Hyperaccumulator Pteris Vittata Research Articles

Impact of metal stress on the production of secondary metabolites in Pteris vittata L. and associated rhizosphere bacterial communities.

Plants adapt to metal stress by modifying their metabolism including the production of secondary metabolites in plant tissues. Such changes may impact the diversity and functions of plant associated microbial communities. Our study aimed to evaluate the influence of metals on the secondary metabolism of plants and the indirect impact on rhizosphere bacterial communities. We then compared the secondary metabolites of the hyperaccumulator Pteris vittata L. collected from a contaminated mining site to a non-contaminated site in Vietnam and identified the discriminant metabolites. Our data showed a significant increase in chlorogenic acid derivatives and A-type procyanidin in plant roots at the contaminated site. We hypothesized that the intensive production of these compounds could be part of the antioxidant defense mechanism in response to metals. In parallel, the structure and diversity of bulk soil and rhizosphere communities was studied using high-throughput sequencing. The results showed strong differences in bacterial composition, characterized by the dominance of Proteobacteria and Nitrospira in the contaminated bulk soil, and the enrichment of some potential human pathogens, i.e., Acinetobacter, Mycobacterium, and Cupriavidus in P. vittata's rhizosphere at the mining site. Overall, metal pollution modified the production of P. vittata secondary metabolites and altered the diversity and structure of bacterial communities. Further investigations are needed to understand whether the plant recruits specific bacteria to adapt to metal stress.

Microbial siderophores and root exudates enhanced goethite dissolution and Fe/As uptake by As-hyperaccumulator Pteris vittata

Arsenic (As) in soils is often adsorbed on Fe-(hydro)oxides surface, rendering them more resistant to dissolution, which is undesirable for phytoremediation of As-contaminated soils. Arsenic hyperaccumulator Pteris vittata prefers to grow in calcareous soils where available Fe and As are low. To elucidate its mechanisms of acquiring Fe and As from insoluble sources in soils, we investigated dissolution of goethite with pre-adsorbed arsenate (AsV; As-goethite) in presence of four organic ligands, including two root exudates (oxalate and phytate, dominant in P. vittata) and two microbial siderophores (PG12-siderophore and desferrioxamine B). Their presence increased As solubilization from As-goethite from 0.03 to 0.27–5.33 mg L−1 compared to the control. The siderophore/phytate bi-ligand treatment released 7.42 mg L−1 soluble Fe, which was 1.2-fold that of the sum of siderophore and phytate, showing a synergy in promoting As-goethite dissolution. In the ligand-mineral-plant system, siderophore/phytate was most effective in releasing As and Fe from As-goethite. Moreover, the continuous plant uptake induced more As-goethite dissolution. The continued release of As and Fe significantly enhanced their plant uptake (from 0.01 to 0.43 mg plant−1 As and 2.7–14.8 mg plant−1 Fe) and plant growth (from 1.2 to 3.1 g plant−1 fw) in P. vittata. Since microbial siderophores and root exudates often coexist in soil rhizosphere, their synergy in enhancing dissolution of insoluble As-Fe minerals may play an important role in efficient phytoremediation of As-contaminated soils.

Phytoextraction of arsenic-contaminated soil with Pteris vittata in Henan Province, China: comprehensive evaluation of remediation efficiency correcting for atmospheric depositions.

Research on the appropriate method for evaluating phytoremediation efficiency is limited. A 2-year field experiment was conducted to investigate phytoremediation efficiency using the hyperaccumulator Pteris vittata on an arsenic (As)-contaminated site. The remediation efficiency was evaluated through the removal rate of As in soils and extraction rate of heavy metals in plants. After 2years of remediation, the concentration of total As in soils decreased from 16.27mgkg-1 in 2012 to 14.58mgkg-1 in 2014. The total remediation efficiency of As was 10.39% in terms of the removal rate of heavy metals calculated for soils, whereas the remediation efficiency calculated from As uptake by P. vittata was 16.09%. Such a discrepancy aroused further consideration on the potential input of As. A large amount of As was brought in by atmospheric emissions, which possibly biased the calculation of remediation efficiency. In fact, considering also the atmospheric depositions of As, the corrected removal rate of As from soil was 16.57%. Therefore, the results of this work suggest that (i) when evaluating the phytoextraction efficiency, the whole input and output cycle of the element of interest in the targeted ecosystem must be considered, and (ii) P. vittata has the potential to be used to remediate As-contaminated soils in Henan Province, China.

Arsenic-enrichment enhanced root exudates and altered rhizosphere microbial communities and activities in hyperaccumulator Pteris vittata

Phytoremediation of arsenic (As)-contaminated soil by hyperaccumulator Pteris vittata is promising. A better understanding of the rhizosphere microbial dynamics that regulate As availability and plant growth is important to optimize the phytoremediation process. In this study, Illumina sequencing of 16S rRNA genes was applied to assess the rhizosphere microbial community structure of P. vittata. Microbial functionality was monitored by soil enzyme activities and MPN-PCR targeting genes of interest. Arsenic (100mgkg−1 AsV) addition to soil significantly increased DOC, root exudates, As and P uptake and the frond biomass of P. vittata. Moreover, As-enrichment significantly increased soil enzyme activities involved in N, P and S cycling and the gene abundance of As transforming bacteria, Fe- and S-reducing bacteria and N and C fixing bacteria in the rhizosphere of P. vittata. Together, the results revealed that the combined selective pressure of As and rhizosphere resulted in stimulation of microbial community, which most likely has a role in reductive dissolution of Fe and S, As and P mobilization, C degradation and fixation, and N fixation. These changes appeared to have a role in mitigation of As toxicity and to promote growth and the As uptake ability of P. vittata under As-enriched conditions.

Intercropped Pteris vittata L. and Morus alba L. presents a safe utilization mode for arsenic-contaminated soil.

Intercropping technology provides income for owners of contaminated soil without increasing environmental risk. Therefore, intercropping of arsenic (As) hyperaccumulator Pteris vittata L. with economic crops is now widely utilized in slightly or moderately As-contaminated farmlands. However, the mechanisms for As mobilization and absorption within the intercropping system are still unclear. To clarify As mobilization and absorption within an intercropping system, portable X-ray fluorescence spectrometry and sequential extraction were utilized to detect the spatial distribution and speciation of As in an intercropped system of P. vittata and cash crop mulberry (Morus alba L.). Compared with the P. vittata monoculture, P. vittata intercropping had higher As concentration, which may have been caused by the efficient exploitation of a greater As source in soil. Compared with the M. alba monoculture, M. alba intercropping had lower As concentration, which may have been caused by the As depletion by P. vittata roots. Spatial distribution of As in the soil indicated a "valley" around the P. vittata roots in both monocultured and intercropped systems, implying that As was depleted around the P. vittata roots. Continuous As extraction confirmed that both P. vittata monoculture and P. vittata and M. alba intercropping can efficiently control the risk of As soil contamination. Moreover, the properties of M. alba leaves were further studied. Mulberry leaves in the intercropping system satisfied the national feed standards. Therefore, intercropping presents a safe utilization mode for As-contaminated soil and can increase the income from silkworm-rearing M. alba leaves, without extra environmental risk.

Micro-distribution of arsenic species in tissues of hyperaccumulator Pteris vittata L.

Arsenic (As) contamination and its harmful consequences have gained increasing attention in research. Phytoextraction, which uses the As hyperaccumulator Pteris vittata L., is a well-established technology adopted in many countries. However, the hyperaccumulation mechanisms of this plant remain controversial. This study investigated the species and the micro-distribution of As species in three P. vittata L. ecotypes after exposure to arsenite (AsIII) and arsenate (AsV) for 7d. Arsenic-accumulating abilities and preferences to As species varied among different ecotypes. The reduction of AsV into AsIII, oxidation of AsIII into AsV, and chelation of AsIII with thiols were all observed in P. vittata. The reduction of As mainly occurred in the rhizoid, whereas oxidation and chelation mainly occurred in the aboveground parts. Correlation analyses showed that the As concentration in pinna was significantly correlated with the AsV percentage in paraxial and abaxial epidermis (positive), AsIII-GSH percentage in paraxial epidermis (positive), and AsIII percentage in paraxial and abaxial epidermis (negative). Results indicated that oxidation and chelation reactions contributed to the accumulation of As in P. vittata.

Arsenic Induced Phytate Exudation, and Promoted FeAsO4 Dissolution and Plant Growth in As-Hyperaccumulator Pteris vittata.

Arsenic hyperaccumulator Pteris vittata (PV) is efficient in taking up As and nutrients from As-contaminated soils. We evaluated the mechanisms used by PV to mobilize As and Fe by examining the impacts of As and root exudates on FeAsO4 solubilization, and As and Fe uptake in four plants: As-hyperaccumulators PV and Pteris multifida (PM), nonhyperaccumulator Pteris ensiformis (PE), and angiosperm plant tomato (Solanum lycopersicum). Phytate and oxalate were dominant in fern plants (>93%), which were 50-83, 15-42, and 0-32 mg kg(-1) phytate and 10-15, 7-26, and 4-12 mg kg(-1) oxalate for PV, PM, and PE respectively, with higher As inducing greater phytate exudation and no phytate being detected in tomato exudates. PV treated with phytate+FeAsO4 had higher As and Fe contents and larger biomass than phytate or FeAsO4 treatment, which were 340 vs 20 and 130 mg kg(-1) As in the fronds and 7900 vs 1600 and 4100 mg kg(-1) Fe in the roots. We hypothesized that As-induced phytate exudation helped PV to take up Fe and As from insoluble FeAsO4 and promoted PV growth. Our study suggests that phytate exudation may be special to fern plants, which may play an important role in enhancing As and nutrient uptake by plants, thereby increasing their efficiency in phytoremediation of As-contaminated soils.

Application of arsenic hyperaccumulator Pteris vittata L. to contaminated soil in Northern China

Phytoextraction of arsenic (As) from contaminated soil using hyperaccumulator Pteris vittata L. has been applied to several contaminated areas in China and USA; however, most of them are located in Southern China. Low temperature during winter is the main reason limiting the application of P. vittata in Northern China. The aim of the current study is to explore the possibility of using P. vittata to remediate As-contaminated soil in Northern China and other areas with similar climate conditions and to obtain appropriate measures to aid in the survival of P. vittata in winter. Four different cover materials were used in the current study; by comparing growth conditions and remediation efficiency, the most suitable cover material for P. vittata was obtained. Results showed that using the combination of plastic film and soil or maize straw significantly improved soil temperature. While, the use of a single layer plastic film was the best choice when considering As accumulation.

Interaction of As and Sb in the hyperaccumulator Pteris vittata L.: changes in As and Sb speciation by XANES.

Arsenic (As) and antimony (Sb) are chemical analogs that display similar characteristics in the environment. The As hyperaccumulator Pteris vittata L. is a potential As-Sb co-accumulating species. However, when this plant is exposed to different As and Sb speciation, the associated accumulating mechanisms and subsequent assimilation processes of As and Sb remain unclear. A 2-week hydroponic experiment was conducted by exposing P. vittata to single AsIII, AsV, SbIII, and SbV or the co-existence of AsIII and SbIII and AsV and SbV. P. vittata could co-accumulate As and Sb in the pinna (>1000mgkg(-1)) with high translocation (>1) of As and Sb from the root to the pinna. P. vittata displayed apparent preference to the trivalent speciation of As and Sb than to the pentavalent speciation. Under the single exposure of AsIII or SbIII, the pinna concentration of As and Sb was 84 and 765% higher than that under the single exposure of AsV or SbV, respectively. Despite the provided As speciation, the main speciation of As in the root was AsV, whereas the main speciation of As in the pinna was AsIII. The Sb in the roots comprised SbV and SbIII when exposed to SbV but was exclusively SbIII when exposed to SbIII. The Sb in the pinna was a mixture of SbV and SbIII regardless of the provided Sb speciation. Compared with the single exposure of As, the co-existence of As and Sb increased the As concentration in the pinna of P. vittata by 50-66%, accompanied by a significant increase in the AsIII percentage in the root. Compared with the single exposure of Sb, the co-existence of Sb and As also increased the Sb concentration in the pinna by 51-100%, but no significant change in Sb speciation was found in P. vittata.

Arsenic uptake, arsenite efflux and plant growth in hyperaccumulator Pteris vittata: Role of arsenic-resistant bacteria.

Bacteria-mediated arsenic (As) transformation and their impacts on As and P uptake and plant growth in As-hyperaccumulator Pteris vittata (PV) were investigated under sterile condition. All As-resistant bacteria (9 endophytic and 6 rhizospheric) were As-reducers except one As-oxidizer. After growing two months in media with 37.5 mg kg(-1) AsV, As concentrations in the fronds and roots were 3655-5389 (89-91% AsIII) and 971-1467 mg kg(-1) (41-73% AsIII), corresponding to 22-52% decrease in the As in the media. Bacterial inoculation enhanced As and P uptake by up to 47 and 69%, and PV growth by 20-74%, which may be related to elevated As and P in plants (r = 0.88-0.97, p < 0.05). Though AsV was supplied, 95% of the As in the bacteria-free media was AsIII, suggesting efficient efflux of AsIII by PV roots (120 µg g(-1) root fw). This was supported by the fact that no AsV was detected in media inoculated with As-reducers while 95% of AsV was detected with As-oxidizer. Our data showed that, under As-stress, PV reduced As toxicity by efficient AsIII efflux into media and AsIII translocation to the fronds, and bacteria benefited PV growth probably via enhanced As and P uptake.

A new method for antimony speciation in plant biomass and nutrient media using anion exchange cartridge

A selective separation method based on anion exchange cartridge was developed to determine antimony (Sb) speciation in biological matrices by graphite furnace atomic absorption spectrophotometry (GFAAS). The selectivity of the cartridge towards antimonite [SbIII] and antimonate [SbV] reversed in the presence of deionized (DI) water and 2mM citric acid. While SbV was retained by the cartridge in DI water, SbIII was retained in citric acid media. At pH 6, SbIII and SbV formed SbIII- and SbV-citrate complexes, but the cartridge had higher affinity towards the SbIII-citrate complex. Separation of SbIII was tested at various concentrations in fresh and spent growth media and plant tissues. Our results showed that cartridge-based Sb speciation was successful in plant tissues, which was confirmed by HPLC–ICP-MS. The cartridge retained SbIII and showed 92–104% SbV recovery from arsenic hyperaccumulator Pteris vittata roots treated with SbIII and SbV. The cartridge procedure is an effective alternative for Sb speciation, offering low cost, reproducible results, and simple Sb analysis using GFAAS.

Uptake of antimonite and antimonate by arsenic hyperaccumulator Pteris vittata: Effects of chemical analogs and transporter inhibitor

Antimonite (SbIII) is transported into plants via aquaglyceroporin channels but it is unknown in As-hyperaccumulator Ptreis vittata (PV). We tested the effects of SbIII analogs (arsenite-AsIII, glycerol, silicic acid-Si, and, glucose), antimonate (SbV) analog (phosphate-P), and aquaglyceroporin transporter inhibitor (silver, Ag) on the uptake of SbIII or SbV by PV gametophytes. PV gametophytes were grown in 20% Hoagland solution containing 65 μM SbIII or SbV and increasing concentrations of analogs at 65–6500 μM for 2 h or 4 h under sterile condition. After exposing to 65 μM Sb for 2 h, PV accumulated 767 mg/kg Sb in SbIII treatment and 419 mg/kg in SbV treatment. SbIII uptake by PV gametophytes was not impacted by glycerol or AsIII nor aquaglyceroporin inhibitor Ag during 2 h exposure. While Si increased SbIII uptake and glucose decreased SbIII uptake by PV gametophytes, the impact disappeared during 4 h exposure. Under P-sufficient condition, P increased SbIII uptake and decreased SbV uptake during 2 h exposure, but the effect again disappeared after 4 h. After being P-starved for 2 weeks, P decreased SbIII with no effect on SbV uptake during 2 h exposure. Our results indicated that: 1) PV gametophytes could serve as an efficient model to study Sb uptake, and 2) unique SbIII uptake by PV may be related to its trait of As hyperaccumulation.

Role of transpiration in arsenic accumulation of hyperaccumulator Pteris vittata L.

Mechanisms of Pteris vittata L. to hyperaccumulate arsenic (As), especially the efficient translocation of As from rhizoids to fronds, are not clear yet. The present study aims to investigate the role of transpiration in the accumulation of As from the aspects of transpiration regulation and ecotypic difference. Results showed that As accumulation of P. vittata increased proportionally with an increase in the As exposure concentration. Lowering the transpiration rate by 28∼67% decreased the shoot As concentration by 19∼56%. Comparison of As distribution under normal treatment and shade treatment indicated that transpiration determines the distribution pattern of As in pinnae. In terms of the ecotypic difference, the P. vittata ecotype from moister and warmer habitat had 40% higher transpiration and correspondingly 40% higher shoot As concentration than the ecotype from drier and cooler habitat. Results disclosed that transpiration is the main driver for P. vittata to accumulate and re-distribute As in pinnae.

Phosphorus solubilization and plant growth enhancement by arsenic-resistant bacteria

Phosphorus is an essential nutrient, which is limited in most soils. The P solubilization and growth enhancement ability of seven arsenic-resistant bacteria (ARB), which were isolated from arsenic hyperaccumulator Pteris vittata, was investigated. Siderophore-producing ARB (PG4, 5, 6, 9, 10, 12 and 16) were effective in solubilizing P from inorganic minerals FePO4 and phosphate rock, and organic phytate. To reduce bacterial P uptake we used filter-sterilized Hoagland medium containing siderophores or phytase produced by PG12 or PG6 to grow tomato plants supplied with FePO4 or phytate. To confirm that siderophores were responsible for P release, we compared the mutants of siderophore-producing bacterium Pseudomonas fluorescens Pf5 (PchA) impaired in siderophore production with the wild type and test strains. After 7d of growth, mutant PchA solubilized 10-times less P than strain PG12, which increased tomato root biomass by 1.7 times. For phytate solubilization by PG6, tomato shoot biomass increased by 44% than control bacterium Pseudomonas chlororaphis. P solubilization by ARB from P. vittata may be useful in enhancing plant growth and nutrition in other crop plants.

Transfer of arsenic and phosphorus from soils to the fronds and spores of arsenic hyperaccumulator Pteris vittata and three non-hyperaccumulators

Concentrations of chemical analogs arsenic (As) and phosphorus (P) were measured in As-hyperaccumulator (Pteris vittata; PV) and three non As-hyperaccumulators (Thelypteris kunthii, Nephrolepsis brownii, and N. falcata) to draw inferences regarding their uptake from soils to roots and translocation to fronds and spores. Frond and root samples of 150 ferns at peak spore maturation were collected with associated soils between July 2012 and July 2014. Arsenic in PV spores (45.4–336 mg kg−1) exceeded soil As (0.60–111 mg kg−1) in all sites and at clean sites (0.60–1.17 mg kg−1) for non-hyperaccumulator spores (1.83–8.60 mg kg−1). In PV, As in fronds and spores correlated positively with soil As (r = 0.71–0.74) with bioconcentration factors (tissue As:soil As) of 14.3–654 and 3.26–53.6 compared to 0.08–0.44 and 0.03–8.37 for three non-hyperaccumulators. However, P in PV spores (1977–4832 mg kg−1) correlated negatively with frond (1028–2439 mg kg−1; r = −0.43) and soil (76.2–170 mg kg−1; r = −0.34) P. PV hyperaccumulates As into fronds and spores from soils with trace As. Since PV spores constituted ~9 % of frond biomass, the elevated spore As may deserve further investigation in their role as a potential health hazard and metal cycling.

Study on the Incineration Experiment to Dispose &lt;i&gt;Ptreis vittata&lt;/i&gt; L&lt;i&gt;.&lt;/i&gt;

Phytoextraction of heavy metal polluted environment generates large quantities of hyperaccumulators. How to dispose hyperaccumulators safely has been one of the most important issues in the phytoextraction field. In this paper, incineration technology was studied in order to investigate the feasibility of incineration technology on disposal of the arsenic-containing hyperaccumulator Pteris vittata L.. The tube furnace experiment examined the changes of arsenic in Pteris vittata L., studied the effects of factors including temperature, time, percentage of CaO on the incineration state. The results show that the most appropriate incineration condition was temperature of 400°C, time of 30min, percentage of CaO of 10%, which solved the problem to reduce biomass of Pteris vittata L. and volatilization of arsenic. The result provide insight into developing harmless disposal or resource-reuse-oriented methods for disposal of the plants used for large scale phytoremediation application.

Chromate and phosphate inhibited each other's uptake and translocation in arsenic hyperaccumulator Pteris vittata L.

We investigated the effects of chromate (CrVI) and phosphate (P) on their uptake and translocation in As-hyperaccumulator Pteris vittata (PV). Plants were exposed to 1) 0.10 mM CrVI and 0, 0.25, 1.25, or 2.50 mM P or 2) 0.25 mM P and 0, 0.50, 2.5 or 5.0 mM CrVI for 24 h in hydroponics. PV accumulated 2919 mg/kg Cr in the roots at CrVI0.10, and 5100 and 3500 mg/kg P in the fronds and roots at P0.25. When co-present, CrVI and P inhibited each other's uptake in PV. Increasing P concentrations reduced Cr root concentrations by 62–82% whereas increasing CrVI concentrations reduced frond P concentrations by 52–59% but increased root P concentrations by 11–15%. Chromate reduced P transport, with more P being accumulated in PV roots. Though CrVI was supplied, 64−78% and 92−93% CrIII were in PV fronds and roots. Based on X-ray diffraction, Cr2O3 was detected in the roots confirming CrVI reduction to CrIII by PV. In short, CrVI and P inhibited each other in uptake and translocation by PV, and CrVI reduction to CrIII in PV roots served as its detoxification mechanism. The finding helps to understand the interactions of P and Cr during their uptake in PV.

CHARACTERIZATION OF ARSENIC UPTAKE IN LIVING PTERIS VITTATA L.

Phytoremediation of arsenic contamination using the hyperaccumulator Pteris vittata is an active area of research. However, the mechanism for the uptake of arsenic in P. vittata remains unclear, due to the absence of methods to monitor this process. This study utilized synchrotron radiation X-ray fluorescence (XRF) spectroscopy and X-ray absorption near-edge structure (XANES) to continuously observe in situ arsenic in living P. vittata. The results showed that the reduction of arsenic(V) to arsenic(III) occurred simultaneously with an increase in frond arsenic, suggesting the reduction affected arsenic uptake. In situ XRF measurements of living P. vittata accurately characterized arsenic uptake but in situ XANES could not be used on live plants without using methods to prevent radiation damage. A novel approach is reported to investigate the changes of elements in plants.

Arsenic enhanced plant growth and altered rhizosphere characteristics of hyperaccumulator Pteris vittata

We investigated the effects of arsenic species on As accumulation, plant growth and rhizospheric changes in As−hyperaccumulator Pteris vittata (PV). PV was grown for 60−d in a soil spiked with 200 mg kg−1 arsenate (AsV−soil) or arsenite (AsIII−soil). Diffusive gradients in thin−films technique (DGT) were used to monitor As uptake by PV. Interestingly AsIII−soil produced the highest PV biomass at 8.6 g plant−1, 27% and 46% greater than AsV−soil and the control. Biomass increase was associated with As−induced P uptake by PV. Although AsIII was oxidized to AsV during the experiment, As species impacted As accumulation by PV, with 17.5% more As in AsIII−soil than AsV−soil (36 vs. 31 mg plant−1). As concentration in PV roots was 30% higher in AsV−soil whereas As concentration in PV fronds was 7.9% greater in AsIII−soil, suggesting more rapid translocation of AsIII than AsV. These findings were important to understand the mechanisms of As uptake, accumulation and translocation by PV.

Pteris vittata continuously removed arsenic from non-labile fraction in three contaminated-soils during 3.5 years of phytoextraction

We evaluated the effectiveness of arsenic (As) hyperaccumulator Pteris vittata to continuously remove As from three contaminated-soils containing 26–126mgkg−1 As over 7 harvests in 3.5 years. Changes in As speciation in soils, amended with P fertilizer (P-soil) or insoluble phosphate rock (PR-soil), were assessed via sequential fractionation. Arsenic in available (soluble+exchangeable), non-labile (bound to amorphous+crystalline Fe/Al oxides), and residual fractions constituted ∼12%, ∼80%, and ∼8% of soil As. Soluble As declined while exchangeable As was unchanged, likely due to replenishment from non-labile As, which accounted for ∼87% of decline in total soil As. Although plant-available As is important, the non-labile As better predicted the frond As concentration in P. vittata, with the correlation being r=0.90 and 0.64 for PR-soils and P-soils. P. vittata removed 44% of soil As from PR-soils compared to 33% from P-soils, suggesting the low-soluble P from PR was more effective than P fertilizer in enhancing As uptake by P. vittata. To facilitate acquisition of P from PR, P. vittata produced larger root biomass to solubilize non-labile As, allowing for more efficient phytoextraction.