Introduction

Detrimental effects of heavy metals on the environment areevident. Soil contaminated with heavy metals is often deprived ofnutrients and microbial diversity, and the high concentration ofheavy metals cause the plants to accumulate these metals or affectthe growth and development of plants [1,2]. The disposal of thesemetals into the soil aggravates soil health problems [3]. Furthermore,these metals when present in different concentrations can be scarce,optimum, or phytotoxic to the plants [4]. Therefore, removal of theheavy metals from the environment by using remediation techniquesis critical.

In environmental science, remediation is a method for reducingor removing the pollutants by acting on the source of contaminationto protect the environment and humans from the harmful effects ofthe contaminants. Returning the contaminated soil to its natural stateis not always possible but necessary. Remediation activities should always be economical and optimized, and the outcome shouldbe balanced amid the benefits, risks, expenditure, and feasibility.Therefore, any acceptable remediation measures can be aptly plannedby understanding the source and nature of contamination, the site,and remediation technologies to be adopted.

Various techniques are available for remediation. The simplestmethod is to remove the uppermost layer of the contaminated soil bydigging and landfilling or capping the contaminated site. However,this method has disadvantages and risks. There is always a possibilitythat the contaminant can leak out during excavation, handling,transporting, and capping, which might contaminate the ground water.In addition, this method is very expensive and laborious. Differenttechniques are available for the remediation of metal-contaminatedsoil, namely chemical, physical, and biological techniques [5]. Thechemical method involves the use of harsh chemicals for chemicalwash, such as leaching of heavy metals using chelating agents [6].Therefore, researchers developed the bioremediation technique, a process by which organic wastes are biologically degraded undercontrolled conditions to an innocuous state or to levels below theconcentration limits established by regulatory authorities [7].

Chemical and physical remediation techniques are costly.According to Glass et al., the cost of land filling for a contaminated site and chemical recycling of contaminants varies between 100 and 500US$/ton, and the cost for electrokinetic monitoring is approximately20–200 US$/ton, whereas the cost involved in phytoextraction is5-40 US$/ton. Therefore, phytoextraction is an effective low-costtechnique for the enhanced remediation of metal-contaminatedsoil [8]. Phytoremediation provides sustainable measures for theremediation of metal-contaminated soil.

Phytoremediation approaches and hyperaccumulation ofmetals in plants

Phytoremediation is defined as the use of plants to remove,transfer, and degrade contaminants in soil, sediment, and water [9].Phytoremediation uses living organisms, particularly plants andmicroorganisms, to reduce, eliminate, transform, and detoxify benignproducts present in soil, sediments, water, and air. Phytoremediationtechnology, a bioremediation method, uses plants as filters foraccumulating, immobilizing, and transforming contaminants to aless harmful form [3].

The term “phytoremediation” is formed by combining the Greek word “phyto” meaning plant and the Latin word “remedium” meaning to restore or clean.

Phytoremediation includes various remediation techniquesthat involve many treatment strategies leading to contaminantdegradation, removal (through accumulation or dissipation), orimmobilization [10].

These remediation techniques may use genetically engineeredor naturally occurring plants for removing contaminants fromthe surrounding environment [11,12]. Utsunamyia and Chaneyreintroduced and developed the method of using hyperaccumulatingplants for extracting metals from contaminated soil [13,14]. Baker etal. reportedly conducted the first field trial on zinc (Zn) and cadmium(Cd) phytoextraction [15].

Types of phytoremediation

Based on contaminants, field conditions, clean-up levelrequired, and plant type, phytoremediation methods can be usedi.e., phytostabilization/phytoimmobilization for reducing mobility ofcontaminant or phytovolatization/phytoextraction for removal of thecontaminant [16].

Phytoremediation approaches involve different plant-basedtechnologies with different modes of action and mechanism. Figure 1 displays the schematic representation of the phytoremediationmechanism. Some of the widely used phytoremediation approaches are as follows:

1. Phytostabilization is the immobilization or precipitation ofcontaminants from soil, groundwater, and mine tailings byplants, thus decreasing their availability.

2. Phytofiltration uses plant roots and other parts to adsorb orabsorb contaminants from the aqueous environment.

3. Phytovolatilization uses plants that can evapotranspiratecontaminants, such as selenium (Se), mercury (Hg), andvolatile hydrocarbons, from soil and groundwater.

4. Phytoextraction is the uptake and concentration of metalsfrom contaminated soil or water directly into the plant tissueand their subsequent removal from the plants.

5. Phytodegradation includes the microbial degradation ofmetals in rhizosphere soil and groundwater.

6. Phytotransformation is the plant uptake of contaminantsfrom water and their conversion into organic compounds,which are less toxic or nontoxic.

7. Vegetative cap uses plants with a unique property ofevaportranspiration, thus preventing the leaching ofcontaminants.

Figure 1: Schematic representation of phytoremediation approaches.

i. Phytostabilization

Phytostabilization involves the use of plants to eliminate thebioavailability of toxic metals in soil [17]. Contaminants in soil areimmobilized by certain hyperaccumulating plants through absorptionand accumulation by roots, adsorption onto roots or precipitationwithin the root zone, and physical stabilization of soil.

Green vegetation is very helpful in controlling soil erosion as plantroots effectively bind the soil. Furthermore, the roots of vegetationfacilitate holding a considerable amount of rain water that returns tothe atmosphere through transpiration. The roots reduce the amount ofheavy metals entering the water table and other water bodies [18]. Tore-establish vegetation at sites where flora have disappeared or beendestroyed due to the presence of high metal concentrations, metaltolerantplant species can be planted, thereby reducing the effectivemigration of contaminants through soil leaching, groundwatercontamination, wind, and transportation of the exposed surface soil[18,19]. Some plants developed metal tolerance during evolutionwhile others may have this ability inherently [20].

Plants selected for phytostabilization preferably should be tolerantto concerned contaminats, hold them in their roots and should resistheavy metal accumulation in their above-ground exposed parts toprevent the entry of heavy metals into the food web [10,21]. Metalaccumulation in plants is measured and expressed in terms of thebio-concentration factor (BF) or accumulation factor (AF) andtranslocation factor (TF) or shoot:root (S:R) ratio [22,23].

Bioconcentration factor (BF) Total element = concentration in the shoot tissueor accumulation factor (AF) Total element concentration in mine tailings

Translocation factor (TF) = Total element concentration in the shoot tissueor shoot:root (S:R) ratio Total element concentration in the root tissue

In a recent study, Agrostis castellana having root bioaccumulationindices >2 and transfer factor < 1 was reported to be a suitable plantfor the phytostabilization of abandoned mine sites in Spain, whichare heavily polluted with heavy metals, such as Zn, copper (Cu), lead(Pb), Cd, and arsenic (As). However, due to substantial heavy metalaccumulation in the above-ground exposed parts of the plant evenat the low transfer factor obserevd, close monitoring and no huntingor grazing in areas under restoration was recommended to preventthe entry of toxic metals into the food chain [24]. Another studyassessed the growth potential of 36 plants belonging to 17 specieson a contaminated site and reported that plants with a high bioconcentrationfactor and a low translocation factor have the ability ofphytostabilization [25]. Of all the plants studied, Phyla nodiflora wasthe most efficient in accumulating Cu and Zn in its shoots, and thuswas appropriate for phytoextraction, whereas Gentiana pennellianawas most suitable for phytostabilization of sites contaminated withPb, Cu, and Zn [25].

To improve the physical and biological characteristics ofcontaminated soil, natural and synthetic supplements were addedduring phytostabilization processes. Thus, phytostabilization istermed as “aided phytostabilization” or “chemophytostabilization.”Changing the pH, increasing organic matter content by addingcompost, adding essential growth nutrients, increasing waterholding capacity, and reducing heavy metal bioavailability facilitatephytostabilization.

Five times reduction was observed in Pb and Zn concentrationsin aerial parts and in the roots of Lolium italicum and Festucaarundinacea, whose growth was greatly improved by the addedcompost [26]. Decreased phytotoxicity index was recorded afteradding compost, cyclonic ashes, and steel shots to an industrialcontaminated sandy soil [27]. Complexing agents, such as citricacid and ethylenediaminetetraacetic acid (EDTA), were shown toinfluence the phytostabilization capacity [28]. Addition of a synthetic(Calcinit + urea + PK14% + calcium carbonate) or organic (cowslurry) compost had a positive response on soil properties, growth,and remediation potential of L. perenne but decreased root-toshoottranslocation factors compared with the control plants [29].In an aided phytostabilization approach, the soil of an ore dustcontaminatedsite in northern Sweden was amended with alkalinefly ashes and peat for reducing the mobility of trace elements andwas vegetated with a mixture consisting of 6 grass and 13 herbspecies. The results showed that the proposed approach significantlyincreased microbial biomass and respiration, decreased microbialstress, and increased key soil enzyme activities [30]. In addition,plant growth-promoting bacteria (PGPB) improved the revegetationof two native species, quailbush and buffalo grass, of mine tailings,minimizing the requirement for compost amendment; however,the results were plant-specific [31]. In a phytostabilization study ofmine soil in France, a mixture of legume species, such as Anthyllisvulneraria, and nonlegume species increased the biomass of the otherspecies, and consequently increased the biomass production of theplant community [32].

Care should be taken so that phytostabilized metals remain inthe soil ecosystem. Because of the change in soil conditions and the degradation of organic matter, a possibility always exists of partialand gradual release and leaching, resulting in the dispersion ofphytostabilized metals to surrounding areas through soil erosion[21]. Therefore, long-term monitoring or “follow-up” programs arerequired in phytostabilization processes to monitor heavy metalmobilization, bioavailability, toxicity, and ecological impact [21].

ii. Phytofiltration

Phytofiltration involves the use of plants for removing pollutantsfrom contaminated surface waters or wastewaters, thus cleaningvarious aquatic environments. When plant roots, seedlings, orexcised plant shoots are used in phytofiltration to adsorb or absorbcontaminants from the aqueous environment, it is termed asrhizofiltration, blastofiltration, and caulofiltration, respectively[33,34]. According to Gardea-Torresdey et al., mechanisms involvedin biosorption include chemisorption, complexation, ion exchange,micro precipitation, hydroxide condensation onto the biosurface,and surface adsorption [35]. Young plants of Berkheya coddii growingin pots on ultramafic soil enriched with Cd, nickel (Ni), Zn, or Pbsubstantially accumulated a considerable amount of these metals,whereas excised shoots in solutions containing the same heavy metalsaccumulated a high amount of these metals in the leaves [34].

In rhizofiltration, terrestrial, rather than aquatic, plants are usedbecause terrestrial plants form extensive fibrous root systems coveredwith root hairs, and therefore have more surface area than the others[10]. Preferably, a plant used for rhizofiltration must accumulateand tolerate high concentrations of metals and should be easy tohandle, have low maintenance cost, and produce minimal secondarywaste requiring disposal. Furthermore, the plants must produce aconsiderable root biomass or have a large root surface area [36].

Various aquatic plants have the potential to remove heavy metalsfrom water, for example, Eichhornia crassipes [37], Hydrocotyleumbellata L. [38], and Lemna minor L. [39]; however, these plantshave limited capacity for rhizofiltration because of their small,slow-growing roots [40]. The high water content in aquatic plantsadds to the problem of drying, composting, and incineration.Despite limitations, E. crassipes (water hyacinth) was effective inremoving trace elements from waste streams [37]. Furthermore,Micranthemum umbrosum is an effective phytofiltrator of As andmoderate accumulator of Cd without any phytotoxic effect [41].The aquatic plants Callitriche stagnalis S., Potamogeton natans L.,and P. pectinatus L. tested in uranium phytofiltration experimentsreduced uranium concentrations in water from 500 to 72.3 μg/L,emphasizing the efficiency of the selected plants in removinguranium from water [42]. The bryophyte Fontinalis antipyreticaand Callitrichaceae members accumulate uranium with preferentialpartitioning in rhizomes/roots, emerging as promising candidates forthe development of phytofiltration [43].

Phytofiltration studies have been conducted on As accumulationby aquatic plants. A study of 18 representative aquatic plantspecies, such as species Ranunculus trichophyllus, R. peltatus subsp.saniculifolius, L. minor, and Azolla caroliniana, and the leaves ofJuncus effusus, reported that these species have a very high potentialfor As phytofiltration when they are introduced into constructedtreatment wetlands or natural water bodies [44].

Terrestrial plants, such as sunflower, Indian mustard, tobacco,rye, spinach, and corn, were studied for their ability to remove Pbfrom effluents, with sunflower exhibiting the greatest ability [45].The roots of Indian mustard (Brassica juncea Czern.) are effectivein removing Cd, chromium (Cr), Cu, Ni, Pb, and Zn [39], whereassunflower (Helianthus annus L.) removes Pb [39], U [46], 137Cs, and90Sr [47] from hydroponic solutions. Cassava (Manihot sculentaCranz) waste biomass was effective in removing two divalent metalions Cd (II) and Zn (II) from aqueous solutions [48].

Sharp dock (Polygonum amphibium), duckweed (L. minor),water hyacinth (E. crassipes), water dropwort (Oenathe javanica),and calamus (Lepironia articulata) are suitable for phytoremediationof polluted water, because sharp dock accumulates N and P in itsshoots, water hyacinth and duckweed are Cd hyperaccumulators,water dropwort is a Hg hyperaccumulator, and calamus is a Pbhyperaccumulator [49].

iii. Phytovolatilization

Phytovolatilization involves the use of plants that uptake metalsfrom soil, biologically convert them in a volatile form, and thenrelease them into the atmosphere by volatilization. Some metalcontaminants, such as As, Hg, and Se, exist naturally in the gaseousform in the environment.

Phytovolatilization can be used for organic pollutants andheavy metals. Furthermore, it has a limitation that it does noteliminate the pollutant completely; it only transfers it from oneform (soil) to another (atmosphere) from where the pollutant canredeposit. Therefore, phytovolatilization is the most controversialphytoremediation technology [33]. Whether the volatilization of theseelements into the atmosphere is safe or harmful remains unknown[50]. Se phytovolatilization has received the most attention to date; therelease of volatile Se compounds from higher plants was first reportedby Lewis et al., who demonstrated that both Se nonaccumulator andaccumulator species volatilize Se [51]. Brassicaceae members canrelease 40 gm Se ha−1 day −1 as various gaseous compounds [52].

B. juncea is effective in removing up to 95% Hg from contaminatedsolutions through volatilization and plant accumulation(phytofiltration) [53]. Most Hg volatilization occurs from the roots,which may have unforeseen environmental effects [53]. Hg uptakeand evaporation are achieved by some bacteria. Researchers areattempting to develop a transgenic plant by transferring the requiredgenes using rDNA technology for environmental restoration.Methylmercury is a strong neurotoxic agent, which is biosynthesizedin Hg-contaminated soil. Bacterial genes, such as merA (for mercuricreductase) and merB (for organomercurial lyase), were transformedinto Arabidopsis thaliana to produce genetically engineered plantscapable of detoxifying organic Hg. Furthermore, these genes, whichare necessary for plants to detoxify organic Hg by converting it tovolatile and less toxic elemental Hg, were expressed in the newlytransformed plants [54]. Bacterial genes, such as those for Hgreductase, have already been successfully transferred into Brassica,tobacco, and yellow poplar trees [55].

iv. Phytoextraction

Phytoextraction, the most commonly recognized phytoremediation technology, is also known as phytoaccumulation,phytoabsorption, or phytosequestration. It involves the use of plantsthat absorb metals from soil and translocate them to harvestableshoots where they accumulate.

Phytoextraction, a specific clean-up technology, cannot beconfused with phytoremediation, which is a concept [33]. Severalplants that may belong to distantly related families, but have thecommon ability to grow on metalliferous soil and accumulateextremely higher levels of heavy metals in the aerial organs thanother plants, without deleterious effects from phytotoxins, aretermed as “hyperaccumulator” [56]. These hyperaccumulator plantsform the basis of phytoextraction. Baker and Brooks reportedthat hyperaccumulators should have a metal accumulation valueexceeding the threshold value of the shoot metal concentration of 1%(Zn and Mn), 0.1% [Ni, cobalt (Co), Cr, Cu, Pb, and aluminium (Al)],0.01% (Cd and Se), or 0.001% (Hg) of the dry weight shoot biomass[15].

Based on its methodology, phytoextraction is generallygrouped into two categories. The first method called continuousphytoextraction involves the use of hyperaccumulating plants,whereas the second method called chelate-induced phytoextractioninvolves the use of high-biomass crop plants and chelating agents[10,21].

In continuous phytoextraction, metal-accumulating plantsare seeded or transplanted into metal-contaminated soil and arecultivated using established agricultural practices. The roots ofgrowing plants absorb metal elements from the soil and translocatethem to the aerial shoots where they accumulate. According to aprevious study, approximately 450 angiosperm species belonging tothe families Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae,Cunouniaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae,Violaceae, and Euphobiaceae [10] have been identified as heavy metal(As, Cd, Co, Cu, Mn, Ni, Pb, Sb, Se, Tl, and Zn) hyperaccumulators todate, accounting for less than 0.2% of all known species [56].

Researchers are continuously searching to find newhyperaccumulators in nature, which remain unidentified,and new reports on these plants continue to accrue [57]. Fewhyperaccumulators (only five species to date) are available for Cd,which is one of the most toxic heavy metals [56]. A study recentlydiscovered a new Cd hyperaccumulator plant Youngia erythrocarpa, afarmland weed [57]. Ni is hyperaccumulated by most taxa (more than75%), and approximately 25% of the discovered hyperaccumulatorsbelong to the family Brassicaceae, and particularly to Thlaspi andAlyssum [56].

Planting and harvesting of hyperaccumulators must be repeatedfor reducing the contamination at a particular site. Furthermore, thetime required depends on the target metal, plant selected, and itsefficacy; the duration of the process can vary from 1 to 20 years [58,59].The success of phytoextraction depends on the ability to produce highbiomass yields and to accumulate high quantities of environmentallycritical metals in the shoot tissue [33,58,60]. For example, Ebbs etal. reported that B. juncea to be more effective in removing Zn andCd from soil than Thlaspi caerulescens (a known hyperaccumulator of Zn), although T. caerulescens accumulated 10 and 2.5 times moreCd and Zn concentration, respectively, in its shoot than B. juncea[61]. B. juncea exhibited this property because it produces 10 timesmore shoot biomass than T. caerulescens. In addition to the highbiomass production capability, the plant must have high toleranceto the targeted metal(s) and be efficient in translocating them fromroots to the harvestable aerial parts of the plant [59]. Recently, therole of symbiotic bacterial species in facilitating plant growth inpoor soil with metal accumulation was observed. A novel species ofRhizobium metallidurans sp. nov., a symbiotic heavy metal-resistantbacterium, was isolated from a Zn-hyperaccumulating A. vulnerarialegume [62]. When these bacteria were inoculated in A. vulneraria,Zn concentration in the shoots increased up to 36% [63].

Chelate-induced phytoextraction is used when metals do notexist in the available form in the soil for sufficient plant uptake;adding chelates or acidifying agents to the metals facilitates theirliberation in the soil solution, thus improving the metal accumulationcapacity and uptake speed of nonhyperaccumulating plants [64]. Inthe past decades, the use of persistent aminopolycarboxylic acids(APCAs), such as EDTA, biodegradable APCAs, ethylene diaminedisuccinate (EDDS), and nitrilo triacetic acid as an alternative toEDTA, and low-molecular-weight organic acids (LMWOA) havebeen used in various phytoextraction experiments [64]. The degreeof chelate-induced extraction depends on several factors, such as thegeochemical fractions of metal in soil, and type and concentration ofchelating agents used [65]. The added chelating agents, however, aretoxic to the plants and have a negative effect on soil microbial growthduring the chelate-induced phytoextraction process [66]. Thereis always a potential risk of leaching of metals to groundwater andthe presence of nondegradable metal-chelating agent complexes incontaminated soil for a long period [67,68]. EDTA, a strong chelatingagent possessing strong complex-forming ability, has been mostextensively studied; however, the interest is now shifted on the usageof biodegradable chelating agents, such as EDDS, a biodegradableisomer of EDTA [65]. EDDS, a naturally occurring substance in soil,is easily decomposed into less detrimental byproducts. EDDS is lessharmful to the environment, can readily solubilize metals from soil,and is highly efficient in inducing metal accumulation in Brachiariadecumbens shoots [69,65].

v. Phytodegradation and phytotransformation

Phytodegradation also known as phytotransformation involvesthe breakdown of contaminants taken up by plants through metabolicprocesses within the plant or the breakdown of contaminantsexternally to the plant through the effect of compounds produced bythe plants [70]. It also includes plant-assisted microbial degradation ofthe contaminants in the rhizosphere region [3,71]. Phytodegradationof organic compounds by plants is reported by many workers [72,73].Caçador and Duarte, reported phytoconversion of Cr (VI) toxic formto the less toxic Cr (III) by halophytes [74]. Various bacterial andfungal microorganisms can facilitate transformation of toxic metalsto their less toxic states. Pseudomonas maltophilia strain, isolatedfrom soil at a toxic waste site in Oak Ridge, Tennessee, was reportedto catalyze the transformation and precipitation various toxicmetal cations and oxyanions [75]. Citric and oxalic acid producing Aspergillus niger, was reported to transform insoluble inorganicmetal compounds ZnO, Zn3(PO4)2 and Co3(PO4)2.to their respectiveorganic insoluble metal oxalates [76].

Pteridophytes as metal hyperaccumulators

Pteris vittata, also known as brake fern, is a perennial, evergreenfern native to China and was the first discovered As hyperaccumulatoras well as the first fern hyperaccumulator [77]. Furthermore, thisfern possesses a remarkable ability for As hyperaccumulation (upto 22,600 mg As kg−1 in its fronds) [77], which is markedly greaterthan most plant species (< 10 mg As kg−1) [78]. Although at a reducedrate, P. vittata is effective in As uptake in the presence of other metals(Ni, Zn, Pb, and Cd); however, its ability to take up other metals islimited [79]. Approximately a dozen of ferns belonging to Pteris andfew from others, such as Pityrogramma calomelanos, were reportedas As hyperaccumulators; however, not all members of Pteris areAs hyperaccumulators [80]. Plasma membranes of the root cells ofP. vittata have a higher density of phosphate/arsenate transportersthan the nonhyperaccumulator P. tremula, which may be a resultof constitutive gene overexpression [81]. As hyperaccumulation byfern depends on the high affinity of the phosphate/arsenate transportsystems to arsenate [82] and the plant’s capability to increase Asbioavailability in the rhizosphere by reducing pH through the rootexudation of high amounts of dissolved organic carbon [83]. Thedecrease in pH increases the amount of water-soluble As that can bereadily taken up by the roots [83,84].