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Enhanced phytoremediation of arsenic contaminated land
Chemosphere 68 (2007) 1906–1912 www.elsevier.com/locate/chemosphere
Enhanced phytoremediation of arsenic contaminated land P. Jankong a, P. Visoottiviseth a
a,*
, S. Khokiattiwong
b
Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Rachataewee, Bangkok 10400, Thailand b Phuket Marine Biological Center, Ministry of Agriculture and Cooperatives, Panwa Cape, Phuket 83000, Thailand Received 20 December 2006; received in revised form 26 February 2007; accepted 26 February 2007 Available online 9 April 2007
Abstract In an attempt to clean up arsenic (As) contaminated soil, the effects of phosphorus (P) fertilizer and rhizosphere microbes on arsenic accumulation by the silverback fern, Pityrogramma calomelanos, were investigated in both greenhouse and field experiments. Field experiments were conducted in Ron Phibun District, an As-contaminated area in Thailand. Soil (136–269 lg As g 1) was collected there and used in the greenhouse experiment. Rhizosphere microbes (bacteria and fungi) were isolated from roots of P. calomelanos growing in Ron Phibun District. The results showed that P-fertilizer significantly increased plant biomass and As accumulation of the experimental P. calomelanos. Rhizobacteria increased significantly the biomass and As content of the test plants. Thus, P-fertilizer and rhizosphere bacteria enhanced As-phytoextraction. In contrast, rhizofungi reduced significantly As concentration in plants but increased plant biomass. Therefore, rhizosphere fungi exerted their effects on phytostabilization. 2007 Elsevier Ltd. All rights reserved. Keywords: Arsenic; Phosphorus-fertilizer; Rhizosphere microbes; Phytoremediation; Phytoextraction; Phytostabilization
1. Introduction The problem of As contamination in the environment is a global concern due to its high toxicity to living organisms and its worldwide distribution. In Ron Phibun District, Nakhon Sri Thammarat Province, Thailand, tin mining activities during the last century caused As contamination of soil and water which ultimately led to human health problems. Although tin mining was prohibited in the late 1980s, the release of arsenopyrite (FeAsS) from the tin ore has left extensive As contamination in many areas of Ron Phibun District. The long-term use of contaminated water for agriculture or direct consumption poses a serious risk of chronic As poisoning among the local population causing diseases such as skin melanoma, cancer and high blood pressure. The problems originating from As contamination still prevail today. Therefore, to protect animal and human health, remediation of the As-contaminated sites has *
Corresponding author. Tel.: +66 2 201 5272; fax: +66 2 354 7161. E-mail address: [email protected] (P. Visoottiviseth).
0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.02.061
become an important issue. Fortunately, the silverback fern (Pityrogramma calomelanos) has been discovered in Thailand as an As-hyperaccumulating fern for phytoextraction of As-contaminated soil (Visoottiviseth et al., 2002). However, a key issue for an effective phytoremediation process, especially phytoextraction, is to enhance pollutant phytoavailability and to sustain adequate pollutant concentrations in the soil solution for plant uptake (Lombi et al., 2001). In addition, candidate plants for the phytoextraction purpose should tolerate the pollutants and produce large amounts of biomass. Besides the ability of the plants, rhizosphere factors are also important for improving phytoremediation efficiency. It has been reported that P-fertilizer, rhizosphere microbes (bacteria and fungi), root exudation, and chelating agents could enhance plant uptake and accumulation of contaminants due to their effects on improving available pollutants for plants (Chen and Cutright, 2002; Cao et al., 2003). For the phytoremediation of As, adding P-fertilizer to As-contaminated soils might affect phytoremediation efficiency due to the physicochemical similarity of the
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912
phosphate ion and arsenate ion (Adriano, 2001). Moreover, because of their role of enhancing plant growth, rhizosphere microbes are responsible for nutrient (N/P) mobilization (Dun˜abeitia et al., 2004). Therefore, the effects of microbes on the availability and transport of As to the plant should be considered for phytoremediation. However, the interactions of As and these rhizosphere factors are not yet well understood (Fitz and Wenzel, 2002; Leung et al., 2006; Trotta et al., 2006). In this study, the effects of P-fertilizer and rhizosphere microbes on plant biomass production and As accumulation in P. calomelanos were investigated with the aim to utilize these factors in their capacity to enhance As remediation of plants in polluted soil. 2. Materials and methods 2.1. Plants and soils Spores of P. calomelanos were collected from Ron Phibun District in southern Thailand. Seedlings were sown from spores in sterilized peat moss. Seedlings at the early sporophyte stage were transplanted individually into sterilized vermiculite. At the age of six months, the seedlings were used to set up experiments. The field experiment was set up in Ron Phibun District. Soil used in the greenhouse experiment was taken from the same site. The soil (0–15 cm depth) had the following properties (dry matter basis): pH (in water) 3.9–6.8, 0.06 dS m 1 EC, 2.54% organic matter, 65 lg g 1 available P, 32 lg g 1 available K, 270 lg g 1 total As (determined by Department of Soil Science, Ministry of Agriculture and Cooperatives, Thailand). The contaminated soil determined in this study contained 136–269 lg As g 1. The soil was air-dried and passed through a 2 mm sieve to remove stones and plant materials. After that, the soil was autoclaved for 45 min to kill native microbial populations. 2.2. P-fertilizer amendment experiments Six hundred grams of sterile soil was placed in each pot for the greenhouse experiment. Two levels of P (0, 100 mg P kg 1 soil as KH2PO4) were applied in the form of a solution to the soil in each pot. The soil was then mixed thoroughly before planting. Seedlings were grown in pots, one plant per pot for 16 wk. There were four replicates for each treatment. The plants were watered daily with tap water and the fertilizer solution was sprayed on the frond every wk to avoid nutrient deficiency. In the field experiment, seedlings were transplanted to each plot (2 m · 2 m). There were two treatments (with and without fertilizer) with eight plants per treatment. Approximately 200 g pellets of fertilizer (N:P:K = 6:18:6) was scattered over the soil of the treatment with fertilizer. At each harvesting time, 4 and 8 wk, four plants were collected.
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2.3. Preparation of inoculum of rhizosphere microbes 2.3.1. Rhizobacterial inoculum Plant roots and soils were collected from As-contaminated land in the Ron Phibun District, Nakhon Sri Thammarat province, southern Thailand. Each soil sample of 5 g was suspended in 20 ml of 0.85% NaCl solution. The soil suspension was left for about 30 min to allow large soil particles to settle out. One milliliter of suspension was added to each of 50 ml of enrichment medium (8 g nutrient broth and 0.5 g yeast extract with 1000 ml distilled water in a 250 ml flask). The culture was then incubated at 30 C on a shaker set at 200 rpm for 7 d. After that, 1 ml of the sample was transferred into 100 ml mineral medium containing arsenate at a concentration of 70 mg l 1 (pH adjusted to 5.6 by HCl) and 2 lg ml 1 of Fungizone (obtained from GIBCO). The mineral medium was prepared by mixing 0.5 g NH4Cl, 0.25 g MgCl2 Æ 6H2O, 0.5 g KH2PO4, 0.5 g beef extract and 0.3 g glucose with 1000 ml distilled water. The cultures were sub-cultured every 2 wk into a fresh medium and incubated under the same conditions. The culture was then centrifuged and rinsed the pellets three times with distilled water. The organisms were then inoculated in 250 ml flasks containing 100 ml of nutrient broth (NB), obtained from Difco, for 48 h on a shaker and then centrifuged at 5500 rpm for 7 min. The pellet containing bacterial cells was suspended in fresh NB and used as rhizobacterial inoculum. 2.3.2. Rhizofungal inoculum Plant roots were washed with tap water, rinsed with sterile deionized water to remove adhered soil, cut into segments (2 cm) and placed on potato dextrose agar (obtained from Difco) plates containing 70 mg l 1 arsenate and antibiotic (Penicillin–Streptomycin at 100 units ml 1 obtained from GIBCO). All plates were incubated at 27 C for 48– 72 h (Visoottiviseth and Panviroj, 2001). Fungal cultures were transferred and grown in 250 ml flasks containing 100 ml of potato dextrose broth (PDB), obtained from Difco, liquid medium and shaken at 150 rpm, 27 C. After 6 d, the mycelia pellets (diameter of 1.3 mm) were harvested by vacuum filtration, washed three times with sterile water, transferred to fresh PDB and used as rhizofungal inoculum. 2.4. Rhizosphere microbe experiments 2.4.1. Greenhouse experiments Sterile soil (600 g) were placed in each sterile pot. Deionised water (100 ml) was added and mixed with the soil. P. calomelanos, at 6-month-old, were acclimatized in Hoagland solution for 1 wk. They were then inoculated with either rhizobacteria or rhizofungi by soaking the plants in the appropriate inoculum for about 30 min. After that, the plants were separately grown in each pot for 6 wk and were watered daily with tap water. There were three
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P. Jankong et al. / Chemosphere 68 (2007) 1906–1912
treatments i.e., rhizobacteria-inoculated, rhizofungi-inoculated and non-inoculated (control), with three replicates per treatment. 2.4.2. Field experiments Six month old P. calomelanos was transplanted into each plot (2 m · 2 m). There were three treatment plots, with eight ferns per plot. Non-inoculated plants were used as a control. The plants were inoculated by soaking in the inoculum suspension for 30 min before planting in the appropriate plot. The plants were harvested after 6 and 12 wk.
2.6. Statistical analysis The data were statistically analyzed by a one-way analysis of variance (ANOVA) for the rhizosphere microbes study and a T-test for the P-fertilizer amendment study. When a significant (P < 0.05) treatment effect was found, the mean values were compared using the Duncan’s multiple range test and student T-test for rhizosphere microorganisms and P-fertilizer amendment study, respectively. 3. Results
2.5. Chemical analysis
3.1. Effects of P-fertilizer on plant biomass and As accumulation
Plant roots and fronds were harvested, washed with tap water, rinsed with deionized distilled water, and oven dried at 50–55 C for 3 d. After that, frond and root biomass were determined and the dried plant material was then ground to powder (mortar/pestle). Samples were prepared for the As concentration analysis by using the dry ashing method. A portion of the dry powder (20–50 mg) was accurately weighed (±0.01 mg) directly into a crucible and mixed with 1.5 ml freshly prepared slurry (30 g Mg(NO3)2, 50 g MgO and 500 ml of distilled water). The mixture was dried overnight at 80 C, and digested in a muffle furnace (200 C for 1 h, 300 C for 1 h and 500 C for 8 h). The residue was dissolved in 6 M HCl (2.5 ml) and the solution mixed with distilled water (2.5 ml). Total arsenic concentrations in these solutions were determined by a Hydride Generation Atomic Absorption Spectrophotometer (HGAAS) using a Perkin–Elmer MHS-20 mercury/hydride system coupled to a Perkin–Elmer 2380 AAS. Arsenic concentrations in the samples were reported on a dry mass basis. The certified reference material MESS3: marine sediment (National Research Council Canada, Ottawa, Canada) was used for quality control as 85 ± 7% recovery, n = 3.
In both the greenhouse and field studies, the addition of P-fertilizer increased the biomass of P. calomelanos significantly (Table 1). For the field study, the beneficial effect of P-fertilizer on root production was clearly shown at 4 wk while the effect on frond production was clearly shown at 8 wk after planting (Table 1). Arsenic concentration in the fronds of P. calomelanos was higher than in the roots as was expected (Table 1). Addition of P-fertilizer increased arsenic concentration in the plant tissues in both the greenhouse and field studies but significantly higher effects were present only in the frond parts (P < 0.05) (Table 1). The translocation factor (TF), the arsenic concentration ratio between frond and root, is also used in this study to measure the ability of plant to translocate As from root to frond. However, the effect of P-fertilizer on TF was not significantly higher than the control (Table 1). Taking into account the dry weight (g) and the arsenic concentrations (lg As g 1) of plants, the arsenic content (lg) in plant organs was calculated and showed the results as in Figs. 1a and 2. The arsenic contents of the P-amendment plants in both the greenhouse and field studies were significantly higher than those of the controls (P < 0.05) (Figs. 1a and 2) due to higher plant
Table 1 Biomass production, arsenic concentration and translocation factor of P. calomelanos with and without (=control) P-fertilizer Experiment
Plant part
Dry biomass (g)
Arsenic concentration (lg As g 1)
Translocation factor (TF) Control
Control
P-fertilization
Control
P-fertilization
P-fertilization
Root Frond Whole plant
0.40 ± 0.02a,* 1.97 ± 0.21a 2.37 ± 0.20a
0.55 ± 0.06a 3.11 ± 0.53b 3.67 ± 0.57b
50.0 ± 6.0a 367 ± 13a
60.0 ± 5.0a 441 ± 22b
7.5 ± 0.7a
7.6 ± 0.9a
Root Frond Whole plant
1.47 ± 0.49a 4.37 ± 0.94a 5.84 ± 1.33a
3.63 ± 0.46b 5.47 ± 0.42a 9.10 ± 0.87a
65.1 ± 7.0a 2360 ± 128a
88.4 ± 18.6a 3822 ± 228b
36.8 ± 2.5a
50.0 ± 16.2a
Root Frond Whole plant
2.41 ± 0.08a 4.88 ± 0.30a 7.28 ± 0.35a
3.30 ± 0.53a 6.37 ± 0.54b 9.67 ± 0.77b
35.4 ± 2.9a 2642 ± 249a
60.3 ± 18.9a 4210 ± 233b
76.5 ± 11.8a
86.6 ± 27.3a
Greenhouse
Field 4 wk
8 wk
* Within each parameter, mean ± SE followed by the same letter is not significantly different among control and P-fertilization for each plant part by Duncan’s multiple range test at the 5% level, n = 3.
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912 1800
a
600
b
Control
Control
P-fertilization
500
As content in root (µg As)
As content (µg As per organ)
a
1200
a
600
a
a
1909
P-fertilization
400
b
300
b 200
a
a
100
0 Root
0
Frond
4 wk 8000 Control
b
b
6000
Control
Rhizofungi
a
4000
a
2000
a
b
a
0 Root
8 wk
40000
Rhizobacteria
As content in frond (µg As)
As content (µg As per organ)
b
32000
b
P-fertilization
b 24000
a 16000
a
8000
Frond
Plant part Fig. 1. Greenhouse study of arsenic content in P. calomelanos: (a) Pfertilizer amendment experiment and (b) rhizosphere microbe experiment. To compare the results within group of plant part, different letters above columns (mean ± SE) for each plant part indicate significant differences between control and P-fertilization (a) and among control, rhizobacteria and rhizofungi inoculation (b) by Duncan’s multiple range test at the 5% level, n = 3.
biomass and tissue arsenic concentration of the treated plants. However, there was no significant effect of P-fertilizer on As content of the root for the greenhouse experiment (Fig. 1a). 3.2. Effects of rhizosphere microbes on plant biomass and As accumulation The rhizosphere microbes increased plant biomass of the fern, P. calomelanos, in both the greenhouse and field experiments (Table 2). However, the effects of the rhizobacteria on increasing plant biomass of the fern were more pronounced than those of the rhizofungi. Arsenic concentration in the fronds of P. calomelanos was higher than in the roots (Table 2). In the greenhouse and field studies, arsenic concentration in the roots and the fronds of rhizofungi-inoculated plants were significantly lower than in the control and rhizobacteria-inoculated plants (P < 0.05). However, when tissue arsenic concentration in the control and rhizobacteria-inoculated
0 4 wk
8 wk
Time Fig. 2. Field study of arsenic content in roots (a) and fronds (b) of P. calomelanos with and without (=control) P-fertilizer. To compare the results within group of harvesting time, different letters above columns (mean ± SE) for each harvesting time indicate significant differences between control and P-fertilization by Duncan’s multiple range test at the 5% level, n = 3.
plants were compared, there were no significant differences (Table 2). Moreover, the rhizobacteria and rhizofungi had no significant effect on TF value in both the greenhouse and field studies (Table 2). Arsenic content in the roots and the fronds of rhizobacteria-inoculated plants were significantly higher than in both the control and rhizofungi-inoculated plants (P < 0.05) (Figs. 1b and 3). However, there were no significant effects of rhizofungi on arsenic content in parts of treated plants compared to the control (Figs. 1b and 3). In particular, at 12 wk after planting in the field study (Fig. 3), arsenic content in the roots of both rhizobacteriaand rhizofungi-inoculated plants were not significantly different to the control. It was noticeable in the field study that arsenic content in the roots of rhizobacteria-inoculated plants at 12 wk was lower than at 6 wk after planting. However, the arsenic content in the fronds of all plants increased markedly from the harvesting time of 6–12 wk in the field study.
a
800 Control
As content in root (µg As)
b
Rhizobacteria 600
Rhizofungi
a
a
400
a
a a
200
0 6 wk
b
70000
As content in frond (µg As)
* Within each parameter, mean ± SE followed by the same letter is not significantly different among control, rhizobacteria and rhizofungi inoculation for each plant part by Duncan’s multiple range test at the 5% level, n = 3.
70.4 ± 4.3a 59.0 ± 10.4a 64.8 ± 7.3a 66.0 ± 19.8a 5148 ± 129a 118 ± 16a,b 6672 ± 490b 134 ± 21b 6762 ± 167b 3.13 ± 0.62a 5.97 ± 0.58a 9.09 ± 0.95a 2.97 ± 0.02a 8.27 ± 0.63b 11.24 ± 0.64a 2.57 ± 0.45a 5.63 ± 0.70a 8.20 ± 1.15a Root Frond Whole plant 12 wk
14.9 ± 0.1a 19.0 ± 0.8b 256 ± 5a 4794 ± 326a 422 ± 12b 6281 ± 219a,b 385 ± 49b 6819 ± 671b 0.707 ± 0.128a,b 0.969 ± 0.174a 1.68 ± 0.29a,b 1.32 ± 0.29b 1.87 ± 0.31b 3.20 ± 0.60b 0.513 ± 0.080a 0.450 ± 0.138a 0.96 ± 0.21a Root Frond Whole plant
198 ± 4a 2941 ± 206a 263 ± 15b 4610 ± 350b 294 ± 4b 4245 ± 88b 0.29 ± 0.01b 0.99 ± 0.10a,b 1.28 ± 0.96b 0.37 ± 0.02c 1.18 ± 0.13b 1.55 ± 0.15b 0.2 ± 0.01a,* 0.70 ± 0.04a 0.90 ± 0.04a Root Frond Whole plant
Control Greenhouse
Field 6 wk
14.9 ± 1.2a 14.5 ± 0.2a
17.8 ± 2.3a
Rhizobacteria
Translocation factor (TF)
Control Rhizofungi Rhizobacteria Control Rhizobacteria
Rhizofungi
Arsenic concentration (lg As g 1) Dry biomass (g) Plant part Experiment
Table 2 Biomass production, arsenic concentration and translocation factor of P. calomelanos inoculated with rhizosphere microbes
18.7 ± 0.9b
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912 Rhizofungi
1910
60000
12 wk
Control
50000
b
Rhizobacteria Rhizofungi
a
40000
a
30000
b
20000 10000
a
a
0 6 wk
12 wk
Time Fig. 3. Field study of arsenic content in roots (a) and fronds (b) of P. calomelanos inoculated with rhizosphere microbes. To compare the results within group of harvesting time, different letters above columns (mean ± SE) for each harvesting time indicate significant differences among control, rhizobacteria and rhizofungi inoculation by Duncan’s multiple range test at the 5% level, n = 3.
4. Discussion 4.1. Effects of P-fertilization on phytoremediation of As-contaminated soil The results of this study showed that P-fertilizer has potential effects on increasing plant biomass and As accumulation in P. calomelanos when grown in arsenic contaminated soil. It is possible that the higher As accumulation in P-amended plants was the result of enhanced mobility of As in the soil which occurred by the replacement of As by P from the soil binding site as a competitive anion exchange (Smith et al., 2002). This mechanism also explained by the study of Quaghebeur and Rengel (2001) which performed on the interactions of As and P in the soil and found that P-fertilizer increased plant uptake of arsenic. However, Fitz and Wenzel (2002) reported that the effects of phosphorus on the uptake and toxicity of arsenic in plants is unpredictable because it depends on plant spe-
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912
cies, chemical speciation of arsenic, growth medium and the experimental conditions. In the case of the As hyperaccumulating fern, Pteris vittata, Caille et al. (2004) found that the addition of phosphate had no significant effect on plant As uptake from naturally As-contaminated soil whereas Cao et al. (2003) showed that phosphate rock amendments increased plant arsenic uptake in two tested soil (chromated-copper-arsenate contaminated soil and As spiked contaminated soil). Despite both experiments using the same plant species and both showing enhanced mobility of As in soil by the addition of phosphate, plant As uptake were still different. This might be related to different phosphate levels in these two experiments which resulting in different As plant uptakes. Cao et al. (2003) used phosphate at a rate of 15 g kg 1 while Caille et al. (2004) used only 50 mg P kg 1 phosphate. In our study, a different hyperaccumulating fern species, P. calomelanos, was used in conjunction with 100 mg P kg 1 soil as KH2PO4 and P-fertilizer and it was found that As accumulation increased in plants. This indicates that the effect of P-addition on As uptake so far is still inconsistent. Since Salt et al. (1998) suggested that phytoextraction relies on plants accumulating high amounts of metal in their above-ground parts, then, based on our results, we can conclude that P-fertilizer is an important factor for improving As-phytoextraction by P. calomelanos. 4.2. Effects of rhizosphere microbes on phytoremediation of As-contaminated soil Besides chemical amendments, biological amendments, such as rhizosphere microbes, have also been investigated for improving metal phytoremediation (Khan, 2005; Go¨hre and Paszkowaski, 2006). The possible benefits of rhizosphere microbes on metal phytoremediation may occur in several ways such as stimulation of plant growth by improving plant nutrition (Vogel-Mikusˇ et al., 2006), metal detoxification/resistance by immobilizing metals in the rhizosphere (Wu et al., 2006) and/or enhancement of metal accumulation in the plant (Arriagada et al., in press). However, there are very few studies on the use of rhizobacteria in phytoremediation of As-contaminated soil. Published papers so far were considered only the ability of bacterial mobilization of As in soil (Turpeinen et al., 1999; Casiot et al., 2003). It has been suggested that As leaching by rhizobacteria under aerobic conditions might be caused by exudation during microbial metabolism (Park et al., 2006). A role of rhizobacteria in the mobilization of insoluble P/nutrients by producing various organic acids might be likely to desorb arsenic as well. Previous studied by our group (Uppanan, 2000) revealed that Alcaligenes spp. isolated from soil in Ron Phibun District could oxidised arsenite to arsenate which plant could uptake via phosphate transporter as mentioned by Meharg and Macnair (1990). For the phytoremediation process, any effects of bacterial activity must apply to the plant. In this study, inocula-
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tion with rhizobacteria did not greatly influence As concentrations in P. calomelanos but greatly enhanced plant biomass especially the above-ground biomass, thus resulting in enhancement of As removal. This result is in accordance with the alternative strategy concept of metal phytoextraction which was suggested by Salt et al. (1998). The effects of rhizofungi on phytoremediation, however, have been shown to differ between distinct fungal isolates, different host plants and different heavy metals (Leyval et al., 1997; Malcova´ et al., 2003). Some reports indicated higher concentrations of metals in plants due to rhizofungi (Whitfield et al., 2003; Arriagada et al., in press), whereas others have found reduced plant concentrations of metals in mycorhizal plants (Shetty et al., 1995; Li and Christie, 2001). In addition, it has also been found that rhizofungi had no significant effect on plant growth and metal accumulation in plants growing in contaminated soil (VogelMikusˇ et al., 2006). In As phytoremediation, there have also been conflicting results regarding the effects of rhizofungi on As uptake by plants. Leung et al. (2006) reported that the addition of rhizofungi enhanced the uptake and accumulation of As in P. vittata. Under the condition of 100 mg As per kg soil, noncolonized plants accumulated 60.4 mg As kg 1 while plants colonized by arbuscular mycorrhizal fungi (AMF) isolated from an As mine accumulated 88.1 g As kg 1 and also enhanced plant growth. On the other hand, Trotta et al. (2006) found that in the same plant species, P. vittata, rhizofungi increased plant growth only in the above ground parts but reduced root As concentration without any effect on frond concentration, therefore resulting in a larger As translocation factor. Moreover, in U and As-contaminated soil, Chen et al. (2006) found that rhizofungi depressed growth of P. vittata particularly at the early stages and had no effect on As concentration in this plant. These results indicated that the effects of rhizofungi on As uptake is inconsistent even though the same plant species was used. In this work, P. calomelanos was used and it was shown that rhizofungi could improved plant biomass but reduce As accumulation. It has been suggested that protection against As accumulation in plants by rhizofungi may occur indirectly by enhancing plant P nutrition and increasing plant growth resulting in a diluting effect on arsenic in the plant, or directly by binding arsenic to the fungal mycelium or roots and immobilization in the rhizosphere (Sharples et al., 2000; Meharg and Hartley-Whitaker, 2002). From our previous work (Visoottiviseth and Panviroj, 2001), Penicillium spp. was the best As-removal fungal species isolated from soil in Ron Phibun District. Our results in this study indicate that the phytoremediation of As-contaminated soil by P. calomelanos can be enhanced by rhizosphere microbes by using rhizosphere bacteria in phytoextraction and rhizofungi in phytostabilization. In future work, the effect of AMF on As phytoremediation by P. calomelanos will be investigated because the occurrence of their structures within roots may directly influence metal uptake in plants.
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Lombi, E., Zhao, F.J., Dunham, S.J., McGrath, S.P., 2001. Phytoremediation of heavy metal contaminated soils: natural hyperaccumulation versus chemically enhanced phytoextraction. J. Environ. Qual. 30, 1919–1926. Malcova´, R., Rydlova´, J., Vosa´tka, M., 2003. Metal-free cultivation of Glomus sp. BEG 140 isolated from Mn-contaminated soil reduces tolerance to Mn. Mycorrhiza 13, 151–157. Meharg, A.A., Hartley-Whitaker, J., 2002. Arsenic uptake and metabolism in arsenic resistant and nonresistant plant species. New Phytol. 154, 29–43. Meharg, A.A., Macnair, M.R., 1990. An altered phosphate uptake system in arsenate-tolerant Holcus lanatus L.. New Phytol. 116, 29–35. Park, J.M., Lee, J.S., Lee, J.-U., Chon, H.T., Jung, M.C., 2006. Microbial effects on geochemical behavior of arsenic in As-contaminated sediments. J. Geochem. Explor. 88, 134–138. Quaghebeur, M., Rengel, Z., 2001.The presence of phosphate in the rhizosphere of Holcus lanatus L. stimulates the uptake of arsenate from kaolinite. In: Arsenic in the Asia–Pacific Region Workshop (Book of Abstracts), 20–23 November 2001, Adelaide, SA, pp. 143–145. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annu Rev Plant Physiol. 49, 643–668. Sharples, J.M., Meharg, A.A., Chambers, S.M., Cairney, J.W.G., 2000. Symbiotic solution to arsenic contamination. Nature 404, 951–952. Shetty, K.G., Hetrick, B.A.D., Schwab, A.P., 1995. Effects of mycorrhizae fertilizer amendments on zinc tolerance of plants. Environ. Pollut. 88, 307–314. Smith, E., Naidu, R., Alston, A.M., 2002. Chemistry of arsenic in soils: II. Effect of phosphorus, sodium and calcium on arsenic sorption. J. Environ. Qual. 31, 557–563. Trotta, A., Falaschi, P., Cornara, L., Minganti, V., Fusconi, A., Drava, G., Berta, G., 2006. Arbuscular mycorrhizae increase the arsenic translocation factor in the As hyperaccumulating fern Pteris vittata L.. Chemosphere 65, 74–81. Turpeinen, R., Pantsar-kallio, M., Haggblom, M., Kairesalo, T., 1999. Influence of microbes on the mobilization, toxicity and biomethylation of arsenic in soil. Sci. Total Environ. 236, 173–180. Uppanan, P., 2000. Screening and Characterization of Bacteria Capable of Biotransformation of Toxic Arsenic Compound in Soil, M.Sc Thesis, Mahidol University, Thailand. Visoottiviseth, P., Panviroj, N., 2001. Selection of fungi capable of removing toxic arsenic compounds from liquid medium. ScienceAsia 27, 83–92. Visoottiviseth, P., Sridokchan, W., Francesconi, K., 2002. The potential of Thai indigenous plant species for phytoremediation of arsenic contaminated land. Environ. Pollut. 118, 453–461. Vogel-Mikusˇ, K., Pongrac, P., Kump, P., Necˇemer, M., Regvar, M., 2006. Colonisation of a Zn, Cd and Pb hyperaccumulator Thlaspi praecox Wulfen with indigenous arbuscular mycorrhizal fungal mixture induces changes in heavy metal and nutrient uptake. Environ. Pollut. 139, 362–371. Whitfield, L., Richards, A.J., Rimmer, D.L., 2003. Effects of mycorrhizal colonization on Thymus polytrichus from heavy metal-contaminated sites in north England. Mycorrhiza 14, 47–54. Wu, S.C., Cheung, K.C., Luo, Y.M., Wong, M.H., 2006. Effects of inoculation of plant growth-promoting rhizobacteria on metal uptake by Brassica juncea. Environ. Pollut. 140, 124–135.
Enhanced phytoremediation of arsenic contaminated land P. Jankong a, P. Visoottiviseth a
a,*
, S. Khokiattiwong
b
Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Rachataewee, Bangkok 10400, Thailand b Phuket Marine Biological Center, Ministry of Agriculture and Cooperatives, Panwa Cape, Phuket 83000, Thailand Received 20 December 2006; received in revised form 26 February 2007; accepted 26 February 2007 Available online 9 April 2007
Abstract In an attempt to clean up arsenic (As) contaminated soil, the effects of phosphorus (P) fertilizer and rhizosphere microbes on arsenic accumulation by the silverback fern, Pityrogramma calomelanos, were investigated in both greenhouse and field experiments. Field experiments were conducted in Ron Phibun District, an As-contaminated area in Thailand. Soil (136–269 lg As g 1) was collected there and used in the greenhouse experiment. Rhizosphere microbes (bacteria and fungi) were isolated from roots of P. calomelanos growing in Ron Phibun District. The results showed that P-fertilizer significantly increased plant biomass and As accumulation of the experimental P. calomelanos. Rhizobacteria increased significantly the biomass and As content of the test plants. Thus, P-fertilizer and rhizosphere bacteria enhanced As-phytoextraction. In contrast, rhizofungi reduced significantly As concentration in plants but increased plant biomass. Therefore, rhizosphere fungi exerted their effects on phytostabilization. 2007 Elsevier Ltd. All rights reserved. Keywords: Arsenic; Phosphorus-fertilizer; Rhizosphere microbes; Phytoremediation; Phytoextraction; Phytostabilization
1. Introduction The problem of As contamination in the environment is a global concern due to its high toxicity to living organisms and its worldwide distribution. In Ron Phibun District, Nakhon Sri Thammarat Province, Thailand, tin mining activities during the last century caused As contamination of soil and water which ultimately led to human health problems. Although tin mining was prohibited in the late 1980s, the release of arsenopyrite (FeAsS) from the tin ore has left extensive As contamination in many areas of Ron Phibun District. The long-term use of contaminated water for agriculture or direct consumption poses a serious risk of chronic As poisoning among the local population causing diseases such as skin melanoma, cancer and high blood pressure. The problems originating from As contamination still prevail today. Therefore, to protect animal and human health, remediation of the As-contaminated sites has *
Corresponding author. Tel.: +66 2 201 5272; fax: +66 2 354 7161. E-mail address: [email protected] (P. Visoottiviseth).
0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.02.061
become an important issue. Fortunately, the silverback fern (Pityrogramma calomelanos) has been discovered in Thailand as an As-hyperaccumulating fern for phytoextraction of As-contaminated soil (Visoottiviseth et al., 2002). However, a key issue for an effective phytoremediation process, especially phytoextraction, is to enhance pollutant phytoavailability and to sustain adequate pollutant concentrations in the soil solution for plant uptake (Lombi et al., 2001). In addition, candidate plants for the phytoextraction purpose should tolerate the pollutants and produce large amounts of biomass. Besides the ability of the plants, rhizosphere factors are also important for improving phytoremediation efficiency. It has been reported that P-fertilizer, rhizosphere microbes (bacteria and fungi), root exudation, and chelating agents could enhance plant uptake and accumulation of contaminants due to their effects on improving available pollutants for plants (Chen and Cutright, 2002; Cao et al., 2003). For the phytoremediation of As, adding P-fertilizer to As-contaminated soils might affect phytoremediation efficiency due to the physicochemical similarity of the
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912
phosphate ion and arsenate ion (Adriano, 2001). Moreover, because of their role of enhancing plant growth, rhizosphere microbes are responsible for nutrient (N/P) mobilization (Dun˜abeitia et al., 2004). Therefore, the effects of microbes on the availability and transport of As to the plant should be considered for phytoremediation. However, the interactions of As and these rhizosphere factors are not yet well understood (Fitz and Wenzel, 2002; Leung et al., 2006; Trotta et al., 2006). In this study, the effects of P-fertilizer and rhizosphere microbes on plant biomass production and As accumulation in P. calomelanos were investigated with the aim to utilize these factors in their capacity to enhance As remediation of plants in polluted soil. 2. Materials and methods 2.1. Plants and soils Spores of P. calomelanos were collected from Ron Phibun District in southern Thailand. Seedlings were sown from spores in sterilized peat moss. Seedlings at the early sporophyte stage were transplanted individually into sterilized vermiculite. At the age of six months, the seedlings were used to set up experiments. The field experiment was set up in Ron Phibun District. Soil used in the greenhouse experiment was taken from the same site. The soil (0–15 cm depth) had the following properties (dry matter basis): pH (in water) 3.9–6.8, 0.06 dS m 1 EC, 2.54% organic matter, 65 lg g 1 available P, 32 lg g 1 available K, 270 lg g 1 total As (determined by Department of Soil Science, Ministry of Agriculture and Cooperatives, Thailand). The contaminated soil determined in this study contained 136–269 lg As g 1. The soil was air-dried and passed through a 2 mm sieve to remove stones and plant materials. After that, the soil was autoclaved for 45 min to kill native microbial populations. 2.2. P-fertilizer amendment experiments Six hundred grams of sterile soil was placed in each pot for the greenhouse experiment. Two levels of P (0, 100 mg P kg 1 soil as KH2PO4) were applied in the form of a solution to the soil in each pot. The soil was then mixed thoroughly before planting. Seedlings were grown in pots, one plant per pot for 16 wk. There were four replicates for each treatment. The plants were watered daily with tap water and the fertilizer solution was sprayed on the frond every wk to avoid nutrient deficiency. In the field experiment, seedlings were transplanted to each plot (2 m · 2 m). There were two treatments (with and without fertilizer) with eight plants per treatment. Approximately 200 g pellets of fertilizer (N:P:K = 6:18:6) was scattered over the soil of the treatment with fertilizer. At each harvesting time, 4 and 8 wk, four plants were collected.
1907
2.3. Preparation of inoculum of rhizosphere microbes 2.3.1. Rhizobacterial inoculum Plant roots and soils were collected from As-contaminated land in the Ron Phibun District, Nakhon Sri Thammarat province, southern Thailand. Each soil sample of 5 g was suspended in 20 ml of 0.85% NaCl solution. The soil suspension was left for about 30 min to allow large soil particles to settle out. One milliliter of suspension was added to each of 50 ml of enrichment medium (8 g nutrient broth and 0.5 g yeast extract with 1000 ml distilled water in a 250 ml flask). The culture was then incubated at 30 C on a shaker set at 200 rpm for 7 d. After that, 1 ml of the sample was transferred into 100 ml mineral medium containing arsenate at a concentration of 70 mg l 1 (pH adjusted to 5.6 by HCl) and 2 lg ml 1 of Fungizone (obtained from GIBCO). The mineral medium was prepared by mixing 0.5 g NH4Cl, 0.25 g MgCl2 Æ 6H2O, 0.5 g KH2PO4, 0.5 g beef extract and 0.3 g glucose with 1000 ml distilled water. The cultures were sub-cultured every 2 wk into a fresh medium and incubated under the same conditions. The culture was then centrifuged and rinsed the pellets three times with distilled water. The organisms were then inoculated in 250 ml flasks containing 100 ml of nutrient broth (NB), obtained from Difco, for 48 h on a shaker and then centrifuged at 5500 rpm for 7 min. The pellet containing bacterial cells was suspended in fresh NB and used as rhizobacterial inoculum. 2.3.2. Rhizofungal inoculum Plant roots were washed with tap water, rinsed with sterile deionized water to remove adhered soil, cut into segments (2 cm) and placed on potato dextrose agar (obtained from Difco) plates containing 70 mg l 1 arsenate and antibiotic (Penicillin–Streptomycin at 100 units ml 1 obtained from GIBCO). All plates were incubated at 27 C for 48– 72 h (Visoottiviseth and Panviroj, 2001). Fungal cultures were transferred and grown in 250 ml flasks containing 100 ml of potato dextrose broth (PDB), obtained from Difco, liquid medium and shaken at 150 rpm, 27 C. After 6 d, the mycelia pellets (diameter of 1.3 mm) were harvested by vacuum filtration, washed three times with sterile water, transferred to fresh PDB and used as rhizofungal inoculum. 2.4. Rhizosphere microbe experiments 2.4.1. Greenhouse experiments Sterile soil (600 g) were placed in each sterile pot. Deionised water (100 ml) was added and mixed with the soil. P. calomelanos, at 6-month-old, were acclimatized in Hoagland solution for 1 wk. They were then inoculated with either rhizobacteria or rhizofungi by soaking the plants in the appropriate inoculum for about 30 min. After that, the plants were separately grown in each pot for 6 wk and were watered daily with tap water. There were three
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P. Jankong et al. / Chemosphere 68 (2007) 1906–1912
treatments i.e., rhizobacteria-inoculated, rhizofungi-inoculated and non-inoculated (control), with three replicates per treatment. 2.4.2. Field experiments Six month old P. calomelanos was transplanted into each plot (2 m · 2 m). There were three treatment plots, with eight ferns per plot. Non-inoculated plants were used as a control. The plants were inoculated by soaking in the inoculum suspension for 30 min before planting in the appropriate plot. The plants were harvested after 6 and 12 wk.
2.6. Statistical analysis The data were statistically analyzed by a one-way analysis of variance (ANOVA) for the rhizosphere microbes study and a T-test for the P-fertilizer amendment study. When a significant (P < 0.05) treatment effect was found, the mean values were compared using the Duncan’s multiple range test and student T-test for rhizosphere microorganisms and P-fertilizer amendment study, respectively. 3. Results
2.5. Chemical analysis
3.1. Effects of P-fertilizer on plant biomass and As accumulation
Plant roots and fronds were harvested, washed with tap water, rinsed with deionized distilled water, and oven dried at 50–55 C for 3 d. After that, frond and root biomass were determined and the dried plant material was then ground to powder (mortar/pestle). Samples were prepared for the As concentration analysis by using the dry ashing method. A portion of the dry powder (20–50 mg) was accurately weighed (±0.01 mg) directly into a crucible and mixed with 1.5 ml freshly prepared slurry (30 g Mg(NO3)2, 50 g MgO and 500 ml of distilled water). The mixture was dried overnight at 80 C, and digested in a muffle furnace (200 C for 1 h, 300 C for 1 h and 500 C for 8 h). The residue was dissolved in 6 M HCl (2.5 ml) and the solution mixed with distilled water (2.5 ml). Total arsenic concentrations in these solutions were determined by a Hydride Generation Atomic Absorption Spectrophotometer (HGAAS) using a Perkin–Elmer MHS-20 mercury/hydride system coupled to a Perkin–Elmer 2380 AAS. Arsenic concentrations in the samples were reported on a dry mass basis. The certified reference material MESS3: marine sediment (National Research Council Canada, Ottawa, Canada) was used for quality control as 85 ± 7% recovery, n = 3.
In both the greenhouse and field studies, the addition of P-fertilizer increased the biomass of P. calomelanos significantly (Table 1). For the field study, the beneficial effect of P-fertilizer on root production was clearly shown at 4 wk while the effect on frond production was clearly shown at 8 wk after planting (Table 1). Arsenic concentration in the fronds of P. calomelanos was higher than in the roots as was expected (Table 1). Addition of P-fertilizer increased arsenic concentration in the plant tissues in both the greenhouse and field studies but significantly higher effects were present only in the frond parts (P < 0.05) (Table 1). The translocation factor (TF), the arsenic concentration ratio between frond and root, is also used in this study to measure the ability of plant to translocate As from root to frond. However, the effect of P-fertilizer on TF was not significantly higher than the control (Table 1). Taking into account the dry weight (g) and the arsenic concentrations (lg As g 1) of plants, the arsenic content (lg) in plant organs was calculated and showed the results as in Figs. 1a and 2. The arsenic contents of the P-amendment plants in both the greenhouse and field studies were significantly higher than those of the controls (P < 0.05) (Figs. 1a and 2) due to higher plant
Table 1 Biomass production, arsenic concentration and translocation factor of P. calomelanos with and without (=control) P-fertilizer Experiment
Plant part
Dry biomass (g)
Arsenic concentration (lg As g 1)
Translocation factor (TF) Control
Control
P-fertilization
Control
P-fertilization
P-fertilization
Root Frond Whole plant
0.40 ± 0.02a,* 1.97 ± 0.21a 2.37 ± 0.20a
0.55 ± 0.06a 3.11 ± 0.53b 3.67 ± 0.57b
50.0 ± 6.0a 367 ± 13a
60.0 ± 5.0a 441 ± 22b
7.5 ± 0.7a
7.6 ± 0.9a
Root Frond Whole plant
1.47 ± 0.49a 4.37 ± 0.94a 5.84 ± 1.33a
3.63 ± 0.46b 5.47 ± 0.42a 9.10 ± 0.87a
65.1 ± 7.0a 2360 ± 128a
88.4 ± 18.6a 3822 ± 228b
36.8 ± 2.5a
50.0 ± 16.2a
Root Frond Whole plant
2.41 ± 0.08a 4.88 ± 0.30a 7.28 ± 0.35a
3.30 ± 0.53a 6.37 ± 0.54b 9.67 ± 0.77b
35.4 ± 2.9a 2642 ± 249a
60.3 ± 18.9a 4210 ± 233b
76.5 ± 11.8a
86.6 ± 27.3a
Greenhouse
Field 4 wk
8 wk
* Within each parameter, mean ± SE followed by the same letter is not significantly different among control and P-fertilization for each plant part by Duncan’s multiple range test at the 5% level, n = 3.
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912 1800
a
600
b
Control
Control
P-fertilization
500
As content in root (µg As)
As content (µg As per organ)
a
1200
a
600
a
a
1909
P-fertilization
400
b
300
b 200
a
a
100
0 Root
0
Frond
4 wk 8000 Control
b
b
6000
Control
Rhizofungi
a
4000
a
2000
a
b
a
0 Root
8 wk
40000
Rhizobacteria
As content in frond (µg As)
As content (µg As per organ)
b
32000
b
P-fertilization
b 24000
a 16000
a
8000
Frond
Plant part Fig. 1. Greenhouse study of arsenic content in P. calomelanos: (a) Pfertilizer amendment experiment and (b) rhizosphere microbe experiment. To compare the results within group of plant part, different letters above columns (mean ± SE) for each plant part indicate significant differences between control and P-fertilization (a) and among control, rhizobacteria and rhizofungi inoculation (b) by Duncan’s multiple range test at the 5% level, n = 3.
biomass and tissue arsenic concentration of the treated plants. However, there was no significant effect of P-fertilizer on As content of the root for the greenhouse experiment (Fig. 1a). 3.2. Effects of rhizosphere microbes on plant biomass and As accumulation The rhizosphere microbes increased plant biomass of the fern, P. calomelanos, in both the greenhouse and field experiments (Table 2). However, the effects of the rhizobacteria on increasing plant biomass of the fern were more pronounced than those of the rhizofungi. Arsenic concentration in the fronds of P. calomelanos was higher than in the roots (Table 2). In the greenhouse and field studies, arsenic concentration in the roots and the fronds of rhizofungi-inoculated plants were significantly lower than in the control and rhizobacteria-inoculated plants (P < 0.05). However, when tissue arsenic concentration in the control and rhizobacteria-inoculated
0 4 wk
8 wk
Time Fig. 2. Field study of arsenic content in roots (a) and fronds (b) of P. calomelanos with and without (=control) P-fertilizer. To compare the results within group of harvesting time, different letters above columns (mean ± SE) for each harvesting time indicate significant differences between control and P-fertilization by Duncan’s multiple range test at the 5% level, n = 3.
plants were compared, there were no significant differences (Table 2). Moreover, the rhizobacteria and rhizofungi had no significant effect on TF value in both the greenhouse and field studies (Table 2). Arsenic content in the roots and the fronds of rhizobacteria-inoculated plants were significantly higher than in both the control and rhizofungi-inoculated plants (P < 0.05) (Figs. 1b and 3). However, there were no significant effects of rhizofungi on arsenic content in parts of treated plants compared to the control (Figs. 1b and 3). In particular, at 12 wk after planting in the field study (Fig. 3), arsenic content in the roots of both rhizobacteriaand rhizofungi-inoculated plants were not significantly different to the control. It was noticeable in the field study that arsenic content in the roots of rhizobacteria-inoculated plants at 12 wk was lower than at 6 wk after planting. However, the arsenic content in the fronds of all plants increased markedly from the harvesting time of 6–12 wk in the field study.
a
800 Control
As content in root (µg As)
b
Rhizobacteria 600
Rhizofungi
a
a
400
a
a a
200
0 6 wk
b
70000
As content in frond (µg As)
* Within each parameter, mean ± SE followed by the same letter is not significantly different among control, rhizobacteria and rhizofungi inoculation for each plant part by Duncan’s multiple range test at the 5% level, n = 3.
70.4 ± 4.3a 59.0 ± 10.4a 64.8 ± 7.3a 66.0 ± 19.8a 5148 ± 129a 118 ± 16a,b 6672 ± 490b 134 ± 21b 6762 ± 167b 3.13 ± 0.62a 5.97 ± 0.58a 9.09 ± 0.95a 2.97 ± 0.02a 8.27 ± 0.63b 11.24 ± 0.64a 2.57 ± 0.45a 5.63 ± 0.70a 8.20 ± 1.15a Root Frond Whole plant 12 wk
14.9 ± 0.1a 19.0 ± 0.8b 256 ± 5a 4794 ± 326a 422 ± 12b 6281 ± 219a,b 385 ± 49b 6819 ± 671b 0.707 ± 0.128a,b 0.969 ± 0.174a 1.68 ± 0.29a,b 1.32 ± 0.29b 1.87 ± 0.31b 3.20 ± 0.60b 0.513 ± 0.080a 0.450 ± 0.138a 0.96 ± 0.21a Root Frond Whole plant
198 ± 4a 2941 ± 206a 263 ± 15b 4610 ± 350b 294 ± 4b 4245 ± 88b 0.29 ± 0.01b 0.99 ± 0.10a,b 1.28 ± 0.96b 0.37 ± 0.02c 1.18 ± 0.13b 1.55 ± 0.15b 0.2 ± 0.01a,* 0.70 ± 0.04a 0.90 ± 0.04a Root Frond Whole plant
Control Greenhouse
Field 6 wk
14.9 ± 1.2a 14.5 ± 0.2a
17.8 ± 2.3a
Rhizobacteria
Translocation factor (TF)
Control Rhizofungi Rhizobacteria Control Rhizobacteria
Rhizofungi
Arsenic concentration (lg As g 1) Dry biomass (g) Plant part Experiment
Table 2 Biomass production, arsenic concentration and translocation factor of P. calomelanos inoculated with rhizosphere microbes
18.7 ± 0.9b
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912 Rhizofungi
1910
60000
12 wk
Control
50000
b
Rhizobacteria Rhizofungi
a
40000
a
30000
b
20000 10000
a
a
0 6 wk
12 wk
Time Fig. 3. Field study of arsenic content in roots (a) and fronds (b) of P. calomelanos inoculated with rhizosphere microbes. To compare the results within group of harvesting time, different letters above columns (mean ± SE) for each harvesting time indicate significant differences among control, rhizobacteria and rhizofungi inoculation by Duncan’s multiple range test at the 5% level, n = 3.
4. Discussion 4.1. Effects of P-fertilization on phytoremediation of As-contaminated soil The results of this study showed that P-fertilizer has potential effects on increasing plant biomass and As accumulation in P. calomelanos when grown in arsenic contaminated soil. It is possible that the higher As accumulation in P-amended plants was the result of enhanced mobility of As in the soil which occurred by the replacement of As by P from the soil binding site as a competitive anion exchange (Smith et al., 2002). This mechanism also explained by the study of Quaghebeur and Rengel (2001) which performed on the interactions of As and P in the soil and found that P-fertilizer increased plant uptake of arsenic. However, Fitz and Wenzel (2002) reported that the effects of phosphorus on the uptake and toxicity of arsenic in plants is unpredictable because it depends on plant spe-
P. Jankong et al. / Chemosphere 68 (2007) 1906–1912
cies, chemical speciation of arsenic, growth medium and the experimental conditions. In the case of the As hyperaccumulating fern, Pteris vittata, Caille et al. (2004) found that the addition of phosphate had no significant effect on plant As uptake from naturally As-contaminated soil whereas Cao et al. (2003) showed that phosphate rock amendments increased plant arsenic uptake in two tested soil (chromated-copper-arsenate contaminated soil and As spiked contaminated soil). Despite both experiments using the same plant species and both showing enhanced mobility of As in soil by the addition of phosphate, plant As uptake were still different. This might be related to different phosphate levels in these two experiments which resulting in different As plant uptakes. Cao et al. (2003) used phosphate at a rate of 15 g kg 1 while Caille et al. (2004) used only 50 mg P kg 1 phosphate. In our study, a different hyperaccumulating fern species, P. calomelanos, was used in conjunction with 100 mg P kg 1 soil as KH2PO4 and P-fertilizer and it was found that As accumulation increased in plants. This indicates that the effect of P-addition on As uptake so far is still inconsistent. Since Salt et al. (1998) suggested that phytoextraction relies on plants accumulating high amounts of metal in their above-ground parts, then, based on our results, we can conclude that P-fertilizer is an important factor for improving As-phytoextraction by P. calomelanos. 4.2. Effects of rhizosphere microbes on phytoremediation of As-contaminated soil Besides chemical amendments, biological amendments, such as rhizosphere microbes, have also been investigated for improving metal phytoremediation (Khan, 2005; Go¨hre and Paszkowaski, 2006). The possible benefits of rhizosphere microbes on metal phytoremediation may occur in several ways such as stimulation of plant growth by improving plant nutrition (Vogel-Mikusˇ et al., 2006), metal detoxification/resistance by immobilizing metals in the rhizosphere (Wu et al., 2006) and/or enhancement of metal accumulation in the plant (Arriagada et al., in press). However, there are very few studies on the use of rhizobacteria in phytoremediation of As-contaminated soil. Published papers so far were considered only the ability of bacterial mobilization of As in soil (Turpeinen et al., 1999; Casiot et al., 2003). It has been suggested that As leaching by rhizobacteria under aerobic conditions might be caused by exudation during microbial metabolism (Park et al., 2006). A role of rhizobacteria in the mobilization of insoluble P/nutrients by producing various organic acids might be likely to desorb arsenic as well. Previous studied by our group (Uppanan, 2000) revealed that Alcaligenes spp. isolated from soil in Ron Phibun District could oxidised arsenite to arsenate which plant could uptake via phosphate transporter as mentioned by Meharg and Macnair (1990). For the phytoremediation process, any effects of bacterial activity must apply to the plant. In this study, inocula-
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tion with rhizobacteria did not greatly influence As concentrations in P. calomelanos but greatly enhanced plant biomass especially the above-ground biomass, thus resulting in enhancement of As removal. This result is in accordance with the alternative strategy concept of metal phytoextraction which was suggested by Salt et al. (1998). The effects of rhizofungi on phytoremediation, however, have been shown to differ between distinct fungal isolates, different host plants and different heavy metals (Leyval et al., 1997; Malcova´ et al., 2003). Some reports indicated higher concentrations of metals in plants due to rhizofungi (Whitfield et al., 2003; Arriagada et al., in press), whereas others have found reduced plant concentrations of metals in mycorhizal plants (Shetty et al., 1995; Li and Christie, 2001). In addition, it has also been found that rhizofungi had no significant effect on plant growth and metal accumulation in plants growing in contaminated soil (VogelMikusˇ et al., 2006). In As phytoremediation, there have also been conflicting results regarding the effects of rhizofungi on As uptake by plants. Leung et al. (2006) reported that the addition of rhizofungi enhanced the uptake and accumulation of As in P. vittata. Under the condition of 100 mg As per kg soil, noncolonized plants accumulated 60.4 mg As kg 1 while plants colonized by arbuscular mycorrhizal fungi (AMF) isolated from an As mine accumulated 88.1 g As kg 1 and also enhanced plant growth. On the other hand, Trotta et al. (2006) found that in the same plant species, P. vittata, rhizofungi increased plant growth only in the above ground parts but reduced root As concentration without any effect on frond concentration, therefore resulting in a larger As translocation factor. Moreover, in U and As-contaminated soil, Chen et al. (2006) found that rhizofungi depressed growth of P. vittata particularly at the early stages and had no effect on As concentration in this plant. These results indicated that the effects of rhizofungi on As uptake is inconsistent even though the same plant species was used. In this work, P. calomelanos was used and it was shown that rhizofungi could improved plant biomass but reduce As accumulation. It has been suggested that protection against As accumulation in plants by rhizofungi may occur indirectly by enhancing plant P nutrition and increasing plant growth resulting in a diluting effect on arsenic in the plant, or directly by binding arsenic to the fungal mycelium or roots and immobilization in the rhizosphere (Sharples et al., 2000; Meharg and Hartley-Whitaker, 2002). From our previous work (Visoottiviseth and Panviroj, 2001), Penicillium spp. was the best As-removal fungal species isolated from soil in Ron Phibun District. Our results in this study indicate that the phytoremediation of As-contaminated soil by P. calomelanos can be enhanced by rhizosphere microbes by using rhizosphere bacteria in phytoextraction and rhizofungi in phytostabilization. In future work, the effect of AMF on As phytoremediation by P. calomelanos will be investigated because the occurrence of their structures within roots may directly influence metal uptake in plants.
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