Phytoremediation of an arsenic-contaminated site using Pteris vittata L.: a two-year study

A field study was conducted to determine the efficiency of Chinese brake fern (Pteris vittata L.), an arsenic hyperaccumulator, on removal of arsenic from soil at an arsenic-contaminated site. Chinese brake ferns were planted on a site previously used to treat wood with chromated copper arsenate (CCA). Arsenic concentrations in surface and profile soil samples were determined for 2000, 2001, and 2002. In both 2001 and 2002, senesced and senescing fronds only, as well as all fronds, were harvested. Frond arsenic concentrations were not significantly different between the three harvests. Compared to senesced fronds, live fronds resulted in the greatest amount of arsenic removal. There were no significant differences in soil arsenic concentrations between 2000, 2001, and 2002, primarily due to the extreme variability in soil arsenic concentrations. However, the mean surface soil arsenic was reduced from 190 to 140 mg kg(-1). Approximately 19.3 g of arsenic were removed from the soil by Chinese brake fern. Therefore, this fern is capable of accumulating arsenic from the CCA -contaminated site and may be competitive, in terms of cost, to conventional remediation systems. However, better agronomic practices are needed to enhance plant growth and arsenic uptake to obtain maximum soil arsenic removal and to minimize remediation time.     

Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress

The sporophyte and gametophyte of Pteris vittata are arsenic hyperaccumulators, however, little is known about the mechanism by which the gametophyte deals with this toxic element. An in vitro system (spores grown in arsenic amended nutrient media) was used to investigate the impact of arsenic on growth of the gametophyte and the role of antioxidative systems in combating As-stress. When mature spores of P. vittata were grown in medium amended with 0-50 mg kg(-1) of arsenic (as arsenate), the arsenic concentration in the gametophyte increased, with increasing arsenate in the media, but did not inhibit the spore germination and biomass development. Increases in the level of antioxidant enzymes, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione-Stransferase) and of ascorbic acid and glutathione probably enabled the gametophyte to withstand the oxidative stress caused by arsenate.     

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Distributions of arsenic and essential elements in pinna of arsenic hyperaccumulator Pteris vittata L.

The distributions of arsenic and 6 essential elements in the pinna of As hyperaccumulator, Pteris vittata L., were studied using synchrotron radiation X-ray fluorescence (SRXRF). Significant correlation between the distribution and mobility of the elements revealed that SRXRF study on the elemental distribution was feasible to inspect the transportations of elements in plants. The distribution of As in the pinna showed that As had great abilities to be transported in xylem vessels and from xylem to mesophyll. The distribution of K, one of the most mobile elements in plants, was similar to that of As, whereas the distributions of Fe and Ca with less mobility in plants were almost opposite to that of As in the pinna.     

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI’s role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.     

Arsenic-resistant bacteria solubilized arsenic in the growth media and increased growth of arsenic hyperaccumulator Pteris vittata L

The role of arsenic-resistant bacteria (ARB) in arsenic solubilization from growth media and growth enhancement of arsenic-hyperaccumulator Pteris vittata L. was examined. Seven ARB (tolerant to 10 mM arsenate) were isolated from the P. vittata rhizosphere and identified by 16S rRNA sequencing as Pseudomonas sp., Comamonas sp. and Stenotrophomonas sp. During 7-d hydroponic experiments, these bacteria effectively solubilized arsenic from the growth media spiked with insoluble FeAsO? and AlAsO? minerals (from < 5 μg L?1 to 5.04-7.37 mg L?1 As) and enhanced plant arsenic uptake (from 18.1-21.9 to 35.3-236 mg kg?1 As in the fronds). Production of (1) pyochelin-type siderophores by ARB (fluorescent under ultraviolet illumination and characterized with thin layer chromatography) and (2) root exudate (dissolved organic C) by P. vittata may be responsible for As solubilization. Increase in P. vittata root biomass from 1.5-2.2 to 3.4-4.2 g/plant dw by ARB and by arsenic was associated with arsenic-induced plant P uptake. Arsenic resistant bacteria may have potential to enhance phytoremediation of arsenic-contaminated soils by P. vittata.     

Effect of Fluoride on Arsenic Uptake from Arsenic-Contaminated Groundwater using Pteris vittata L.

High-arsenic groundwater in inland basins usually contains high concentrations of fluoride. In the present study, the effects of fluoride on arsenic uptake by Pteris vittata and on arsenic transformation in growth media were investigated under greenhouse conditions. After P. vittata was hydroponically exposed to 66.8 μM As (V) in the presence of 1.05 mM F^? in the form of NaF, KF, or NaF+KF for 10 d, no visible toxicity symptoms were observed, and there were not significant differences in the dry biomass among the four treatments. The results showed that P. vittata tolerated F^? concentrations as high as 1.05 mM but did not accumulate fluoride in their own tissues. Arsenic uptake was inhibited in the presence of 1.05 mM F^?. However, in hydroponic batches with 60 μM As (III) or 65 μM As (V), it was found that 210.6 and 316.0 μM F^? promoted arsenic uptake. As(III) was oxidized to As(V) in the growth media in the presence and absence of plants, and F^? had no effect on the rate of As(III) transformation. These experiments demonstrated that P. vittata was a good candidate to remediate arsenic-contaminated groundwater in the presence of fluoride. Our results can be used to develop strategies to remediate As-F-contaminated water using P. vittata.     

Hyperaccumulation of arsenic by callus,sporophytes and gametophytes of <Emphasis Type="Italic">Pteris vittata</Emphasis> cultured<Emphasis Type="Italic"> in vitro</Emphasis>

The callus of Pteris vittata was induced from gametophytes generated from spores in vitro, and grew rapidly with periodical medium change. Arsenic tolerance and accumulation of P. vittata callus were compared with those of Arabidopsis thaliana callus. Cell death was not detected in P. vittata callus even at arsenate concentrations up to 2 mM; however, A. thaliana callus died at low (0.2 mM) arsenate concentrations. Meanwhile, P. vittata callus accumulated almost three times more As than A. thaliana callus when exposed to 0.2 mM arsenate. About 60% of the total As was removed when 7.5 g of P. vittata callus was cultured on 150 ml of half-strength MS liquid medium containing 450 μg As for 2 days. Furthermore, P. vittata callus, sporophytes, and gametophytes all grew well under 1 mM of arsenate and accumulated 1,250; 1,150 and 2,180 mg kg⁻¹ dry weight As when grown on 2 mM arsenate for 15 or 30 days. The characteristics of non-differentiated cells, large biomass, ease of culture, good synchronization, and excellent As sequestering, make the callus of P. vittata a new ideal system to study the mechanisms of As hyperaccumulation and phytoremediation in As-contaminated groundwater.     

Influence of the arbuscular mycorrhizal fungus Glomus mosseae on uptake of arsenate by the As hyperaccumulator fern Pteris vittata L.

We report for the first time some effects of colonization by an arbuscular mycorrhizal (AM) fungus (Glomus mosseae) on the biomass and arsenate uptake of an As hyperaccumulator, Pteris vittata. Two arsenic levels (0 and 300 mg As kg^–1) were applied to an already contaminated soil in pots with two compartments for plant and hyphal growth in a glasshouse experiment. Arsenic application had little or no effect on mycorrhizal colonization, which was about 50% of root length. Mycorrhizal colonization increased frond dry matter yield, lowered the root/frond weight ratio, and decreased frond As concentration by 33–38%. Nevertheless, transfer of As to fronds showed a 43% increase with mycorrhizal colonization at the higher soil As level. Frond As concentrations reached about 1.6 g kg^–1 (dry matter basis) in non-mycorrhizal plants in the As-amended soil. Mycorrhizal colonization elevated root P concentration at both soil As levels and mycorrhizal plants had higher P/As ratios in both fronds and roots than did non-mycorrhizal controls.     

Development of suitable hydroponics system for phytoremediation of arsenic-contaminated water using an arsenic hyperaccumulator plant Pteris vittata

In this study, we found that high-performance hydroponics of arsenic hyperaccumulator fern Pteris vittata is possible without any mechanical aeration system, if rhizomes of the ferns are kept over the water surface level. It was also found that very low-nutrition condition is better for root elongation of P. vittata that is an important factor of the arsenic removal from contaminated water. By the non-aeration and low-nutrition hydroponics for four months, roots of P. vittata were elongated more than 500 mm. The results of arsenate phytofiltration experiments showed that arsenic concentrations in water declined from the initial concentrations (50?μg/L, 500?μg/L, and 1000?μg/L) to lower than the detection limit (0.1?μg/L) and about 80% of arsenic removed was accumulated in the fern fronds. The improved hydroponics method for P. vittata developed in this study enables low-cost phytoremediation of arsenic-contaminated water and high-affinity removal of arsenic from water.     

Role of trichome of Pteris vittata L. in arsenic hyperaccu-mulation

Heavy metal pollution of soils, caused by various anthropogenic sources, is a major environmental problem. Due to its cost-effectiveness and environ-mental friendliness, phytoremediation of arsenic-con- taminated soils has attracted more and more attention. An arsenic (As) hyperaccumulator, Chinese brake (Pteris vittata L.) was discovered by Chen et al. in China[1]. The field phytoremediation in Chenzhou City, Hunan Province has been successfully carried out by Chen et al. since 2000[2,3].…     

Effects of nutrients on arsenic accumulation by arsenic hyperaccumulator Pteris vittata L.

This research investigated the effects of various nutrients on arsenic (As) removal by arsenic hyperaccumulator Pteris vittata L. in a Hoagland nutrient solution (HNS). The treatments included different concentrations of Ca and K in 20% strength of HNS, different strengths of HNS (10, 20 and 30%), different strengths of HNS (10 and 20%) with and without CaCO₃, and different concentrations of Ca, K, NO₃, NH₄, and P in 20% strength of HNS. The plants were grown in nutrient solution containing 1 mg As L^?1 for 4 weeks except the Ca/K experiment where the plants were grown in nutrient solution containing 10 or 50 mg As L^?1 for 1 week. Adding up to 4 mM Ca or 3 mM K to 20% strength HNS significantly (P < 0.05) increased plant arsenic accumulation when the solution contained 10 mg As L^?1. Plant arsenic removal was reduced with increasing Ca and K concentrations at 50 mg As L^?1. Lower strength of HNS (10%) resulted in the greatest plant arsenic removal (79%) due to lower competition of P with As for plant uptake. Addition of CaCO₃ to 20% strength of HNS significantly increased arsenic removal by P. vittata. Among the nutrients tested, NO₃ and CaCO₃ were beneficial to plant arsenic removal while NH₄, P and Cl had adverse effects. This experiment demonstrated that it is possible to optimize plant arsenic removal by adjusting nutrients in the growth medium.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Chelator-assisted phytoextraction of arsenic,cadmium and lead by Pteris vittata L. and soil microbial community structure response

Abstract

Using biodegradable chelators to assist in phytoextraction may be an effective approach to enhance the heavy-metal remediation efficiencies of plants. A pot experiment was conducted to investigate the effects of ethylenediamine disuccinic acid (EDDS), citric acid (CA), and oxalic acid (OA) on the growth of the arsenic (As) hyperaccumulator Pteris vittata L., its arsenic (As), cadmium (Cd), and lead (Pb) uptake and accumulation, and soil microbial responses in multi-metal(loid)-contaminated soil. The addition of 2.5-mmol kg^?1 OA (OA-2.5) produced 26.7 and 14.9% more rhizoid and shoot biomass, respectively compared with the control, while EDDS and CA treatments significantly inhibited plant growth. The As accumulation in plants after the OA-2.5 treatment increased by 44.2% and the Cd and Pb accumulation in plants after a 1-mmol kg^?1 EDDS treatment increased by 24.5 and 19.6%, respectively. Soil urease enzyme activities in OA-2.5 treatment were significantly greater than those in the control and other chelator treatments (p?<?0.05). A PCR–denatured gradient gel electrophoresis analysis revealed that with the addition of EDDS, CA and OA enhanced soil microbial diversity. It was concluded that the addition of OA-2.5 was suitable for facilitating phytoremediation of soil As and did not have negative effects on the microbial community.     

超富集植物蜈蚣草中砷化学形态的EXAFS研究

采用同步辐射扩展X射线吸收精细结构(SREXAFS)技术研究了超富集植物蜈蚣草(PterisvittataL.)中As的化学形态及其在转运过程中的变化。结果表明,蜈蚣草中的As主要以As(Ⅲ)与O配位的形态存在。As(V)被植物吸收后,很快转化为As(Ⅲ),其转化过程主要发生在根部。As(Ⅲ)向地上部转运的过程中价态基本不变。在植物的根部和部分叶柄中存在少量与As-GSH相似的As-S结合方式,但是在As含量最高的羽叶中基本上未发现这种结合方式。与需要提取和分离过程的化学方法相比,采用EXAFS方法研究植物中的砷形态不需经过预分离或化学预处理就可以直接测定植物样品中元素的化学形态,因此可以避免样品预处理过程对As形态的干扰,并获得可靠的砷化学形态方面的信息。     

Relationship between plant phosphorus status and the kinetics of arsenate influx in clones ofdeschampsia cespitosa (L.) beauv. that differ in their tolerance to arsenate

Uptake kinetics of arsenate were determined in arsenate tolerant and non-tolerant clones of the grassDeschampsia cespitosa under differing root phosphorus status to investigate the mechanism controlling the suppression of arsenate influx observed in tolerant clones. Influx was always lower in tolerants compared to non-tolerants. Short term influx of arsenate by the high affinity uptake system in both tolerant clones was relatively insensitive to root phosphorus status. This was in contrast to the literature where the regulation of the phosphate (arsenate) uptake system is normally much more responsive to plant phosphorus status. The low affinity uptake system in both tolerant and non-tolerant clones, unlike the high affinity uptake system, was more closely regulated by root phosphate status and was repressed to a much greater degree under increasing root phosphorus levels than the high affinity system.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

Phytoremediation of arsenic contaminated soil by Pteris vittata L. I. Influence of phosphatic fertilizers and repeated harvests

A greenhouse experiment was conducted to evaluate the effectiveness of diammonium phosphate (DAP), single superphosphate (SSP) and two growing cycles on arsenic removal by Chinese Brake Fern (Pteris vittata L.) from an arsenic contaminated Typic Haplustept of the Indian state of West Bengal. After harvest of Pteris vittata the total, Olsen's extractable and other five soil arsenic fractions were determined. The total biomass yield of P. vittata ranged from 10.7 to 16.2 g pot(-1) in first growing cycle and from 7.53 to 11.57 g pot(-1) in second growing cycle. The frond arsenic concentrations ranged from 990 to 1374 mg kg(-1) in first growing cycle and from 875 to 1371 mg kg(-1) in second growing cycle. DAP was most efficient in enhancing biomass yield, frond and root arsenic concentrations and total arsenic removal from soil. After first growing cycle, P. vittata reduced soil arsenic by 10 to 20%, while after two growing cycles Pteris reduced it by 18 to 34%. Among the different arsenic fractions, Fe-bound arsenic dominated over other fractions. Two successive harvests with DAP as the phosphate fertilizer emerged as the promising management strategy for amelioration of arsenic contaminated soil of West Bengal through phyotoextraction by P. vittata.     

Phytoremediation of arsenic contaminated soil by Pteris vittata L. II. Effect on arsenic uptake and rice yield

A greenhouse experiment evaluated the effect of phytoextraction of arsenic from a contaminated soil by Chinese Brake Fern (Pteris vittata L.) and its subsequent effects on growth and uptake of arsenic by rice (Oryza sativa L.) crop. Pteris vittata was grown for one or two growing cycles of four months each with two phosphate sources, using single super phosphate (SSP) and di-ammonium phosphate (DAP). Rice was grown on phytoextracted soils followed by measurements of biomass yield (grain, straw, and root), arsenic concentration and, uptake by individual plant parts. The biomass yield (grain, straw and rice) of rice was highest in soil phytoextracted with Pteris vittata grown for two cycles and fertilized with diammonium phosphate (DAP). Total arsenic uptake in contaminated soil ranged from 8.2 to 16.9 mg pot(-1) in first growing cycle and 5.5 to 12.0 mg pot(-1) in second growing cycle of Pteris vittata. There was thus a mean reduction of 52% in arsenic content of rice grain after two growing cycle of Pteris vittata and 29% after the one growing cycle. The phytoextraction of arsenic contaminated soil by Pteris vittata was beneficial for growing rice resulted in decreased arsenic content in rice grain of <1 ppm. There was a mean improvement in rice grain yield 14% after two growing cycle and 8% after the one growing cycle of brake fern.     

蜈蚣草砷超富集机制及其在砷污染修复中的应用

蕨类植物蜈蚣草能够从土壤中吸收砷,并储存于地上部分羽叶的液泡中。蜈蚣草具有高效的抗氧化系统,以降低砷的毒害;其砷酸还原系统和液泡区隔化是蜈蚣草进行砷解毒和砷超富集的重要机制。本文综述了目前蜈蚣草砷超富集机制研究的主要进展,并对其在修复砷污染环境的应用中进行了讨论。