Root Exudate Enhanced Contaminant Desorption - American

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Root Exudate Enhanced Contaminant Desorption: An Abiotic Contribution to the Rhizosphere Effect Gregory H. LeFevre,† Raymond M. Hozalski, and Paige J. Novak* Department of Civil Engineering, University of Minnesota, 500 Pillsbury Drive S.E., Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: Despite reports in the literature of superior contaminant degradation in the root-zone of plants, this phenomenon, known as the rhizosphere effect, is poorly understood. We investigated whether root exudates could enhance desorption of residual pollutants, thus improving bioavailability and subsequent biodegradation potential. Root exudates were harvested from three species of hydroponically grown plants, and artificial root exudates (AREs) were created using a literature recipe. Aliquots of the exudates were metabolized by soil bacteria to investigate whether biotransformed exudates exhibited different chemical characteristics or had different effects on contaminant bioavailability than ‘raw exudates.’ Slurries of naphthalene-aged soil containing raw exudates had a significantly lower soilwater distribution coefficient (Kd) than slurries with metabolized exudates or no-exudate controls, exhibiting median reductions of 50% and 55%, respectively. Raw exudates had a significantly lower surface tension while not increasing overall solubility, indicating the presence of surface-active compounds below the critical micelle concentration; this is a newly observed mechanism of the rhizosphere effect. Exudate samples were characterized by specific UV absorbance, spectral slope, fluorescence index, and excitation−emission matrices. Substantial changes in organic carbon character pre- and postmetabolism, and between harvested exudates and AREs, suggest that AREs are not chemically representative of plant root exudates. Overall, we present evidence that enhanced contaminant desorption in the presence of exudates provides an abiotic contribution to the rhizosphere effect.

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conditions for aerobic biodegradation.10 Plants also release root exudates that may act as contaminant analogues and either precondition the microbial community or specifically induce contaminant degradation.1,2,11−14 Finally, plant root exudates contain large quantities of simple carbohydrates that tend to stimulate overall bacterial populations1,2,11,15,16 or facilitate cometabolic degradation of contaminants.17 Current research does not indicate the extent to which each of these mechanisms may influence overall contaminant degradation. A mechanism upon which little research has focused is the potential for raw or metabolized plant root exudates to abiotically alter the bioavailability of hydrophobic contaminants in the rhizosphere. Exudates contain substances such as amino acids, organic acids, sugars, phenolic compounds, polysaccharides, and humic compounds,18 some of which are known to increase the desorption of hydrophobic organic compounds from soils.19 There is also some evidence that compounds in root exudates or their metabolized derivatives, such as glycerolipids and glycoproteins, have surface-active properties.14,19,20 Furthermore, soil bacteria that inhabit the rhizo-

INTRODUCTION Many persistent organic pollutants are known to degrade more rapidly and completely in the rhizosphere, the soil area that has been physically, chemically, or biologically altered by the presence of plant roots.1−4 This phenomenon is known as the ‘rhizosphere effect.’2−5 Researchers, for example, have measured significantly lower polycyclic aromatic hydrocarbon (PAH) residual in vegetated compared to unvegetated experimental systems.6 In addition, residual sorbed PAH concentrations in planted soils are typically a function of proximity to plant roots, with concentrations decreasing nearer the root.7,8 Because of the complex symbiotic relationships between plant roots and their associated bacteria and mycorrhizal fungi, combined with the inherent physical difficulty involved in studying the rhizosphere, the reasons for enhanced contaminant loss in the rhizosphere are not completely understood.1−5,9 Nevertheless, the performance and reliability of vegetated pollution control systems, such as phytoremediation, monitored natural attenuation, and stormwater bioretention cells, could be optimized through more efficient design if the fundamental mechanisms involved in the rhizosphere effect were better characterized. There are multiple hypotheses that can explain enhanced contaminant degradation in the rhizosphere. Plant roots can increase oxygen levels in soils and may therefore improve © 2013 American Chemical Society

Received: Revised: Accepted: Published: 11545

May 31, 2013 September 16, 2013 September 18, 2013 September 18, 2013 dx.doi.org/10.1021/es402446v | Environ. Sci. Technol. 2013, 47, 11545−11553

Environmental Science & Technology

Article

sphere are known producers of biosurfactants,14 and there is a substantial body of work demonstrating the ability of biosurfactants to increase contaminant desorption.21 Because bioavailability of persistent organic contaminants often limits long-term degradation,5 enhanced desorption in the rhizosphere could be a critical mechanism for facilitating phytoremediation success, and one that could be easily promoted in the field. Indeed, Gao et al.22 reported that artificial root exudates (AREs) enhanced the desorption of phenanthrene and pyrene from soil, and others23,24 have observed similar results through addition of organic acids that are likely present in exudates. Nevertheless, real plant root exudates are known to be complex1,25,26 and may vary substantially from AREs in function and character. Only one study found24 that real plant root exudates (from select agricultural crops) impacted contaminant sorption in soils, in this case sorption of DDT. At present, no work has investigated how subsequent exudate metabolism by soil bacteria may impact desorption. Furthermore, direct comparisons of organic carbon character between AREs and harvested exudates are lacking. In this work, we examined the effect of both raw and metabolized plant-harvested and artificial root exudates on naphthalene sorption and solubility. The effects of these exudates on naphthalene biodegradation were also studied. Naphthalene, a PAH, was chosen as a model contaminant because it is a pollutant commonly found in the environment, is moderately hydrophobic (logKow = 3.3), and sorbs readily to organic matter in soil. The specific objectives of this research were to (1) determine if and how raw or metabolized exudates enhanced naphthalene desorption from aged soil and (2) characterize the raw and metabolized plant-harvested and artificial root exudates to better understand their effect on contaminant sorption or degradation.

be sterile. This allowed the most realistic exudation pattern25 and better emulated the type of exudates (minimally degraded) that would be likely to reach nearby soil in the environment. Flasks were covered with aluminum foil to maintain dark conditions and prevent algal growth in the Hoagland’s solution. A set of fluorescent grow lights with a 16 h light/8 h dark period32 was used to grow the plants hydroponically for three months, at which point the Hoagland’s solution containing the root exudates was harvested. Periodically, exudate samples were collected for analysis, and the balance of the sample volume plus transpiration losses was replaced with 50%-strength Hoagland’s solution. Exudates harvested from the hydroponically grown plants are referred to as ‘harvested exudates’. Artificial root exudates (AREs) were prepared according to Joner et al.34 (Table S1), filter-sterilized through a prerinsed 0.22 μm nitrocellulose filter (Millipore #SA1J789H5), and diluted with sterile Hoagland’s solution to 10% original concentration prior to use. This diluted solution is simply referred to as ‘AREs’ throughout. AREs were stored in a 1-L baked amber flask capped with an autoclaved lid at 4 °C in the dark. Harvested raw exudates and AREs were characterized for dissolved organic carbon (DOC), UV 254 , specific UV absorbance (SUVA), spectral slope, and fluorescence (excitation−emission matrices, EEMs; fluorescence index, FI) as described below. ‘Raw’ exudates are defined herein as those exudates filtered from the hydroponic media (or created, for AREs) that have not yet undergone a specific metabolism step with inoculated bacteria (see below); some minimal degradation of ‘raw’ exudates is likely to have occurred while in contact with the plant roots, however. Production of Metabolized Root Exudates. To produce metabolized exudates, the raw harvested exudates and AREs were incubated with soil bacteria in batch cultures. Soil bacteria from a bioretention site on the University of Minnesota campus were live-extracted as described in the Supporting Information and frozen at −70 °C in 25% (v/v) glycerol for later use. Glycerol stocks of the soil bacteria were thawed and inoculated (10 μL) into R2A broth.35 The cells were grown for 24 h at room temperature (22 °C). Cells from this enrichment were washed in PBS,35 their density was measured optically, and then they were inoculated into the harvested exudates and AREs. These solutions were covered with aluminum foil and placed on a shaker table (room temperature, 120 rpm) for 9 days. Although no extracted culture can fully capture the microbial diversity of the rhizosphere, the exposure duration of the exudates to a mixed soil community was assumed to metabolize the bioavailable fraction in a similar manner to that expected in soil. The metabolized exudates or AREs were then filtered through precombusted (550 °C) Whatman 0.7 μm GF/F filters (Cat # 1825; refs 36 37) and stored in the dark at 4 °C. Metabolized exudates and AREs were characterized for DOC, UV254, SUVA, spectral slope, and fluorescence as described below. Soil Partitioning. Soil partitioning experiments were used to determine if desorption was enhanced in the presence of raw or metabolized exudates. Soil, described elsewhere38 (total organic carbon = 3.25 ± 0.37%), was aged for 17 months in the dark at room temperature in an aqueous solution containing excess solid naphthalene to ensure saturation. Approximately 3 g of the aged soil was weighed and added to 17 mL of a solution containing raw or metabolized exudates and four 3mm diameter baked glass beads to assist with mixing in a

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METHODS Production of Root Exudates. Plants that represent three major families of vegetation were chosen for exudate production and harvest: grass (Family: Poaceae; Cord Grass, Spartina pectinata), sedge (Family: Cyperaceae; Porcupine Sedge; Carex hystricina), and legume (Family: Fabaceae; Purple Prairie Clover, Dalea purpurea). Grasses are often used for phytoremediation applications,1,2 sedges are common in moist soil/wetland areas, and legumes are known to have unique root-rhizosphere properties that may enhance hydrocarbon remediation.27 These plants are also recommended for use in bioretention areas, which may receive organic contaminants such as petroleum hydrocarbons via stormwater.28−30 All plants were grown by Glacial Ridge Growers (Glenwood, MN). A hydroponic system (Figure S1) was used to harvest root exudates. Replicate plants (3 replicates per species) were maintained in separate hydroponic systems for a total of 9 individual plants. Teflon tubes fitted with a Luer-lock valve were inserted into the exit ports of 1-L vacuum flasks for exudate collection. The flasks were filled with filter-sterilized half-strength Hoagland’s nutrient solution for hydroponic growth (Sigma H2395; e.g., refs 31−33); 4-L flasks were used for the sedges to accommodate the large root mass. Prior to growing hydroponically, the root mass of each plant was cleaned of soil by hand washing with deionized water, after which it was suspended in the hydroponic solution either by autoclaved polyurethane foam plugs (grasses or clover) or friction (sedges). The hydroponic systems were not designed to 11546

dx.doi.org/10.1021/es402446v | Environ. Sci. Technol. 2013, 47, 11545−11553


Article

carbon-free 20 mL vial. The vial was filled headspace free and sealed with a PTFE-lined stopper. The solution consisted of raw or metabolized exudates (harvested and AREs) diluted to 3.0 mg/L DOC with half-strength Hoagland’s solution and 50 mM NaN3 (added as a biocide). This exudate concentration is environmentally relevant for rhizosphere conditions26 (see also Results and Discussion) and allowed normalization between individual plants in the hydroponic systems such that all tested DOC concentrations were the same. The vials were covered with opaque tape and placed on an end-over-end rotator for 18 days. Preliminary kinetics tests showed that this provided sufficient time for the soil-solution mixture to reach equilibrium (Figure S3). After 18 days, the vials were removed from the rotator and allowed to settle overnight. The aqueous fraction from each vial was removed and centrifuged in a 20 mL polystyrene tube at 2500 rcf ( × g) for 6 min, and the supernatant was analyzed for naphthalene as described below. The solids remaining in the vial were solvent-extracted using hexane and analyzed for sorbed naphthalene via gas chromatography (GC) as described previously,30,38 with a naphthalene extraction efficiency of 100.1% ± 12.5%. Replicate (n = 3) experiments were conducted on each individual replicate hydroponic plant system (i.e., n = 9 for sedge, n = 9 for grass, n = 9 for clover) for both the raw and metabolized exudates. Nine replicates were conducted for the experiments using the raw AREs, the metabolized AREs, and half-strength Hoagland’s solution (control). Solubility. The ability of the exudates (raw or metabolized) to alter naphthalene solubility was investigated. The following were added to vials such that no headspace remained: 0.1 g of solid naphthalene (Fisher), 8 mL of a solution containing raw or metabolized exudates (harvested and AREs, diluted to 3.0 mg/L DOC with half-strength Hoagland’s solution containing 50 mM NaN3 added as a biocide), and four 3-mm diameter baked glass beads to aid in mixing. The vials were sealed with PTFE-lined stoppers and placed on an end-over-end rotator for 5 days. After 5 days the aqueous naphthalene concentration was quantified as described below. Replicate (3) experiments were conducted on each individual replicate hydroponic plant system (i.e., n = 9 for sedge, n = 9 for grass, n = 9 for clover) for both the raw and metabolized exudates. Six replicate experiments were also conducted for each of the following: the raw AREs, the metabolized AREs, half-strength Hoagland’s solution as a control, and Milli-Q water as a reference value. Analytical Methods. Quantification of Dissolved Organic Carbon. Samples were filtered through precombusted (550 °C oven) Whatman 0.7 μm GF/F filters (Cat # 1825; refs 36 and 37) into carbon-free glass vials. The filtered samples were analyzed for organic carbon using a GE Sievers 900 Portable TOC Analyzer with an operating range of 0.03 ppb to 50 ppm and detection limit of 240 ppb. Surface Tension. Surface tension of liquids was measured using a Krüss K105T digital tensiometer. Liquid temperatures were measured (18−23 °C), and the surface tension values were then corrected39 to a temperature of 20 °C. The standard error for a set of replicate measurements (n = 4) was ±0.47%. Aqueous Naphthalene Concentration. Aqueous naphthalene was quantified via high performance liquid chromatography using an Agilent 1200 with UV/vis detector fitted with a Supleco Ascentis RP-Amide column (Cat# 565324-U; 15 cm × 4.6 mm, 5 μm). The eluent, consisting of 80% HPLC-grade (>99.9%) acetonitrile and 20% ultrapure water, was supplied at a flow rate of 1.0 mL/min. The injection volume was 20 μL.

The naphthalene peak signal was at 218 nm with a reference wavelength of 360 nm. The elution time was 3.8 min. The method detection limit35 for naphthalene was 0.57 μg/L. Spectral Measurements. Filtered samples were analyzed for ultraviolet/visible light absorbance from 200 to 600 nm using a Shimadzu UV-1601 PC spectrophotometer and a quartz cuvette. SUVA was determined by normalizing the UV254 by the DOC concentration.40 The spectral slope coefficient value was calculated37 using a least-squares regression fit of the absorbance values over the 300−600 nm range with a reference wavelength of 400 nm, using the following: aλ = a λoe s(λo − λ)

where aλ is the measured absorbance at the given wavelength, aλo is the absorbance at the reference wavelength, s is the spectral slope coefficient, λ is the wavelength at the given absorbance reading, and λo is the reference wavelength value. Optical Biomass Density Measurements. Biomass density was determined optically using a Beckman DU 530 UV/vis spectrophotometer. The following established relationship41 was used to estimate dry biomass concentration: W = 9929(1 −

1 − 0.7347A 660 )

where W is the dry biomass (mg/L), and A660 is the absorbance at 660 nm. Fluorescence Measurements. Excitation−emission matrices (EEMs) were generated on a Jobin-Yvon Horiba Floromax 3 fluorometer with xenon lamp using the method of Cory and McKnight.42 EEMs were measured in ratio mode with an excitation range of 240 to 600 nm (at 5 nm intervals) and emission measurements over the range of 320 to 550 nm (at 2 nm intervals) with an integration time of 0.25 s. Samples were first filtered through precombusted GF/F filters36 and then analyzed for UV254; strongly absorbing samples (UV254 > 0.3 Abs) were diluted by half to avoid inner-filter effects.42 All samples were equilibrated and run at room temperature. Lamp intensity and cuvette contamination were assessed daily, as was the water-Raman peak using a fresh Milli-Q water blank. EEMs are expressed in Raman units (measured value less the nanopure water blank value divided by the water-Raman area). The fluorescence index (FI) was calculated from the EEM measurements and is the ratio of the emission intensity at a wavelength of 470 nm to that at 520 nm, at an excitation of 370 nm.37 Data Analysis. Either a one-way analysis of variance (ANOVA) or a Student’s t test was conducted to assess systematic differences between multiple groups or between two groups, respectively. A Tukey-Kramer post-test was used to perform comparisons if ANOVA revealed significant differences (p < 0.05). Nonparametric analysis (Wilcoxon rank-sum, Wilcoxon matched-pairs tests, or Kruskal−Wallis tests) was employed if data were distributed in a significantly non-normal/ non-lognormal manner (determined via the Shapiro-Wilk normality test; α = 0.05). All statistical analysis was conducted in GraphPad Prism (version 5.1).

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RESULTS AND DISCUSSION Raw Exudates Alter Contaminant Desorption. The raw exudates were able to enhance desorption of naphthalene compared to the metabolized exudates or the control. Soilwater partitioning coefficient (Kd, L/kg) values for naphthalene-aged soil were significantly lower for the raw exudates than 11547



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Interestingly, although compounds in the raw exudates resulted in a significant decrease in Kd and surface tension, there was no concomitant increase in naphthalene solubility in the raw exudate samples (Figure 3, p = 0.9253). This observed

for the metabolized exudates at a 97% confidence level (p = 0.0292), with a median reduction of 50% (Figure 1). The raw

Figure 1. Summary of measured naphthalene soil−water partitioning coefficient values (Kd, L/kg). Kd values are pooled as raw and metabolized exudates with control (50%-strength Hoagland’s solution) and presented with summary statistics. Exudate origin for each point is shown for reference, but vegetation types were not significantly different (p > 0.1). The gray bars indicate geometric mean for each category.

Figure 3. Solubility of aqueous naphthalene in solutions of raw and metabolized exudates and comparison with control (Hoagland’s media) and DI water as a reference. Error bars indicate 95% confidence interval (n = 9 for sedge, grass, clover; n = 6 for AREs, Hoagland’s, and water). Presence of exudates did not alter solubility (p = 0.9253).

exudates were significantly lower than the control at a 93% confidence level (p = 0.0672) with a median reduction of 55%, whereas the metabolized exudates showed no difference from the control (p = 0.8301). Results in Figure 1 are pooled into either raw or metabolized exudates; Kd was not significantly affected by exudate source (p > 0.1; Figure 1). This enhanced desorption was likely due to the presence of low concentrations of surface-active compounds, as suggested by lower surface tension measurements in the raw exudate samples compared to the control or pure water43 (Figure 2). The raw exudates had a moderately (approximately 7%) but significantly (p = 0.0180) lower surface tension compared to the control, whereas the metabolized exudates were not different from the control (p = 0.1310). These surface-active compounds were likely degraded during the bacterial metabolism of the exudates.

behavior is consistent with the presence of surface-active compounds below the critical micelle concentration (CMC), where desorption (mobilization) of hydrophobic compounds is enhanced through the reduction of surface and interfacial tension without affecting the bulk aqueous solubility of the same compound43,44 (solubilization). Surfactant activity leading to reductions in surface tension is dependent upon concentration of surface-active compounds until the CMC is reached, whereupon solubility changes occur with no further reductions in surface tension.44 Because contaminants such as PAHs are not typically bioavailable when adsorbed to organic matter in soil or when present in the micelle phase,45 root exudate enhanced desorption may promote in situ biodegradation. Indeed, the observed desorption effects would likely be magnified for high molecular weight PAHs or PCBs because the effects of surface-altering compounds tend to be more pronounced for larger and less polar molecules.45−47 There is evidence in the literature of root exudates containing surface-active compounds such as glycerolipids and glycoproteins.14 Low molecular weight organic acids, sometimes found in root exudates, can also make certain contaminants more bioavailable by chelating cations present in soil and destabilizing organic matter-solid mineral complexes, thus releasing organic matter into solution.48,49 Compounds in the exudates may also compete with contaminants for adsorption binding sites on soil organic matter.19,20 It is unclear which of these mechanisms occurred in our system; nevertheless, the decreased Kd values observed in the presence of raw exudates suggest that a similar enhanced desorption effect could occur in the rhizosphere prior to the biological processing and biodegradation of root exudates.

Figure 2. Surface tension of the raw and metabolized exudates with Hoagland’s media as control. Center bars indicate mean ±95% confidence interval. The literature value for pure water surface tension at 20 °C is 72.8 mN/m (ref 39). 11548



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Table 1. Characterization of Raw and Metabolized Exudates by Origina Metabolized Exudateb

Raw Exudate Average

Std Dev

Average

Std Dev

% Change

Dissolved Organic Carbon (mg L−1)

Sedge Grass Clover 1/10 AREs

4.78 7.59 9.22 35.2

1.41 5.62 2.24 NA

0.77 7.09 3.10 18.7

0.10 7.49 0.50 NA

−84 −7 −66 −47

Spectral Slope Coefficient


0.0016 0.0060 0.0082 0.0136

2.0 × 10−4 4.6 × 10−3 4.0 × 10−3 NA

0.0181 0.0184 0.0200 0.0137

3.5 × 10−4 2.3 × 10−3 2.8 × 10−4 NA

1045 315 239 0.5

Fluorescence Index (FI)


1.3833 1.3870 1.4022 2.0383

2.14 × 10−2 7.30 × 10−3 3.25 × 10−2 NA

1.3988 1.4079 1.3993 1.5268

2.23 × 10−2 1.18 × 10−2 1.51 × 10−2 NA

1.1 1.5 −0.25 −25.1

UV 254

sedge grass clover 1/10 AREs

0.134 0.268 0.196 0.222

0.012 0.148 0.022 NA

0.135 0.315 0.208 0.556

0.021 0.137 0.001 NA

0.5 24 13 150

SUVA Specific UV Absorbance (L mg−1 m−1)

sedge grass clover 1/10 AREs

3.05 4.16 2.21 0.63

1.27 2.00 0.54 NA

17.8 6.94 6.79 2.97

3.93 3.76 1.15 NA

521 71 218 371

a

The average and standard deviation are based upon single instrument measurements of three biological replicates for the plant exudates (single values only for laboratory-created AREs). Full data for each biological replicate are located in the SI. bThe standard deviation and mean for the metabolized clover does not include sample 1 due to potential contamination (see text for details).

Summaries of the exudate DOC characterization tests indicate substantial changes to the exudates following bacterial metabolism and between the harvested exudates and the AREs (Table 1). Following metabolism, the SUVA and UV254 values increased significantly (p = 0.0098 and p = 0.0098, respectively). Because aromaticity is positively correlated with SUVA,40,54,55 it is likely that the most aliphatic fractions of the exudates degraded and either aromatic byproducts were generated or the aromatic portions of the exudates were retained following bacterial metabolism;56 however, such structural changes were not directly measured by other chemical methods. Some research indicates that the affinity of hydrophobic organic compounds for aliphatic portions of organic matter can be greater than that of the aromatic components.56 In this regard the DOC characterization is consistent with the results of our desorption work, showing that as exudate aromaticity increases with metabolism, the affinity of the naphthalene for the exudates decreases and the naphthalene remains bound to the soil. The metabolized exudates had a significantly higher spectral slope than the raw exudates (p < 0.0001). The sedge had the greatest increase in spectral slope, followed by the grass, clover, and AREs, respectively. These results agree with the SUVA values for the metabolized exudates. Given that both measurements are related to aromaticity,36 this was expected. Spectral slope is independent of the organic carbon concentration and is an indication of the extent of DOC diagenesis and DOC source.36 Lower slope values indicate a terrestrial DOC source, whereas higher slopes tend to correspond with autochthonous DOC sources.36 The spectral slope data for the raw and metabolized exudates was again consistent with expectations, as metabolized exudates

In addition to enhancing desorption, raw exudates may provide a desirable carbon and energy source for bacteria that competes with contaminant degradation through a variety of mechanisms, including repression at the enzyme activity or transcription level.50,51 For example, Kamath et al.51 showed that root-derived substances could repress the expression of the naphthalene dioxygenase gene while simultaneously promoting greater total contaminant degradation rates by increasing overall microbial activity. Exudates can serve as carbon and energy sources for bacteria to nonspecifically increase microbial populations, which tends to enhance the overall degradation of target contaminants and their metabolites.1,4,5,7,14,52,53 In naphthalene biodegradation batch tests using the raw and metabolized exudates (described in the SI), we observed no definitive evidence of substrate competition, with exudate addition (at concentrations equal to the abiotic experiments) having no significant effect on naphthalene biodegradation rate. Characterization of Harvested and Artificial Root Exudates. The organic carbon levels for the raw exudates varied considerably between each plant type (Table 1). The AREs (diluted to 10% with Hoagland’s solution, as previously described) contained substantially higher DOC than any of the harvested plant root exudates. After being metabolized, the DOC of the exudates decreased significantly (p = 0.0117), as expected. The metabolized Clover 1 sample was not included in further data analysis because the dramatic increase in DOC following metabolism corroborated noted visual observations that primary production or contamination occurred in this sample. DOC and spectral slope values for the raw exudates collected from the plant roots through time are shown in the Figure S5 and Figure S6, respectively. 11549



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Figure 4. Example excitation−emission matrices (EEM) for harvested plant root exudates (top; sample Grass 2) and artificial root exudates (below). Plot A is the harvested raw exudate and Plot B is the metabolized exudate; red dashed circle highlights shift from the humic region to the microbiologically derived region following metabolism. Plot C is the raw ARE and Plot D is the metabolized ARE. Note character and intensity differences between the AREs and harvested exudates and following metabolism. Note that by convention, all plots have an equal number of contour intervals to accent variations in relative shape; therefore, the scales for each plot vary. Full EEM data are in the SI.

shift from the humic region to the microbiologically derived region following metabolism. This is consistent with the observations made regarding spectral slopes, SUVA values, and more generally, DOC concentrations. Furthermore, both the raw and metabolized AREs differ substantially from the plantharvested exudate EEM profiles (Figure 4). It appears that the AREs are less represented in the humic zone than the harvested exudates, suggesting that AREs contain more labile carbon forms than those that are found naturally in the exudates from plant roots. Indeed, as the characterization data illustrates, an important finding of this research was that the AREs differ markedly from the harvested plant root exudates in terms of their organic carbon character. This difference was observed with multiple quantitative (DOC, SUVA, spectral slope, FI) and qualitative (EEMs) characterization approaches. For the experiments conducted, no significant differences (p > 0.1) were observed between the AREs and the three types of harvested plant root exudates (both raw and metabolized) regarding their impact on naphthalene desorption, surface tension, or solubility. Nevertheless, because the structure and character of organic carbon is known to affect both biological and abiotic processes,36,42,58,59 it is possible that the significant differences in the organic carbon character between the AREs and plant root exudates could result in different behavior in other circumstances. AREs used in this study were created using an established literature recipe34 and have been used by others to simulate exudates in the rhizosphere (e.g., refs 22, 60). Despite ease of preparation

were hypothesized to exhibit a more weathered and autochthonous signature. There was no significant systematic difference observed in the fluorescence index values of the raw and metabolized exudates (p = 0.4596). The raw ARE FI value, however, was much larger than that of the raw harvested exudates and also decreased substantially upon metabolism. For natural organic matter in aquatic systems, higher FI values (>1.4) indicate microbiologically derived carbon sources, whereas low FI values (50,000 mg/L as TOC). For example, the CMC for the biosurfactant dirhamnolipid is approximately 50 mg/L; above this concentration, hydrocarbon solubility changes will be observed.47 Given the observable effect even at relatively low ambient concentrations, the total influence of plant root deposition and exudates at the field scale could be substantial. Plant roots have a profound impact on local soil conditions by changing water flux and oxygenation,10 increasing soil porosity, and altering the microbial community through the release of exudates;1 exudate-enhanced desorption may also influence in situ contaminant degradation. Previous research has also indicated the value of increased vegetation densities and rooting depths for remediation purposes;7,8,16,30,65 this work further supports this conclusion.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 612-626-9846. Fax: 612-626-7750. E-mail: [email protected]. Present Address

† Department of Civil & Environmental Engineering, Stanford University, 473 Via Ortega, Stanford, CA 94305.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Patrick McNamara (Marquette University) for his careful reading of this manuscript and members of the James Cotner lab (University of Minnesota), especially Meghan Jacobson, for providing use of their fluorometer and operating instructions. This material is based upon work supported by the National Science Foundation under Grant No. DGE-0504195. Additional funding provided by a grant from the University of Minnesota Water Resources Center, an NSF Graduate Research Fellowship (GHL), and a University of Minnesota Graduate School Fellowship (GHL).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional method details, quality assurance/control, biodegradation batch tests, full exudate characterization data (DOC, UV254, SUVA, surface tension, spectral slope coefficients, FI, EEMs), DOC/spectral slope coefficient measurements through time. This material is available free of charge via the Internet at http://pubs.acs.org. 11551



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Root Exudate Enhanced Contaminant Desorption - American

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