the possibility of its efux across the plasma membrane back into the external solution, a short uptake period of 20 min was used (Han et al., 2005). Fig. 3(a) shows that the uptake of Cd (II) increased rapidly when Cd(II) in solution rose from 0 to 0.1 mg/L, then a slower rise in the uptake curve occurred while the Cd(II) in the uptake solution increased to 5 mg/L. This indicates that concentration-dependent uptake for Cd(II) inux in the roots of A. microphylla cv. MH3 was characterized by a smooth, non-saturated curve that approached linearity at a low Cd(II) concentration. Cd(II) uptake kinetics may be characterized with a modied MichaelisMenten equation: V = aC + Vmax C Km + C (1)

Cd remaining in Azolla (g/g.d.wt)

300 250 200 150 100 50 0 0 60 120 180 240 300 360 420 480

Uptake time (min)

Fig. 2. (a) Effect of desorption time on the uptake of Cd(II) by A. microphylla cv. MH3: ( ) roots; ( ) leaves. (b)Time course of Cd(II) uptake by A. microphylla cv. MH3 after desorption with 5 mM ice-cold CaCl2 for 40 min: ( ) roots; ( ) intact A. microphylla cv. MH3; ( ) leaves.

Cd(II) content in the roots of A. microphylla cv. MH3 compared to that in A. caroliniana Willd, However, the former has a larger root biomass, there was no statistically signicant difference regarding Cd(II) content in the intact plant. A. microphylla cv. MH3 was used in the following experiments. 3.2. Desorption time and time course of Cd(II) uptake by A. microphylla cv. MH3 Because the roots of A. caroliniana Willd and A. microphylla cv. MH3 grow downward, the nutrient solution and the downward leaves of both species come into direct contact with the surface of the nutrient solution. Therefore, we need to verify whether Cd(II) is directly taken up by the downward leaves or transported from the roots. In this study, 5 mM ice-cold CaCl2 (Hart et al., 1998) was chosen as the reagent to desorb the extracellular Cd(II) after 4 h uptake of A. microphylla cv. MH3 in uptake solution; the results are shown in Fig. 2(a). Our results indicated that after desorption in 5 mM ice-cold CaCl2 for 40 min, the Cd(II) content in the roots declined from 839.1 to 335.4 g/g for A. microphylla cv. MH3. This implies that a majority of root Cd(II) was bound with free space and cell-wall, which can be desorbed during the rst 30 min. Similar results were reported by Lasat et al. (1996) and Han et al. (2005), suggesting that one mechanism for accumulating Cd(II) through the roots can be termed exchange adsorption. This is explained by the reversibly bound Cd(II) in the roots being readily exchanged when it is exposed to desorption solution. The exchange sites were

where V is the uptake rate, C is the Cd(II) concentration in uptake solution, a is parameter characterizing the linear part of the uptake rate, and Vmax and Km represent the maximum uptake rate and MichaelisMenten rate constant of the saturable component of Eq. (1), respectively. The linear components are hypothesized to represent cell-well-bound metal ions remaining after desorption, while the saturable component represents true uptake across the plasma membrane. In this study, the modied MichaelisMenten equation was also employed to t experimental data and resolved it into saturable and linear components (R = 0.999) and shown in Fig. 3(b). The values of Km and Vmax were found to be 0.23 mg/L and 16.49 g/(g.f.wt h), respectively. A high Vmax value suggested that there might be a higher density of Cd(II) transporters per unit membrane area in the roots of A. microphylla cv. MH3 (He et al., 2007). 3.4. Contribution of Ca(II) channel and Zn(II) transporters to Cd(II) inux into A. microphylla cv. MH3 In this section the control experiment shows that there is no signicant impact on the growth and physiology of the Azolla when blocking of the Ca(II) channel or Zn(II) deciency in the 4 h uptake time occurs. Fig. 4 shows the Cd(II) uptake was partly inhibited by Ca(II) channel blocker of 0.2 mM La(NO3 )3 treatment. Cd(II) contents in the intact A. microphylla cv. MH3, its leaves and roots decreased by 39%, 37%, and 41%, respectively, compared to the con-

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25 20

15 10 5 0 0 1 2 3 4 5

Cd in solution (mg/L)

30 25 20 15 10 5 0 0

Cd in solution (mg/L)
Fig. 3. Concentration-dependent kinetics of Cd(II) uptake by A. microphylla cv. MH3 roots: (a) Effect of initial concentration of Cd(II) in the Cd uptake solution (means SE, n = 3); (b) Deconvolution of the overall kinetic curves into liner and saturable components according to MichaelisMenten equation.

of the Ca pathways role in Cd uptake and translocation by the hyperaccumulator Sedum alfredii has been reported by Lu et al. (2010). Their study suggests that Cd uptake and translocation in the hyperaccumulator S. alfredii plants is positively associated with Ca pathway. However, this disagreed with Han et al. (2006), who indicated that all blockers of Ca(II), Na(I), K(I) channels had no statistically signicant inhibition effects on Cd(II) inux into maize roots, implying that Cd(II) uptake through Ca(II), Na(I), K(I) cation channel is unlikely to occur. This difference was probably due to different plant cultivars used in that study. La(III) is a broad type of Ca(II) channel blocker, but also blocks non-selection cation channels, anion channels and general metabolism. The decrease in Cd(II) uptake by A. microphylla cv. MH3 under La(III) could result either from competitive uptake between La(III) and Cd(II) through Ca(II) channels, or from the blockage of Ca(II) channels. These channels are also the channels of Cd(II) inux into A. microphylla cv. MH3. However, further research is needed to verify what mechanism is responsible for Ca(II) channel inhibition. The presence of Zn(II) can control the levels of Cd(II) entering the plant not only by controlling its intracellular levels, but also by replacing the toxic Cd(II) (Aravind and Prasad, 2003). Cd(II) and micronutrients such as Cu(II), Zn(II), Fe(II) and possibly Mn(II) are present in reasonable amounts at the site in various forms, and have a common transport site (Cataldo et al., 1983). Cd(II) and Zn(II) may share similar uptake and transport systems in the Cd (II) hyperaccumulator of Arabidopsis halleri (Kpper et al., 2000). In addition, Cd(II) and Zn(II) are both metals as found on the periodic table and exhibit similar chemical properties and environmental behaviours. Furthermore, Zn(II), unlike Cd(II) is an essential plant micronutrient, so Cd(II) inux into plants would occur through Zncarrier. Han et al. (2006) also indicated that zinc deciency exerted a stimulatory effect on Cd(II) uptake in maize roots, suggesting that Cd(II) was most probably taken up into the maize roots via Zn(II) transporter protein. However, our study indicated that Zn(II) deciency markedly enhanced Cd(II) uptake by A. microphylla cv. MH3, enabling us to conclude that the Cd(II) inux into this fern was mediated at least in part by Ca2+ channels and most potentially governed by Zn(II) transporters. 4. Conclusions Cd(II) accumulation by A. microphylla cv. MH3 increased when uptake time and initial Cd(II) concentration rose. A modied form of the MichaelisMenten equation t the concentration-dependent kinetics of Cd(II) uptake by roots, and the values of Km and Vmax were found to be 0.23 mg/L and 16.49 g/(g.f.wt h), respectively. The use of La(III) as Ca(II) channel blocker signicantly inhibited Cd(II) inux, and Zn(II) deciency markedly increased Cd(II) uptake. These results indicate that the uptake of Cd(II) through this oating aquatic fern was mediated at least in part by Ca(II) channels, and zinc transporters. Acknowledgement This study was nancially supported by the MinJiang Fellowship from Fujian Normal University, P.R. China. References
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trol. It also can be seen from Fig. 4 that Zn(II) deciency markedly increased Cd(II) uptake by 57%, 59% and 40% in the intact A. microphylla cv. MH3 and leaves and roots, respectively, when compared to the control. The results suggested that Ca(II) channel blocker partly inhibited Cd(II) uptake, and the Ca(II) channels also serve as channels of Cd(II) inux into A. microphylla cv. MH3. A similar observation

Cd influx into Azolla roots [(g/g.f.wt ) /h]

500 450 400

Cd in Azolla roots (g/g.f.wt)

Control Control - Zn (II) Control + La (III)

Cd in Azolla (g/g.d.wt)

350 300 250 200 150 100 50 0 Intact Azolla Leaves Roots

Fig. 4. Inuences of a Ca2+ channel blocker of La3+ and Zn(II) deciency on Cd(II) uptake by A. microphylla cv. MH3.

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