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Phytoremediation: synergistic use of plants and bacteria to clean up the environment
Biotechnology Advances 21 (2003) 383 – 393 www.elsevier.com/locate/biotechadv
Phytoremediation: synergistic use of plants and bacteria to clean up the environment Bernard R. Glick * Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
Abstract Phytoremediation is a relatively new approach to removing contaminants from the environment. It may be defined as the use of plants to remove, destroy or sequester hazardous substances from the environment. Unfortunately, even plants that are relatively tolerant of various environmental contaminants often remain small in the presence of the contaminant. To remedy this situation, plant growth-promoting bacteria that facilitate the proliferation of various plants especially under environmentally stressful conditions may be added to the roots of plants. These bacteria have been selected to lower the level of growth-inhibiting stress ethylene within the plant and also to provide the plant with iron from the soil. The net result of adding these bacteria to plants is a significant increase in both the number of seeds that germinate and the amount of biomass that the plants are able to attain, making phytoremediation in the presence of plant growth-promoting bacteria a much faster and more efficient process. D 2003 Elsevier Inc. All rights reserved. Keywords: Phytoremediation; Plants; Bacteria
1. The problem: environmental contamination The removal from the environment of many potentially toxic compounds is complicated by the numerous classes and types of these chemicals. For example, many soils are contaminated with one or more metals, radioactive or inorganic compounds. Of these, the metals may include lead, zinc, cadmium, selenium, chromium, cobalt, copper, nickel and mercury; the radioactive compounds may be uranium, cesium or strontium; and the other
* Tel.: +1-519-888-4567; fax: +1-519-746-0614. E-mail address: [email protected] (B.R. Glick). 0734-9750/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0734-9750(03)00055-7
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inorganic compounds might include arsenic, sodium, nitrate, ammonia or phosphate. Soil may become polluted with high concentrations of metals by either a natural phenomenon such as proximity to an ore body, or as a consequence of industrial activities. The remediation of heavily metal-contaminated soils often involves excavation and removal of soil to ‘‘secured’’ landfills, a technology that is expensive and requires site restoration. As an alternative, in the past few years, several groups of scientists have begun to develop technological approaches to using certain plants to remove metal contaminants from the soil. In addition to the above-mentioned inorganic compounds, soils and water systems may also be contaminated with organic compounds including chlorinated solvents like trichloroethylene; explosives such as trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-hexahydrotriazine (RDX); petroleum hydrocarbons including benzene, toluene and xylene (BTX), polyaromatic hydrocarbons (PAHs); and pesticides such as atrazine and bentazon. While many of these compounds can be metabolized by some soil bacteria, this process is usually slow and inefficient, in part as a consequence of the relatively low numbers of these degradative microorganisms in soil. However, there is mounting evidence that the biodegradation of recalcitrant organic compounds in the soil is enhanced around the roots of plants.
2. Phytoremediation: a solution to environmental contamination? One recently developed method of environmental clean-up is called phytoremediation (Cunningham and Berti, 1993; Kumar et al., 1995; Raskin et al., 1994, 1997). This procedure may be defined as the use of plants to remove, destroy or sequester hazardous substances from the environment. Phytoremediation of metals and other inorganic compounds may take one of several forms: phytoextraction, the absorption and concentration of metals from the soil into the roots and shoots of the plant; rhizofiltration, the use of plant roots to remove metals from effluents; phytostabilization, the use of plants to reduce the spread of metals in the environment; or phytovolatilization, the uptake and release into the atmosphere of volatile materials such as mercury- or arsenic-containing compounds. Phytoremediation of organic compounds may occur by phytostabilization; phytostimulation, the stimulation of microbial biodegradation in the rhizosphere, the area around the roots of plants; or by phytotransformation, the absorption and degradation of organic contaminants by the plant. Following the testing of a large number of different plants, a number of plants that can naturally accumulate large amounts of metal have been identified and are being studied for the phytoremediation of metals that are present in the environment. These plants, called hyperaccumulators, are often found growing in areas with elevated metal concentrations in the soil. Unfortunately, in the presence of very high concentrations of metals, even hyperaccumulating plants attain only a small size. That is, high concentrations of metals are inhibitory to the growth of plants, even those plants that are capable of hyperaccumulating metals. Depending upon the amount of metal at a particular site and the type of soil at that site, it could take 15– 20 years to completely remove the metal from the soil and thereby remediate the site, even with hyperaccumulating plants. This is a timeframe that is usually considered to be too slow for practical application.
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A number of different types of plants are effective at stimulating the degradation of organic molecules in the rhizosphere. Typically, these plants all have extensive and fibrous roots, which form an extended rhizosphere; these plants include many common grasses as well as corn, wheat, soybean, peas and beans. In addition, several varieties of trees can take up and degrade some organic contaminants. For example, plants with phytotransformation activity may contain nitroreductases, which are useful for degrading TNT and other nitroaromatics, dehalogenases for the degradation of chlorinated solvents and pesticides, and laccases that can degrade anilines such as triaminotoluene.
3. Plant growth-promoting bacteria Beneficial free-living soil bacteria are generally referred to as plant growth-promoting rhizobacteria and are found in association with the roots of many different plants (Glick et al., 1999). The high concentration of bacteria around the roots, i.e., in the rhizosphere, presumably occurs because of the presence of high levels of nutrients (especially small molecules such as amino acids, sugars and organic acids) that are exuded from the roots of most plants, and can then be used to support bacterial growth and metabolism (Whipp, 1990; Bayliss et al., 1997; Penrose and Glick, 2001). Plant growth-promoting bacteria can positively influence plant growth and development in two different ways: indirectly or directly (Glick et al., 1999). The indirect promotion of plant growth occurs when these bacteria decrease or prevent some of the deleterious effects of a phytopathogenic organism. Bacteria can directly promote plant growth by providing the plant with a compound that is synthesized by the bacterium or by facilitating the uptake of nutrients from the environment by the plant. Plant growthpromoting bacteria may: fix atmospheric nitrogen and supply it to plants; synthesize siderophores which can solubilize and sequester iron from the soil and provide it to plant cells; synthesize several different phytohormones including auxins and cytokinins which can enhance various stages of plant growth; have mechanisms for the solubilization of minerals such as phosphorus which then become more readily available for plant growth; and contain enzymes that can modulate plant growth and development (Brown, 1974; Davison, 1988; Kloepper et al., 1989; Lambert and Joos, 1989; Patten and Glick, 1996; Glick et al., 1998). A particular bacterium may affect plant growth and development using any one, or more, of these mechanisms and a bacterium may utilize different mechanisms under different conditions. For example, bacterial siderophore synthesis is likely to be induced only in soils that do not contain sufficient levels of iron. Similarly, bacteria do not fix nitrogen when sufficient fixed nitrogen is available. 3.1. The role of ACC deaminase in the reduction of plant ethylene In higher plants, ethylene is produced from L-methionine via the intermediates, Sadenosyl-L-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC; Yang and Hoffman, 1984). The enzymes involved in this metabolic sequence are SAM synthetase, which catalyzes the conversion of methionine to SAM (Giovanelli et al., 1980); ACC synthase, which is responsible for the hydrolysis of SAM to ACC and 5V-
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methylthioadenosine (Kende, 1989); and ACC oxidase, which metabolizes ACC to ethylene, carbon dioxide and cyanide (John, 1991). In 1978, an enzyme capable of degrading ACC was isolated from Pseudomonas sp. strain ACP (Honma and Shimomura, 1978). Since then, ACC deaminase has been detected in the fungus, Penicillium citrinum (Honma, 1993), and in a number of bacterial strains (Klee and Kishore, 1992; Jacobson et al., 1994; Glick et al., 1995; Campbell and Thomson, 1996; Burd et al., 1998; Belimov et al., 2001; Ghosh et al., 2003; Ma et al., 2003) and in the yeast, Hansenula saturnus (Minami et al., 1998), all of which originated from the soil either as soil sample isolates or as microbes typically found in the soil. This enzyme cleaves the plant ethylene precursor, ACC, to produce ammonia and a-ketobutyrate. Glick et al. (1998) proposed that microorganisms that contain the enzyme ACC deaminase can all act to promote plant growth since they can act as a sink for ACC and thereby lower ethylene levels in a developing or stressed plant. Ethylene is important for normal plant development in plants as well as for their response to stress (Deikman, 1997). Ethylene is important during the early phase of plant growth; it is required by many plant species for seed germination, and the rate of ethylene production increases during germination and seedling growth (Abeles et al., 1992). However, high levels of ethylene lead to inhibition of root elongation. A model was developed whereby plant growth-promoting bacteria that possess the enzyme ACC deaminase and are bound to seeds or roots of seedlings, can reduce the amount of plant ethylene and the extent of its inhibition on root elongation (Glick et al., 1998). In this model, the plant growth-promoting bacteria bind to the surface of either the seed or root of a developing plant; in response to tryptophan and other small molecules in the seed or root exudates (Whipp, 1990; Bayliss et al., 1997; Penrose and Glick, 2001), the plant growth-promoting bacteria synthesize and secrete indole acetic acid (IAA) (Patten and Glick, 1996, 2002), some of which may be taken up by the plant. This IAA, together with endogenous plant IAA, can either stimulate plant growth or induce the synthesis of ACC synthase, which converts SAM to ACC. A portion of the ACC produced by this latter reaction is exuded from seeds or plant roots along with other small molecules normally present in seed or root exudates (Bayliss et al., 1997; Penrose and Glick, 2001). The ACC in the exudates is taken up by the bacteria and is subsequently converted by ACC deaminase to ammonia and a-ketobutyrate. This decreases the amount of ACC outside the plant so that the plant must exude increasing amounts of ACC in order to maintain the equilibrium between internal and external ACC levels. Thus, in the first instance, the bacteria induce the plant to synthesize more ACC than it would otherwise need and, secondly, it stimulates the exudation of ACC from the plant. As a result of the ACC deaminase-containing bacteria lowering the ACC level within the plant and acting as a sink for ACC, the amount of ethylene that is produced by the plant is also reduced. When tested, strains of ACC deaminase-containing plant growth-promoting bacteria were found to reduce the amount of ACC that was detectable by HPLC, and hence the ethylene levels in canola seedlings were also lowered (Penrose et al., 2001). Moreover, ACC deaminase-containing plant growth-promoting bacteria promote root elongation in a variety of (ethylene sensitive) plants (Hall et al., 1996). There is also a considerable decrease in the levels of ‘‘stress ethylene’’—the accelerated biosynthesis of ethylene
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associated with biological and environmental stresses, and pathogen attack (Morgan and Drew, 1997)—in plants grown in the presence of ACC deaminase-containing plant growth-promoting bacteria. The deleterious effects of flooding on tomato plants (Grichko and Glick, 2001) were decreased and the shelf life of the petals of ethylene sensitive cut flowers was prolonged following treatment with ACC deaminase-containing plant growthpromoting bacteria (Nayani et al., 1998). Moreover, biocontrol strains of bacteria carrying ACC deaminase genes were able to more effectively protect plants against various phytopathogens: cucumber plants were protected against Pythium damping-off, and potato slices, exposed to Erwinia caratovora in a small sealed bag, were protected against Erwinia soft rot (Wang et al., 2000). In addition, canola seedlings grown in the presence of high levels of nickel, produced much less ethylene when the seeds were inoculated with an ACC deaminase-containing nickel-resistant plant growth-promoting strain that (Burd et al., 1998). It appears that the ‘‘stress ethylene’’ produced in each of these situations, and the damage caused by it, was reduced by the activity of ACC deaminase that lowered the level of ethylene produced by the plant.
4. The use of plant growth-promoting bacteria in phytoremediation 4.1. Metals While plants grown on metal contaminated soils might be able to withstand some of the inhibitory effects of high concentrations of metals within a plant, two features of most plants could result in a decrease in plant growth and viability. That is, in the presence of high levels of metals, most plants (i) synthesize stress ethylene and (ii) become severely depleted in the amount of iron that they contained. Moreover, plant growth-promoting bacteria may be used to relieve some of the toxicity of metals to plants. This could occur in two different ways. As indicated earlier, the use of ACC deaminase-containing plant growth-promoting bacteria would be expected to decrease the level of stress ethylene in a plant growing in soil that contained high levels of metal. In addition, plants are able to take up and utilize complexes between bacterial siderophores and iron. Plant siderophores bind to iron with a much lower affinity than bacterial siderophores so that in metal contaminated soils a plant is unable to accumulate a sufficient amount of iron unless bacterial siderophores are present. Our primary objective was the development of a phytoremediation system that could be used to help to remove nickel from soil as inexpensively and as quickly as possible. Prior to this work, there were reports in the scientific literature that indicated that Brassica juncea (Indian mustard) was a nickel-hyperaccumulating plant and could be used for this purpose. However, preliminary laboratory experiments indicated that the growth of Indian mustard, and the related plant Brassica campestris (canola), which could also accumulate high levels of nickel and other metals, was significantly inhibited by the presence of moderate amounts of nickel in the soil. In an effort to overcome the inhibition of plant growth by nickel, a bacterium was isolated from a nickel contaminated soil sample; the bacterium was (i) nickel-resistant, (ii) able to grow at the cold temperatures (i.e., 5– 10 jC) that one expects to find in nickel contaminated soil environments in Canada and (iii) an active producer of ACC deaminase (Burd et al., 1998). In total, there were approximately
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4 103 nickel-resistant bacteria per gram dry weight of soil, or about 1% of the total bacterial population that was culturable on Tris-buffered low-phosphate medium. In order to isolate plant growth promoting bacteria, all of the nickel-resistant isolates were tested for the ability to grow on minimal medium with ACC as the sole source of nitrogen (Glick et al., 1995). Approximately 7% of the nickel tolerant strains also had the ACC+ phenotype. Finally, nickel-resistant bacterial strains that were also able to grow on ACC were tested for the ability to produce siderophores. Based on the idea that bacterial siderophores might facilitate the uptake of iron by plants, the best siderophore producing strain (designated SUD165) was selected for subsequent study. This strain was characterized by fatty acid analysis as Kluyvera ascorbata. In laboratory tests, it was ascertained that K. ascorbata SUD165 not only had the above-mentioned traits but could also promote plant growth in the presence of high levels of nickel (Burd et al., 1998; Ma et al., 2001). At all levels of nickel tested (i.e., 1 – 6 mM Ni2 +), using both a low and a high level of bacterial cell treatment (cell suspension absorbance at 600 nm of 0.025 or 0.50), with both canola and tomato plants, with both roots and shoots, and both in gnotobiotic growth pouches and in pots with soil, the addition of K. ascorbata SUD165 significantly decreased the toxicity of the added nickel. Moreover, the protective effect of K. ascorbata SUD165 increased as the density of the cell suspension increased. While the bacterium K. ascorbata SUD165 did not change the amount of nickel taken up per milligram dry weight of either roots or shoots and thus had no influence on amount of nickel accumulated by the plant, it did lower the amount of ethylene that was evolved by plants treated with nickel in comparison to treat with nickel in the absence of the bacterium. The simplest explanation of the data is that the bacterium protects the plant against the inhibitory effects of nickelinduced stress ethylene formation. To improve the performance of K. ascorbata SUD165, the bacterium was grown on a minimal medium that did not contain any measurable iron. Of the tens of thousands of bacteria plated, only a few colonies were able to grow under these conditions. It was reasoned that these bacteria probably contained a spontaneous mutation that caused the overproduction of the bacterial siderophores. This siderophore overproduction enabled the bacterium to sequester a sufficient amount of iron, even though the iron was present at extremely low levels, to permit these bacteria to grow. The amount of siderophores produced by each spontaneous mutant was quantified and the strain that produced the highest level of siderophores (about 100-fold more than the wild-type) was designated K. ascorbata SUD165/26 and selected for additional study (Burd et al., 2000). When the wild-type bacterium and the siderophore overproducing mutant were tested in the laboratory, as expected both of them were observed to promote the growth of tomato, canola and Indian mustard plants in the presence of inhibitory levels (generally 2 mM) of nickel, lead or zinc. In addition, the siderophore overproducing mutant decreased the inhibitory effect of the added metal on plant growth significantly more than the wildtype bacterium. Heavy metal contamination of soil is often associated with iron-deficiency in a range of different plant species (Mishra and Kar, 1974). The low iron content of plants that are grown in the presence of high levels of heavy metals generally results in these plants becoming chlorotic since iron deficiency inhibits both chloroplast development and chlorophyll biosynthesis (Imsande, 1998). Moreover, iron deficiency causes the plant to synthesize stress ethylene.
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Once they have bound iron, microbial iron-siderophore complexes can be taken up by plants and thereby serve as an iron source for plants (Bar-Ness et al., 1991). It was therefore reasoned that the best way to prevent plants from becoming chlorotic in the presence of high levels of heavy metals was to provide them with an associated siderophore-producing bacterium that could provide a sufficient amount of iron to the plant. Taken together, these results suggest a dual role for bacteria that facilitate plant growth in the presence of heavy metals. On the one hand, the bacteria lower the level of stress ethylene in the plant thereby allowing the plant to develop longer roots and thus better establish itself during early stages of growth (Burd et al., 1998; Glick et al., 1998). On the other hand, once the seedling is firmly established in the soil, the bacterium helps the plant to acquire sufficient iron for optimal plant growth, in the presence of levels of heavy metals that might otherwise make the acquisition of iron difficult (Burd et al., 2000). When the siderophore overproducing mutant was tested in the field with soil that had been contaminated with nickel over a period of many years, it was observed that both the number of Indian mustard seeds that germinated in the nickel-contaminated soil, and the size that the plants were able to attain was increased by 50 –100% by the addition of the bacterium to the soil. While additional laboratory and field testing of the selected bacterium is necessary, at this stage, the data look sufficiently promising to consider commercialization. 4.2. Arsenate Given the toxicity of arsenate to most plants, the development of a phytoremediation scheme for the detoxification of arsenate-contaminated soils is not a simple matter. For example, unlike what was observed with nickel, lead and zinc, arsenate-resistant plant growth-promoting bacteria did not significantly protect plants from arsenate inhibition. On the other hand, transgenic canola plants expressing a bacterial ACC deaminase under the control of the cauliflower mosaic virus 35S promoter, which ensures that the transgene will be expressed constitutively, were significantly more resistant to the toxic effects of arsenate than were wild-type canola plants (Nie et al., 2002). Following growth in the presence of arsenate, there were significant differences between transgenic and non-transformed canola. Regardless of the presence or absence of plant growth-promoting bacteria, a maximum of about 25% of the non-transformed seeds germinated in the presence of arsenate. By contrast, about 70% of the transgenic canola seeds germinated under the same conditions. Although a small ethylene pulse is important in breaking seed dormancy in many plants, too much ethylene can inhibit plant seed germination (Bewley and Black, 1985). In the presence of arsenate, ACC deaminase may enhance the process of germination by hydrolyzing any excess ACC that forms as a consequence of the stress, hence lowering the inhibitory level of ethylene in seeds. The fresh and dry weights of canola roots and shoots, and the shoot chlorophyll contents further support the hypothesis that lowering ethylene levels protects the plant against arsenate inhibition. When grown in the presence of arsenate, the fresh and dry weights of roots and shoots of transgenic canola, especially when they were treated with the ACC deaminase-containing plant growth-promoting bacterium Enterobacter cloacae CAL2, were much higher than with non-transformed canola. Other properties of this
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bacterial strain, in addition to ACC deaminase activity, may contribute to this result—the bacterium synthesizes IAA, siderophores and antibiotics, all of which may stimulate plant growth. In this regard, antibiotic-secreting plant growth-promoting bacterial strains can inhibit the proliferation and subsequent invasion of phytopathogens, hence protecting plants, already debilitated by arsenate in the soil, from further damage. When biomass is considered in calculating the arsenate accumulation, each transgenic canola plant accumulates approximately four times as much arsenate, on a dry-weight basis, as non-transformed canola. The higher rate of germination of transgenic canola also contributes to the total amount of arsenate accumulation. The significant increase of arsenate accumulation made by transgenic canola in conjunction with plant growthpromoting bacteria makes phytoremediation much more efficient. However, despite the improved performance of transgenic versus wild-type canola, it is unlikely that this result can be the basis of a large-scale process that is useful for the phytoremediation of arsenate contaminated soils. 4.3. Organic contaminants Polycyclic aromatic hydrocarbons are a particularly recalcitrant group of contaminants and are known to be highly persistent in the environment. There are many sources for PAH contamination in soils including creosote, fossil fuel processing and steel production. It is expensive and time consuming to remediate persistent contaminants such as PAHs from soils, and the techniques used to remediate PAH-contaminated soils tend to be inefficient. Physical removal of PAH-contaminated soil and washing of those soils with solvents is expensive, and has met with mixed results. Bioreactors have been attempted, however the contaminated soils must still be brought to the reactor for the cleanup. This is expensive, and it damages the natural structure and texture of the soil. In situ microbial remediation (i.e., bioremediation) has been attempted, but it is difficult to generate sufficient biomass in natural soils to achieve an acceptable rate of movement of hydrophobic PAHs (which are often tightly bound to soil particles) to the microbes where they can be degraded. As well, PAHs are very stable compounds, and the initial oxidation step is biologically slow and metabolically expensive. Thus, in most situations, microbial remediation alone is probably too slow to be a realistic approach to this problem. In addition, relatively few microorganisms can use high molecular weight PAHs as a sole carbon source. More recently, there have been some improvements in the strategies for bacterial remediation of contaminated soil, including inoculation with bacteria that were selected from PAH contaminated sites, or supplementing contaminated soils with nutrients (Suthersan, 2002). Nonetheless, there has only been limited success with these techniques. For bioremediation to be effective, the overall rate of PAH removal and degradation must be accelerated above current levels. One way to achieve this is to increase the amount of biomass in the contaminated soil. For this reason, the use of higher plants (i.e., phytoremediation) has received considerable attention (Cunningham et al., 1995; Cunningham and Ow, 1996). The advantages of phytoremediation compared to other approaches are: (1) it preserves the natural structure and texture of the soil; (2) energy is primarily derived from sunlight; (3) high levels of biomass in the soil can be achieved; (4) it is low in cost; and (5) it has the
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potential to be rapid. Although using plants for remediation of persistent contaminants may have advantages over other methods, many limitations exist for the large-scale application of this technology. For example, many plant species are sensitive to contaminants including PAHs so that they grow slowly and it is time consuming to establish sufficient biomass for meaningful soil remediation. In addition, in most contaminated soils, the number of microorganisms is depressed so that there are not enough bacteria either to facilitate contaminant degradation or to support plant growth. To remedy this situation, both degradative and plant growth-promoting bacteria may be added to the plant rhizosphere. Phytoremediation (i.e., degradation of organics in the presence of plants) alone is not significantly faster than bioremediation (i.e., where biodegradation of the organics is by microorganisms independent of plants) for removal of PAHs that include three rings or less (Huang, El-Alawi, Penrose, Glick and Greenberg, submitted for publication), although phytoremediation outperformed bacterial treatment with respect to removal of the larger, more strongly soil bound PAHs. Cultivating plants together with plant growth-promoting bacteria allowed the plants to germinate to a much greater extent, and then to grow well and rapidly accumulate a large amount of biomass. In addition, the plant growth-promoting bacteria in these experiments increased PAH removal; this is probably due to an alleviation of a portion of the stress imposed upon the plant by the presence of the PAHs. The plant growth-promoting bacteria increased seed germination and plant survival in heavily contaminated soils, decreased the plant dry weight to fresh weight ratio, increased the plant water content, helped plants to maintain their chlorophyll contents and chlorophyll a/b ratio, and promoted plant root growth. As a consequence of the treatment of plants with plant growth-promoting bacteria, the plants provide a greater sink for the contaminants since they are better able to survive and proliferate.
5. Conclusions Although phytoremediation has received considerable attention recently, and there are an increasing number of reports suggesting that it should become the technology of choice for the clean up of various types of environmental contamination, this technology is still in its infancy and it has yet to be used commercially to any extent. Nevertheless, it is predicted to account for approximately 10 – 15% of the growing environmental remediation market by the year 2010. However, to realize the full potential of this technology, it is necessary for plants to grow as large as possible in the presence of various environmental contaminants. One way to achieve this goal is to utilize plant growth-promoting bacteria to facilitate the growth of the plants used for phytoremediation.
Acknowledgements Work from the author’s laboratory was supported by grants from the Natural Science and Engineering Research Council, CRESTech (a province of Ontario Centre of
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Excellence) and Inco. The following individuals contributed to the work reviewed here: Genrich Burd, Donna Penrose, Varvara Grichko, Lin Nie, George Dixon, Wenbo Ma, Bruce Greenberg, XiaoDong Huang, Jiping Li, Saleh Shah and Cheryl Patten.
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Imsande J. Iron, sulfur, and chlorophyll deficiencies: a need for an integrative approach in plant physiology. Physiol Plant 1998;103:139 – 44. Jacobson CB, Pasternak JJ, Glick BR. Partial purification and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Can J Microbiol 1994;40:1019 – 25. John P. How plant molecular biologists revealed a surprising relationship between two enzymes, which took an enzyme out of a membrane where it was not located, and put it into the soluble phase where it could be studied. Plant Mol Biol Rep 1991;9:192 – 4. Kende H. Enzymes of ethylene biosynthesis. Plant Physiol 1989;91:1 – 4. Klee HJ, Kishore GM. Control of Fruit Ripening and Senescence in Plants. United States Patent Number: 5,702,933; 1992. Kloepper JW, Lifshitz R, Zablotowicz RM. Free-living bacterial inocula for enhancing crop productivity. Trends Biotechnol 1989;7:39 – 43. Kumar PBAN, Dushenkov V, Motto H, Raskin I. Phytoextraction: the use of plants to remove heavy metals. Environ Sci Technol 1995;29:1232 – 8. Lambert B, Joos H. Fundamental aspects of rhizobacterial plant growth promotion research. Trends Biotechnol 1989;7:215 – 9. Ma W, Zalec K, Glick BR. Effects of the bioluminescence-labeling of the soil bacterium Kluyvera ascorbata SUD165/26. FEMS Microbiol Ecol 2001;35:137 – 44. Ma W, Sebestianova S, Sebestian J, Burd GI, Guinel F, Glick BR. Prevalence of 1-aminocyclopropaqne-1carboxylate in deaminase in Rhizobia spp. Antonie van Leeuwenhoek 2003;83:285 – 91. Minami R, Uchiyama K, Murakami T, Kawai J, Mikami K, Yamada T, et al. Properties, sequence, and synthesis in Escherichia coli of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus. J Biochem 1998;123:1112 – 8. Mishra D, Kar M. Nickel in plant growth and metabolism. Bot Rev 1974;40:395 – 452. Morgan PW, Drew CD. Ethylene and plant responses to stress. Physiol Plant 1997;100:620 – 30. Nayani S, Mayak S, Glick BR. The effect of plant growth promoting rhizobacteria on the senescence of flower petals. Ind J Exp Biol 1998;36:836 – 9. Nie L, Shah S, Burd GI, Dixon DG, Glick BR. Phytoremediation of arsenate contaminated soil by transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae CAL2. Plant Physiol Biochem 2002;40:355 – 61. Patten CL, Glick BR. Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol 1996;42:207 – 20. Patten CL, Glick BR. The role of bacterial indoleacetic acid in the development of the host plant root system. Appl Environ Microbiol 2002;68:3795 – 801. Penrose DM, Glick BR. Levels of 1-aminocyclopropane-1-carboxylic acid (ACC) in exudates and extracts of canola seeds treated with plant growth-promoting bacteria. Can J Microbiol 2001;47:368 – 72. Penrose DM, Moffatt BA, Glick BR. Determination of 1-aminocyclopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings. Can J Microbiol 2001;47:77 – 80. Raskin I, Kumar PBAN, Dushenkov S, Salt DE. Bioconcentration of heavy metals by plants. Curr Opin Biotechnol 1994;5:285 – 90. Raskin I, Smith RD, Salt DE. Phytoremediation of metals: using plants to remove pollutants from the environment. Curr Opin Biotechnol 1997;8:221 – 6. Suthersan SS. Natural and enhanced remediation systems. Boca Raton: CRC Press; 2002. p. 239 – 67. Wang C, Knill E, Glick BR, De´fago G. Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growthpromoting and disease-suppressive capacities. Can J Microbiol 2000;46:898 – 907. Whipp JM. Carbon utilization. In: Lynch JM, editor. The rhizosphere. Chichester, UK: Wiley; 1990. p. 59 – 97. Yang SF, Hoffman NE. Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 1984;35:155 – 89.
Phytoremediation: synergistic use of plants and bacteria to clean up the environment Bernard R. Glick * Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
Abstract Phytoremediation is a relatively new approach to removing contaminants from the environment. It may be defined as the use of plants to remove, destroy or sequester hazardous substances from the environment. Unfortunately, even plants that are relatively tolerant of various environmental contaminants often remain small in the presence of the contaminant. To remedy this situation, plant growth-promoting bacteria that facilitate the proliferation of various plants especially under environmentally stressful conditions may be added to the roots of plants. These bacteria have been selected to lower the level of growth-inhibiting stress ethylene within the plant and also to provide the plant with iron from the soil. The net result of adding these bacteria to plants is a significant increase in both the number of seeds that germinate and the amount of biomass that the plants are able to attain, making phytoremediation in the presence of plant growth-promoting bacteria a much faster and more efficient process. D 2003 Elsevier Inc. All rights reserved. Keywords: Phytoremediation; Plants; Bacteria
1. The problem: environmental contamination The removal from the environment of many potentially toxic compounds is complicated by the numerous classes and types of these chemicals. For example, many soils are contaminated with one or more metals, radioactive or inorganic compounds. Of these, the metals may include lead, zinc, cadmium, selenium, chromium, cobalt, copper, nickel and mercury; the radioactive compounds may be uranium, cesium or strontium; and the other
* Tel.: +1-519-888-4567; fax: +1-519-746-0614. E-mail address: [email protected] (B.R. Glick). 0734-9750/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0734-9750(03)00055-7
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inorganic compounds might include arsenic, sodium, nitrate, ammonia or phosphate. Soil may become polluted with high concentrations of metals by either a natural phenomenon such as proximity to an ore body, or as a consequence of industrial activities. The remediation of heavily metal-contaminated soils often involves excavation and removal of soil to ‘‘secured’’ landfills, a technology that is expensive and requires site restoration. As an alternative, in the past few years, several groups of scientists have begun to develop technological approaches to using certain plants to remove metal contaminants from the soil. In addition to the above-mentioned inorganic compounds, soils and water systems may also be contaminated with organic compounds including chlorinated solvents like trichloroethylene; explosives such as trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-hexahydrotriazine (RDX); petroleum hydrocarbons including benzene, toluene and xylene (BTX), polyaromatic hydrocarbons (PAHs); and pesticides such as atrazine and bentazon. While many of these compounds can be metabolized by some soil bacteria, this process is usually slow and inefficient, in part as a consequence of the relatively low numbers of these degradative microorganisms in soil. However, there is mounting evidence that the biodegradation of recalcitrant organic compounds in the soil is enhanced around the roots of plants.
2. Phytoremediation: a solution to environmental contamination? One recently developed method of environmental clean-up is called phytoremediation (Cunningham and Berti, 1993; Kumar et al., 1995; Raskin et al., 1994, 1997). This procedure may be defined as the use of plants to remove, destroy or sequester hazardous substances from the environment. Phytoremediation of metals and other inorganic compounds may take one of several forms: phytoextraction, the absorption and concentration of metals from the soil into the roots and shoots of the plant; rhizofiltration, the use of plant roots to remove metals from effluents; phytostabilization, the use of plants to reduce the spread of metals in the environment; or phytovolatilization, the uptake and release into the atmosphere of volatile materials such as mercury- or arsenic-containing compounds. Phytoremediation of organic compounds may occur by phytostabilization; phytostimulation, the stimulation of microbial biodegradation in the rhizosphere, the area around the roots of plants; or by phytotransformation, the absorption and degradation of organic contaminants by the plant. Following the testing of a large number of different plants, a number of plants that can naturally accumulate large amounts of metal have been identified and are being studied for the phytoremediation of metals that are present in the environment. These plants, called hyperaccumulators, are often found growing in areas with elevated metal concentrations in the soil. Unfortunately, in the presence of very high concentrations of metals, even hyperaccumulating plants attain only a small size. That is, high concentrations of metals are inhibitory to the growth of plants, even those plants that are capable of hyperaccumulating metals. Depending upon the amount of metal at a particular site and the type of soil at that site, it could take 15– 20 years to completely remove the metal from the soil and thereby remediate the site, even with hyperaccumulating plants. This is a timeframe that is usually considered to be too slow for practical application.
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A number of different types of plants are effective at stimulating the degradation of organic molecules in the rhizosphere. Typically, these plants all have extensive and fibrous roots, which form an extended rhizosphere; these plants include many common grasses as well as corn, wheat, soybean, peas and beans. In addition, several varieties of trees can take up and degrade some organic contaminants. For example, plants with phytotransformation activity may contain nitroreductases, which are useful for degrading TNT and other nitroaromatics, dehalogenases for the degradation of chlorinated solvents and pesticides, and laccases that can degrade anilines such as triaminotoluene.
3. Plant growth-promoting bacteria Beneficial free-living soil bacteria are generally referred to as plant growth-promoting rhizobacteria and are found in association with the roots of many different plants (Glick et al., 1999). The high concentration of bacteria around the roots, i.e., in the rhizosphere, presumably occurs because of the presence of high levels of nutrients (especially small molecules such as amino acids, sugars and organic acids) that are exuded from the roots of most plants, and can then be used to support bacterial growth and metabolism (Whipp, 1990; Bayliss et al., 1997; Penrose and Glick, 2001). Plant growth-promoting bacteria can positively influence plant growth and development in two different ways: indirectly or directly (Glick et al., 1999). The indirect promotion of plant growth occurs when these bacteria decrease or prevent some of the deleterious effects of a phytopathogenic organism. Bacteria can directly promote plant growth by providing the plant with a compound that is synthesized by the bacterium or by facilitating the uptake of nutrients from the environment by the plant. Plant growthpromoting bacteria may: fix atmospheric nitrogen and supply it to plants; synthesize siderophores which can solubilize and sequester iron from the soil and provide it to plant cells; synthesize several different phytohormones including auxins and cytokinins which can enhance various stages of plant growth; have mechanisms for the solubilization of minerals such as phosphorus which then become more readily available for plant growth; and contain enzymes that can modulate plant growth and development (Brown, 1974; Davison, 1988; Kloepper et al., 1989; Lambert and Joos, 1989; Patten and Glick, 1996; Glick et al., 1998). A particular bacterium may affect plant growth and development using any one, or more, of these mechanisms and a bacterium may utilize different mechanisms under different conditions. For example, bacterial siderophore synthesis is likely to be induced only in soils that do not contain sufficient levels of iron. Similarly, bacteria do not fix nitrogen when sufficient fixed nitrogen is available. 3.1. The role of ACC deaminase in the reduction of plant ethylene In higher plants, ethylene is produced from L-methionine via the intermediates, Sadenosyl-L-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC; Yang and Hoffman, 1984). The enzymes involved in this metabolic sequence are SAM synthetase, which catalyzes the conversion of methionine to SAM (Giovanelli et al., 1980); ACC synthase, which is responsible for the hydrolysis of SAM to ACC and 5V-
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methylthioadenosine (Kende, 1989); and ACC oxidase, which metabolizes ACC to ethylene, carbon dioxide and cyanide (John, 1991). In 1978, an enzyme capable of degrading ACC was isolated from Pseudomonas sp. strain ACP (Honma and Shimomura, 1978). Since then, ACC deaminase has been detected in the fungus, Penicillium citrinum (Honma, 1993), and in a number of bacterial strains (Klee and Kishore, 1992; Jacobson et al., 1994; Glick et al., 1995; Campbell and Thomson, 1996; Burd et al., 1998; Belimov et al., 2001; Ghosh et al., 2003; Ma et al., 2003) and in the yeast, Hansenula saturnus (Minami et al., 1998), all of which originated from the soil either as soil sample isolates or as microbes typically found in the soil. This enzyme cleaves the plant ethylene precursor, ACC, to produce ammonia and a-ketobutyrate. Glick et al. (1998) proposed that microorganisms that contain the enzyme ACC deaminase can all act to promote plant growth since they can act as a sink for ACC and thereby lower ethylene levels in a developing or stressed plant. Ethylene is important for normal plant development in plants as well as for their response to stress (Deikman, 1997). Ethylene is important during the early phase of plant growth; it is required by many plant species for seed germination, and the rate of ethylene production increases during germination and seedling growth (Abeles et al., 1992). However, high levels of ethylene lead to inhibition of root elongation. A model was developed whereby plant growth-promoting bacteria that possess the enzyme ACC deaminase and are bound to seeds or roots of seedlings, can reduce the amount of plant ethylene and the extent of its inhibition on root elongation (Glick et al., 1998). In this model, the plant growth-promoting bacteria bind to the surface of either the seed or root of a developing plant; in response to tryptophan and other small molecules in the seed or root exudates (Whipp, 1990; Bayliss et al., 1997; Penrose and Glick, 2001), the plant growth-promoting bacteria synthesize and secrete indole acetic acid (IAA) (Patten and Glick, 1996, 2002), some of which may be taken up by the plant. This IAA, together with endogenous plant IAA, can either stimulate plant growth or induce the synthesis of ACC synthase, which converts SAM to ACC. A portion of the ACC produced by this latter reaction is exuded from seeds or plant roots along with other small molecules normally present in seed or root exudates (Bayliss et al., 1997; Penrose and Glick, 2001). The ACC in the exudates is taken up by the bacteria and is subsequently converted by ACC deaminase to ammonia and a-ketobutyrate. This decreases the amount of ACC outside the plant so that the plant must exude increasing amounts of ACC in order to maintain the equilibrium between internal and external ACC levels. Thus, in the first instance, the bacteria induce the plant to synthesize more ACC than it would otherwise need and, secondly, it stimulates the exudation of ACC from the plant. As a result of the ACC deaminase-containing bacteria lowering the ACC level within the plant and acting as a sink for ACC, the amount of ethylene that is produced by the plant is also reduced. When tested, strains of ACC deaminase-containing plant growth-promoting bacteria were found to reduce the amount of ACC that was detectable by HPLC, and hence the ethylene levels in canola seedlings were also lowered (Penrose et al., 2001). Moreover, ACC deaminase-containing plant growth-promoting bacteria promote root elongation in a variety of (ethylene sensitive) plants (Hall et al., 1996). There is also a considerable decrease in the levels of ‘‘stress ethylene’’—the accelerated biosynthesis of ethylene
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associated with biological and environmental stresses, and pathogen attack (Morgan and Drew, 1997)—in plants grown in the presence of ACC deaminase-containing plant growth-promoting bacteria. The deleterious effects of flooding on tomato plants (Grichko and Glick, 2001) were decreased and the shelf life of the petals of ethylene sensitive cut flowers was prolonged following treatment with ACC deaminase-containing plant growthpromoting bacteria (Nayani et al., 1998). Moreover, biocontrol strains of bacteria carrying ACC deaminase genes were able to more effectively protect plants against various phytopathogens: cucumber plants were protected against Pythium damping-off, and potato slices, exposed to Erwinia caratovora in a small sealed bag, were protected against Erwinia soft rot (Wang et al., 2000). In addition, canola seedlings grown in the presence of high levels of nickel, produced much less ethylene when the seeds were inoculated with an ACC deaminase-containing nickel-resistant plant growth-promoting strain that (Burd et al., 1998). It appears that the ‘‘stress ethylene’’ produced in each of these situations, and the damage caused by it, was reduced by the activity of ACC deaminase that lowered the level of ethylene produced by the plant.
4. The use of plant growth-promoting bacteria in phytoremediation 4.1. Metals While plants grown on metal contaminated soils might be able to withstand some of the inhibitory effects of high concentrations of metals within a plant, two features of most plants could result in a decrease in plant growth and viability. That is, in the presence of high levels of metals, most plants (i) synthesize stress ethylene and (ii) become severely depleted in the amount of iron that they contained. Moreover, plant growth-promoting bacteria may be used to relieve some of the toxicity of metals to plants. This could occur in two different ways. As indicated earlier, the use of ACC deaminase-containing plant growth-promoting bacteria would be expected to decrease the level of stress ethylene in a plant growing in soil that contained high levels of metal. In addition, plants are able to take up and utilize complexes between bacterial siderophores and iron. Plant siderophores bind to iron with a much lower affinity than bacterial siderophores so that in metal contaminated soils a plant is unable to accumulate a sufficient amount of iron unless bacterial siderophores are present. Our primary objective was the development of a phytoremediation system that could be used to help to remove nickel from soil as inexpensively and as quickly as possible. Prior to this work, there were reports in the scientific literature that indicated that Brassica juncea (Indian mustard) was a nickel-hyperaccumulating plant and could be used for this purpose. However, preliminary laboratory experiments indicated that the growth of Indian mustard, and the related plant Brassica campestris (canola), which could also accumulate high levels of nickel and other metals, was significantly inhibited by the presence of moderate amounts of nickel in the soil. In an effort to overcome the inhibition of plant growth by nickel, a bacterium was isolated from a nickel contaminated soil sample; the bacterium was (i) nickel-resistant, (ii) able to grow at the cold temperatures (i.e., 5– 10 jC) that one expects to find in nickel contaminated soil environments in Canada and (iii) an active producer of ACC deaminase (Burd et al., 1998). In total, there were approximately
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4 103 nickel-resistant bacteria per gram dry weight of soil, or about 1% of the total bacterial population that was culturable on Tris-buffered low-phosphate medium. In order to isolate plant growth promoting bacteria, all of the nickel-resistant isolates were tested for the ability to grow on minimal medium with ACC as the sole source of nitrogen (Glick et al., 1995). Approximately 7% of the nickel tolerant strains also had the ACC+ phenotype. Finally, nickel-resistant bacterial strains that were also able to grow on ACC were tested for the ability to produce siderophores. Based on the idea that bacterial siderophores might facilitate the uptake of iron by plants, the best siderophore producing strain (designated SUD165) was selected for subsequent study. This strain was characterized by fatty acid analysis as Kluyvera ascorbata. In laboratory tests, it was ascertained that K. ascorbata SUD165 not only had the above-mentioned traits but could also promote plant growth in the presence of high levels of nickel (Burd et al., 1998; Ma et al., 2001). At all levels of nickel tested (i.e., 1 – 6 mM Ni2 +), using both a low and a high level of bacterial cell treatment (cell suspension absorbance at 600 nm of 0.025 or 0.50), with both canola and tomato plants, with both roots and shoots, and both in gnotobiotic growth pouches and in pots with soil, the addition of K. ascorbata SUD165 significantly decreased the toxicity of the added nickel. Moreover, the protective effect of K. ascorbata SUD165 increased as the density of the cell suspension increased. While the bacterium K. ascorbata SUD165 did not change the amount of nickel taken up per milligram dry weight of either roots or shoots and thus had no influence on amount of nickel accumulated by the plant, it did lower the amount of ethylene that was evolved by plants treated with nickel in comparison to treat with nickel in the absence of the bacterium. The simplest explanation of the data is that the bacterium protects the plant against the inhibitory effects of nickelinduced stress ethylene formation. To improve the performance of K. ascorbata SUD165, the bacterium was grown on a minimal medium that did not contain any measurable iron. Of the tens of thousands of bacteria plated, only a few colonies were able to grow under these conditions. It was reasoned that these bacteria probably contained a spontaneous mutation that caused the overproduction of the bacterial siderophores. This siderophore overproduction enabled the bacterium to sequester a sufficient amount of iron, even though the iron was present at extremely low levels, to permit these bacteria to grow. The amount of siderophores produced by each spontaneous mutant was quantified and the strain that produced the highest level of siderophores (about 100-fold more than the wild-type) was designated K. ascorbata SUD165/26 and selected for additional study (Burd et al., 2000). When the wild-type bacterium and the siderophore overproducing mutant were tested in the laboratory, as expected both of them were observed to promote the growth of tomato, canola and Indian mustard plants in the presence of inhibitory levels (generally 2 mM) of nickel, lead or zinc. In addition, the siderophore overproducing mutant decreased the inhibitory effect of the added metal on plant growth significantly more than the wildtype bacterium. Heavy metal contamination of soil is often associated with iron-deficiency in a range of different plant species (Mishra and Kar, 1974). The low iron content of plants that are grown in the presence of high levels of heavy metals generally results in these plants becoming chlorotic since iron deficiency inhibits both chloroplast development and chlorophyll biosynthesis (Imsande, 1998). Moreover, iron deficiency causes the plant to synthesize stress ethylene.
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Once they have bound iron, microbial iron-siderophore complexes can be taken up by plants and thereby serve as an iron source for plants (Bar-Ness et al., 1991). It was therefore reasoned that the best way to prevent plants from becoming chlorotic in the presence of high levels of heavy metals was to provide them with an associated siderophore-producing bacterium that could provide a sufficient amount of iron to the plant. Taken together, these results suggest a dual role for bacteria that facilitate plant growth in the presence of heavy metals. On the one hand, the bacteria lower the level of stress ethylene in the plant thereby allowing the plant to develop longer roots and thus better establish itself during early stages of growth (Burd et al., 1998; Glick et al., 1998). On the other hand, once the seedling is firmly established in the soil, the bacterium helps the plant to acquire sufficient iron for optimal plant growth, in the presence of levels of heavy metals that might otherwise make the acquisition of iron difficult (Burd et al., 2000). When the siderophore overproducing mutant was tested in the field with soil that had been contaminated with nickel over a period of many years, it was observed that both the number of Indian mustard seeds that germinated in the nickel-contaminated soil, and the size that the plants were able to attain was increased by 50 –100% by the addition of the bacterium to the soil. While additional laboratory and field testing of the selected bacterium is necessary, at this stage, the data look sufficiently promising to consider commercialization. 4.2. Arsenate Given the toxicity of arsenate to most plants, the development of a phytoremediation scheme for the detoxification of arsenate-contaminated soils is not a simple matter. For example, unlike what was observed with nickel, lead and zinc, arsenate-resistant plant growth-promoting bacteria did not significantly protect plants from arsenate inhibition. On the other hand, transgenic canola plants expressing a bacterial ACC deaminase under the control of the cauliflower mosaic virus 35S promoter, which ensures that the transgene will be expressed constitutively, were significantly more resistant to the toxic effects of arsenate than were wild-type canola plants (Nie et al., 2002). Following growth in the presence of arsenate, there were significant differences between transgenic and non-transformed canola. Regardless of the presence or absence of plant growth-promoting bacteria, a maximum of about 25% of the non-transformed seeds germinated in the presence of arsenate. By contrast, about 70% of the transgenic canola seeds germinated under the same conditions. Although a small ethylene pulse is important in breaking seed dormancy in many plants, too much ethylene can inhibit plant seed germination (Bewley and Black, 1985). In the presence of arsenate, ACC deaminase may enhance the process of germination by hydrolyzing any excess ACC that forms as a consequence of the stress, hence lowering the inhibitory level of ethylene in seeds. The fresh and dry weights of canola roots and shoots, and the shoot chlorophyll contents further support the hypothesis that lowering ethylene levels protects the plant against arsenate inhibition. When grown in the presence of arsenate, the fresh and dry weights of roots and shoots of transgenic canola, especially when they were treated with the ACC deaminase-containing plant growth-promoting bacterium Enterobacter cloacae CAL2, were much higher than with non-transformed canola. Other properties of this
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bacterial strain, in addition to ACC deaminase activity, may contribute to this result—the bacterium synthesizes IAA, siderophores and antibiotics, all of which may stimulate plant growth. In this regard, antibiotic-secreting plant growth-promoting bacterial strains can inhibit the proliferation and subsequent invasion of phytopathogens, hence protecting plants, already debilitated by arsenate in the soil, from further damage. When biomass is considered in calculating the arsenate accumulation, each transgenic canola plant accumulates approximately four times as much arsenate, on a dry-weight basis, as non-transformed canola. The higher rate of germination of transgenic canola also contributes to the total amount of arsenate accumulation. The significant increase of arsenate accumulation made by transgenic canola in conjunction with plant growthpromoting bacteria makes phytoremediation much more efficient. However, despite the improved performance of transgenic versus wild-type canola, it is unlikely that this result can be the basis of a large-scale process that is useful for the phytoremediation of arsenate contaminated soils. 4.3. Organic contaminants Polycyclic aromatic hydrocarbons are a particularly recalcitrant group of contaminants and are known to be highly persistent in the environment. There are many sources for PAH contamination in soils including creosote, fossil fuel processing and steel production. It is expensive and time consuming to remediate persistent contaminants such as PAHs from soils, and the techniques used to remediate PAH-contaminated soils tend to be inefficient. Physical removal of PAH-contaminated soil and washing of those soils with solvents is expensive, and has met with mixed results. Bioreactors have been attempted, however the contaminated soils must still be brought to the reactor for the cleanup. This is expensive, and it damages the natural structure and texture of the soil. In situ microbial remediation (i.e., bioremediation) has been attempted, but it is difficult to generate sufficient biomass in natural soils to achieve an acceptable rate of movement of hydrophobic PAHs (which are often tightly bound to soil particles) to the microbes where they can be degraded. As well, PAHs are very stable compounds, and the initial oxidation step is biologically slow and metabolically expensive. Thus, in most situations, microbial remediation alone is probably too slow to be a realistic approach to this problem. In addition, relatively few microorganisms can use high molecular weight PAHs as a sole carbon source. More recently, there have been some improvements in the strategies for bacterial remediation of contaminated soil, including inoculation with bacteria that were selected from PAH contaminated sites, or supplementing contaminated soils with nutrients (Suthersan, 2002). Nonetheless, there has only been limited success with these techniques. For bioremediation to be effective, the overall rate of PAH removal and degradation must be accelerated above current levels. One way to achieve this is to increase the amount of biomass in the contaminated soil. For this reason, the use of higher plants (i.e., phytoremediation) has received considerable attention (Cunningham et al., 1995; Cunningham and Ow, 1996). The advantages of phytoremediation compared to other approaches are: (1) it preserves the natural structure and texture of the soil; (2) energy is primarily derived from sunlight; (3) high levels of biomass in the soil can be achieved; (4) it is low in cost; and (5) it has the
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potential to be rapid. Although using plants for remediation of persistent contaminants may have advantages over other methods, many limitations exist for the large-scale application of this technology. For example, many plant species are sensitive to contaminants including PAHs so that they grow slowly and it is time consuming to establish sufficient biomass for meaningful soil remediation. In addition, in most contaminated soils, the number of microorganisms is depressed so that there are not enough bacteria either to facilitate contaminant degradation or to support plant growth. To remedy this situation, both degradative and plant growth-promoting bacteria may be added to the plant rhizosphere. Phytoremediation (i.e., degradation of organics in the presence of plants) alone is not significantly faster than bioremediation (i.e., where biodegradation of the organics is by microorganisms independent of plants) for removal of PAHs that include three rings or less (Huang, El-Alawi, Penrose, Glick and Greenberg, submitted for publication), although phytoremediation outperformed bacterial treatment with respect to removal of the larger, more strongly soil bound PAHs. Cultivating plants together with plant growth-promoting bacteria allowed the plants to germinate to a much greater extent, and then to grow well and rapidly accumulate a large amount of biomass. In addition, the plant growth-promoting bacteria in these experiments increased PAH removal; this is probably due to an alleviation of a portion of the stress imposed upon the plant by the presence of the PAHs. The plant growth-promoting bacteria increased seed germination and plant survival in heavily contaminated soils, decreased the plant dry weight to fresh weight ratio, increased the plant water content, helped plants to maintain their chlorophyll contents and chlorophyll a/b ratio, and promoted plant root growth. As a consequence of the treatment of plants with plant growth-promoting bacteria, the plants provide a greater sink for the contaminants since they are better able to survive and proliferate.
5. Conclusions Although phytoremediation has received considerable attention recently, and there are an increasing number of reports suggesting that it should become the technology of choice for the clean up of various types of environmental contamination, this technology is still in its infancy and it has yet to be used commercially to any extent. Nevertheless, it is predicted to account for approximately 10 – 15% of the growing environmental remediation market by the year 2010. However, to realize the full potential of this technology, it is necessary for plants to grow as large as possible in the presence of various environmental contaminants. One way to achieve this goal is to utilize plant growth-promoting bacteria to facilitate the growth of the plants used for phytoremediation.
Acknowledgements Work from the author’s laboratory was supported by grants from the Natural Science and Engineering Research Council, CRESTech (a province of Ontario Centre of
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Excellence) and Inco. The following individuals contributed to the work reviewed here: Genrich Burd, Donna Penrose, Varvara Grichko, Lin Nie, George Dixon, Wenbo Ma, Bruce Greenberg, XiaoDong Huang, Jiping Li, Saleh Shah and Cheryl Patten.
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