Published in J. Environ. Qual. 34:207-216 (2005).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Bioremediation and Biodegradation
Effect of Root Death and Decay on Dissipation of Polycyclic Aromatic Hydrocarbons in the Rhizosphere of Yellow Sweet Clover and Tall Fescue
Zakia D. Parrisha,
M. Katherine Banksb,* and
A. Paul Schwabc
a Department of Soil and Water, Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT 06511
b School of Civil Engineering, Purdue University, West Lafayette, IN 47907
c Department of Agronomy, Purdue University, West Lafayette, IN 47907
* Corresponding author (kbanks{at}purdue.edu)
Received for publication February 26, 2004.
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ABSTRACT
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A 12-mo greenhouse study was conducted to evaluate the contribution of root death and decay on the dissipation of polycyclic aromatic hydrocarbons (PAHs) in rhizosphere soil. The contaminated soil was previously treated by land-farming, but residual PAHs remained after treatment. Tall fescue (Festuca arundinacea Schreb.) and yellow sweet clover (Melilotus officinalis Lam.) were the target plants. To specifically evaluate the effect of root decay on contaminant dissipation, plants were treated with glyphosate, a broad spectrum herbicide, to induce root decay. Although tall fescue treatments had the highest root and shoot biomass and root surface area, this plant did not result in the highest contaminant degradation rates. Significant differences were noted between treatments for seven PAHs, with the active yellow sweet clover resulting in 60 to 75% degradation of these compounds. Induced root death and decay did not produce a significant enhancement of PAH degradation. The PAH microbial degrader populations in the vegetated treatments were more than 100 times greater than those in the unvegetated control. The phospholipid fatty acid (PLFA) structural group profile shifted over the growing period, indicating a change in the community structure. In conclusion, phytoremediation was shown to be an effective polishing tool for PAH-affected soil previously subjected to biological treatment.
Abbreviations: MPN, most probable number • PAH, polycyclic aromatic hydrocarbon • PLFA, phospholipid fatty acid
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INTRODUCTION
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POLYCYCLIC AROMATIC HYDROCARBONS are potentially carcinogenic compounds often present in high concentrations in manufactured gas plant residues. These compounds are hydrophobic and decrease in solubility with increasing molecular weight. Although PAHs in soils contaminated with these residues are usually resistant to degradation, phytoremediation holds promise as a secondary treatment approach for residual concentrations after completion of conventional remediation (Cunningham et al., 1996; Ferro et al., 1999). However, the mechanisms responsible for the removal of PAHs during phytoremediation have not been clearly defined.
Plants may enhance rates of PAH degradation by stimulating microbial growth and activity, and/or by increasing PAH bioavailability in the rhizosphere (Binet et al., 2000). The rhizosphere, the soil directly adjacent to the root, is a zone of increased microbial activity and numbers. The presence of plants may influence carbon dioxide concentration, pH, osmotic potential, redox potential, oxygen concentration, and moisture content in soil (Anderson et al., 1993). Plant characteristics such as species, age, root type, soil type, and the weathered age of the contaminant will determine the structure of microbial communities in the rhizosphere (Sylvia et al., 1998).
The effect of plant roots on PAH degradation has not been thoroughly assessed. In the rhizosphere, the size of root impact zone is determined by the concentration and type of carbon exudates (Taiz and Zeiger, 1998), and expands with increasing root hairs, root overlap, and fibrous roots (Sylvia et al., 1998). The degradation of complex compounds, such as PAHs, may be enhanced by the presence of root exudates because of increased interaction between microbes, nutrients, and contaminants (Reilley et al., 1996; Cunningham et al., 1996). Fine roots are in constant flux in the rhizosphere because root death and production occur simultaneously. The dead fine roots serve nutrient and energy sources for soil microorganisms and fauna (Vogt et al., 1998). Under periods of stress, roots require large amounts of carbon to continue efficient maintenance respiration (Janssens et al., 2002; Eissenstat and Van Rees, 1994). A "reoccurring rhizosphere," the result of a buildup of labile organic carbon from root exudates and root turnover, is believed to exist in the area adjacent to the coarse roots due to the constant flux in soil carbon (Sanchez and Bursey, 2002). Fine root dynamics and phenolic content in mulberry (Morus spp.) saplings were evaluated by Leigh et al. (2002) who observed an average root turnover of 58% over one growing season. Phenolic compounds that accumulate in the cell vacuoles during normal root activity were released during root turnover into the soil and reached a maximum of 38 g/kg dry weight of dead root.
Low molecular weight PAHs (three or fewer rings) have been effectively bioremediated, although the degradation of the higher molecular weight compounds may be limited by their low aqueous solubility and high rates of sorption to the soil. However, there have been reports of bacterial degradation of four-, five-, and six-ring PAHs (Kanaly and Harayama, 2000; Mueller et al., 1997; Juhasz et al., 1997; Heitkamp et al., 1988). With increasing residence time, microbial mineralization of soil PAHs decreases (Hatzinger and Alexander, 1995; Erickson et al., 1993). Aging is the chemical–physical entrapment of a chemical in soil micropores or the soil organic matrix where humin pore sizes range from 2 to 360 nm and is believed to be responsible for the decline in degradation over time (Alexander, 2000; Malekani et al., 1997; Alexander, 1995). Before biodegradation can occur, PAHs must be released from soil sequestration into the aqueous phase (Potter et al., 1999).
A surge in degradation rates has been observed in phytoremediation systems during the senescence period of the plant life cycle. Banks et al. (2000) reported a correlation between the decrease of established clover roots and the degradation of total petroleum hydrocarbons in the fall season. Of note is that plant roots may exude large amounts of phenolic compounds as they begin to senesce and enter winter dormancy (Hegde and Fletcher, 1996). Also, plant root exudates may provide a source of carbon and energy for microbes, resulting in enhanced metabolism and/or cometabolism of PAHs. Miya and Firestone (2001) reported enhanced phenanthrene degradation due to addition of slender oat (Avena barbata Pott ex Link) root exudates and root debris. Plants may respond to chemical stresses by increasing or changing exudation, which modifies the rhizosphere microflora (Walton et al., 1994). Reilley et al. (1996) reported that pyrene degradation rates increased in rhizosphere soil and were at the highest when rhizosphere organic acids were added to the soil. Fletcher and Hedge (1995) screened seventeen perennial plant species for the production of phenolic compounds, such as flavonoids, which act as cometabolites in contaminant degradation. They found that mulberry, crabapple, and osage orange synthesize these compounds in large quantities and were able to support the growth of PCB-degrading bacteria. Another investigation revealed that the roots of sweet clover produced large amounts of coumarin (1% by dry wt.), a naturally occurring benzo[a]pyrene derivative, which also may stimulate PCB degradation on release (Fletcher, personal communication, 2000). Dead and decaying roots not only provide additional carbon to the soil, but also produce air-filled macropores increasing oxygen diffusion (Ferro et al., 1999).
Particularly of interest in this study is the use of phytoremediation as a secondary polishing approach for PAH-contaminated soil previously treated by land-farming. Land-farming has been used for decades by the petroleum industry, because target hydrocarbons have been shown to be effectively metabolized by microbial populations in the soil when enhanced by amendments and tilling (Chaineau et al., 1996). This practice of spreading contaminated soils onto soil surfaces, adding fertilizer or biosolids, and tilling, was the first technique used for soil and sludge bioremediation (Taylor and Viraraghavan, 1999; Loehr and Webster, 1996a). Reductions in PAH concentrations typically occur after an initial period of rapid degradation of the two- and three-ring PAHs followed by a very slow rate of change (Sayles et al., 1999; Loehr and Webster, 1996a). Removal of total PAH ranges between 50 and 70%, however, dissipation for five- and six-ring compounds is variable (Sayles et al., 1999; Guerin, 1999). Loehr and Webster (1996b) conducted a case study in which contaminated soil from a creosote wood treatment site was land-farmed for 18 mo, followed by establishment of a warm-season grass for two growing seasons. The untreated controls and land-farmed plots had total PAH concentrations of 2630 and 1020 mg/kg, respectively, after the land-farming phase was complete. However, in the grass-treated plots, an additional 81% reduction in total PAH concentration to 195 mg/kg was observed.
The objectives of this study were to investigate whether phytoremediation can enhance contaminant degradation when used as a secondary biological treatment approach and to evaluate the effect of root decay on PAH dissipation. Root turnover and decay have a significant influence on microbial activity, which may be the primary means of contaminant degradation due to large increases in labile carbon that stimulate microbial population surges. The plant life cycle, particularly root death and decay, may significantly contribute to the degradation process.
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MATERIALS AND METHODS
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Site Description
The USEPA initiated a remediation project at an abandoned manufactured gas plant site in Bedford, Indiana, to compare the efficacy of several biological treatment technologies. Manufactured gas production began around the turn of the century in the U.S. Midwest and continued for approximately 30 yr (Khodadoust et al., 2000; Battelle, 1999). The contaminated soil at this site contained PAH concentrations (including five- and six-ring compounds) that vary from 400 to 5000 mg/kg total PAHs and increase with soil depth, up to 5 m. The four technologies under investigation were in situ phytoremediation, ex situ compost or biopile, ex situ land-farming, and natural attenuation. The land-farming treatment for the excavated material was conducted throughout the entire 3-yr study (Battelle, 1999). Municipal wastewater biosolids were used as the amendment along with monthly tilling for the land-farming operation. Treated soil from the Bedford manufactured gas plant site that did not meet the cleanup objectives after land-farming for one year was used in this study to evaluate phytoremediation as a polishing method for removal of residual PAHs.
Soil Analyses
Soils were sampled before the initiation of the experiment and analyzed for post-land-farming PAH content (Table 1). Additional samples were analyzed by MDS Harris Laboratories (Lincoln, NE) for soil physical and chemical properties. The soil was a sandy loam, with an organic matter content of 5% and initial available nitrogen and phosphorus concentrations of 123 and 95 mg/kg, respectively (Table 2). The total extractable material was measured to determine the amount of contaminant-based carbon on which fertilization rates should be based. The soil total extractable material was determined using a modification of the shaking extraction procedure developed by Schwab et al. (1999). Two grams of soil was weighed into a 30-mL centrifuge tube to which 10 mL of a 50:50 (v/v) methylene chloride to acetone mix (Fisher Scientific, Hampton, NH) was added. The vial was mechanically shaken for 30 min and centrifuged for 10 min at 4000 rpm, and the supernatant was transferred to a clean bottle. This process was repeated twice, homogenizing and weighing the extract resulting from the three cycles. The supernatant was evaporated to dryness in a preweighed vial, and the total extractable material was determined by weighing the resulting residue. The mean total extractable material concentration was 11200 mg/kg soil.
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Table 1. Physicochemical properties and initial polycyclic aromatic hydrocarbon concentrations in (post land-farmed) soil.
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Greenhouse Experiment Design
Tall fescue, yellow sweet clover, and an unvegetated control were the soil treatments in this study. The soil was homogenized and approximately 6 kg was placed in 12-L pots. Tall fescue and yellow sweet clover were grown in separate pots and seeded at the normal seeding rates of 22.4 and 16.8 kg/ha, respectively. Immediately before the initiation of the experiment, which began once both seed types had germinated, soil samples were collected from each pot for analyses.
For each of the two plant species, one set of pots at three different time intervals was sprayed with 2% glyphosate (Roundup; Monsanto, St. Louis, MO) to induce plant death. Glyphosate is a broad-spectrum herbicide. Four replicate pots of each species were sprayed at 3, 6, and 9 mo after the experiment was initiated to allow ample time for plant decay before the 6-, 9-, and 12-mo destructive sampling. The plants were sprayed twice with 2% glyphosphate over a period of 1 wk, and all plants were dead by the end of 2 wk. All other treatments, including the controls (12 total per treatment), were not exposed. Thus, there was a total of five treatments: tall fescue, tall fescue + glyphosate, yellow clover, yellow clover + glyphosate, and unvegetated controls. Therefore, there were five plant treatments, three sampling times, and four replicates for a total of 60 pots. Figure 1
is a schematic depicting the glyphosate treatments and destructive sampling. At each sampling time, soil and plants were collected and analyzed for PAH concentration, microbial enumeration, plant biomass, and plant root surface area.
The initial total extractable material analysis, which quantifies solvent-extractable carbon, was used to determine the amount of nutrients to be added on a C to N to P ratio of 100:20:5. Nitrogen and phosphorus were applied in split applications in the form of urea and monobasic potassium phosphate, KH2PO4 (Sigma-Aldrich, St. Louis, MO), at a rate of 240 mg N/pot and 63 mg P/pot, respectively, biweekly to all of the treatments in a 250-mL solution. The plants were watered on an as-needed basis to minimize the generation of leachate.
Sample Collection
For each sampling period, plant shoots and roots were removed, separated, and analyzed by weight. A plant root sample was collected from the pot for surface area analysis. For the soil analyses, a soil sample was removed from the plant roots, homogenized, directed through a 2-mm sieve, and used for PAH analysis, most probable number (MPN) quantification, and phospholipid fatty acid (PLFA) analysis. A sample of any leachate generated after irrigation was assessed for PAH concentration.
Soil Polycyclic Aromatic Hydrocarbon Analysis
Soils were analyzed for PAHs by soil extraction and gas chromatography (GC) with a flame ionization detector (FID). Fifteen of the 16 USEPA Priority PAHs were extracted from soil using a shaking extraction procedure (Schwab et al., 1999). Two grams of soil were weighed into a 30-mL centrifuge tube, to which 10 mL of a 50:50 (v/v) methylene chloride to acetone mix (Fisher Scientific) and 1000 µL of 1000 mg/L tetracosane matrix spike (Sigma-Aldrich) was added. The vial was mechanically shaken for 30 min and centrifuged for 10 min at 4000 rpm, and the supernatant was transferred to a clean bottle. This process, except for the addition of the matrix spike, was repeated twice, homogenizing and weighing the extract resulting from the three cycles. A 1.5-mL aliquot of extract was transferred into a 2-mL GC vial along with 5 µL of 1000 mg/L androstane (Sigma-Aldrich) as an internal standard. Each sample was capped and stored at 4°C until analyzed by GC–FID.
Gas Chromatography
The PAH concentrations in the extracts were quantified using gas chromatography with a capillary column and flame ionization detector. The column was an HP-5 fused silica capillary column (Hewlett-Packard, Palo Alto, CA), with an inside diameter of 320 µm, a length of 30 m, and a 0.25-µm film thickness. The carrier gas was helium, delivered at 1160 µL/s; the fuel gas was hydrogen, 750 µL/s; nitrogen was the make-up gas at a flow rate of 750 µL/s; and zero-grade air was the oxidant at a flow of 7000 µL/s. The detector and injection port temperatures were 350 and 250°C, respectively. The splitless injection volume of 2 µL was delivered by a 10-µL syringe. The initial oven temperature was maintained at 40°C for 2 min, then increased at 10°C/min to 320°C, and maintained at 320°C for 5 min. The PAH concentrations in the samples were measured using integrated GC areas and converted to concentrations using standard calibration curves.
Microbial Enumeration
The microbial population was quantitatively enumerated using the PAH degrader MPN procedure developed by Wrenn and Venosa (1996). A 5-g soil sample from each treatment was added to a 60-mL autoclaved bottle containing 45 mL of a 2% sodium pyrophosphate solution. The soil solution was mechanically shaken for 30 min and allowed to settle for 10 min. A PAH substrate mixture dissolved in pentane (Fisher Scientific) was filtered and added to rows 1 through 11 of sterile, 96-well microtiter plates (50 µL/well). The substrate consisted of 2 µg/L of phenanthrene and 0.2 µg/L of naphthalene, pyrene, and anthracene (Sigma-Aldrich). The pentane was allowed to evaporate so that the PAHs were deposited onto the well surfaces. After the pentane evaporated, 180 µL of a growth medium solution containing Bushell–Haas (B–H) broth and 0.85% NaCl was added.
Once the soil solution settled, supernatant was added to the rows based on a dilution series. The plates were wrapped in plastic wrap, which allowed air movement while minimizing evaporation, and incubated at 25°C for 3 wk. Positive wells turned yellow or brown due to the accumulation of the partial oxidation products of aromatics (Wrenn and Venosa, 1996). The MPN was calculated using the USEPA computer program MPN Calculator (Version 4.04; USEPA, 1993).
Microbial populations from time zero and after 12 mo of plant growth were evaluated qualitatively using PLFA–fatty acid methyl ester (FAME) analysis (Microbial Insights, Rockford, TN). Total lipids were extracted and separated by column chromatography into polar lipid fatty acids, which were derivatized into fatty acid methyl esters. Fatty acid methyl ester was quantified using GC–mass spectrometry (MS) to verify the fatty acid structures. Four different characterizations were developed to assess the PLFA profile: biomass, community structure, diversity, and physiological status. Biomass, as measured by PLFA, is proportional to the number of cells because phospholipid analysis only quantifies viable cells. Six structural groups were used to develop the community structure analysis, which are distinguishable between phylogenic groups of microbes.
Plant Analyses
Plant biomass was separated into roots and above soil surface biomass (shoots). The roots and shoots were weighed, dried at 65°C in a preweighed paper bag for 48 h, and weighed again. The roots collected for surface area analysis were cleaned in a 1:3 (v/v) sodium hexametaphosphate to water solution and stored in a 1:4 (v/v) ethanol to water solution until digitally scanned. The scanned image was processed using WinRhizo 3.10b software (Regent Instruments, 1998) to estimate the root surface area. The root biomass measurements were mathematically adjusted to include the sample of roots collected for surface area analysis.
Statistical Analysis
Statistical significance of the treatment effects was evaluated using CoStat software (COHORT, 2002) based on n = 4 and p < 0.05. The error bars represent the least significant difference (LSD) as determined by the Student–Newman–Keuls test. Results shown for the MPN, plant biomass, and root surface area analyses for each treatment are the average value of the four replicates, with the error bars representing one standard deviation.
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RESULTS
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Polycyclic Aromatic Hydrocarbon Degradation
The percentages of each of the 15 priority PAHs degraded in the five treatments are shown in Table 3. Significant differences in concentration (p < 0.05, n = 4) existed between treatments for seven of the PAHs. At the end of 12 mo, the active yellow sweet clover treatment resulted in 70.9, 72.2, and 75.5% degradation of 1,2-benz[a]anthracene, fluoranthene, and pyrene, respectively, which was significantly greater than the other four treatments. The percent of benzo[a]pyrene and anthracene degraded in the four plant treatments were not statistically different from one another at the 9- and 12-mo sampling periods, although all were higher than the unvegetated control, which had concentration reductions of 36.8 and 50.3%, respectively. The unvegetated control resulted in significantly less degradation of chrysene and phenanthrene than the two yellow sweet clover treatments, which had greater than 60% reduction for both compounds.
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Table 3. Percent degradation of individual polycyclic aromatic hydrocarbon (PAH) compounds by each of the five treatments.
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Microbial Enumeration
At the beginning of the experiment, the PAH degrader numbers in the five treatments were not significantly different from one another. However, the microbial numbers did statistically differ over the course of the greenhouse study (Fig. 2)
. During the 6- and 9-mo sampling periods, the degrader populations fluctuated, probably as a consequence of subtle changes in the rhizosphere, which may reflect the heterogenous nature of the contaminated soil. After 12 mo of plant growth, the PAH degrader populations in the vegetated treatments were more than 100 times greater than that in the unvegetated control. Significant differences were found between the two tall fescue treatments and the glyphosate-treated yellow sweet clover.
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Fig. 2. Polycyclic hydrocarbon degraders in soil after phytoremediation with yellow sweet clover and tall fescue. Soil was pretreated by land-farming. Error bars represent one standard deviation (n = 4). MPN, most probable number. TFG, tall fescue with glyphosate; TFA, active tall fescue; SCG, yellow sweet clover with glyphosate; SCA, active yellow sweet clover; UC, unvegetated control.
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The viable microbial biomass ranged between 17000 and 32000 pmol PLFA per gram dry weight of soil (3.5 x 108 and 6.4 x 108 cells per gram dry weight of soil) for the five treatments at the 0- and 12-mo sampling periods (Table 4). The PLFA analysis indicates that the unvegetated control at time zero had higher levels of biomass than the other treatments at the same sampling period. After 12 mo, the biomass content in the control decreased and was no longer significantly different than the glyphosate-treated yellow sweet clover, although still elevated. Both of the glyphosate treatments had higher microbial biomass than their respective active treatments. The number of viable microbial cells per gram of dry weight of soil for each of the samples followed the same pattern as the PLFA analysis. The PLFA structural groups present in each treatment at time zero and after 12 mo of plant growth are used to classify bacteria and fungi according to their fatty acid profiles. Over 12 mo, the monosaturates, representative of Gram-negative bacterial populations, decreased in all five treatments; however, they remained the dominant population, which is characteristic of most soils. Another pattern across treatments was the increase in the terminally branched, mid-chain branched, and polyunsaturates, which are representative firmicutes, actinobacteria, and eukaryotes, respectively. The terminally and mid-chain branched populations are Gram-positive bacteria, which resulted in a greater shift the community structure in the glyphosate treatments than their active counterparts. The prokaryote to eukaryote ratios increased slightly over the course of the study for both yellow sweet clover treatments and significantly increased in the unvegetated control by 37% (Table 4).
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Table 4. Viable microbial biomass expressed as picomoles phospholipid fatty acid (PLFA) per gram dry weight of soil, cells per gram dry weight of soil, and fatty acid community structural groups expressed as a percent of total.
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Shoot and Root Biomass
The active tall fescue treatment had significantly higher shoot biomass at all three sampling periods (Table 5). After 12 mo, there was little to no difference in shoot biomass between the glyphosate-treated and active yellow sweet clover treatments. The clover was repeatedly treated with glyphosate with little effect. Apparently, mature clover plants that are growing slowly require a more concentrated solution for effective control (Monsanto, 2002). As expected, the glyphosate-treated tall fescue had considerably less shoot biomass than the active tall fescue, at 93.1 and 217 g of dry shoot per pot, respectively. However, the glyphosate-treated tall fescue had notably greater shoot biomass than that of both yellow sweet clover treatments, which were 26.3 and 22.6 g of dry shoot biomass per pot, respectively. As predicted (Sims and Overcash, 1983), no target PAHs were identified in the root or shoot biomass samples. There also were no PAHs detected in the minimal leachate generated throughout the study period.
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Table 5. Average plant root and shoot biomass, root to shoot ratio, and root surface area after phytoremediation of contaminated soil.
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No roots remained after 6 mo in the glyphosate-treated tall fescue and yellow sweet clover pots; however, there were roots remaining in those treatments after 9 and 12 mo (Table 5). The active tall fescue had greater root biomass than the other treatments, although it was not statistically different from that of the glyphosate-treated tall fescue at 12 mo. Several of the glyphosate yellow sweet clover replicates sacrificed during the 9-mo sampling formed nodules even though fertilizer was applied bimonthly.
There were no differences after 12 mo in the root surface areas between treatments planted with the same plant species, despite the application of glyphosate to the designated pots. Both tall fescue treatments had well over 10000 cm2 of root surface area, which was significantly greater than that of the two yellow sweet clover treatments.
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DISCUSSION
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Degradation rates for PAHs in the unvegetated controls were consistently lower than the vegetated treatments, suggesting that land-farming coupled with phytoremediation is superior to land treatment alone. Sayles et al. (1999) observed significant differences in the two-, three-, and four-ring PAHs and total PAH concentration in soil from a wood treatment plant that was land-farmed for 25 wk. In particular, 1,2-benz[a]anthracene, fluoranthene, and pyrene concentrations were reduced by 54, 69, and 67%, respectively. Similar results were noted by Guerin (1999), who observed reductions in the same compounds by 46, 52, and 29% over 17 wk of treatment. However, Sayles et al. (1999) did not observe any significant removal of the five- and six-ring compounds. Of note is that the concentration of benzo[a]pyrene, a five-ring PAH, was statistically lower in the planted treatments in this study after a phyto-polishing step was implemented.
The induction of root decay did not result in increased PAH degradation. The active yellow sweet clover treatment resulted in reductions approximately 10% greater than the glyphosate treatments. Of note is that nodules were observed in the root zone of the active yellow sweet clover at the 9-mo sampling event. Nodules primarily form under nitrogen-limited conditions, although the activity of the nodules in this study was not assessed and no nodules were found during the following sampling period. Hutchinson et al. (2001) found a direct correlation between nitrogen and phosphorus availability and total petroleum hydrocarbon (TPH) dissipation. Fine root turnover can enhance the ability of soil microorganisms to degrade organic compounds (Olson et al., 2003). These large flushes of carbon can result in the rapid depletion of available inorganic nutrients (Margesin et al., 2000), further increasing the need for fertilizer. Breedveld and Sparrevik (2000) observed a significant increase in soil PAH mineralization and a decrease in total organic carbon (TOC) levels as the result of nutrient additions.
Vegetated soils had significantly higher numbers of PAH degraders than the unvegetated control (Fig. 2). Muratova et al. (2003) observed higher microbial numbers in PAH-contaminated soil planted with alfalfa (Medicago sativa L.) and reed [Phalaris australis (Cav.) Trin. ex Steud.] than in unplanted soil. In this study, the vegetated treatments did not have higher microbial biomass (Table 5); however, there was a shift in the structural group profile over time (Table 4). The PLFA profiles provided an indication of changes in the biological diversity of microbial communities and groups of microbes. The firmicutes and actinobacteria populations increased over the course of the study, indicating that the rise in Gram-positive bacterial population may have been due to conditions more favorable to growth. Several Gram-positive bacteria, including Mycobacterium, Rhodococcus, and Paenibacillus, are capable of degrading, and, in some cases, mineralizing PAHs in soil and sediments (Daane et al., 2001; Hamann et al., 1999; Meyer et al., 1999; Bouchez et al., 1997; Mueller et al., 1997; Kästner et al., 1994; Pichinoty et al., 1986). A treatment-based change in the microbial community structure also was noted during a 16-wk study conducted by Joner et al. (2002).
A correlation between total PAH degradation and root surface area was observed for the active yellow sweet clover treatment (r2 = 0.92); however, no relationships were observed for the other planted treatments (Fig. 3 and 4)
. Strong correlations were observed between the root and shoot biomass, as well as the root surface area and root biomass for the active yellow sweet clover, with r2 values of 0.98 and 0.94, respectively (Fig. 5 and 6)
. The root to shoot biomass ratios (RSRs) increased during the first sampling periods for the glyphosate-treated and active tall fescue and the glyphosate-treated yellow sweet clover, and declined during the latter sampling periods (Table 5). The application of glyphosate had the largest effect on root growth between the 9- and 12-mo sampling periods, resulting in RSRs close to one. The active tall fescue experienced a natural root turnover, most likely stress induced, during this same period, also resulting in nearly a 1:1 root to shoot ratio.
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Fig. 3. Total polycyclic aromatic hydrocarbon (PAH) concentration as a function of root surface area using active tall fescue and yellow sweet clover treatments.
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Fig. 4. Total polycyclic aromatic hydrocarbon concentration as a function of root surface area using glyphosate-treated tall fescue and yellow sweet clover.
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Fig. 6. Relationship between root biomass and root surface area of active tall fescue and yellow sweet clover.
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A relationship between the change in concentration of the seven individual compounds that were significantly degraded in the planted treatments and the change in root surface area and root biomass was evaluated to determine degradation rate constants during active root growth (Table 6). The 9- to 12-mo time period was not evaluated because both root biomass and root surface area of the active plant species declined, indicating the roots were under stress and not actively growing. The correlations between the change in PAH concentration and the change in root surface area for the active planted treatments resulted in very low slopes ranging from 2.0 x 10–6 to –1.1 x 10–3 (mg/kg)/cm2, indicating that there was no clear link between the degradation of the individual PAHs and the root surface area. The rate constants for the change in PAH concentration as a function of the change in the root biomass for the active planted treatments were more significant (–0.0002 to –0.084 [mg/kg]/g).
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Table 6. Changes in polycyclic aromatic hydrocarbon concentrations as affected by root characteristics during active root growth.
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The effect of the root decay induced in the glyphosate-treated plants was evaluated by assessing the correlation between the change in PAH concentration as a result of root decay and the change in the decay in root biomass and root surface area (Table 7). Higher rate constants were observed for yellow sweet clover, indicating a stronger relationship for this plant, particularly between PAH concentration and root biomass. Data from the 9- to 12-mo sampling periods, when the active plants experienced a natural root turnover, were included in the decay correlations and the active yellow sweet clover measurements were similar to those of the glyphosate-treated clover.
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Table 7. Changes in polycyclic aromatic hydrocarbon (PAH) concentrations as affected by root characteristics during period of root decay after application of glyphosate.
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CONCLUSIONS
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This research supports the notion that phytoremediation is a feasible treatment approach for PAH-contaminated soil. This method may be particularly applicable as a polishing tool for previously treated material. Tall fescue treatments resulted in the highest root and shoot biomass, and root surface area, but did not produce the highest contaminant degradation rates. Contrary to our hypothesis on the effect of root decay on PAH dissipation, the active yellow sweet clover treatment resulted in the highest degradation rates. Differences were observed between treatments for seven PAHs, with sweet clover resulting in 60 to 75% degradation of those compounds. Root death and decay did not prove to have a major effect on PAH degradation in this study. The PAH degraders in the vegetated treatments were significantly greater than those in the unvegetated control.
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ACKNOWLEDGMENTS
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This research was supported by the USEPA SITE Program.
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REFERENCES
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ABSTRACT
INTRODUCTION
