I. Remediation of environmental contaminants via engineering and biological methods
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- I. Remediation of environmental contaminants via engineering and biological methods
- II. Phytoremediation with transgenics
- III. Endophyte-assisted phytoremediation
- IV. Concluding remarks
Industrial and military activities have led to widespread contamination of the environment, including thousands of sites, termed SuperFund sites, that are severely polluted. The concentrations of the contaminants can vary from highly toxic concentrations from an accidental spill to barely detectable concentrations that, after long-term exposure, can be detrimental to human health (Alexander, 1999).
The cost of cleaning up contaminated sites is extremely high. In the USA alone, $6–8 billion is spent annually in remediation efforts, with global costs in the range of $25–50 billion (Glass, 1999; Tsao, 2003). Engineering methods for the remediation of contaminated sites include excavation, transport, soil washing, extraction, pumping and treating of contaminated water, addition of reactants such as hydrogen peroxide or potassium permanganate, and incineration. A serious consequence of the high cost of remediation technologies is that polluted commercial properties are often abandoned rather than cleaned up. There are over 500 000 of these so-called brownfields in the USA.
Another popular clean-up method involves augmented bioremediation with the addition of specific microbial strains known to degrade the pollutant. Bacteria and fungi collectively can utilize a vast range of organic molecules. But for bioremediation using microbes at a particular site to be successful, many conditions must be met. These include the ability of the microbes with the desired metabolic activity to survive in that environment, the accessibility or bioavailability of the chemical, and the presence of inducers to activate expression of the necessary enzymes. Many organic pollutants are recalcitrant to degradation and cannot be used as sole carbon sources. The pollutants are sometimes metabolized by enzymes with other natural substrates; therefore, these substrates sometimes need to be present in order for the genes to be expressed. This requirement is problematic if the inducing chemical is itself a harmful pollutant, such as phenol. Bioremediation also depends on the presence of sufficient carbon and energy sources. Often, thousands of gallons of a food source such as molasses must be pumped down into the site to allow bacterial growth. The use of microorganisms in engineered bioremediation systems has had mixed success. A review of this broad and active field is beyond the scope of this review; a recent book provides an excellent overview of bioremediation of xenobiotics, petroleum, BTEX (benzene, toluene, ethylbenzene, and xylene), explosives, and heavy metals (Fingerman & Nagabhushanum, 2005).
Phytoremediation is the use of plants to treat contaminated sites. This technology has been extensively reviewed (for recent reviews see Schnoor et al., 1995; Salt et al., 1998; Meagher, 2000; Dietz & Schnoor, 2001; McCutcheon & Schnoor, 2003; Newman & Reynolds, 2004; Suresh & Ravishankar, 2004; Pilon-Smits & Freeman, 2006). Phytoremediation takes advantage of the natural ability of plants to extract chemicals from water, soil, and air using energy from sunlight. Some of the advantages and disadvantages are listed in Table 1. Its primary advantage is that it is approximately 10 times less expensive than conventional strategies (Chappell, 1998). Table 2 illustrates these cost differences between phytoremediation and other technologies. Another benefit involves safety issues. Plants act as soil stabilizers, minimizing the amount of contaminated dust that could leave the site and enter the surrounding neighborhoods. With phytoremediation it is also easier to monitor the site. Unlike bioremediation with microbes, phytoremediation is easily visible; the condition of the plants can be visually monitored, and samples of plant tissue can be tested for the presence of the pollutant over time. Other advantages of phytoremediation over the engineering or bioremediation methods include the possibility of a useful product such as wood, pulp, or bioenergy (Stanton et al., 2002) that could help finance the clean-up. Plants also supply nutrients for rhizospheric bacteria that may also aid in remediation of the pollutants. Finally, phytoremediation also provides wildlife habitat. For example, poplar (Populus spp.) tree plantations can harbor an abundance of birds and small mammals (Moser et al., 2002), and willow (Salix spp.) thickets can provide the stopover sites for c. 60 migrating bird species (Kuzovkina & Quigley, 2005).
Table 1. Advantages and disadvantages of phytoremediation (Chappell, 1998)
|Less costly than mechanical methods||Limited to shallow contaminants|
|Passive, solar-driven||Phytotoxicity effects of contaminants|
|High public acceptance||Slower than mechanical methods|
|Retains topsoil||Unknown effects of biodegradation products|
|Less secondary waste generation||Potential for contaminants to enter the food chain|
Table 2. Estimates of phytoremediation costs versus costs of established technologies (Chappell, 1998)
|Contaminant||Phytoremediation costs||Estimated cost using other technologies||References|
|Metals||$80 per cubic yard||$250 per cubic yard||Black (1995)|
|Petroleum||$70 000 per site||$850 000 per site||Jipson (1996)|
|Lead||$50 000 per acre||$1.2 million per acre||Plummer (1997)|
|Radionuclides in surface water||$2–$6 per thousand gallons treated||None listed||Richman (1996)|
|1 ha to a 15-cm depth (various contaminants)||$2500–$15 000||None listed||Cunningham et al. (1996)|
Phytoremediation has been used to treat a variety of pollutants including metals, petroleum, solvents, explosives, polycyclic aromatic hydrocarbons, and other organic contaminants. For an extensive listing of phytoremediation projects, see the US Environmental Protection Agency (EPA) website: http://www.cluin.org/products/phyto/. Phytoremediation involves different processes depending on the type of pollutant. Remediation of metals presents a distinct challenge because the pollutants cannot be metabolized but must instead be translocated to the foliage where they are more easily removed by harvesting the upper parts of the plant, or are volatilized such as in the case of mercury. Phytoextraction refers to this method of removal of contaminants from the soil and translocation to the foliage. Phytoextraction is an effective means of remediating a site because it reduces the overall mass to be treated from tons of widespread contaminated soil to plant tissue that can be dried to a small volume. To be effective, the concentration of the metal in the harvestable part of the plants must be higher than the concentration in the soil so that the volume of the hazardous plant waste is less than the volume of the contaminated soil. Unlike engineering methods that would remove the fertile topsoil, phytoremediation would not reduce the fertility of the site (Robinson et al., 2000). Plants that are especially good at concentrating the pollutants are termed hyperaccumulators. A metal hyperaccumulator is defined as a plant that can concentrate the metals to a level of 0.1% for nickel, cobalt, copper, and lead, 1% for zinc, and 0.01% for cadmium (Baker et al., 2000). For example, Pteris vittata (Chinese brake fern) efficiently hyperaccumulates arsenic in its fronds which can be effectively harvested (Zhang et al., 2002). Arsenic is a lethal poison that is released into the environment from natural processes in certain geographical areas and through the use of arsenic-based chemicals. Pteris vittata can effectively remove this metalloid from soil. For example, in soil contaminated with arsenic at a concentration of 97 ppm, the older fronds of the fern had arsenic concentrations of up to 3894 µg per gram of tissue. Less than 168 µg arsenic per gram was found in the root tissue. More than 95% of the arsenic removed from the soil by the fern was translocated to the aboveground biomass. Unfortunately, this plant species grows well only in warm, humid environments with mild winters (N. Peck, pers. comm.). Another hyperaccumulator is Thlaspi caerulescens, which can concentrate cadmium, a highly toxic and probably carcinogenic metal (Vido, 2001), in the above-ground tissues at concentrations 1000 times higher than the normal toxic concentration of only 1 ppm (Brown et al., 1995). In this work by Brown et al. (1995), the plants were exposed to 200 µM cadmium and accumulated 1140 mg kg−1. In work using Agrobacterium rhizogenes-induced root cultures of T. caerulesens, up to 10 600 µg cadmium per gram of dry weight of roots was accumulated (Nedelkoska & Doran, 2000). The mechanism of uptake of cadmium and zinc by this member of the Brassicaceae family has been well studied and involves a highly expressed metal transporter (Pence et al., 2000). The transporter gene, ZNT1, encodes a high-affinity zinc/low-affinity cadmium transporter, as demonstrated in yeast. The zinc/cadmium pumping ATPase was recently purified directly from T. caerulescens and was shown to transport both zinc and cadmium (Parameswaran et al., 2007). Although the research on these hyperaccumulating species is promising, the species themselves are too small and slow-growing for some phytoremediation applications (Ebbs et al., 1997). For this reason, high-biomass crops such as poplar and willow are being studied for phytoremediation of metals. Poplar and willow are not hyperaccumulators as they do not concentrate metals to high concentrations, but, because of their greater biomass and deep root systems, they are also effective remediators of metal contamination. In a review by Pulford and Watson, willow was specifically suggested for phytoremediation of heavy-metal contaminated lands because the method requires the ability of a plant to re-grow readily after its shoots have been harvested, a distinctive trait of willow (Pulford & Watson, 2003). The ‘bioconcentration factor’ (BCF) refers to the metal concentration in plant tissues relative to the metal concentration in the substrate, and a value greater than 1 means that the plant actively concentrates the metal. In a review of cadmium accumulation by willow, it was reported that BCFs vary widely among different willow species, from as low as 0.05–16.8 in woody stems to up to 27.9 in foliage (Dickinson & Pulford, 2005). Given the substantial genetic diversity of willow, with over 450 Salix species, this variability is not too surprising. Willow plants (Salix matsudana × Salix alba NZ1040) grown in soil contaminated with cadmium at concentrations commonly found in agricultural sites fertilized with cadmium-containing fertilizers accumulated cadmium in the above-ground tissues with BCFs of c. 10 (Robinson et al., 2000). The authors concluded that, by extrapolation, it would take only one planting of willow to remove the amount of cadmium from a field treated for 100 yr with cadmium-based fertilizers. However, as the experiment was conducted using plants introduced to pots only 2 months after the cadmium was added, and the bioavailability in a natural system would be much lower, it would probably take more than one planting. In a recent paper by French et al., Salix, Populus, and Alnus were compared in a study on remediation of brownfield land (French et al., 2006). Of five willow clones, all concentrated copper, and four of them also concentrated cadmium and zinc to concentrations up to 13 times higher than the soil concentrations. From a field trial of Salix viminalis for phytoextraction of cadmium and zinc, the authors calculated that it would take decades to decontaminate the site with this species, because the extraction efficiency decreased with time (Hammer et al., 2003). In general, willows, as fast-growing, deep-rooted, high-biomass trees and shrubs, hold much promise for remediation of cadmium – a serious metal pollutant – but there is still room for improvement in the bioconcentration factors and their consistency over time.
Unlike phytoextraction, phytodegradation involves the metabolic degradation of organic pollutants. In this process, plants break down the pollutant through either internal or secreted enzymes. Phytodegradation of chlorinated hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), and explosives has been studied most extensively. Other compounds, such as PCBs, have also been studied but with less success. Trichloroethylene (TCE), one of the most common groundwater pollutants, is a known hepatotoxin and carcinogen (Bruning & Bolt, 2000). Hybrid poplars (Populus trichocarpa × Populus deltoides) take up and degrade TCE, producing the same TCE metabolites as mammals (Newman et al., 1997; Gordon et al., 1998). In a controlled field study with hybrid poplar, the trees removed over 99% of the added TCE (Newman et al., 1999). Less than 9% of the TCE taken up was transpired, as detected by leaf bag experiments. In order to determine if poplar cells have an inherent ability to degrade TCE, or if microorganisms are responsible for the degradation, studies were conducted with suspensions of pure poplar culture cells. When these poplar culture cells were dosed with TCE, the same metabolites were seen as those in the whole plant (Newman et al., 1997; Shang et al., 2001; Shang & Gordon, 2002). Experiments with both poplar culture cells and whole plants demonstrated that the primary metabolite, trichloroethanol, is glycosylated, as happens in mammalian systems (Shang et al., 2001). Other plant species are also able to take up and metabolize TCE, such as the tropical leguminous tree Leucaena leucocephala (Doty et al., 2003) and sweet potato plants Ipomoea batatas (Z. Khan and S. L. Doty, unpublished). Having suitable species available for both temperate and tropical zones broadens the range of application.
Plants are also capable of phytodegradation of other common environmental pollutants, including carbon tetrachloride (CT) and perchloroethylene. Anaerobic degradation of CT by soil microbes can lead to production of carcinogenic chloroform; by contrast, plants are capable of metabolizing CT aerobically using a cytochrome P450 enzyme (Wang et al., 2002b). In a controlled field study in which poplar trees were watered with 12–15 mg l−1 CT over a 6-yr period, CT was taken up and dechlorinated (Wang et al., 2004b). There was no significant evapotranspiration of CT, nor increased accumulation of chloride ion in the dosed trees compared with undosed ones. But chloride ions had built up in the root zone. Because soil microbes from the site did not dechlorinate CT, the authors concluded that the trees had taken up and dechlorinated CT, and then exported the excess chloride ions into the soil. Poplar trees were also effective in remediating perchloroethylene (C. A. James et al., unpublished). In a recent field study, nearly all of this pollutant was removed and metabolized, with over 95% of the chlorine recovered as free innocuous chloride, showing effective dechlorination of the perchloroethylene. As with the CT study, the free chloride accumulated in the rhizosphere.
Polycyclic aromatic hydrocarbons (PAHs), another group of widespread environmental pollutants, are released as a waste product during energy extraction processes. Sixteen PAHs are listed as Priority Pollutants by the US EPA because of their carcinogenic properties and prevalence. The levels of PAHs in the environment are increasing (Washington Department of Ecology). Phytodegradation of PAHs occurs to some extent. Because PAHs are lipophilic, adsorption to root surfaces may be an important first step in phytoremediation (Schwab et al., 1998; Burken & Schnoor, 1998). Research by several laboratories has demonstrated that there are wide differences in the abilities of different plant species to reduce PAH concentrations (Trenck & Sandermann, 1979), and that plants themselves can degrade PAHs (Harms, 1996). Phytoremediation used as a ‘polishing step’ after other methods for clean-up of PAH contamination was especially successful (Parrish et al., 2004). Wittig, Ballach, and Kuhn conducted a three-part investigation in the use of poplar cuttings for PAH removal (Wittig et al., 2003; Ballach et al., 2003; Kuhn et al., 2004). Populus nigra cuttings in containers of sand with nutrient solution containing PAHs caused a reduction in the amounts of a range of PAHs, including anthracene, phenanthrene, pyrene, fluoranthene, chrysene, and benzo[a]pyrene. Recently, an extensive field study was conducted using poplar trees to reduce the PAH concentration in groundwater (Widdowson et al., 2005). Results showed concentrations to fall at the time the poplar roots reached the saturated zone, approx. 1 yr after planting; a variety of factors including rhizospheric microorganisms, plant uptake, phytovolatilization, and biodegradation contributed to the decrease in PAH concentration. In none of the studies to date have the plants completely removed PAHs from contaminated areas.
Another important class of environmental pollutants for which plants can be used for remediation is explosives including trinitrotoluene (TNT) and Royal Demolition Explosive (RDX; hexahydro-1,3,5-trinitro-1,3,5-triazine). TNT is toxic to humans, causing aplastic anemia and hepatitis (Rosenblatt, 1980). RDX is less toxic but does affect the central nervous system (Rosenblatt, 1980). More than 100 military bases and explosives-manufacturing facilities in the USA are contaminated with these chemicals. The groundwater at these sites is contaminated, increasing the hazard that the health risk will spread beyond the military bases (Rivera et al., 1998). Research with aquatic plants demonstrated that TNT can be metabolized in the absence of microorganisms (Hughes et al., 1997). Both poplar and willow have been used in munitions remediation research. Hybrid poplar (P. deltoides × P. nigra) was able to take up TNT from hydroponic solution, but the trees only translocated c. 10% of it to the foliage (Thompson et al., 1998). In a study comparing phytoremediation of TNT by hybrid willow (Salix clone EW-20) and Norway spruce (Picea abies), it was shown that both tree species readily metabolized TNT (Schoenmuth & Pestemer, 2004). In this study, the trees were exposed to 5.2 mg TNT per kg soil. After 2 months, 3–14% of the radiolabeled TNT was translocated to the aboveground tissues. Another important explosive is RDX. Poplar tissue cultures and leaf extracts exposed to 20 mg l−1 of this explosive mineralized 17% of the RDX to carbon dioxide when exposed to light (van Aken et al., 2004b). RDX uptake was also studied in the aquatic plant Myriophyllum aquaticum and in hairy root cultures of Catharanthus roseus (Bhadra et al., 2001). In this study, plants exposed to 5 mg l−1 RDX took up c. 75% of the chemical. A serious problem with phytoremediation of TNT and RDX is that the contaminated soil and water at military firing ranges can contain concentrations of these chemicals that are phytotoxic. Obviously, only healthy and actively growing plants would be effective in taking up pollutant and metabolizing it fully.
Although much research has been done to demonstrate the success of phytoremediation, resulting in its use on many contaminated sites, the method still lacks wide application. Its primary disadvantage when compared with engineering methods is that it is often considered too slow or only seasonally effective. Regulatory agencies often require significant progress in remediation to be made in only a few years, making most phytoremediation applications unsuitable. Plant species with the ability to treat a particular pollutant are often either unable to grow under the environmental conditions of the contaminated site or are too small to be useful, such as many of the hyperaccumulators. In some contaminated sites, the pollutants can be at phytotoxic concentrations, as in the case of TNT at military firing ranges, or recalcitrant to degradation by plants, as in the case of PAHs. For these reasons, attention has recently focused on ways to enhance the phytoremediation capacity of plants using either transgenic methods or endophytes.