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TECHNICAL REPORTS

Bioremediation and Biodegradation

Comparison of Plant Families in a Greenhouse Phytoremediation Study on an Aged Polycyclic Aromatic Hydrocarbon–Contaminated Soil

^a Dep. of Chemical and Biological Engineering, Colorado State Univ., Fort Collins, CO 80523
^b Dep. of Biology, Colorado State Univ., Fort Collins, CO 80523
^c Dep. of Microbiology, Immunology, and Pathology, Colorado State Univ., Fort Collins, CO 80523

^* Corresponding author (kenneth.reardon{at}colostate.edu).

Received for publication September 14, 2006.

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, recalcitrant, and potentially carcinogenic pollutants. Plants and their associated rhizosphere microbes can promote PAH dissipation, offering an economic and ecologically attractive remediation technique. This study focused on the effects of different types of vegetation on PAH removal and on the interaction between the plants and their associated microorganisms. Aged PAH-polluted soil with a total PAH level of 753 mg kg^–1 soil dry weight was planted with 18 plant species representing eight families. The levels of 17 soil PAHs were monitored over 14 mo. The size of soil microbial populations of PAH degraders was also monitored. Planting significantly enhanced the dissipation rates of all PAHs within the first 7 mo, but this effect was not significant after 14 mo. Although the extent of removal of lower-molecular-weight PAHs was similar for planted and unplanted control soils after 14 mo, the total mass of five- and six-ring PAHs removed was significantly greater in planted soils at the 7- and 14-mo sampling points. Poaceae (grasses) were the most effective of the families tested, and perennial ryegrass was the most effective species; after 14 mo, soils planted with perennial ryegrass contained 30% of the initial total PAH concentration (compared with 51% of the initial concentrations in unplanted control soil). Although the presence of some plant species led to higher populations of PAH degraders, there was no correlation across plant species between PAH dissipation and the size of the PAH-degrading population. Research is needed to understand differences among plant families for stimulating PAH dissipation.

Abbreviations: M_m, Molecular mass • MPN, most probable number • PAH, polycyclic aromatic hydrocarbons

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
PHYTOREMEDIATION, as defined by Dietz and Schnoor (2001), is the use of vegetation for the in situ treatment of contaminated soils, sediments, and/or ground water. It has gained acceptance over the last decade as a cost-effective, noninvasive, and complementary technology for engineering-based remediation methods (Pilon-Smits, 2005). Phytoremediation has been applied successfully in cleaning up sites contaminated with a variety of organic compounds, such as the solvent trichloroethylene, the herbicide atrazine, the explosive trinitrotoluene, and petroleum hydrocarbons including oil, gasoline, benzene, toluene, and polycyclic aromatic hydrocarbons (Pilon-Smits, 2005 and references therein). In most cases, the mechanisms involved in phytoremediation of organic contaminants are poorly understood, and little information is available on the efficacy of different plant species.

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic contaminants whose structure consists of fused aromatic rings (Olson et al., 2003). PAHs tend to sorb to soil and thus are not very bioavailable, nor are they translocated effectively in plants. The lower molecular mass (M_m) PAHs are less hydrophobic (log K_ow 3–5) and more water soluble than the high M_m PAHs; thus, they are fairly biodegradable under aerobic conditions (Juhasz and Naidu, 2000; Karthikeyan and Kulakow, 2003; Olson et al., 2003). High M_m PAHs (log K_ow > 5) are much less bioavailable and undergo very slow aerobic biodegradation. Their aerobic biodegradation typically is limited to co-metabolism. For example, naphthalene, a two-ringed PAH, may induce the activity of oxygenase enzymes responsible for the initial degradation of high M_m PAHs. Consequently, low M_m PAHs are more rapidly degraded, whereas high M_m PAHs are recalcitrant in the environment (Dietz and Schnoor, 2001; Olson et al., 2003).

Vegetation can affect the removal of soil PAHs in several ways. Although plant uptake and transport of PAHs seems unlikely due to their physical–chemical characteristics, plants may stimulate PAH degradation through interactions with the soil microbial population (bacteria and fungi). The growth and metabolic activity of these micro-organisms can be substantially enhanced by root-released compounds, which include primary and secondary plant metabolites (Juhasz and Naidu, 2000; Kuiper et al., 2001, 2004; Olson et al., 2003). Rhizodeposition into the soil can include root exudates and lysates from root turnover. A forensic examination of vegetation at a former sludge basin has established that the local vegetative community can successfully invade a polluted site (Olson and Fletcher, 2000). Over time, the initial pioneer species were replaced as a result of plant succession (Olson et al., 2001). In this forensic study, the soil around plant roots contained significantly lower PAH levels than unvegetated bulk soil, and different plant species were found to affect PAH levels to different extents. These studies demonstrate the importance of vegetation and of plant species selection. Better knowledge of the characteristics of different plant families (and individual plant species), with respect to their capacity to facilitate PAH dissipation, would be beneficial for the development of more efficient phytoremediation strategies.

In recent years, several other PAH phytoremediation studies have been performed. In many cases, non-aged, PAH-spiked soil was used for phytoremediation experiments in the field, greenhouse, or laboratory (Jin et al., 1999; Huang et al., 2004a,b; Smith et al., 2006). However, in a study using aged creosote-contaminated soil, Allard et al. (2000) concluded that the age of the contamination is the limiting factor for its degradation. Therefore, the use of soils to which PAHs were freshly added is likely to yield results that do not correspond to real field situations.

The goal of this study was to obtain further insights into the effects of vegetation on PAH removal, including the relative effectiveness of different plant species and the interaction between the plant and its associated rhizosphere microbial population. Eighteen different plant species, representing eight families and a diversity of habitats, were tested for their capacity to stimulate PAH dissipation over a 14-mo period. Several of these plant species were shown in other studies to be effective in phytoremediation of organic contaminants via phytostimulation (Olson et al., 2001; Huang et al., 2004a,b; Joner et al., 2004; Johnson et al., 2005; Parrish et al., 2005a,b). In addition to a time-dependent characterization of 17 different soil PAHs, the PAH-degrading rhizosphere microbial community was monitored, allowing the assessment of any correlation between plant species and the abundance of microorganisms involved in PAH degradation. Another key feature of this study was the use of aged, not spiked, PAH-contaminated soil to better represent field sites and thus provide results that are more meaningful for the design of phytoremediation strategies.

	Materials and Methods

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
Experimental Soil
The aged PAH-contaminated soil used for this study was obtained from the US Coast Guard Housing Area at the Alameda Naval Air Station (NAS) (Alameda, CA). Alameda NAS comprises 2479 acres of property, which consist of a combination of bay land and mud flats. Before its acquisition by the US Army (1930), at least two large industrial sites, a borax-processing plant and an oil refinery, were located on the Eastern end of the Alameda NAS. In 1997, the Alameda NAS was closed and currently belongs to the city of Alameda. The soil was received in two barrels (209 L each), which were thoroughly homogenized using a Mini-Microenfractionator (H&H Ecosystems, North Bonneville, WA). This device uses a large impeller and air flows to intensely mix soil. The homogenized soil was tested for general soil properties by the Soil, Water, and Plant Testing Laboratory, Colorado State University (Fort Collins, CO). The soil characteristics are listed in Table 1. The mixed soil was distributed into 2-L plastic pots, in which plant seeds were sown directly.

Gas Chromatography
A gas chromatograph (Hewlett-Packard 5890 GC) equipped with a flame-ionization detector (GC-FID) and autosampler was used to determine PAH concentrations in extracted samples. A Restek Rtx-5 ms capillary column (30 m by 0.32 mm) with a 1.0-µm film thickness was used to separate PAHs. The splitless injector was maintained at a constant 280°C. The initial oven temperature was 60°C; after 1 min the temperature increased at 20°C/min to 200°C and then increased at 6°C/min to 310°C, where it was held for 3 min. The detector temperature was maintained constant at 300°C. Ultrapure hydrogen, oxygen, and helium (carrier gas) were used throughout the analyses.

Microbial Enumeration
The PAH-degrading microbial population was enumerated using the most-probable number (MPN) procedure described by Wrenn and Venosa (1996). Soil dilutions in a range of 10^–1 to 10^–11 were prepared using sterile, double-distilled water. Ten microliters of a four-PAH solution (10 g L^–1 phenanthrene, 1.0 g L^–1 chrysene, 1.0 g L^–1 benzo(a)pyrene, and 1.0 g L^–1 pyrene in pentane) was pipetted into each well of a 96-well plate, and the pentane was allowed to evaporate. Each well was then filled with 180 L of Bushnell-Haas Broth medium (1.0 g L^–1 KH₂PO₄, 1.0 g L^–1 K₂HPO₄, 1.0 g L^–1 NH₄NO₃ g L^–1, 0.2 MgSO₄•7H₂O, 0.05 g L^–1 FeCl₃, 0.02 g L^–1 CaCl₂•2H₂O). Twenty microliters of each soil dilution was then added to each well. The 96-well plates were allowed to incubate at room temperature for 3 wk with the lid on. PAH degradation resulted in a yellow-brown color, which was detected by eye and recorded as a positive score for the well. These scores were used to estimate the MPN of PAH-degrading microbes in the soil as described by Wrenn and Venosa (1996).

Statistical Analyses
Statistical analyses (Student's t test, correlation analysis) were performed using JMP-IN software (SAS Institute Cory, NC).

	Results

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
Effects of Vegetation on Total Soil PAH Concentrations
All 18 plant species germinated and grew well in the polluted soil, which had a total initial PAH level of 753 mg kg^–1 soil DW. No phytotoxicity symptoms, such as chlorosis or necrosis, were observed throughout this study. At the end of the study, the pots were fully vegetated, and the roots had spread throughout the 2-L pots.

Polycyclic aromatic hydrocarbon concentrations in planted and unplanted soils are listed in Table 3 and presented in Fig. 1 (by plant family) and Fig. 2 (by PAH ring number); the complete data set is available as supplementary material. Under unplanted (control) conditions, the total soil PAH concentration decreased from 753 mg kg^–1 soil DW at the beginning of the this study (t₀) to 542 mg kg^–1 soil DW at the first sampling time (t₁, 220 d after planting) and to 385 mg kg^–1 soil DW at the second sampling point (t₂, 430 d after planting), as compared with the level at t₀ (Fig. 1A). This decrease in PAH concentration was likely due to the activity of the soil microbial community. Leaching was not considered to be a significant factor because these PAHs are not very water-soluble (Log K_ow 3–5) and because the pots were watered from below.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Total concentration (mg kg–1 soil DW) of different classes of polycyclic aromatic hydrocarbons (PAH) in soil planted with species from different plant families, sampled at different times (t1, 220 d after planting; t2, 430 d after planting). The horizontal line denotes the concentration at t0. (A) Total PAH concentration; (B) 2- to 4-ringed PAHs; and (C) 5- to 6-ringed PAHs. Total PAH concentration corresponds to the sum of the averages of individual PAH concentrations. *Statistical differences at p < 0.05 (Student's t test) as compared with unplanted soil. The plant species used from each family are listed in Table 2. The 2- to 4-ring PAHs are naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, and chrysene. The 5- to 6-ringed PAHs are benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)acephenanthrylene, benzo(a)pyrene, indol(1,2,3-cd)pyrene, dibenzo(ah)anthracene, and benzo(ghi)perylene.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Polycyclic aromatic hydrocarbon (PAH) removal 220 d (t1) and 430 d (t2) after planting, comparing unplanted and planted treatments, and within planted treatments comparing monocotyledon with dicotyledon plant species. The different panels represent different classes of PAH. (A) Two-ring: naphthalene. (B) Three-ring: acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene. (C) Four-ring: fluoranthene, pyrene, benzo(a)anthracene, chrysene. (D) Five-ring: benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)acephenanthrylene, benzo(a)pyrene. (E) Six-ring: indol(1,2,3-cd)pyrene, dibenzo(ah)anthracene, benzo(ghi)perylene. (F) Total PAH. *Statistical differences at p < 0.05 (Student's t test) as compared with unplanted soil.

Relationship between PAH Type and the Effect of Vegetation
High M_m PAHs tend to be more recalcitrant because they are less bioavailable; therefore, it is of interest to compare the effects of planting between low M_m PAHs (2–4 rings) and high M_m PAHs (5–6 rings). At t₀, phenanthrene (a three-ring PAH) and pyrene and fluoranthene (four-ring PAHs) were the most abundant PAHs. The concentration of low M_m PAHs at t₀ was 547 mg kg^–1 soil DW and decreased in unplanted soil to 358 mg kg^–1 soil DW at t₁ and to 241 mg kg^–1 soil DW at t₂ (Fig. 1B). In contrast, the first significant decrease in the concentration of high M_m PAHs in unplanted soil from the t₀ level (205 mg kg^–1 soil DW) was at t₂ with 158 mg kg^–1 soil DW (Fig. 1C). This relatively small decrease in high M_m PAHs illustrates their recalcitrance to microbial degradation on the time scale of this study. Planting enhanced the rate of dissipation of low M_m PAHs: At t₁, soils planted with Asclepiadaceae, Asteraceae, Geraniaceae, Polygonaceae, and Poaceae had significantly lower total PAH levels compared with unplanted soil (decrease of 30%) (Fig. 1B). This general stimulatory effect of vegetation on PAH remediation was no longer significant at t₂ (Fig. 1B). Similarly, the concentration of high M_m PAHs in soils planted with these five families was significantly lower at t₁ than in unplanted soil (decrease of 20%) (Fig. 1C) but not at t₂ (Fig. 1C). Thus, the overall effects of planting on PAH dissipation were not dependent on the molecular mass of the PAH.

Comparison of Plant Species for PAH Dissipation
Plant species within families were not significantly different in terms of their ability to stimulate PAH dissipation. However, when plant species within the same families were pooled and families were compared, differences became apparent. Among the plant families tested, the Poaceae seemed to be the most effective at stimulating PAH dissipation. Among the species tested from this plant family, big bluestem (Andropogon gerardii) and perennial ryegrass (Lolium perenne) were most effective. For example, dissipation of total PAHs in ryegrass-planted soil was 40% greater than in unplanted soil (supplementary data). On the other end of the spectrum was lemon mint (Monocarda citriodora); soils in which this plant were grown contained higher levels of total PAHs (852 mg kg^–1 soil DW, t₁) compared with unplanted soil (542 mg kg^–1 soil DW, t₁), perhaps through inhibition of PAH-degrading microorganisms. At t₁, Poaceae-planted soils had significantly lower concentrations of each individual PAH as compared with unplanted soil. At t₂, this positive effect of grasses on PAH removal was much less pronounced: Only five of the PAHs were still lower in Poaceae-planted soils than in unplanted soil, and the differences were smaller. Among the dicotyledon families tested, the Asteraceae and Geraniaceae seemed to be most effective at stimulating PAH dissipation (Table 3).

Polycyclic aromatic hydrocarbon dissipation decreased with increasing number of aromatic rings in unplanted and planted soils (Fig. 2). The concentrations of two-ring PAHs decreased in unplanted and planted soils and with monocotyledon and dicotyledon species (Fig. 2A). In planted soils, 88 to 92% of these PAHs had dissipated at t₁, a significantly higher level than the 70% reduction observed in unplanted soils. At t₂, all treatments (planted and unplanted) yielded a 90 to 92% reduction in two-ring PAH levels. A similar pattern and contribution of monocotyledon and dicotyledon species was observed for three- and four-ring PAHs except that the decrease from the initial concentration was less (58–66% for three-ringed PAHs and 35–45% for four-ringed compounds in planted soils at t₁) (Fig. 2B, C). The dissipation of five-ring PAHs was higher for planted (up to 37%) than unplanted soil (16%) at t₁ (Fig. 2D). At t₂, the pooled planted treatments also had significantly reduced more five-ring PAHs (41%) than bare soil (35%); this effect was mainly due to the monocotyledons because PAH levels in soils planted with those species decreased by 47%. Relative to unvegetated control soils, planting had a pronounced and significant stimulatory effect on the dissipation of six-ring PAHs at t₁ (Fig. 2E). Although bare soil had only a 2% reduction of this recalcitrant class of PAH at t₁, planted soils had concentrations that were 15% lower on average, and concentrations in monocotyledon-planted soils were 26% lower. At t₂, the planted treatments had dissipated significantly more six-ringed PAHs than the unplanted soil (up to 35% for monocotyledon-planted soils vs. 21% for unplanted soil).

Vegetation and the Abundance of PAH-Degrading Microbes
A possible explanation for the stimulatory effect of planting on PAH dissipation is that the plants promote the concentration and activity of PAH-degrading microbes. At t₁, soils planted with red clover and kleingrass had the highest MPN of PAH-degraders, with kleingrass-planted soil containing greater than 100-fold more PAH degraders than unplanted soil (Fig. 3A ). On average, planted soils had 10-fold more PAH degraders than unplanted soil after 220 d, and only soils planted with lemon mint and big bluestem had lower levels of PAH degraders than unplanted soil. At t₂, soils planted with kleingrass and sunflower contained the largest populations of PAH degraders (100-fold higher than unplanted soils) (Fig. 3B). On average, planted soils contained fourfold more PAH degraders than unplanted soils after 430 d, and only soils planted with stiff goldenrod, big bluestem, and tall fescue had lower MPNs than the control soil. A comparison between the MPNs at t₁ and t₂ (Fig. 3C) reveals a significant increase in the PAH-degrading population in seven of the planted soils, including a 15-fold increase in soil planted with sunflower. The MPN values in five other planted soils increased slightly (to a similar extent as the unplanted soil), and the PAH-degrading population was found to decrease between t₁ and t₂ in six planted soils. There were no obvious trends relating plant species/families and their effect on PAH degrader abundance. There was also no significant correlation between MPN of PAH degraders and PAH removal across the different plant species for t₁ or t₂.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Most probable numbers (MPN) of polycyclic aromatic hydrocarbon–degrading microbes in soil planted with different plant species 220 d (A, t1) and 430 d (B, t2) after planting. The fold-change in MPN between t1 and t2 is shown in (C) and was calculated by subtracting MPN (t1) from MPN (t2) and dividing by MPN (t1). The horizontal lines denote unplanted control soil. The numbers underneath the bars indicate the plant species as listed in Table 2.

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

From this study, it is clear that planting significantly stimulated the dissipation of PAHs from aged contaminated soil. This vegetation effect was predominantly observed in the first 220 d after planting and was most pronounced for monocotyledon species. Although the concentrations of larger PAHs decreased to a lesser extent in all treatments, the stimulatory effect of vegetation was most pronounced for these more recalcitrant high M_m PAHs. In planted and unplanted soils, the reduction of two-ringed PAHs was the largest (up to 90%). This is not surprising because the two- and three-ringed PAHs are known to be good candidates for microbial biodegradation (Juhasz and Naidu, 2000; Kuiper et al., 2001, 2004). The four-ringed PAHs phenanthrene, fluoranthene, and pyrene were the most abundant PAHs at all three sampling points. Although Joner et al. (2004) reported that the dissipation of four-ringed PAHs was the highest, in this study there was a clear inverse correlation between PAH molecular mass and dissipation. This difference may be due to different soil characteristics and/or different indigenous soil microbial populations. The high M_m PAHs, present at relatively low concentrations at all sampling times, had low extents of dissipation, a result that might be explained by low bioavailability (Joner et al., 2004; Parrish et al., 2005b).

Overall, our results are in good agreement with those from previous studies (Pradhan et al., 1998; Hutchinson et al., 2001; Chen et al., 2003; Parrish et al., 2004). In each of these earlier 6- to 12-mo studies, PAH-contaminated soils were planted with two or three plant species, and an unplanted control was monitored in parallel. In the study by Pradhan et al. (1998), the reduction in total PAHs was 57% after 6 mo; in the Hutchinson et al. (2001) study, the decrease was 49% after 6 mo and 57 to 68% after 12 mo; and Parrish et al. (2004) reported a decrease in total PAH levels of 9 to 24% in 12 mo. In the study by Chen et al. (2003), the reduction in pyrene was 35% after 6 mo. In all of these studies, several grasses were used, and in several of them one dicotyledon species was included (clover or alfalfa). Although in the study by Pradhan et al. (1998) alfalfa and switchgrass performed equally well, Parrish et al. (2004) observed that the two grass species (tall fescue and annual ryegrass) performed better than yellow sweet clover (24, 15, and 9% of PAHs removed, respectively), which is more in agreement with our findings. As in our studies, the planted treatments performed better than the unplanted controls in the earlier studies. Hutchinson et al. (2001) found no significant differences between the treatments until 12 mo (i.e., none at the 6-mo sampling point), whereas in our study the most pronounced differences between the treatments were found in the first 7 mo. Our study differs from those previously reported in that we tested many more plant species (18) representing a broader range of plants (eight families). Also, the concentrations of the different PAH compounds are reported in the present study to provide insights on the effectiveness of planting and specific plant species for each of 17 PAHs.

The mechanisms responsible for the stimulatory effects of vegetation on PAH dissipation require further study. Plant root–released compounds likely play a key role in this process by acting as carbon source to the general soil microbial population (Singer et al., 2003) and/or by activating microbial genes involved in the degradation of pollutants with similar structure (Olson et al., 2003). A study conducted using mulberry showed that root turnover in the rhizosphere significantly enhanced the number of PCB degraders, not only by providing the substrates to sustain bacterial growth, but also by increasing oxygen diffusivity in the soil, which is essential for the activity of the oxygenase enzymes involved in the aerobic degradation of this class of aromatic contaminants (Leigh et al., 2002). In contrast, a study performed with yellow sweet clover and tall fescue on the effect of root death and decay on the dissipation of PAHs in the rhizosphere found no major effect on PAH degradation, although increased numbers of PAH degraders were found in the planted soils (Parrish et al., 2005a). It is also possible that certain root-released compounds act as biosurfactants that make PAHs more bioavailable, an effect that would be more significant for the high M_m species. A combination of changes in the soil chemical and physical properties due to root turnover, higher PAH bioavailability, and an increased number of total microbes and of PAH degraders may explain the positive effect of planting on the phytoremediation efficiency of PAH contaminated soils. Finally, it is conceivable that some of the more volatile PAHs were outgassed from the soils via root disturbance and aeration of the soils (Peck and Hornbuckle, 2004).

Planting aged PAH-contaminated soils was mainly effective at stimulating PAH dissipation during the first 220 d after planting, whereas at the second sampling point (430 d after planting), almost no difference in PAH dissipation was observed when comparing planted and unplanted soils, except for high M_m PAHs. Several explanations could be advanced, one being a decrease in the population of PAH degraders over time. This could be due to a lack of nutrients because they have been used for plant and microbial growth. Hutchinson et al. (2001) showed a positive correlation between the addition of inorganic fertilizer (C, N, and P) and PAH dissipation rate after 1 yr in an aged contaminated soil. In our study, however, the MPN of PAH-degrading microorganisms was higher at t₂ than at t₁ for several dicotyledons (Helianthus annuus, Lotus corniculata, Geranium viscosissimum, Monocarda citriodora, and Rumex crispus) and monocotyledons (Agropyron smithii and Lolium perenne). Alternatively, as the root density increased, this may have led to a supraoptimal level of a certain root released compounds, inhibiting the rate of microbial PAH degradation. The age of the contaminant also may have limited its biodegradation (Allard et al., 2000). Using a similar time frame to the one described here and nonspiked, aged PAH-contaminated soil, Parrish et al. (2005b) concluded that the aging process is the most relevant factor in the dissipation of PAHs.

Selection of Plant Species and PAH Remediation Strategies
Certain plant families have been widely used in PAH phytoremediation studies and have proven to be successful in PAH removal. The most common are tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) from the Poaceae and yellow sweetclover (Melilotus officinalis) and red clover (Trifolium pratense) from the Fabaceae (Huang et al., 2004a,b; Joner et al., 2004; Johnson et al., 2005; Parrish et al., 2005a,b). In our study, which compared eight plant families comprising 18 plant species with diverse habits and morphologic characteristics, the Poaceae emerged as the most effective at stimulating PAH dissipation, whereas the Fabaceae were among the least effective. Among the dicotyledons, the Asteraceae, Geraniaceae, and Polygonaceae stimulated PAH dissipation most and thus show promise for use in PAH remediation, together with grasses. Indeed, Helianthus annuus (Asteraceae) was reported to successfully colonize a PAH-contaminated former sludge basin (Olson and Fletcher, 2000). Because forbs on average reach a larger root depth than grasses (Olson et al., 2001), they may be valuable species to include in a clean-up strategy for a contaminated soil. These differences among dicotyledon families illustrate the importance of plant selection when planning remediation strategies.

Why would certain plant species, or even plant families, be more effective than others in stimulating PAH dissipation? Based on visual observation, all plants grew well and showed no visual phytotoxicity symptoms. Therefore, tolerance does not seem to have been a factor. Soils planted with Poaceae species were found to have the largest decrease in total PAHs concentration. Poaceae (grasses) are known to have a dense fibrous root system, and, based on other studies (not shown), the root biomass of the Poaceae was likely significantly larger than that of the dicotyledon species. A larger root surface is expected to stimulate microbial density and activity by providing a surface area for adhesion and metabolic sources of root-derived carbon. Indeed, two grasses were among the species with the highest number of PAH degraders, as judged from MPN assay. Huang et al. (2004a) suggested that it may be a strategy of Poaceae to become more tolerant to PAH by favoring root growth over shoot growth. Any transport of low M_m PAHs would be limited, and the shoots would be more protected. As for the observed differences between dicotyledon families, a possible explanation is that different species likely differ in the composition of their root-released compounds, which may include stimulatory secondary plant compounds and/or biosurfactants that make PAHs more bioavailable in the rhizosphere (Olson et al., 2003). More research is needed to further investigate these plant–microbe interactions involved in phytostimulation of PAH dissipation.

Planting with Lamiaceae and Fabaceae species initially yielded the least positive (or even a negative) effect on the rate of PAH dissipation. However, in the last 210 d of the study, the largest decrease in PAH concentration of all treatments was observed in soils planted with these species. In the same period between t₁ and t₂, there was an increase in the MPN of PAH degraders in soils planted with lemon mint. Gilbert and Crowley (1997) showed that plant-derived terpenes from spearmint (Lamiaceae) induced PCB degradation by Arthrobacter, suggesting that a similar compound may have been released into the soils planted with lemon mint. It is not clear why the effect of these species was different in the first and second periods of this study; perhaps they need to reach a certain age in their life cycle before the stimulatory effect on PAH dissipation is realized.

Another potential factor in the positive effect of the Fabaceae (Legumes) between t₁ and t₂ may be their association with rhizobia. Having the ability to fix atmospheric N₂ could be an advantage in relation to other plant species because PAH-contaminated soils are generally nutrient poor, and often the availability of nitrogen is limited (Hutchinson et al., 2001). The higher availability of N may have stimulated plant and microbial activity in the pots of the Fabaceae. However, a positive change in MPN between t₁ and t₂ was not found for most Fabaceae-planted soils.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
Planting is an effective means of increasing PAH dissipation rates for all PAHs, with the most pronounced effects relative to unplanted control soil noted within the first 7 mo (for all PAHs) and for the high M_m PAHs (at t₁ and t₂). Some plant families are more effective than others, and the Poaceae (grasses) were found to be the most effective of the eight families tested. The presence of some plant species led to higher populations of PAH degraders. There was no correlation across plant species between PAH dissipation effectiveness and the size of the PAH-degrading population, suggesting that other factors such as bioavailability might play a key role. Additional research is needed to understand why some plants are better than others for stimulating PAH dissipation under ecological and environmentally relevant time frames.

	ACKNOWLEDGMENTS

 
We thank Bryan Page and Kerry Hale for technical assistance. We gratefully acknowledge the support of Al Venosa and Al Bourquin. Funding for this project was obtained from the USEPA through the Great Plains Rocky Mountain Hazardous Substance Research Center (Project SP99-1).

	NOTES

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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.

P.E. Olson and A. Castro contributed equally to this work.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 

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