Prospects of Field Crops for Phytoremediation of Contaminants
Poonam, Renu Bhardwaj, Resham Sharma, Neha Handa, Harpreet Kaur, Ravdeep Kaur, Geetika Sirhindi and A.K. Thukral
Anthropogenic activities have led to increased pollution of soil all over the world. These pollutants can be either organic (e.g., PCBs, PAHs, fertilizers, pesticides) or inorganic pollutants including various heavy metals (e.g., Cd, Cu, As, Zn, Hg, Pb). Phytoremediation is a green technology in which plants are used to clean up pollutants from water and soil. This environmentally friendly and cost-effective technology is now focusing on higher plants with large biomass that have a high tolerance to pollutants. Due to low shoot and root growth of hyperaccumulator plants, phytoremediation study has moved toward the high biomass species such as herbaceous field crops. Field crops may have low metal concentrations, but they compensate this with their high biomass yield. Various amendments, such as use of chelating agents, plant growth-promoting bacteria, plant growth-promoting hormones, and mycorrhizae, can be used to increase the phytoremediation potential of field crops. Molecular techniques used to produce transgenic plants also show promise for the efficient use of field crops for phytoremediation. Thus, due to the higher growth potential of field crops compared to hyperaccumulators, phytoremediation efficiency should be thought of as a future significant remediation tool.
Keywords
phytoremediation; contaminants; hyperaccumulators; field crops; stress
19.1 Introduction
Soil is being degraded as a result of industrial, agricultural, and civil activities worldwide, and there is no way to avoid exposure of it to toxic chemicals and metals. Soil contamination, whether diffused or localized, causes loss of various soil functions and also leads to contamination of surface and groundwater. Diffusion from the atmosphere, flowing water, and eroded soil itself are the main sources of soil pollution. Other than this, the application of pesticides and fertilizers, sewage sludge, dust from smelters, industrial waste, and inefficient watering practices of agricultural lands causes contamination (Schwartz et al., 2001; Passariello et al., 2002).
Brown fields, a term given to contaminated soils (French et al., 2006), are mostly associated with abandoned industrial plants, accidental release of pollutants, and inappropriate municipal and industrial waste disposal sites. Risk of contamination from the mining industry, which is a foremost cause of land degradation, is associated with sulfur and metal-bearing tailing sites and with the use of chemicals required in the refining processes (EEA, 2003).
There are various organic contaminants, primarily petroleum hydrocarbons, aromatic hydrocarbons, polynuclear aromatic hydrocarbons (PAHs), nitroaromatic compounds (NACs) and chloroaromatics, and so on, and inorganic pollutants including heavy metals such as cadmium (Cd), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb), zinc (Zn), and arsenic (As). According to a 2003 European Economic Area (EEA) report, metal accounts for more than 37% of the contamination, followed by mineral oil (33.7%), PAHs (13.3%), and others.
The term “soil remediation” means returning the soil to a form of ecological stability together with the establishment of the plant communities it supported prior to disturbance (Allen, 1988). Conventional technologies of soil remediation include soil washing with chemicals and addition of lime, phosphate, calcium carbonate, and more as needed (Ebbs et al., 1998; Krebs et al., 1999; Chen et al., 2000); however, most of them are not ecofriendly and sustainable.
Phytoremediation is an integrated multidisciplinary approach for the cleaning of contaminated soils (Cunningham and Ow, 1996; Maurice and Lagerkvist, 2000). It is a group of technologies that uses plants to reduce, remove, degrade, or immobilize environmental pollutants, and its aim is to restore polluted sites so that they are usable. In the past, this technique has been applied to various pollutants in small-scale, laboratory studies. The pollutants treated include heavy metals, chlorinated solvents, polychlorinated benzenes (PCBs), PAHs, organophosphate insecticides, radionuclides, explosives, and surfactants (Khan et al., 2004).
Some plant species can accumulate higher concentrations of pollutants without showing toxicity (Klassen et al., 2000; Bennet et al., 2003); such plants must possess few properties, excrete a large concentration of pollutants into roots, translocate pollutants into surface biomass, and produce a significant quantity of plant biomass. Thus, plants with these properties give promising results in ecofriendly clean-up strategies. Field crops are economically grown plants and include food crops (e.g., rice, wheat, common bean, maize), oil crops (e.g., sunflower, mustard, rapeseed, groundnut), and bioenergy and fodder crops. Field crops in this context are very valuable as they have a short life cycle and produce a large amount of biomass, thus, compensating for their low uptake with these properties. Therefore, present-day research focuses on identifying plant species with higher potential for phytoremediation as well as developing new methods of phytoremediation.
19.2 Contaminants in soil, water, and plants
Heavy metals and metalloids are the major contaminants that accumulate in soil through emissions from industrial areas, disposal of metal wastes, mine tailings, animal manures, pesticides, sewage sludge, coal combustion residues, atmospheric deposition, wastewater irrigation, and spillage of petrochemicals (Khan et al., 2008; Zhang et al., 2010). The heavy metals found mostly at contaminated sites include Zn, Cu, Cr, Pb, As, Cd, Ni, and Hg. Metals unlike organic contaminants do not undergo degradation by microbes and chemicals (Kirpichtchikova et al., 2006). After introduction into soil, their total concentration persists for a long time (Adriano, 2003). Contamination of soil by heavy metals poses a threat to the ecosystem and humans through the food chain, ingestion, or contact with soil, drinking of groundwater, land tenure problems, and food insecurity due to reduction in usable land for agricultural production (McLaughlin et al., 2000a,b; Ling et al., 2007).
A variety of approaches can be used for the remediation of contaminated soil. The technologies have been broadly classified by the US Environmental Protection Agency (EPA) into two categories: (1) containment remedies and (2) source control (Maslin and Maier, 2000; McLaughlin et al., 2000a,b). Containment remedies involve the construction of caps, liners, and vertically engineered barriers (VEB) for the prevention of contaminant migration. Source control includes ex situ and in situ treatment technologies. Ex situ treatment technologies involve the removal or excavation of contaminated soil from the site, whereas in in situ treatment technologies there is no need to excavate contaminated soil; it is treated at its original site.
The selection of any remediation technology depends on a number of factors, according to Wuana and Okieimen (2011), including: (1) long-term effectiveness, (2) cost, (3) general acceptance, (4) commercial availability, (5) applicability to mixed wastes/organics and heavy metals, (6) applicability to high metal concentrations, (7) volume reduction, (8) toxicity reduction, and (9) mobility reduction. Reliable methods to detect environmental pollutants, their dynamics and fate, are required to evaluate their impact on soil quality and living organisms. Techniques used to monitor volatile and semivolatile pollutants in soil include physicochemical techniques such as solid phase micro-extraction (SPME), followed by analysis by GC-MS, use of bioindicators, and use of sensing technology (e.g., electronic nose) (Cesare and Macagnano, 2013).
The increase in human population has raised the quantity of waste and introduced many different types of pollutants into water bodies; these were not considered pollutants earlier but are now seen as harmful to the environment and public health. The pollutants include pharmaceuticals, toxins, hormones, viruses, and endocrine-disrupting chemicals (Xagoraraki and Kuo, 2008). Heavy metals (e.g., Cd, Pb, Mn, Fe, Zn, Cu) are also major contaminants of water (Opaluwa et al., 2012). Human activity is the major source of most of the water pollutants, whereas some amount of them are added by natural activities such as volcanic eruptions.
The main anthropogenic activities that cause water pollution include agricultural waste, livestock waste, industrial chemical waste, pesticides, fertilizers, mine drainage, untreated municipal sewage, spillage of petroleum products, spent solvents, and so forth. Once pollutants are discharged into any of the surface water bodies or the groundwater, they enter the water cycle. Pollutants may also undergo physical, biological, and chemical transformations (Xagoraraki and Kuo, 2008). The contaminants in water bodies, such as heavy metals, are also bioaccumulated in the flora and fauna of that region and so enter into the food chain. The contaminated water, whether used for drinking, irrigation, or other purposes, may lead to many health issues. For example, chemical pollutants can damage functional systems (e.g., immune system and nervous system) and major organs (e.g., kidney and liver), and pathogenic microorganisms in the water lead to gastrointestinal problems.
Increased cancer risk is also a major threat posed by enhanced concentrations of pollutants in drinking water (Xagoraraki and Kuo, 2008). An atomic absorption spectrophotometer (AAS) is used to assess the presence and amount of heavy metals in polluted water (Opaluwa et al., 2012). Adsorbents, such as activated carbon, can be used to remove heavy metals from contaminated water; however, it is an expensive material. So, instead of using commercial activated carbon, researchers used materials (e.g., sawdust, chitosan, mango leaves, coconut shell) that were inexpensive, had a high-adsorption capacity, and were locally available (Renge et al., 2012).
Contaminants enter plants when they are grown in soil that has various types of them, such as heavy metals, or when irrigated with polluted water containing contaminants. The plants show growth reduction, altered metabolism, metal accumulation, and lower biomass production (Nagajyoti et al., 2010). Some metals (e.g., Mn, Cu, Zn, Co, and Cr) are important for plant metabolism in trace amounts. When these metals are present in bioavailable forms and in excess, they become toxic to plants. Few heavy metals are very toxic to metal-sensitive plants, so result in growth inhibition and may also cause death of the organisms. The uptake of heavy metals does not show a linear increase with an increasing metal concentration. A number of factors affect the uptake of heavy metals by plants, which includes the growing environment. Some examples are soil aeration, soil moisture, soil pH, temperature, competition between plant species, type and size of plants, plant root systems, type of leaves, the elements available in the soil, and plant energy supply to roots and leaves (Yamamato and Kozlowski, 1987).
Metal contamination affects various biochemical and physiological processes in plants such as carbon dioxide fixation, gaseous exchange, respiration, and nutrient absorption. The toxic effects of six heavy metals—Mn, Cd, Cr, Hg, Co, and Pb—were studied on Zea mays by Ghani (2010). Cd was found to be the most toxic and Cr to be the least toxic metal. The phytotoxicity of the six heavy metals was found in this order: Cd>Co>Hg>Mn>Pb>Cr. Heavy metals in plants lead to production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide radicals, and hydroxyl radicals. The ROS can oxidize biological molecules, lead to major cellular damages, and ultimately cell death. Hydroxyl radicals produced in the DNA proximity can remove or add hydrogen atoms to the DNA backbone or bases, respectively (Pryor, 1988). This resulted in 104–105 DNA base modifications in a cell in one day (Ames et al., 1991). Fe2+ ions, free in solution or coordinated with ring nitrogens or complexed to a phosphate residue, were involved in these DNA alterations mediated by the hydroxyl radical (Luo et al., 1994).
Metal ions also lead to oxidative modification of proteins and free amino acids (Stadtman, 1993). The oxidation in proteins most commonly occurs at arginine, histidine, methionine, proline, cysteine, and lysine residues. Transition metals (e.g., iron) and oxygen lead to lipid peroxidation and damage to biological membranes. Plants cope in a number of ways with metal toxicity. The ROS generated in leaf cells are removed by enzymes of the antioxidant system of plants such as ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT). Proline is reported to detoxify active oxygen in Cajanus cajan and Brassica juncea under heavy metal stress (Alia et al., 1995).
19.3 Phytoremediation: a green technology
A staggering number of anthropogenic activities that involve industrial discharges from electroplating, tanneries, municipalities, smelting, refining, and so on pose a significant threat to the quality of the ever-fragile ecosystem (Gisbert et al., 2003; Liu et al., 2005). These controlled and uncontrolled actions have resulted in the addition of toxic xenobiotic compounds, both organic and inorganic in nature, inclusive of many heavy metals and other toxic elements that have intruded into food chains via soil; they cause innumerable allergies and life-threatening diseases in humans and animals (Zornoza et al., 2002; Liu et al., 2005). Their highly toxic concentration has destroyed soil properties and rendered large tracts of agricultural land unfit for cultivation. There are a number of ex situ soil cleanup techniques based on the physicochemical theory (e.g., volatilization, vitrification, excavation, soil washing, soil incineration, chemical extraction, solidification, and landfills) for contaminant removal; however, these are very costly, inconvenient, and sometimes further damage soil properties (Meagher, 2000; Liu et al., 2013).
To address this major quandary, an in situ and novel approach to reach a solution that is natural and safe is phytoremediation, often referred as Green/Clean Technology. In other words, this approach is the use of special plants, known as hyperaccumulators, for the extraction of contaminants from the environment or for lowering their toxicity. These plants possess extraordinary abilities for degradation and removal of many obstinate xenobiotics (both organic and inorganic) and act as a sink for their accumulation, thus are referred to as “green livers” (Ismail, 2013). Subsequently, using it has instigated hope for many environmental enthusiasts and multinational coporations, thus making phytoremediation a very popular field (Salt et al., 1998).
For each one of the processes, there are two possible strategies for exercising this green cleanup technique. The first involves decontamination using hyperaccumulator plants that are capable of accumulating 50 to 100 times more contaminants as compared to normal plants via roots (Pulford and Watson, 2003). The second alternative is to use normal plants as pollutant scavengers coupled with changes in soil environment, either by increasing bioavailability and stabilization of contaminants or by using biotechnological approaches. Compared to the methods used earlier, phytoremediation turned out to be inexpensive, environmentally safe, aesthetically pleasing, and apt for decontaminating large tracts of otherwise unusable agricultural soils.
For the preceding reasons, phytoremediation should be viewed as a long-term remediation solution because many cropping cycles may be needed, spanning several months to years, to determine the contaminants. Thus, remediation using plants can be seen as a competitive and far superior approach to existing conventional technologies for decontaminating “spiked” sites. Still, there are several factors that influence the use of phytoremediation as a green technology such as plants’ biomass accumulation, responsiveness of the plants to agricultural practices (e.g., harvesting and cropping), availability of the metal, and so on (Paz-Alberto and Sigua, 2013). Therefore, there is a need to establish effective monitoring and evaluation methods for in situ field remediation (Gerhardt et al., 2009). In addition, it is important to understand the long-term implications of green plant technology in sequestering environmental pollutants.
There are many types of phytoremedial processes, among them: (1) rhizofiltration, adsorption and precipitation of contaminants present near the plant root zone; (2) phytoextraction, also known as phytoaccumulation in which the contaminant is translocated by plant roots to aboveground parts; (3) phytostabilization i.e., by using plants, contaminants are transformed into an immobilized form; (4) phytodegradation, also known as phytotransformation, where contaminants are broken down into less toxic forms inside the plants or in the rhizosphere; and (5) phytovolatalization, the uptake of a contaminant and transpiration of it into the atmosphere. Table 19.1 shows the detailed mode of action of these phytoremedial processes and various examples of plants on which studies have been done.
Table 19.1
Key Phytoremediation Processes in Different Plant Species
| Phytoremediation Mechanism | Mode of Action | Plant Species and Interaction |
| Rhizofiltration | Absorption, precipitation, and concentration of heavy metal ions into the roots from the polluted effluents | Aquatic macrophytes known for metal filtration (Eichhornia crassipes a, Hydrocotyle umbellate b, Lemna minor, c etc.) |
| Phytoextraction | Metal uptake from soil/water, translocation via xylem, and sequestration in aerial plant parts | A total of 400 hyperaccumulator species, 90 belonging to Brassicaceae itself d,e |
| Phytostabilization | Absorption, precipitation, complex formation, and metal valence reduction for fixing the metal, stabilizing the soil and preventing toxic leachate formation in a short span of time (immobilization of metal to a less toxic form) | Mercury stabilization to least toxic forms in Brassica juncea L. f |
| Phytodegradation | Optimum use of metabolic capabilities of plants and rhizoshphere microbes to break down soil contaminants mostly organic in nature via different enzymes (e.g., dehalogenases, oxygenases, and reductases) | Trinitrotoluene (TNT) breakdown via Nicotiana tabaccum g |
| Phytovolatilization | Uptake and transpiration of contaminants into the atmosphere (evaporates or vaporizes). | Selenium/mercury vaporization via Brassica juncea L. h |
19.4 Field crops as hyperaccumulators and their potential for phytoremediation
Field crops are grown on a large scale for consumption purposes. They are usually annual with a life cycle of 3 to 5 months. Crop plants can be used for phytoremediation because they have high biomass production and can easily adapt to the changing environment (Keller et al., 2003; Meers et al., 2005; Ciura et al., 2005). To be regarded as successful, phytoremediative agents of crop plants must be able to tolerate, as well as accumulate, significant amounts of pollutants (Angelova et al., 2011). Such crops can also have a commercial use as fodder if pollutant accumulation does not exceed critical levels for livestock (Murillo et al., 1999). However, certain crops have the capability to accumulate high concentrations of heavy metals thereby making them unfit for consumption. Further, this wasteful biomass can be used for reextracting the accumulated metals by a process called phytomining. Other than food crops, bioenergy crops have a great potential for phytoremediation because they can be used for both energy production and environmental cleanup.
To successfully carry out phytoremediation with field crops, it is necessary for the plants to be able to accumulate significant levels of pollutants. Thus, hyperaccumulator field crops can be considered as the most favorable contenders for phytoremediation. Hyperaccumulators have the unique ability to actively take up large amounts of pollutants especially metals, 100-fold higher than nonaccumulator plant species (Yang et al., 2005). All hyperaccumulators are tolerant to toxic substances; however, they are very different from the category of tolerant plants that can exclude pollutants from entering plant systems. Therefore, tolerant species may also include nonaccumulators.
Concentration of a pollutant that is taken up by the crop plant system varies according to different species. Hyperaccumulator plants possess a greater potential to absorb pollutants from the soil, faster translocation from roots to shoots, and better mechanisms of sequestration of contaminants as compared to nonaccumulators (Rascio and Navari-Izzo, 2011). Among various types of pollutants, heavy metal pollution has been studied widely, and hyperaccumulation mechanisms in plants have also been established in detail. To absorb metals from the soil, the crop plants either release ligands to bind metals or acidify the rhizosphere with the help of plasma membrane proton pumps (Peer et al., 2006).
The soluble metals enter the root system by symplast or apoplast. Further, to enter the vascular system of the plant, metals use pumps and channels of essential elements. The metals via xylem sap are translocated and deposited in the leaves of the plant. After deposition in the leaves, the metals are detoxified by forming complexes to chelates present in the cellular system. Finally, the chelated metal in the cell is sequestered to an organelle where it is unable to interfere with normal cellular mechanisms; usually, they are sequestered in the vacuole. However, these chelated metal complexes can also remain bound to the cell wall or in some cases are volatilized (Peer et al., 2006).
More than 500 species of plants belonging to families, such as Brassicaceae, Asteraceae, Fabaceceae, Caryophyllaceae, Poaceae, Euphorbiaceae, and others, have been reported to accumulate metals in large quantities; those that perform maximum hyperaccumulators belong to Brassicaceae and Asteraceae (Ebbs et al., 1997; Sarma, 2011). Table 19.2 contains a list of food crops that have potential for phytoremediation.
Table 19.2
Food Crops with Probable Role in Phytoextraction of Heavy Metals
| Plant Species | Common Name | Family | Metal Affinity | Reference(s) |
| Brassica carinata | Ethiopian mustard | Brassicaceae | Cd, Cr, Cu, Ni, Pb, Zn | Marchiol et al. (2004) |
| Brassica juncea | Indian mustard | Brassicaceae | Ag, Cr, Cd, Cu, Ni, Pb, Zn, Mn, Se | McCutcheon and Schnoor (2003); Bennett et al. (2003); Marchiol et al. (2004); Clemente et al. (2005); Haverkamp et al. (2007) |
| Brassica napus | Rapeseed | Brassicaceae | Cr, Hg, Pb, Se, Zn, Cu | McCutcheon and Schnoor (2003); Marchiol et al. (2004) |
| Festuca spp. | – | Poaceae | Cu, Zn | Alvarez et al. (2003) |
| Glycine max | Soybean | Fabaceae | As, Cd, Cu, Pb, Zn | Fellet et al. (2007) |
| Helianthus annus | Sunflower | Asteraceae | Cr, Cu, Zn, As, Cd, Co, Pb | McCutcheon and Schnoor (2003); Fellet et al. (2007); Marchiol et al. (2007) |
| Hordeum vulgare | Barley | Poaceae | Al, As, Cu, Zn, Pb | McCutcheon and Schnoor (2003); Soriano and Fereres (2003) |
| Lolium perenne | Perrenial ryegrass | Poaceae | Cu, Pb, Zn | Alvarenga et al. (2009) |
| Medicago sativa | Alfalfa | Fabaceae | Cr, Pb, Cu, Cd, As | McCutcheon and Schnoor (2003); Pajuelo et al. (2007) |
| Oryza sativa | Rice | Poaceae | Cu, Pb, Zn | Murakami and Ae (2009) |
| Phaseolus vulgaris | Common bean | Fabaceae | As, Cu, Pb, Zn | Luo et al. (2005, 2008) |
| Pistia stratiotes | Water lettuce | Araceae | Cr, Cd, Hg, Cu | Sen et al. (1987); McCutcheon and Schnoor (2003) |
| Pisum sativum | Sweet pea | Fabaceae | Pb | Chen et al. (2004) |
| Raphanus sativus | Radish | Brassicaceae | Cd, Cr, Cu, Ni, Pb, Zn | Marchiol et al. (2004) |
| Sorghum bicolor | Sorghum | Poaceae | As, Cd, Co, Cu, Pb, Zn | Fellet et al. (2007); Marchiol et al. (2007) |
| Triticum secalotriticum | – | Poaceae | As, Cd, Cu, Pb, Zn | Soriano and Fereres (2003) |
| Vicia faba | Horse bean | Fabaceae | Al | McCutcheon and Schnoor (2003) |
| Zea mays | Maize | Poaceae | As, Cd, Cu, Pb, Zn | Luo et al. (2005); Fellet et al. (2007) |
Source: Modified from Vamerali et al. (2010).
19.5 Facilitated phytoextraction in crops
Phytoextraction is the uptake of contaminants from the environment into plants and then storage of them in harvestable plant parts. Using various chelating/reducing agents—microbial secretions and soil exudations and increasing contaminant bioavailability, mobility, and uptake into the hyperaccumulator system—has been well documented in the literature for three decades. The soil amendments increase metal diffusion in the soil solution, keeping them in bioavailable form and stabilizing the metal ions. Chelating agents occur naturally in hyperaccumulators in the form of “Natural Chelating Agents,” such as low-molecular-weight organic acids (LMWOAs), amino acids, ferretins, nicotianamine, and so on, released by crop roots into the rhizosphere or secreted and accumulated in cell cytoplasm, which makes the ions of both nutrients and contaminants more mobile and compliable. Sequestration is followed by dissociation of the metal–chelator bond because plants take up the metal and release the chelator back into the soil (see Figure 19.1).
The process continues with the subsequent intra- and intercellular activation of natural chelating responses and sequestration in cell cytoplasm, followed by chelator replenishment to the rhizosphere. Source: Modified from Bhardwaj et al. (2013).
Hyperaccumulator crop plants have built-in chelator molecules, such as phytochelatins (PC), metallothioneins (MT), root exudations, and others, rich in amino acids, organic acids, ferritins, and phytins that remediate metal in the plant’s proximity. Alternatively, soil amendments, both biodegradable and nonbiodegradable, can be added to the growth medium to facilitate metal and/or contaminant removal, particularly in commercialized phytoremediation practices (Bucheli-Witschel and Egli, 2001; Jiang et al., 2004). Either way, facilitating the contaminant removal process by employing chelate–metal kinetics is one of the most popular phytoremedial practices in use at present.
19.5.1 Chelating agents
Chelating agents can be naturally tapped or used as soil modifications to enhance the availability of heavy metals to the field crops. “Chelate” usually refers to a complex between metal and a chelating agent, not the agent itself (Nowack and Van Briesen, 2005). Several examples have been reported across field crops (e.g., Pisum sativum, Brassica juncea, and Zea mays) for metal remediation via chelants/chelators (Bucheli-Witschel and Egli, 2001). Natural chelators are mostly proteins or nonproteins by chemical nature. A number of synthetic chelating agents—for example, aminopolycarboxylic acids, ethylenediamine tetraacetic acid (EDTA), ethelynediaminedisuccinic acid (EDDS), nitriloacetic acid (NTA), ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (GEDTA), and diethylenetriamine pentaacetic acid (DTPA)—have been used frequently to increase the mobility and transport of heavy metals from contaminated soil via field crops. However, these persist in the soil and may also cause toxicity, and so are not reliable. A more environmentally friendly approach is the use of biodegradable amino acids/LMWOA(s) as chelators; this is a promising approach in phytoextraction research.
19.5.1.1 Amino acids
Chelation is also mediated by metal-binding peptides or amino acids (Salt et al., 1998). Amino acids (e.g., cysteine, glycine, histidine, proline) are nitrogen-donating ligands that have a high affinity for metal ions (Rauser, 1999). Generally, amino acids are used to enhance nutrient availability to garden crops; therefore this concept was thought to be applicable to metals as well. Due to its ability to act as a trident ligand via carboxylato, amine, and imadazole groups, histidine is considered a versatile ligand and one of the most important free amino acids. Callahan et al. (2006) observed how free histidine increases Ni tolerance and capacity to translocate it to the shoot region by chelation.
Krämer et al. (1996) reported that Ni exposure to Alyssum lesbiacum increased total amino acid content; in particular, free histidine content showed an increase of up to 36 times. Extended X-ray absorption fine structure analysis revealed histidine–nickel (His-Ni) complexes in vivo. Further, exogenous application of free histidine increased Ni tolerance and translocation from root to shoot in a nonhyperaccumulator (Alyssum montanum). Overexpression of A. lesbiacum ATP-PRT cDNA increased the free histidine pool 15-fold in the shoot tissues in transgenic Arabidopsis (Kerkeb and Kramer, 2003). Research evidence also is needed to verify the role of other amino acids (e.g., L-glycine) as metal chelators.
19.5.1.2 Phytins
Many ligands are produced by crop plants for binding heavy metal ions such as Cd, Cu, Ni, and Zn. One of them is a phosphorus-rich complex called Zn-phytates. Phytin or phytate is a mixed salt of myoinositol hexaphosphoric acid or phytic acid. It is variably distributed throughout the protein matrix, or confined in condensed masses called globoids. In vitro studies indicated that those phytic acids possess iron-chelating properties (Minihane and Rimbach, 2002).
Van Steveninck et al. (1994) conducted electron probe microanalysis of fractured, quench-frozen root specimens of common crop species. They found that a substantial amount of Zn can be bound as Zn phytate (myo-inositol kis-hexaphosphate) within small vacuoles of cells in the root elongation zone of lucerne, soybean, lupins, tomato, rapeseed, cabbage, radish, maize, and wheat when exposed to high levels of Zn (80–300 μM). Lou et al. (2007) conducted greenhouse experiments to study the effects of chelating agents on the growth and metal accumulation of Pteris vittata L., Vetiveria zizanioides L., and Sesbania rostrata L. in soil polluted with As, Cu, Pb, and Zn using five chelating agents—EDTA, HEDTA, NTA, OA, and phytic acid (PA). The results with PA-induced uptake were substantially significant.
19.5.1.3 Organic acids
Organic acids are low-molecular-weight (−CHO) containing compounds, which are widely distributed among all organisms. They carry a negative charge, according to the dissociation properties and presence of a number of carboxylic acids, which allows the displacement of anions from the soil matrix and the complexation of metal cations in solution. These are involved in various soil processes such as detoxification of metals by plants, microbial proliferation in the rhizosphere, dissolution of soil minerals, and mobilization and nutrient uptake by microorganisms and plants (Marschner, 1995).
Citric, ferulic, fumaric, lactic, malic, oxalic, propionic, succinic, sinapic, and tartaric acids are the most common low-molecular-weight organic acids (LMWOAs) found in the soil (Raskin et al., 1997; Dakora and Phillips, 2002). Under favorable environmental conditions, the concentration of LMWOAs is low in the soil. Primarily, organic acid formation occurs in the soil through metabolism by microbes, degradation of canopy detritus, and so on (Oburger et al., 2009). These are known to be key players of abiotic stress tolerance, nutrient deficiencies, and interaction between plant and microbes that operate at the root–soil interface. Many recent reports indicate that under environmental stress, the biosynthesis, accumulation, transport, and exudation by roots of plant/microbial interactions with organic acids is greatly increased (Fernández et al., 2012; Tan et al., 2013). With respect to heavy metal/metalloid/radionuclide uptake via organic acids, some important reports are discussed next.
Huang et al. (1998) found organic acid increased uranium extraction in Indian mustard and Chinese cabbage. Similarly, increased chromium accumulation in tomato by organic acids was observed. The authors found that Brassica juncea may accumulate uranium (Ur) in its shoots mediated by citric acid application up to a level of 5000 mg/kg. When considering their application as a soil amendment for phytoextraction purposes, LMWOA(s) have a potential advantage over substances, such as EDTA and DTPA, in that they are more readily degraded in the soil and their persistence in soil is pretty short-lived. These function as natural chelating agents and are very efficient in the solubilization of mineral soil components such as heavy metals (Wasay et al., 1998; Hens and Hocking, 2002).
Spartina maritima was evaluated for phytoextraction potential for several heavy metals in salt marshes via three different LMWOAs (i.e., citric, malic, and acetic acid). Acetic acid turned out to be the most effective metal-extracting organic acid with many metals (Duarte et al., 2011). The role of LMWOAs (citric acid and succinic acid) from root exudations in the rhizosphere sediments of three mangrove plant species showed that the concentration of them was altered with the varying levels of PAH contamination.
Among the mangrove species, Bruguiera gymnorrhiza attained the highest root biomass and concentration of LMWOAs and also was the most efficient in removing PAHs (Wang et al., 2013). Citric, fumaric, malic, and α-cetoglutaric acids were studied for accumulation in Sesuvium portulacastrum and Brassica juncea determined by the HPLC technique in shoots, roots, and xylem saps for exposure to lead. For both species, a positive correlation was observed between lead and citrate concentrations in xylem sap. Accumulation of citric acid in xylem and shoots of S. portulacastrum indicated its high potential to translocate and accumulate this metal in shoots, suggesting their possible use to remediate Pb-polluted soils (Ghanaya et al., 2013). Thus, after taking an overview of low-molecular-weight organic acids, it has been observed that these are feasible chelators in the context to soil remediation through plants (Agnello et al., 2013).
19.5.2 Growth-promoting bacteria and mycorrhizae
Plant growth-promoting rhizobacteria and mycorrhizal associations contribute positively in ameliorating the phytoremediation efficiency of contaminated soils. These soil microbes play a pivotal role at the plant–rhizosphere interface. Rhizospheric soil microbes enhance the effectiveness of the phytoremedial process in two ways: (1) directly facilitating phytoextraction by enhancing root-to-shoot metal translocation or facilitating phytostabilization of the contaminant by lowering its mobility or availability in rhizosphere; and (2) indirectly facilitating phytoremediation by enhancing plant growth and biomass, increasing the survival of plants in the contaminated environment, improving acquisition and recycling of nutrients, and controlling plant pathogens (Rajkumar et al., 2012).
19.5.2.1 Mycorrhizae
Mycorrhizae are the mutualistic association between fungi and roots of plants. Almost 80% of terrestrial plants have symbiotic associations with mycorrhizae fungi (Sylvia, 2005). There are many types of mycorrhizal associations: arbuscular mycorrhizas, ectomycorrhizas, orchid mycorrhizas, and ericaceous mycorrhizas. Of these the most widespread is the association between roots of terrestrial plants and arbuscular mycorrhizal fungi (AMF) (Marques et al., 2009). AMF assist phytoremedial efficiency in numerous ways—for example, by improving plant root and shoot growth and biomass, up-regulating nutrient and water uptake by plants, and increasing plant survival under environmental stress (Smith and Read, 2008; Denton, 2007). AMF associations also enhance phytoextraction of metals by increasing root-to-shoot metal translocation.
Bhaduri and Fulekar (2012) reported AMF inoculation increased the phytoremediation potential of Ipomoea aquatic by increasing Cd accumulation in plant tissues. AMF protect plants from damage caused by organic pollutants by regulating translocation and distribution in rhizodermis then in shoots (Fester, 2013). Arbuscular mycorrhizal fungi also have a stimulatory effect on organic contaminants, degrading bacteria living in rhizosphere (Alarcon et al., 2008). Yu et al. (2011) and Zhou et al. (2009) found that AMF associations have increased the biodegradation of PAHs by Lolium multiflorum and Medicago sativa plants. Malachowska-Jutsz and Kalka (2010) reported that AMF associations helped in the phytoremediation of petroleum contaminants by Triticum aestivum.
19.5.2.2 Plant growth-promoting rhizobacteria
Plant growth-promoting rhizobacteria (PGPRs) are the beneficial rhizosphere inhabiting soil bacteria (Kloepper and Schroth, 1978). Depending on their association with the host plant, there are two categories of PGPRs: intracellular and extracellular. Intracellular PGPRs are like nodule-forming bacteria that inhabit the inside of plant cells. Extracellular PGPRs (e.g., Burkholderia, Pseudomonas, and Bacillus) are free-living bacteria that live outside plant cells (Tak et al., 2013). They are present in bulk in the rhizosphere of hyperaccumulator plants inhabiting metal-contaminated soils (Naees et al., 2011).
Inoculation of PGPRs in contaminated sites has been reported to enhance the total phytoremedial efficiency of field crops. Inoculation of Pseudomonas fluorescence improved plant growth and cadmium extraction in inoculated plants in comparison to noninoculated plants (Heshmatpure and Rad, 2012). PGPRs also have been isolated from organic pollutant contaminated soils and found to have rhizodegradation potential. Pseudomonas is one of the prominent groups of rhizobacteria involved in organic contaminant degradation (Glick, 2010). Symbiotic association between Rhizobium and alfalfa is reported to have a stimulatory effect on other soil microflora and polyaromatic hydrocarbon degradation (Teng et al., 2011).
PGPRs enhance plant growth and phytoremediation efficiency as follows:
1. by secreting plant growth-promoting substances such as indole-3-acetic acid (IAA), cytokinin, and gibberellins (Glick, 2012);
2. by excreting stress-alleviating metabolites such as 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) (Glick, 1995; Rajkumar et al., 2006);
3. by altering metal bioavailability by secreting chelators such as siderophores and organic acids, altering soil pH, and through oxidation/reduction reactions to enhance their accumulation (Ma et al., 2011; Rajkumar et al., 2012); and
4. by solubilizing nutrients such as phosphorus and nitrogen fixation (Glick, 2012).
Various studies have demonstrated the beneficial interaction of PGPRs and field crops. Sheng and Xia (2006) found that inoculation of cadmium-resistant, rhizosphere-competent bacterial strains in Brassica napus increased root and shoot biomass. Similarly, inoculation of PGPRs in Brassica juncea enhanced the growth and biomass production when grown in Pb-Zn mine tailings and Ni polluted soil (Wu et al., 2006, Zaidi et al., 2006). Ding (2012) found that the interaction of Medicago osativa and soil microbes degraded benzo[a]pyrene in soil and decreased the accumulation of pollutant inside plant tissues.
19.5.3 Plant growth regulatory substances
Plant growth regulatory substances (PGRs) are the phytohormones (i.e., auxins, cytokinins, gibberellins, ethylene, and abscisic acid). The new class of phytohormones includes brassinosteroids, salicylic acid, jasmonic acid, and stringolactones. These are essential for normal growth, development, and reproduction of plants. PGRs-mediated phytoremediation is known as phytohormone-assisted phytoremediation (Barbafieri and Tassi, 2011). Phytohormones stimulate plant growth by regulating various intercellular processes (Hadi et al., 2010). Phytohormones serve as one of the effective strategies for improving phytoremediation by boosting plant biomass and root and shoot growth, increasing plant tolerance toward contaminants, and enhancing metal acquisition and accumulation (Ouzounidou and Ilias, 2005; Tassi et al., 2008). Application of both crude and commercially available plant growth regulators increased metal uptake stress tolerance in pearl millet (Firdaus-e-Bareen et al., 2012).
19.5.3.1 Auxins
The auxin group is one of the master phytohormones that regulate plant cell division, organ differentiation, growth, and maturation (Trewavas, 2000). Auxins regulate cation uptake and cation fluxes (Vamerali et al., 2011). IAA is the most commonly occurring natural auxin, while other auxins, such as indole-3-butyric acid (IBA), 4-chloro-IAA, and phenylacetic acid, also occur (Machackova et al., 2008).
Very few studies have been conducted to check the potential of phytohormones for phytoremediation. IAA treatment increased lead accumulation in Medicago sativa roots (López et al., 2005). When administered to Sedum alferdii hyperaccumulating and nonhyperaccumulating ecotypes, IAA treatment increased lead accumulation 2.7-fold more in hyperaccumulating ecotype than in nonhyperaccumulating ecotype (Liu et al., 2007). Du et al. (2011) observed that IAA treatment increased phytoextraction potential of lead by zinc/cadmium hyperaccumulator Picris divaricata. Thus, auxin treatment can be used for the phytoremediation of polymetallic-contaminated sites. Chouychai (2012) found IBA treatment alleviated lindane and alpha endosulfan toxicity in Brassica chinenses by increasing its root and shoot fresh weights. Thus, auxin treatment may alleviate organochlorine phytotoxicity.
19.5.3.2 Cytokinins
Cytokinins (Ck) regulate plant growth and development by playing a critical role in cell division and differentiation, shoot initiation, leaf expansion, delay of senescence, bud formation, growth of lateral buds, chlorophyll synthesis, control of shoot–root balance, and transduction of nutritional signals. Cks can also stimulate crop productivity and plant resistance to various environmental stresses (Sakakibara, 2006; Tassi et al., 2008). Transpiration rate is one of the key processes regulating the success of phytoremediation efficiency (Rock, 2003). Cytokinin treatment can increase the acquisition of metal by accelerating the transpiration rate (Tassi et al., 2008). Appliction of Cks has been found to increase stomatal aperture in leaves of Tradescantia and Paphiopedilum tonsum (Irving et al., 1992) and the transpirational rate in leaves of Helianthus, Hordeum, Brassica, Triticum, Avena, and Vigna (Pharmavati et al., 1998; Pospíšilová et al., 2000). Tassi et al. (2008) found that Ck treatment can effectively improve phytoextraction of heavy metals by up-regulating metal uptake, increasing biomass production, and improving the plant transpiration rate. Simultaneous application of Ck and ammonium thiosulfate enhances mercury uptake and translocation in Brassica juncea and Helianthus annuus up to 248% and 232% by increasing plant biomass and the evapotranspiration rate (Cassina et al., 2012).
19.5.3.3 Brassinosteroids
Brassinosteroids (BRs) are polyhydroxysteroids that regulate various physiological responses in plants, including germination, seedling photomorphogenesis, root and stem elongation, leaf bending and epinasty, vascular differentiation, male fertility, induction of ethylene biosynthesis, timing of senescense and flowering, activation of photosynthesis, and resistance to biotic and abiotic stresses (Clouse and Sasse, 1998; Krishna, 2003; Bajguz and Hayat, 2009). Brassinosteroids alleviate heavy metal stress in plants (Bajguz, 2011). BR-assisted phytoremediation can improve the phytoremediation potential of inorganics by increasing germination, improving crop yield, increasing stress tolerance, facilitating phytostabilization and phytoextraction, and increasing root apparatus. BRs can assist phytoremediation of organics by increasing their degradation, growth, and biomass under contaminant stress (Barbafieri and Tassi, 2011).
19.5.4 Molecular techniques
The molecular techniques applied to plants mainly include genetic engineering to enhance tolerance, accumulation, and metabolism of pollutants. Many genes have been identified from various organisms, especially bacteria and yeasts, that are involved in the acquisition, allocation, and detoxification of pollutants (Ehrlich, 1997). Thus, genetic engineering of plants may help improve the phytoremediation efficiency of plants.
A full-length OsCs1 gene encoding for citrate synthase has been isolated; it is induced under Al toxicity in Oryza sativa. Transgenic tobacco lines containing OsCS1 genes showed increased citrate reflux and enhanced aluminum tolerance (Han et al., 2009). Up-regulated expression of γ-glutamylcysteine synthetase or glutathione synthetase in transgenic Brassica juncea resulted in the higher accumulation of and tolerance to some metals (e.g., Cd, Cr, and As) (Reisinger et al., 2008). Chen et al. (2013) used a one gene transgenic approach to enhance arsenic tolerance and accumulation in Arabidopsis thaliana. A key arsenite, As(III), antiporter PvACR3 present in As hyperaccumulator fern, Pteris vittata, was expressed in Arabidopsis thaliana derived by promoter CaMV 35S. Transgenic plants have shown a many-fold increase in As tolerance. Arsenic methylation and volatilization have been induced due to expression of bacterial arsenite S-adenosylmethyltransferase in transgenic plants (Xiang-Yan et al., 2011).
Matsui et al. (2013) isolated a transcription factor, AtPHR1, regulator of inorganic phosphate starvation response in Arabidopsis thaliana. This factor was made to overexpress in garden plants (i.e., Torenia, Petunia, and Verbena) and these transgenic plants resulted in the hyperaccumulation of inorganic phosphate in leaves and accelerated phosphate absorption rates from hydropnic solutions. Transgenic plants with the bacterial genes used in polychlorinated biphenyl (PCB) degradation showed effective removal of PCBs from contaminated sites (Novakova et al., 2009).
Bacterial mercuric ion reductase (mer A) and organomercurial lyase (mer B) genes are used in genetically engineered plants for the phytoremediation of mercury. Various plant species, such as poplar, rice, tobacco, peanut, Arabidopsis, and Chlorella, have been modified with these genes. These genetically modified plants grow well in organic and inorganic mercury-contaminated soils and accumulate Hg in roots (Ruiz and Daniell, 2009). Transgenic plants that overexpress the bacterial mercury reductase have exhibited a high tolerance to organic mercury (Bizilly et al., 2003) and effectively enhanced the volatilization of ionic mercury (Haque et al., 2010).
19.6 Conclusion and future prospects
The presence of several contaminants limits the use of polluted sites; such contaminants include various organic and inorganic compounds. The remediation of these sites can be achieved through a newly developed, low-cost green technology known as phytoremediation in which plants are used to clean the contaminants. A few plant species with a higher tolerance for contaminants have been identified. The hyperaccumulator species have some limitations such as poor growth rate and difficulty in practical applications. Thus, field crops are seen as a reliable alternative to hyperaccumulators. To elucidate the limitations of field crops for phytoremediation, such as low uptake and translocation of target pollutants and for activation of unavailable pollutants in soil, various improvement methods should be employed. To achieve this, chelators can be used to enhance the bioavailability of pollutants and phytohormones can be applied to increase the growth rate. Use of molecular and biotechnological approaches and advanced agricultural practices may be helpful to the advancement of phytoremediation procedures.