soil remediation and plants || phytoremediation

63Soil Remediation and Plants. http://dx.doi.org/10.1016/B978-0-12-799937-1.00003-6Copyright © 2015 Elsevier Inc. All rights reserved.

Phytoremediation: A Promising Strategy on the Crossroads of Remediation

Arif Tasleem Jan, Arif Ali and Qazi Mohd. Rizwanul HaqDepartment of Biosciences, Jamia Millia Islamia, New Delhi, India

INTRODUCTION

In the era of industrialization, developmental progress marked with the tech-nological advancement has raised serious environmental challenges as they bypassed the safety demands of the natural environment (Bennett et al., 2003). Although, a number of natural processes (volcanoes, forest fires, etc.) release different pollutants into the environment, anthropogenic activities are believed to be the major cause of environmental pollution. Pollutants of various kinds, particularly heavy metals that arise as a result of accidental or process spill-age, disposal of untreated industrial waste and by sludge application to agri-cultural soils, have contributed significantly to the deterioration of land and water resources, as is evident from changes in ecosystem processes. Amid their distribution along with potential deleterious effects on human health, their con-tamination presents a black picture of a complex industrialized hi-tech society. Being unable to degrade by soil microbiota, their evacuation from the polluted site generally relies on immobilization or physical removal. Besides being expensive and non-specific, use of physicochemical techniques for the treat-ment of soil pollutants has rendered land useless for the growth of plants as the population of microbes associated with various biological activities such as nitrogen fixation are greatly affected during the process of decontamination.

Continued from the past, progressive deterioration of environmental quality resulting from the discharge of untreated industrial effluents and sewage has severely threatened human and environmental health. Metal pollutants that pres-ent a continuing threat to mankind and to its surroundings need to be addressed in order to reverse their effects so as to maintain the balance of the system. Sensing chemicals in the environment and responding to changes in their concentrations so as to bring the system back into balance is the fundamental principle behind

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64 Soil Remediation and Plants

the process of bioremediation. In the simplest sense, the goal of bioremediation (use of biological agents) is to attenuate pollutants by transforming them to less toxic or to innocuous products. Over the years, the success of bioremediation technology has emerged as the most advantageous clean-up technique for the treatment of pollutants that have polluted soil and water resources, with bacteria being the most important organism in the context of immobilization or the trans-formation of metal pollutants (Malik, 2004; Fu and Wang, 2011).

METAL POLLUTANTS AND HUMAN HEALTH

Since the commencement of the industrial revolution, redistribution of met-als in the environment is gradually ending with the build-up in terrestrial and aquatic habitats. They are not just part of our long industrial heritage, how-ever; but are finding increasing uses in areas as diverse as medicine, electronics, catalysis and the generation of nuclear power. Being non-degradable and thus persistent, metal pollutants with known effects on human health represent wide-spread pollutants of great concern. Toxicity of metals is generally represented in terms of their concentration, inability to undergo degradation completely by any methods, ability to get transformed to more toxic forms under certain envi-ronmental conditions and by their ability to get accumulated in the food chain that hamper normal physiological activities and ultimately endanger human life ( McLaughlin et al., 1999; Nair et al., 2008). Given our long and intimate asso-ciation with metals, they present a great concern both at personal and at public health levels as their disturbed hazardous concentrations result in toxicity and sometimes even in lethal effects as they escape control mechanisms such as homeostasis, transport, compartmentalization and binding to specific cell con-stituents. Toxicity of metal compounds is accredited to their prevalence in close proximity to humans which facilitates their access into living systems.

Mercury (Hg), a transition metal belonging to group 12 (IIB) of the periodic table, exists in three valence states: elemental or metallic form (Hg0), inorganic (Hg2+ or Hg+) and organic forms (R-Hg+ or R-Hg-X). Despite the fact that all forms are toxic, the organic form is considered to be the most toxic and the elemental the least toxic. The detrimental fate of organomercurial compounds in humans is endorsed to its hydrophobic character, a property which it employs in crossing the placental and blood–brain barriers (Jan et al., 2009; Singh et al., 2011). Transport of mercury within the body of humans is attributed to molecu-lar mimicry, a mechanism by which metal compounds compete with endog-enous substrates in getting entry into different cells and tissues. Having high affinity for thiol groups of enzymes and proteins, both organic and inorganic mercury cross membranes with different abilities, which leads to their inactiva-tion (Hajela et al., 2002; Gupta and Ali, 2004). Compared to organic mercury, inorganic mercury exerts its toxic consequences mainly on getting methyl-ated to methylmercury compounds that lead to increases in the intracellular calcium level rapidly from the extracellular calcium pools, thereby hampering

65Chapter | 3 A Promising Strategy on the Crossroads of Remediation

production of neurotransmitters and creating serious imbalances in the develop-ment of the brain (Pedersen et al., 1999).

Arsenic (As), a group 15 (V) member of the periodic table, exists as metal-loid arsenic (As0), arsenite (As3+), arsenate (As5+) and as an arsine gas (As3−). However, arsenic compounds are generally categorized as organic (non-toxic) as well as inorganic (toxic) forms. Inorganic arsenic predominantly exists in trivalent (As3+) and pentavalent (As5+) forms, with trivalent compounds having the property to get absorbed more rapidly due to their high lipid solubility and, thus, being more toxic than pentavalent ones (Kaur et al., 2009). They are inter-convertible depending on the redox status of the environment; with pentavalent arsenic more stable and predominant under aerobic conditions and trivalent species under anaerobic conditions (Duker et al., 2005). Arsenic exerts its toxic consequences soon after binding to proteins such as haemoglobin, whereby it gets redistributed to organs such as liver, kidney, lung, spleen and intestines. However, unlike other metals (mercury, cadmium, etc.) that get biomagnified through the food chain, arsenic does not possess the property to get biomagni-fied. Arsenate (As5+), a phosphate analogue competing with phosphates for adenosine triphosphate, results in adenosine diphosphate monoarsine forma-tion by substituting arsenic for phosphate. Soon after its formation, it interferes with essential cellular processes such as oxidative phosphorylation by hamper-ing adenosine triphosphate (ATP) synthesis (Jomova et al., 2011). Compared to arsenate, toxicity of arsenite (As3+) is accredited to its propensity to bind sulphydryl groups of dihydrolipoic acid (a pyruvate dehydrogenase cofactor associated with the conversion of pyruvate to acetyl coenzyme A), thereby resulting in the reduction in citric acid cycle activity. In addition to that, bind-ing of arsenite (As3+) to sulphydryl groups is associated with the inhibition of gluconeogenesis and production of glutathione, responsible for protecting cells against oxidative stress (Jomova et al., 2011).

Lead (Pb) is a persistent and common environmental contaminant. Inorganic Pb can be absorbed following inhalation, oral and dermal exposure through the respiratory tract, as well as through the gastrointestinal tract primarily in the duo-denum (ATSDR, 2007). Nutritional status of iron and calcium contributes as a risk factor for Pb intoxication, as their deficiency increases retention of ingested Pb (Ruff et al., 1996). By inhibiting the activities of enzymes involved in haem biosynthesis, particularly δ-aminolevulinic acid dehydratase, it is known to amend the haematological system. Increased circulating aminolevulinic acid, a weak γ-aminobutyric acid (GABA) agonist, decreases GABA release by presyn-aptic inhibition (Hernberg and Nikkanen, 1970; Millar et al., 1970). In blood, Pb is primarily found in red blood cells, where it destabilizes the cellular membranes of red blood cells (RBC). By decreasing the cell membrane fluidity and increas-ing the rate of erythrocyte haemolysis, it leads to the development of anaemia (Needleman, 2004; Bellinger and Bellinger, 2006). Ability of lead to mimic cal-cium or perturb calcium homeostasis makes lead a powerful neurotoxin having diverse impacts on the central nervous system (ATSDR, 2007).


In addition to interfering with cellular functions, metal intoxication also involves generation of reactive oxygen (ROS) and nitrogen species (RNS) that induces a complex series of downstream adaptive and reparative events, driven by oxidative and nitrative stress. ROS includes free radicals such as hydroxyl (OH.), superoxide (O2

. −), peroxyl (RO2.) and alkoxyl (RO.), along with cer-

tain non-radical species such as peroxynitrite (ONOO−), H2O2, etc., which are either oxidizing agents or get easily converted into radicals. It is plausible that an increase in ROS/RNS production due to exogenous stimuli alters cellular func-tions through direct modifications of biomolecules (proteins, nucleic acids and lipids) (Jan et al., 2011). In view of the essentiality of redox homeostasis, induc-tion of oxidative stress due to fluctuations in the defence machinery appears to be a remarkable phenomenon by which metals cause toxicity in living organisms.

MICROBIAL-BASED REMEDIATION

Besides being responsible for redistribution to the aquatic and terrestrial ecosystems, metals from anthropogenic sources contribute more towards a wide range of toxic effects in microbial communities. Their toxic consequences pref-erentially vary with the species available for interactions with the microflora in their natural environment. Despite being toxic, microbes flourish in metal- polluted locations apparently by adapting themselves through a variety of resis-tance mechanisms that are both active as well as incidental, with frequencies ranging from a few per cent in pristine environments to nearly 100 % in heav-ily polluted environments (Gadd and Griffith, 1978; Gadd, 2010). Microbes with their symbiotic associations to each other and higher organisms, contrib-ute actively to ecological phenomena that involve metal transformations hav-ing beneficial or detrimental consequences with respect to the human context (Gadd, 2010). Natural attenuation that, as a whole, depends on natural processes for transformation or immobilization of metal contaminants is believed to be the major process involved in the reduction/transformation of contaminant con-centrations, spurred by the addition of nutrients (biostimulation) or through the addition of bacterial strains with desired catalytic capabilities (bioaugmenta-tion) that enhance the resident microbial population’s ability to transform con-taminants (Rosenberg et al., 1992; Tyagi et al., 2011). However, for deciding whether a particular approach is appropriate for the clean-up of a contaminated site, it is necessary to have knowledge of degradative capabilities of native as well as the organisms that could be added against the particular contaminants so as to enhance their remediation from the site.

Microbes being prime mediators of biochemical cycling are ubiquitous and, as such, are well adapted to exist on substances that are often present in the environment at toxic concentrations. They interact with metals, alter their physical and chemical state by making changes in metal speciation that lead to a decrease or increase in their mobility, so as to enhance their survival in metal-contaminated sites (Gadd, 1992). Among various strategies through which


bacteria resist toxicity of metals, important ones include: (1) toxic metal may get sequestered on cell wall components or by intracellular metal binding pro-teins and peptides such as metallothioneins, phytochelatins, etc.; (2) changing uptake pathways which block uptake of a metal; (3) enzymatic conversion of the metal to a less toxic form and (4) lowering the intracellular concentration of the metal by a specific efflux system or by intracellular compartmentalization (Mowll and Gadd, 1984; White and Gadd, 1998; Gadd, 2004).

As microorganisms inhabiting native habitats evolved towards ecological fit-ness rather than to biotechnological competence, cleaning contaminated sites by bacteria through the process of natural selection is a challenging task. In order to broaden the horizon of capacity for biological systems towards creation of supe-rior microbial chemical factories, it seems necessary to go beyond the genetic confines of microorganisms for the assembly of biosynthetic pathways for both natural and unnatural compounds so as to exploit them for bioremediation pur-poses (Doong et al., 1998; Mohan and Pittman, 2007; Chauhan and Jain, 2010; Patel et al., 2010). Recent developments in the genetic engineering approach that have begun to uncover the endogenous detoxification system in microbes, along with the ways to genetically engineer microbes, can be employed in mak-ing inroads successfully into this environmental clean-up technology.

ENHANCING BIOREMEDIATION THROUGH GENETIC ENGINEERING

Considerable interest in exploring metal resistance mechanisms in bacteria serves to highlight the credentials that are particularly advantageous for their practical application in dealing with unfavourable metal burdens in nature. As the efficacy of a desired in situ catalytic activity (biodegradation) depends on the presence of microflora possessing desired catalytic activity at the target site, it is equally manifested that one key enzyme may or may not be present and, if present, might be harboured by only a small proportion of the microbial popula-tion. Therefore, with the commencement of the genetic era, a better understand-ing of microorganisms’ natural transformation abilities at the genetic level has accelerated the progress to genetically engineer microbes so as to achieve the factual expression of genes that are necessary for the detoxification of toxic pollutants. Natural processes that involve mutations, DNA shuffling, horizontal gene transfers, etc. have a significant contribution in the formation of hybrid genes and metabolic operons (Van der Meer, 1997). However, compared to nat-ural attenuation, engineered bioremediation relies on accelerating the growth of microorganisms as well as on optimizing the environment in which they carry out the detoxification reactions. Using the genetic engineering approach, bac-terial surface display of metal-binding peptides and proteins to remove metal pollutants from water and waste water had a significant impact in making it an increasingly active research area with a wide range of biotechnological and industrial applications.


Surface Expression of Novel Metal Binding Peptides and Proteins

Besides offering higher metal-binding capacity and/or specificity and selectivity for different metal ions, surface expression of novel metal binding peptides and proteins has enabled rapid binding and, as such, their immobilization for improved metal resistance of growing cultures (Pazirandeh et al., 1998). Sur-face exposure of metal binding peptides and proteins is fortuitous and relatively efficient as it improves metal binding properties of microorganisms that are employed in various systems for their removal from the environment. Depend-ing on the degrading capability of the indigenous microbes and the extent of contamination of the site to be treated, the decision to implement either or both of these techniques for bioremediation seems essential.

From the previous figures, it is quite apparent that compared to fast grow-ers that result in a large heap of unwanted biomass, an optimal clean-up agent for use in bioremediation would be one that displays maximum catalytic ability with a minimum cell mass. It has readily been found that unwelcome biomass produced by fast growers provides a palatable niche for protozoa to flourish which prevents normal microflora from growing beyond a certain level (Iwasaki et al., 1993). One major issue pertaining to bacterial species being propagated as monoculture under optimized controlled environment for use in the bioremedia-tion process is that they may not be the ones performing the bulk of biodegra-dation in natural niches. To be exploited in the process of bioremediation, it is necessary that they might establish themselves, interact with other bacteria in unknown ways as they are expected to be hindered in their activities by exposure to a multitude of poorly controllable external factors, some of which place them under considerable stress (Timmis and Pieper, 1999; Sayler and Ripp, 2000). Knowing that genetically modified organisms (GMOs) are generally exposed to unfavourable conditions that often place them under stress, conceptualization to genetically engineer bacteria for characteristics such as stability without selec-tion, minimal physiological burden, small size, non-antibiotic selection markers, minimal lateral transfer of cloned genes to indigenous organisms and traceabil-ity of specific genes seems necessary so as to enhance their exploitation for use in bioremediation in order to meet the growing demands for their eventual applications in the field.

PHYTOREMEDIATION

Despite the fact that whether the introduced strains are fast growers or slow growers, the introduction of GMOs, particularly those which are raised in labo-ratory conditions with modified metabolic machinery at the start point, faces problems in colonization, niche specificity and poor adaptation of its tran-scriptional machinery. Whether fast growers or slow growers, introduction of foreign microbes, no matter whether they are recombinant or not, into an unfa-miliar territory in itself poses a threat of ecological imbalance (Lindow et al., 1989). Keeping in view the difficulties that might arise on introducing GMOs,


expression of bacterial genes in plant carriers seems a quite reasonable approach that is believed to open a new perspective as a promising alternative aimed at environmental clean-up. The use of genetically modified plants generated by ectopic expression of bacterial genes to achieve remediation of polluted sites has several advantages over microbial-based remediation: spontaneous deliv-ery of catalysts well below the soil surface through roots and methods for the extensive agricultural dispersion of seeds are already optimized and importantly chances for the horizontal transfer of genes seem less probable.

Owing to its ‘green’ approach, exploiting plants to remediate environmentally toxic compounds is expanding its dimensions by gaining a significant amount of public attention. Plants possessing traits such as being fast growing, having a deep root system, having a high biomass, being easy to harvest, tolerant and having the ability to accumulate a wide range of heavy metals in its aerial and harvestable parts have commonly been exploited for use in phytoremediation. However, to date, no plant has been found that fulfils all the above criteria as most hyperaccumulator plants have slow growth rates and are low biomass producers; whereas, plants that show rapid growth have been found to be sensitive to metals and as such are known to accumulate only low concentrations in their shoots. Researchers have found that plants that generally grow in areas commonly con-taminated with metal pollutants, belonging to Brassicaceae and Fabaceae fami-lies, possess the characteristics of hyperaccumulators (Gleba et al., 1999). To date, 400 hyperaccumulator plant species possessing the characteristics of hyper-accumulators from 22 families have been identified, with the Brassicaceae family alone containing 87 hyperaccumulating species with the widest range of metal accumulation properties from 11 genera (Baker and Brooks, 1989).

Based on the method by which remediation of metal contaminants is achieved, phytoremediation has been broadly categorized into the following areas:

Phytoextraction: use of metal accumulators to remove metals upon concen-trating them in the harvestable parts from the soil.Rhizoremediation: use of microorganisms that are associated with plants to eliminate the contaminant from the soil.Phytostabilization: reducing the environmental burden of pollutants by stabi-lizing them through the use of plants.Phytovolatilization: conversion of toxic pollutants to innocuous products by employing genetic engineering of genes from microbial origin.

Phytoextraction

Over the last few decades, remediation of sites contaminated with metal pollutants by plants has gained much attention over the microbial-based remediation strat-egies (Kramer, 2005; Pilon-Smits, 2005; Doty, 2008). Although not exclusive, various phytoremediation strategies can be employed simultaneously in order to achieve remediation against the particular pollutant at a given time. However, for


any remediation strategy to be effective in eliminating the toxic pollutant, basic criteria settles on the type and form of the contaminant present at that site. Despite the fact that soluble fractions of various pollutants are generally present as ions or unionized organometallic complexes, heavy metals also exist in colloidal, ionic, particulate and dissolved phases, with high affinity for humic acids, organic clays and oxides coated with organic matter (Connell and Miller, 1984; Elliot et al., 1986). Preferentially, their solubility in the environment (soil and groundwater) is controlled by pH, cation exchange capacity, oxidation state and the amount of metal present at that site along with the redox potential of the system (Connell and Miller, 1984; Elliot et al., 1986; Baker and Walker, 1990; McNeil and Waring, 1992; Martinez and Motto, 2000). In the rhizospheric region, bioavailability of metals is affected up to a greater extent by various plant and/or microbial activi-ties that affect their uptake by plants via altering their mobility and bioavailability (Ma et al., 2011; Miransari, 2011; Aafi et al., 2012; Yang et al., 2012). Presence of lipophilic compounds in plant exudates or lysates has an effect either in increas-ing their solubility in water or in promoting growth of biosurfactant-producing microbial populations. Owing to the beneficial roles that they are known to play in metal mobilization, use of beneficial microbes possesses several advantages over chemical amendments such as the fact that metabolites produced by microbes are biodegradable, less toxic and can be produced in situ at rhizosphere soils. Furthermore, their bioavailability can be enhanced by chelators such as sidero-phores, organic acids and phenolics of plant and microbial origin associated with the release of metal cations from soil particles followed by its uptake by plants. Besides contributing to plant growth, microbial processes and/or activities in the rhizosphere soils enhance the effectiveness of phytoremediation either by: (1) enhancing metal translocation (facilitate phytoextraction) or by reducing metal bioavailability in the rhizosphere (phytostabilization) and (2) by indirect promo-tion of phytoremediation achieved either by conferring resistance to plants and/or enhancing their biomass production so as to achieve remediation of pollutants up to a greater extent (Glick, 2010; Kuffner et al., 2010; Rajkumar et al., 2010; Babu and Reddy, 2011).

Despite being permissive, a metal after mobilization into soil solution has to be captured by the cells of the root. Following adherence, transport of metal ions is guided by transporters, particularly channel proteins and/or by H+-coupled carrier proteins that are embedded in the membrane. On getting inside, metals undergo precipitation to carbonate, sulphate or phosphate precipitates for their immobilization through extracellular (apoplastic) and intracellular (symplas-tic) compartments (Raskin et al., 1997). Flow of substances through the apo-plastic pathway is relatively unregulated as substances flow without crossing membranes. However, subsequent to sequestration inside root cells, symplastic transport of ions involves movement into the stele followed by release into the xylem on crossing the casparian strip. On gaining entry into the root, metals are either stored in the root or translocated to the shoots. Metal ions are trans-ported actively as free ions or as metal–chelate complexes across the tonoplast


(Cataldo and Wildung, 1978). Being important with respect to metal ion stor-age, metals are often chelated either by organic acid or phytochelatins. Inside the vacuole, formation of insoluble precipitates followed by their compartmen-talization occurs as a mechanism of resistance against the damaging effect of metals (Cunningham et al., 1995).

Among the various approaches that are employed to achieve phytoreme-diation, phytoextraction of metals and metalloids is considered to be the most challenging due to the high quantity of pollutants present. Phytoextraction is broadly categorized into: chelate-assisted phytoextraction (induced phytoex-traction) and long-term phytoextraction (continuous phytoextraction), of which chelate-assisted phytoextraction is more acceptable and presently being imple-mented commercially.

Chelate-Assisted PhytoextractionChelate-assisted phytoextraction involves the addictive use of metal chelating agents in combination with non-accumulator plants, bearing far less ability to accumulate metal than hyperaccumulators, but with high biomass potential so as to increase the soluble metal fraction. It involves the additive function of two basic processes: transport of metals to the harvestable shoot upon release into soil solution. It has been observed that use of fast-growing tree species fulfils the need for high biomass production aided by chemicals in order to achieve high metal accumulation. Chelates such as ethylenediaminetetraacetic acid (EDTA) are accredited with the function to increase the formation of soluble metal-EDTA within the plants that allows their movement from root system towards shoot, where actual accumulation of metals occurs as metal–EDTA complex. Transport of metal–chelate complexes that occur through xylem to shoots appears to play an important role in the accumulation of chelate-assisted metal complexes in plants (Blaylock et al., 1997; Huang et al., 1997). Inside the plants, movement of metal–chelate complex, besides relying on efficient capil-lary plumbing system, relies on the high-surface-area collection system pro-vided by the roots. Movement of metals as a metal–chelate complex to shoots is followed by the retention of metal–chelate complex as water evaporates via the transpiration stream.

In contrast to soils that possess only a trace amount of various elements, metalliferous soils containing high concentrations of elements have been found to vary widely in their effects on different plant species. The total amount of metal removed from a site is generally calculated as a product concentration of metals in harvested plant material to total harvested biomass. Despite the fact that certain plant species posses an inherent capacity to sequester high concentrations of metals in the above-ground tissues, addition of chelates have been found to have a profound effect in enhancing metal accumulation up to a greater extent. Studies carried by Huang et al. (1997) and Blaylock et al. (1997) reported rapid accumulation of lead (> 1% of shoot dry bio-mass) in the shoots. Based on the observation that high biomass crops (Indian


mustard, corn and sunflower) could be ‘induced’ to accumulate high concen-trations of lead, it is generally apprehended that these strategies would lead the way in designing strategies that can be employed to achieve phytoextrac-tion of other toxic metals using appropriate chelates (Huang and Cunningham, 1996; Blaylock et al., 1997; Huang et al., 1997). Meers et al. (2007) reported that the use of ethylene diamine disuccinate, a naturally occurring chelator with a high degree of biodegradability, on soil sediments contaminated with Zn and Cd, enhances metal uptake to a significant extent by willows grown on the contaminated soils (Meers et al., 2007). On comparing the effects of different chelates, it was observed that metal accumulation efficiency is directly related to the affinity of chelate that is used to achieve the decontami-nation of available metal. From the results of various studies, it is concluded that for efficient phytoextraction, chelates possessing a high affinity for the metal of interest should be used, such as EDTA for lead, ethylene glycol tet-raacetic acid for cadmium and citrate for uranium (Blaylock et al., 1997). Following harvest, metal-enriched plant residue can either be disposed off as hazardous material or, if economically feasible, used for metal recovery.

Continuous PhytoextractionMetal chelate uptake in plants is correlated with stress, which is sometimes followed by plant death. However, it is not clear whether stress is necessary for induction or whether it simply reflects the accumulation of high concentra-tions of synthetic chelate in the plant. Despite the beneficial effect in terms of increasing the efficiency of phytoextraction, it has been observed that chemical amendments (e.g. EDTA) are not only phytotoxic, but also exert their toxic consequences on beneficial soil microorganisms known to play important roles in plant growth and development (Ultra et al., 2005; Evangelou et al., 2007; Muhlbachova, 2009). In an alternative approach, accumulation of metals can be achieved based on the specialized physiological processes occurring over the complete growth cycle in plants. Unlike induced metal uptake, continuous phy-toextraction is achieved under the genetic and physiological capacity of plants specialized to accumulate, translocate and resist high amounts of metals. There are reports where the removal of metals such as zinc, cadmium and nickel along with arsenic and chromium by hyperaccumulating plants that grow on soils rich in heavy metals has been achieved by continuous phytoextraction (Baker and Brooks, 1989). Dissecting the mechanism that is employed to achieve hyperac-cumulation of metals will help in understanding the biological mechanisms by which plants become superior for the remediation of soils. As a type of manage-ment strategy, phytoextraction for metal contaminated sediment is thought to address efficiency, duration, leaching, food chain contamination and cost ben-efits. However, its application is limited by low biomass production and insuf-ficiently high metal uptake into plant tissue along with the lack of knowledge regarding minimum amendments that are required (e.g. compost, irrigation) to support long-term plant establishment.


Rhizoremediation

As an alternative strategy, degradation of contaminants that is achieved by microorganisms inhabiting the rhizosphere holds great potential for the reme-diation of contaminated soil (Kuiper et al., 2004). As an essentiality for rhi-zoremediation, the association commonly referred to as the ‘rhizosphere effect’ shows great potential where nutrients in the form of root exudates, oxygen and favourable redox conditions to soil microorganisms provided by plants results in increased bacterial diversity, population density and activity compared with bulk soil (Molina et al., 2000).

RhizofiltrationRhizofiltration that involves the use of plants (both terrestrial and aquatic) to absorb, concentrate and precipitate contaminants from polluted aqueous sources with low contaminant concentration in their roots finds its application in dealing with effluents discharged from industries and agricultural run-off as well as acid mine drainage. It is more commonly used for lead (Pb), cadmium (Cd), copper (Cu), nickel (Ni), zinc (Zn) and chromium (Cr), which are primarily retained within the roots (Chaudhry et al., 1998). As a cost-effective technology, it has been used for the treatment of surface or groundwater containing significant proportions of heavy metals such as Cr, Pb and Zn (Kumar et al., 1995; Ensley, 2000). Its commercialization is driven by its technical advantages in its applica-bility to many metals, besides possessing the ability to treat high volumes with less need for toxic chemicals, reduced volume of secondary waste and the pos-sibility of recycling (Dushenkov et al., 1995; Kumar et al., 1995). Being advan-tageous in its ability for use in in situ as well as ex situ applications, studies performed on plants such as sunflower, Indian mustard, tobacco, rye, spinach and corn have highlighted its application in the removal of lead from effluent, with sunflower having the greatest ability.

RhizodegradationAmong the various strategies that are employed to achieve remediation of pol-luted sites, rhizodegradation involves use of plants to stimulate the microbial community near the root–soil interface, in order to enhance the degradation of recalcitrant compounds in the soil. In this type of association, plants are known for their contribution in making contaminants of the soil more bioavailable through release of a series of low molecular weight organic acids, carbon and nitrogen compounds to nourish microbes in the rhizosphere and by releasing exudates that enhance degradation of soil contaminants through induction of biochemical pathways within bacteria (Leigh et al., 2002; White et al., 2003). Kuiper and colleagues (2004) described a two-step enrichment approach where bacteria after harvesting from the roots of plants growing in contaminated sites are enhanced for their biodegradation potential and are then made to re-colonize plant roots.


Successful rhizosphere colonization depends not only on interactions between the plants and the microorganisms of interest but also on interactions with other rhizospheric microorganisms and the environment. In the past two decades, several techniques such as visualization of bacteria that carry the luxAB genes encoding bacterial luciferase, the green fluorescent protein and/or another reporter gene along with in situ hybridization assays using fluorescent probes have been devised to track colonization of roots by bacteria (Broek et al., 1998; Tombolini et al., 1999; Ramos et al., 2000; Sharma et al., 2013). These techniques have been found to be highly advantageous in illustrating the intro-duced microorganisms that are often unable to compete with indigenous micro-organisms or are unable to establish in high numbers at the rhizosphere region (Rattray et al., 1995; Lubeck et al., 2000). In a major boost to this strategy, some bacteria have emerged with the strategies to out-compete other microorganisms by delivering toxins, using extremely efficient nutrient utilization systems or by physical exclusion (Lugtenberg et al., 1991).

Phytostabilization

At times, when there seems no immediate effort to clean up sites polluted by met-als as they are not of high priority on a remediation agenda, traditional means such as in-place inactivation, a remediation technique that employs use of soil amend-ments to immobilize or fix metals, are employed to reduce metal toxicity (Berti and Cunningham, 2000). Phytostabilization (also known as phytorestoration) is a plant-based remediation technique that is aimed at reducing the risk of metal pollutants by stabilizing them through formation of a vegetative cap at the plant rhizosphere, where sequestration (binding and sorption) processes immobilize metals so as to make them unavailable for livestock, wildlife and human exposure (Munshower, 1994; Cunningham et al., 1995; Wong, 2003). Unlike other phy-toremediative techniques, the goal of phytostabilization is not to remove metal contaminants from a site, but rather to stabilize them and reduce the risk to human health and the environment. Phosphate fertilizers, organic matter, iron or manga-nese oxyhydroxides, natural or artificial clay minerals being the most prominent soil amendments fulfil the criteria of fixing metals rapidly following incorpora-tion and/or making chemical alterations that are very long lasting, if not perma-nent. Besides being poor translocators of metal contaminants to above-ground plant tissues that could be consumed by humans or animals, characteristics of plants that are employed for phytostabilization include easy to establish and care for, quickly grown, produce dense canopies and root systems and be tolerant of metal contaminants and other site conditions. In being less expensive, less envi-ronmentally evasive and easy to implement, phytostabilization is considered to be more advantageous than other soil-remediation practices (Berti and Cunningham, 2000). For more heavily contaminated soils, phytostabilization aims at stabilizing the contaminated sites with tolerant plants in order to reduce the risk of erosion and leaching of these pollutants to water bodies.


PhytovolatilizationPhytovolatilization is aimed at transforming organic and inorganic contaminants that are taken along with water to volatile gaseous species within the plant fol-lowed by their eventual release into the atmosphere at comparatively low concen-trations (Mueller et al., 1999). To date, the phytovolatilization approach has been used primarily for the removal of mercury, where Hg2+ is transformed into less toxic Hg0. Recent efforts to achieve remediation of mercury were aimed at insert-ing bacterial Hg ion reductase genes into plants such as Arabidopsis thaliana L. and tobacco (Nicotiana tabacum L.) so as to achieve phytovolatilization of mercury to a greater extent (Rugh et al., 1996; Heaton et al., 1998; Rugh et al., 1998; Bizily et al., 1999). Although this remediation approach has added benefits of minimal site disturbance, less erosion and no need to dispose of contaminated plant material, it is still considered as the most controversial of all phytoremedia-tion technologies as release of mercury into the environment is likely to be recy-cled by precipitation and then redeposit back into the ecosystem (Henry, 2000).

Insights into Genetic Engineering Approaches for Phytoremediation

For any plant to be employed in the process of phytoremediation, it needs to fulfil the basic criteria of being tolerant to various metal pollutants. Since the beginning of the genetic era, there is now ample data available regarding the expression of various genes that are believed to impart resistance through vari-ous mechanisms of plants and, more particularly, bacteria. In order to enhance the metal accumulation property of plants, current remediation strategies are particularly aimed at enhancing the metal accumulation property following its transport from the soil to the roots and translocation to shoots in plants. For any particular strategy to be effective, it is necessary to have a thorough understand-ing of the mechanisms that are operating within the hyperaccumulators that are employed in acquisition and translocation, followed by accumulation so as to achieve remediation of polluted sites. Feasibility in employing any strategy to genetically engineer a rapidly growing non-accumulator in order to enhance it for the property that hyperaccumulators generally harbour requires quite good understanding of the biological processes that are operating within the hyperac-cumulators. Unfortunately, apart from the mechanisms of tolerance, these bio-logical processes are not well understood. For phytoremediation to succeed, current research needs to be targeted at improving existing lines of phytoex-tracting plants for high metal acquisition, high-biomass and hyperaccumulation both in hyperaccumulating as well as in nonaccumulating plants.

Metal Sequestering Proteins and PeptidesFollowing mobilization, metal uptake and its accumulation in plants is affected by concentration and affinities of chelating molecules as well as by the pres-ence and selectivity of transport activities. For enhancing metal uptake and, as


such, its accumulation, three approaches to genetically engineer plants have been envisioned: increasing the number of uptake sites, altering the specific-ity of uptake systems so as to reduce the competition by unwanted cations and increasing the number of intracellular high-affinity binding sites so as to enhance the sequestering capacity of the plants. Among them, increasing the number of intracellular binding sites drives the passage of transition metal ions across the plasma membrane followed by its sequestration that enhances accumulation. Metallothioneins (MT) are cysteine-rich, low-molecular-weight metal-binding proteins endowed with a wide range of functional capabilities in a biosystem (Hamer, 1986; Sousa et al., 1998). They possess high metal content (predominantly Zn, Cu or Cd), bound by sulphur atoms in thiolate clusters, besides having highly conserved cysteine residues (18–23) that bind metal ions and sequester them in the biologically inactive form (Vasak, 2005; Bell and Vallee, 2009). Since establishing its role in metal detoxification, plant metallo-thioneins are grouped into four subfamilies, MT1, MT2, MT3, MT4, with dif-ferent expressions for fulfilling different functions during development in plant tissues (Cobbett and Goldsbrough, 2002). It was observed that expression of MT1a and MT1b in roots of non-accumulator plants, like A. thaliana and pop-lar were high during exposure to Cd, Cu and Zn (Garcia-Hernandez et al., 1988; Zhou and Goldsbrough, 1994; Kohler et al., 2004), while as in Thlaspi caerule-scens, levels of expression of MT1 mRNA were constitutively higher in leaves than in roots (Roosens et al., 2005). Although MT2b is not considered as the major player in the process of metal tolerance, increased expression of MT2b was found to be associated with Cu tolerance and increased As translocation from root to shoot in A. thaliana (Schat et al., 1996; Grispen et al., 2009). In non-accumulator plants, expression of MT3 genes increases on exposure to Cu and as such is known to play an important role in the maintenance of Cu homeo-stasis under conditions of high Cd and Zn in the cytoplasm (Guo et al., 2003; Kohler et al., 2004; Roosens et al., 2004). This suggestion is supported by the discovery of a small cavity that is adapted for Cd chelation in the MT3 protein by A. thaliana as compared with the analogous protein from T. caerulescens (Roosens et al., 2004, 2005). In A. thaliana, it is reported that MT4 plays an important role in metal homeostasis during seed development and seed germi-nation rather than in metal decontamination (Guo et al., 2003).

Research performed on microorganisms has resulted in providing valuable models to understand and engineer plants for tolerance to metal as the number of potential gene targets in microorganisms is much higher and more diverse than in plants (Silver and Misra, 1988). Among the various mechanisms that are involved in detoxification and transformation of metals, engineering plants for proteins and peptides that possess the potential to efficiently immobilize metals has emerged as a technique that is useful for their removal from the environment (Beveridge and Murray, 1980). Transgenic plants expressing metallothionein genes usually exhibit an increased metal tolerance as was reported in trans-genic tobacco expressing the MT gene from Silene vulgaris (Zimeri et al., 2005;


Zhigang et al., 2006; Gorinova et al., 2007). In another study, expression of MT1 from Brassica rapa L. in the chloroplasts of A. thaliana showed increased tolerance to Cd and to oxidative stress (Kim et al., 2007). These observations were complementary to observations in A. thaliana mutants lacking MT1a and MT2b; the double mutant had normal Cu tolerance but Cu accumulation in roots was reduced by 30% (Guo et al., 2008). As overexpression of MTs result in enhanced metal accumulation, it offers a promising strategy to achieve remedia-tion of metals at contaminated sites.

Similarly, Histidine (His), a free amino acid present at high concentration in the roots of hyperaccumulators, forms stable complexes with Ni, Zn and Cd (Kramer et al., 1996; Haydon and Cobbett, 2007). Compared to non-accumulator Alyssum montanum, hyperaccumulators such as Alyssum lesbiacum show a larger pool of His available for chelation in roots. In the hyperaccumulator A. lesbiacum, concentration of His is increased by Ni treatment without inducing the expression of the genes involved in the biosynthetic pathway (Persans et al., 1999; Ingle et al., 2005). Overexpression of ATP-phosphoribosyltransferase, the first enzyme of the His biosynthetic pathway in transgenic A. thaliana plants, increases toler-ance but not the accumulation (Ingle et al., 2005).

Engineering for Improved Enzyme ActivitiesOne major challenge to overcome the major hurdles associated with phytoreme-diation is to construct genetically modified plants that, besides possessing the ability to tolerate high shoot metal concentrations, should produce large biomass in order to keep the required number of cropping cycles to a minimum. In addition to the enhancement of the metal accumulation properties via genetic engineer-ing, a thorough understanding of the biological processes that are involved in metal transformation after acquisition from soil seems equally essential. As metal tolerance is a key prerequisite for phytoremediation, the cellular mechanism for/behind metal tolerance that involves the detoxification of metal ions, via chelation or transformation to a less toxic or easier to handle form followed by compart-mentalization, is apprehended as essential to achieve the desired fate. It is gener-ally estimated that microorganisms that act as valuable models for understanding and engineering metal tolerance in higher plants can provide valuable information regarding the number of known potential gene targets that can be engineered into plants to achieve decontamination of the target site (Silver and Misra, 1988). With the exception of mammalian metallothionein and phytochelatin synthase, plants have broadly been engineered with genes of microbial origin for their tolerance via accumulation and decontamination against the wide range of contaminants (Mejare and Bulow, 2001). In view of the possibility to increase the remedia-tion potential of plants, research carried out by Meagher’s group has demon-strated enhanced mercury (Hg) tolerance and phytovolatilization in transgenic plants expressing E. coli mercuric ion reductase (merA) and organomercury lyase (merB) genes (Meagher and Heaton, 2005). In the last decade, more progress as a step towards enhancement of remediation potential includes transformation of


aquatic plants for Hg phytovolatilization in wetlands, transformation in the fast-growing tree eastern cottonwood (Populus deltoides) as well as in the chloroplast genome of tobacco, with both merA and merB genes (Ruiz et al., 2003; Czako et al., 2006; Lyyra et al., 2007). Chloroplast transformation is much preferred over the nuclear transformation as it prevents escape of transgene via pollen to related species, high levels of foreign gene expression and engineering multiple genes or pathways in a single transformation event.

CONCLUSIONS

The metals being toxic at higher concentrations posses the ability to replace essential metals in pigments or enzymes, thereby disrupting their function, in addition to oxidative stress via the formation of free radicals. An increase in the concentration of metal pollutants is on the rise as the paradigm shifts more towards industrialization. Rapid industrialization with faulty waste dis-posal, increased anthropogenic activities and modern agricultural practices have resulted in an increase in the pollution of the environment. Recognition of the ecological and human health hazards has necessitated the urge for management and remediation of contaminated sediments so as to minimize ecological risks and risks associated with food chain contamination. Compared to the conven-tional treatment methods, the employment of various phytoremediation strate-gies to clean up soil and water pollution is gaining a lot of importance because of its cost effectiveness and high efficiency of detoxification and because it is environmentally friendly. On the contrary to microbial-based remediation strat-egies, plant-based remediation technology appears to be a promising alternative in its applicability to address the challenges in terms of minimum requirements for an external energy source for growth, in possessing an extensive root system that reaches every crevice and covers a large area in the soil, where it functions as a pump driven by solar energy to extract and concentrate elements from the soil. Besides that, this technology also offers the flexibility for developing non-destructive desorption techniques for biomass regeneration and/or quantitative metal recovery. Despite being a promising alternative in the management and remediation of contaminated sediments, phytoremediation still requires fur-ther development and optimization in order to improve traits that will lead to improvement in its applicability in the restoration of polluted sites.

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soil remediation and plants || phytoremediation

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