Environ. Sci. Technol. 2003, 37, 2371-2375
Rhizosphere Gradients of Polycyclic Aromatic Hydrocarbon (PAH) Dissipation in Two Industrial Soils and the Impact of Arbuscular Mycorrhiza ERIK J. JONER* AND CORINNE LEYVAL Laboratoire des Interactions Microorganismes-Mine´rauxMatie`re Organique, dans les Sols (LIMOS) - CNRS, FRE 2440, H. Poincare´ University, P.O. Box 239, F-54506 Vandoeuvre-les-Nancy Ce´dex, France
Phytoremediation of organic pollutants depends on plant-microbe interactions in the rhizosphere, but the extent and intensity of such rhizosphere effects are likely to decrease with increasing distance from the root surface. We conducted a time-course pot experiment to measure dissipation of polycyclic aromatic hydrocarbons (PAHs) in the rhizosphere of clover and ryegrass grown together on two industrially polluted soils (containing 0.4 and 2 g kg-1 of 12 PAHs). The impact of the fungal root symbiosis arbuscular mycorrhiza (AM) on PAH degradation was also assessed, as these fungi have previously improved plant establishment on PAH-polluted soils and enhanced PAH degradation in spiked soil. The two soils behaved differently with respect to the time-course of PAH dissipation. The less polluted and more highly organic soil showed low initial PAH dissipation rates, with small positive effects of plants after 13 weeks. At the final harvest (26 weeks), the amounts of PAHs extracted from nonplanted pots were higher than the initial concentrations. In parallel planted pots, PAH concentrations decreased as a function of proximity to roots. The most polluted soil showed higher initial PAH dissipation (25% during 13 weeks), but at the final harvest PAH concentrations had increased to values between the initial concentration and those at 13 weeks. An effect of root proximity was observed for the last harvest only. The presence of mycorrhiza generally enhanced plant growth and favored growth of clover at the expense of ryegrass. Mycorrhiza enhanced PAH dissipation when plant effects were observed.
Eh, partial pressures of O2/CO2 and other gases, moisture, etc. Rhizosphere technology has been applied to bioremediation of organic pollutants through the use of plants for accelerating degradation rates (2), and the presence of plants has positive effects on degradation of a wide range of compounds including simple phenolics, aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), trichloroethylene, chlorinated aromatics, insecticides, and explosives (3-7). The positive effects of roots on biodegradation may be derived from the additional input of easily degradable C that induces diauxic growth or cometabolic processes (5, 8). Root exudation varies both qualitatively and quantitatively between plant species, as a function of plant age, according to mycorrhizal status, and nutrient deficiency (9-12). Other parameters that have profound influences on phytoremediation of organics include rooting density, fertilization, and watering. As for the soil, its texture, structure, organic matter content, hydrophobicity, etc. will influence the diffusion of exudates into the soil and mineral nutrients toward the roots. Thus, the extension of the rhizosphere may vary for welldescribed parameters such as C diffusion and nutrient depletion (13, 14). The question of how far a rhizosphere effect on degradation of organic pollutants may extend has however never been approached, but the preferential use of plants with fibrous root systems for rhizodegradation indicates that it is rather narrow. Arbuscular mycorrhiza (AM) is a ubiquitous symbiosis between soil fungi and roots of most herbaceous plant species that permits the host plant to exploit nutrients in the soil outside the rhizosphere through fungal transport. The involved fungi also have a certain capacity to affect the soil through extrusion of H+ ions (15) and protein/glycoprotein deposition (16). The latter, together with hyphal entanglement, also improves soil aggregation (17). Extraradical AM hyphae are fed with C from the host plant and may reach densities of >100 m in 1 g of soil (18). They are also ephemeral and thus represent a flux of C from roots to soil that is one of the most important C sources for microorganisms outside the rhizosphere (19). The impact of mycorrhizas, and particularly AM, is starting to draw attention in the context of phytoremediation of organics (20-22), and recent results have shown that these symbioses may be crucial for both plant establishment on contaminated sites (23) and for degradation of PAHs (24). The aims of the present study were thus to measure the rhizosphere effect on dissipation of PAHs in two contrasting industrial soils and to assess the impact of arbuscular mycorrhiza by comparing the performance of mycorrhizal and nonmycorrhizal plants.
Materials and Methods Introduction The rhizosphere is the soil volume surrounding a root where physical, chemical, and/or biological parameters have been modified by the presence of the root. The rhizosphere is most commonly distinguished from the bulk soil by a depletion of immobile nutrients (NH4+, H2PO4-/HPO4-2, and certain micronutrients) and an elevated microbial activity based on readily available carbon derived from root exudation and other root depositions (1). Other soil parameters may also be altered directly or indirectly by the roots, such as pH, * Corresponding author phone: +47 6494 9191; fax: +47 6494 2980; e-mail:
[email protected]. Current address: Norwegian Forest Research Institute, Hogskoleveien 12, N-1432 Aas, Norway. 10.1021/es020196y CCC: $25.00 Published on Web 04/16/2003
2003 American Chemical Society
A pot experiment with a full factorial design was set up with five replicates per treatment (two soils; 0.4 and 2 g PAH kg-1, three planting conditions; unplanted, planted nonmycorrhizal and planted mycorrhizal, and two harvest times; 13 and 26 weeks). The soil with the lowest PAH content (soil 1) was collected from the surface horizon (0-30 cm) outside an old coal factory in central UK and supported a mixed grass/clover vegetation. The soil with the highest PAH content (soil 2) was collected by the proprietor of an abandoned coke plant in northern France and contained occasional ruderal plants. Additional soil data are given in Table 1. Both soils were dried to ca. 10% water content, sieved to 50% of available water had been lost by evapotranspiration. Pots were kept in a growth chamber with 300-350 µmol m-2 s-1 PAR (16 h day-1), at 22/17 °C day/night temperature, and a relative humidity of 60-70%. Prior to the harvests (13 or 26 weeks after sowing) pots were left without watering for 2 days. At harvest, shoots were cut at the soil surface, dried, and weighed. The upper 2-5 mm of soil in each pot, and the soil that did not adhere to roots, was discarded (approximately 10-25% of the soil mass). Roots and remaining soil were further separated by gently crushing the soil and shaking the roots, and the portion of soil obtained in this manner was classified as loosely attached to roots (70-80% of soil mass). Soil that required continued, vigorous rubbing and shaking of the root system was classified as strongly attached to roots (2-10% of the soil). After this sequential soil separation by shaking, the intact root system was washed in a large beaker with 0.5 L deionized water, and 2372
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TABLE 2. Root Dry Weight, Root Density in Soil, and Mycorrhizal Colonization after 13 and 26 Weeks Phytoremediation of Two Industrial Soils without (NM) and with (Myc) Arbuscular Mycorrhizaa
harvest 13 weeks
PAH level mycorrhizal in soil status low high
26 weeks
low high
root mass (g)
colonized root lengthb root length density clover ryegrass (cm cm-3) (%) (%)
NM Myc NM Myc
0.39 c 0.52 bc 0.15 c 0.32 c
19.9 abc 23.2 ab 7.3 c 17.6 abc
0 34 b 0 36 b
0 26 ab 0 11 a
NM Myc NM Myc
0.51 bc 0.84 ab 0.34 c 0.93 a
17.6 abc 19.7 abc 11.7 bc 30.5 a
0 66 c 0 59 c
0 25 ab 0 36 b
a Values followed by the same letter within a column are not significantly different (p > 0.05, n ) 5). b Colonization data were tested after log(x+1) transformation, including data for both plant species.
the adhering soil was recovered on a Whatman GF/A glass microfiber filter (this fraction was classified as rhizoplane soil; 0.5-3% of soil mass). Assuming that these portions of soil had formed consecutive cylindrical layers around the roots, an average thickness of each layer was estimated based on soil mass and density, mean root diameter, and the total root length of each pot. Washed roots were cut in 0.5-1 cm pieces, mixed under a jet of water, and blotted dry, and a subsample was taken for measurements of total and mycorrhiza-colonized root length using trypan blue staining (25) and a line intersect method (26). Roots of ryegrass and clover were assessed separately, using morphological traits to distinguish roots of the two plants. PAHs were extracted by Soxhlet extraction [15 and 5 g air-dry soil for soil 1 and soil 2, respectively, or soil+microfiber filter cut into pieces (ca. 0.5 cm2), 120 mL chloroform, 4 h] and quantified using a 3400 CX Varian gas chromatograph (programmable sample injector set between 25 and 300 °C at 180 °C min-1; oven temperatures: 70-150 °C at 10 °C min-1 and 150-300 °C at 6 °C min-1) coupled with a mass spectrometer (ION TRAP Saturn III, Varian) fitted with a 30 m DB5 MS column. The mass spectrometer was operated at 70 eV in electron impact mode, quantifying 12 PAHs by single ion monitoring at an ion trap temperature of 220 °C. Tests were run to check if PAHs were lost during filtration of rhizoplane soil (filtrate subject to liquid-liquid extraction and GC-MS analysis), and if soil fractionation introduced a bias with respect to CaCO3 content. Only minor differences in CaCO3 content were found and corrected for. Treatment effects were tested by ANOVA, and means were compared by Fishers PLSD test for unplanned comparisons using the software StatView (Abacus Concepts, Inc. Berkeley, CA).
Results Shoot dry weights of plants grown on soil 1 (initial PAH concentration of 400 mg kg-1) were higher than those grown on soil 2 (initial PAH concentration of 2000 mg kg-1) (data not shown), but root dry weights were comparable and did not vary between mycorrhizal and nonmycorrhizal treatments except for soil 2 at the last harvest (Table 2). This pattern was the same when comparing root length densities. Mycorrhizal colonization was observed in mycorrhizainoculated treatments only, and colonized roots comprised from 11 to 66% of the total root length. No significant differences were observed for mycorrhizal colonization between the two soils, but colonization increased with time for both plants species in both soils, except ryegrass in soil 1. At the end of the experiment both soils supported plants
TABLE 3. PAH Concentrationsa in Two Soils as a Function of Proximity to Roots Measured after 13 and 26 Weeks of Cultivation of Mycorrhizal and Nonmycorrhizal Plants
soil sample start no plants nonmycorrhizal loosely adhering strongly adhering mycorrhizal loosely adhering strongly adhering no plants nonmycorrhizal loosely adhering strongly adhering rhizoplane soil mycorrhizal loosely adhering strongly adhering rhizoplane soil
soil 1 ∑12 PAH (mg kg-1) 405
b
SEM
soil 2 ∑12 PAH (mg kg-1)
SEM
18
2030
a
64
13 Weeks 348 c 12
1494
cd
50
315 327
cd cd
25 13
1577 1801
bcd ab
99 21
311 298
cd d
10 13
1539 1777
bcd b
56 97
26 Weeks 460 a 19
1763
b
47
477 413 275
a b d
19 20 18
1382 1689 1149
de bc ef
96 153 164
435 362 222
ab bc e
12 24 13
1042 1182 655
f ef g
62 92 57
a Values followed by the same letter within a column are not significantly different (p >0.05, n ) 5).
with a moderate to strong mycorrhizal colonization. Both frequency and intensity of colonization were higher in cloverroots compared to roots of ryegrass for both soils and at both harvests, which is a common observation also in nonpolluted soils due to the lower mycotrophy (mycorrhiza dependence) of fast-growing grasses (27). Total PAH concentrations in unplanted soils decreased to 87% and 73% of the initial concentrations during the first 13 weeks of the experiment for soil 1 and soil 2, respectively (Table 3). After 26 weeks, these respective values were up to 116% and 87%. The PAH concentration in soil supporting plants were unaffected by proximity to roots and the mycorrhizal status of these roots after 13 weeks. For soil 1, only samples of strongly adhering soil in the mycorrhizal treatment had PAH concentrations that were lower (298 mg kg-1) than in the unplanted control treatment (348 mg kg-1). For soil 2, there were no differences in PAH concentrations between samples adhering loosely and strongly to roots nor between mycorrhizal and nonmycorrhizal treatments. The PAH concentrations were higher (1777-1801 mg kg-1) in soil adhering strongly to roots compared to nonplanted soil (1494 mg kg-1). Total PAH concentration in nonplanted soil at the final harvest (26 weeks) was higher (460 mg kg-1) than the initial concentration (405 mg kg-1) for soil 1 and intermediate (1763 mg kg-1) between the initial value (2030 mg kg-1) and the concentration at 13 weeks (1494 mg kg-1) for soil 2. Samples of soil 1 adhering loosely to roots had similar PAH concentrations (477 mg kg-1) to corresponding nonplanted soil, whereas strongly adhering soil and rhizoplane soil had lower (362-413 mg kg-1) and substantially lower concentrations (222-275 mg kg-1), respectively. A reduction in PAH concentrations was observed in the mycorrhizal treatment in the case of rhizoplane soil. For soil 2, there were substantially lower PAH concentrations in all rhizosphere samples (11821382 mg kg-1) compared to nonplanted soil (1763 mg kg-1), except in the case of strongly adhering soil of the nonmycorrhizal treatment. The mycorrhizal treatment had lower total PAH concentrations than the nonmycorrhizal treatment for all three levels of sampling with respect to root proximity. When the PAH concentrations of individual samples were
FIGURE 1. PAH concentration in soil as a function of root proximity for (a) soil 1 and (b) soil 2 after cultivating clover and ryegrass in the presence or absence of arbuscular mycorrhiza for 26 weeks. Dotted and unbroken lines represent logarithmic curves fitted to the data for each soil and two mycorrhizal treatments. Horizontal lines indicate the PAH concentration in unplanted controls. plotted against a calculated average distance to roots, curves representing rhizosphere gradients of PAH concentrations could be fitted to the data of the two soils at the last harvest (Figure 1). These curves showed steeper gradients in the case of soil 1 but larger differences between mycorrhizal and nonmycorrhizal treatments in the case of soil 2. The steepest gradients of individual PAHs were observed for light (MW < 180) PAHs, whereas larger molecules were less affected by roots (phenanthrene and benzo[a]pyrene given as examples in Figure 2).
Discussion Phytoremediation of organics is based on the beneficial effects of roots on degradation. A multitude of changes occur in soil in the presence of roots that may be apprehended as changes in its chemical characteristics, modified microbial composition, and enhanced microbial activity. The single most important factor that causes microbial changes around roots is the input of large quantities of readily available organic substrates in the form of root exudates. These exudates diffuse into the soil where they gradually disappear due to radial dilution and microbial consumption. A substrate gradient thus exists that, along with other gradients, are likely to result in corresponding degradation gradients of recalcitrant molecules for which phytoremediation is effective. The present report is to our knowledge the first demonstration that such gradients exist, exemplified by 12 nonvolatile PAHs and two industrially polluted soils. Relating soil PAH concentration to root proximity, rather than reporting concentrations in homogenized samples from planted treatments, avoided any confounding effects of differences in root length density between treatments. Further, the distinction of soil more or less intimately assoVOL. 37, NO. 11, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Mean phenanthrene (Phe) and benzo[a]pyrene (BaP) concentration in soil as a function of root proximity for (a) soil 1 and (b) soil 2. Sampling, treatments, and symbols as in Figure 1 (bars are SEM, n ) 5). ciated with roots permitted the demonstration of a high PAH degradation potential in rhizoplane and strongly adhering rhizospheric soil, that would not have been apprehended if these small soil volumes (comprising 0.5-3% and 2-10% of the total soil volume, respectively) had been diluted with nonrhizospheric soil, as in bulked samples from planted soil. The finding that PAH dissipation is higher around arbuscular mycorrhizal roots than around nonmycorrhizal roots is another important result of this study which confirms our previous results obtained with an experimentally contaminated soil (24). In our view, this may have two possible explanations: Either (1) mycorrhiza modifies root physiology (enzyme activity, exudation, longevity) in a manner that stimulates PAH degradation, either by root derived enzymes or by rhizosphere organisms, or (2) mycorrhizal colonization affects root surface properties or rhizosphere soil properties that act on PAH availability through adsorption. The former explanation can be supported by several observations reported in the literature: First, mycorrhiza enhances the level of hydrogen peroxide in roots (28) and the activity of oxidative enzymes in roots and rhizosphere soil (29), which may lead to enhanced oxidation of PAHs around mycorrhizal roots. Second, mycorrhizal colonization results in quantitative (10) and qualitative (30) changes in root exudation, which in turn modify the microbial community colonizing the rhizosphere of mycorrhizal roots (31, 32). This mycorrhiza-associated microflora may be more competent in PAH degradation than that of nonmycorrhizal roots, simply by chance, or due to mycorrhizal enhancement of aromatic compounds in rhizosphere soil or roots (33, 34) that in turn induce degradation of more complex compounds with comparable chemical structure (35). 2374
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The latter explanation (modified root or rhizosphere properties resulting in adsorption) implies that the root surface or the rhizosphere soil of mycorrhizal and nonmycorrhizal plants are different. Effects of arbuscular mycorrhiza on root surface characteristics have to our knowledge not been investigated. The rhizosphere of mycorrhizal plants is however known to be enriched in organic matter (36), and arbuscular mycorrhiza has striking effects on average root longevity, as it enhances the proportion of higher order laterals with a short life span (37). Root senescence is usually preceded by a suberization that renders them more hydrophobic, and this process may potentially interfere with PAH availability through sorption. Considering that the half-life of ryegrass roots at a soil temperature of 20 °C is about 1 month (38), an experiment that lasts 6 months may implicate quantitatively important interactions between PAHs and dead roots. Short-term spiking experiments with high degradation rates have shown that adsorption of PAHs to roots is negligible (7, 39). PAHs do, however, have a strong affinity for adsorption on organic matter and may on a longer time scale thus accumulate on root debris and other types of organic matter in the rhizosphere, the quantity of which may be increased by mycorrhiza. Plant water uptake induces a mass flow of PAHs toward the roots (40) into a zone of enhanced microbial activity and conditions that favor PAH degradation but that also contains ample sites for adsorption on organic matter. The rhizosphere may thus be a sink for organic pollutants both through degradation and adsorption to organic matter. Which of these mechanisms that prevails could not be determined in the present experiment, as our PAH extractions/analyses, unlike 14C studies, only detect the parent compounds and no transformed PAHs. For the same reason, we cannot explain the apparent increase in PAH concentrations that was occasionally observed. These fluctuations may be due to biologically mediated desorption of PAHs that could not be extracted by Soxlhet extraction at the start of the experiment. More stringent extraction procedures (e.g. SFE, Soxtherm, pyrolysis-GC-MS) may have answered to this. Even in samples taken the furthest away from roots, lower PAH concentrations were found in the mycorrhizal treatment of one of the soils. These samples were taken on an average 0.6 mm from the surface of the roots, which is still within an expected diffusion range of root-derived C. Outside this range (normally beyond 1-2 mm), one may expect to see possible effects of mycorrhizal hyphae in a volume of soil that is unaffected by roots (41). The root densities encountered in the present experiment did however render the maximum inter-root distance smaller than this, and a pure hyphal effect could therefore not be assessed. Reduced concentrations of PAHs in the soil samples furthest away from roots still indicate that a hyphal effect may exist, which holds good promise for the application of mycorrhiza in polluted field soils where root densities are far lower than in pot experiments. Addition of mineral nutrients to PAH contaminated soil has frequently resulted in enhanced rates of PAH degradation (42-44). During growth, roots absorb and reduce the amounts of bioavailable mineral nutrients such as ammonium and phosphate down to levels where they may be limiting microbial activity and growth (45-47). This nutrient depletion only concerns the rhizosphere where gradients are formed due to slow diffusion to replenish ions absorbed by the roots (13, 48). In mycorrhizal plants, N and P depletion also extend several centimeters beyond the rhizosphere due to hyphal uptake of nutrients that are transported back to feed the host plant (49, 50), but this hyphal depletion is commonly less exhaustive than root depletion (41, 51). In return, nutrient depletion in the rhizosphere can be less exhaustive around mycorrhizal than around nonmycorrhizal roots due to symbiotic feedback inhibition of root uptake (52). In the
present experiment, mineral nutrients were added weekly, so that mineral nutrient depletion in the rhizosphere would only temporarily be limiting microbial degradation. A question that remains to be answered is to what extent permanent nutrient depletion zones would affect biodegradation of PAH in the rhizosphere. The dissipation gradients observed in the absence of mycorrhiza were far narrower (e0.3 mm) than e.g. gradients in root exudation of organic acids and plant enzymes or the depletion zones of the most diffusion limited mineral nutrients (which are commonly g2 mm, see e.g. refs 41, 53, 54), and they were established only upon prolonged plant growth. It thus seems that the extent of the rhizosphere in a phytoremediation context can be very different from what we are used to from plant physiology and agronomy. This is important to take into account e.g. when choosing plants for phytoremediation purposes, and it may partially explain why plants with dense, fine roots such as the Gramineae are among the plant species that perform well for phytoremediation of several organics. The dramatic increase in PAH dissipation observed in the rhizosphere of plants inoculated with mycorrhiza further demonstrates that the rhizosphere concept is lacking a crucial component if these ubiquitous root symbionts are not taken into account.
Acknowledgments We thank Drs. D. Johnson and S. P. McGrath of the IACR Rothamsted, UK, for providing soil 1 and J. M. Portal for performing the PAH analyses. This work was funded by the European Commission (ENV4-CT97-0602).
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Received for review September 25, 2002. Revised manuscript received March 7, 2003. Accepted March 19, 2003. ES020196Y
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