Environ. Sci. Technol. 2001, 35, 2773-2777
Rhizosphere Effects on Microbial Community Structure and Dissipation and Toxicity of Polycyclic Aromatic Hydrocarbons (PAHs) in Spiked Soil E R I K J . J O N E R , * ,† A N D E R S J O H A N S E N , ‡ ANDREAS P. LOIBNER,§ MARY ANN DELA CRUZ,§ OLIVER H. J. SZOLAR,§ JEAN-MARIE PORTAL,† AND CORINNE LEYVAL† Centre de Pe´dologie Biologique, CNRS, FRE 2111 associated with the H. Poincare´ University, 17 rue N. D. des Pauvres, B.P. 5, F-54501 Vandoeuvre-les-Nancy Ce´dex, France, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Fredriksberg C, Denmark, and Institute for Agrobiotechnology, Konrad Lorenz Strasse 20, A-3430 Tulln, Austria
Phytoremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs) has so far neglected the possible role of the ubiquitous symbiotic associations between plant roots and fungi known as arbuscular mycorrhizas. A time course laboratory experiment with clover and ryegrass grown on spiked [500 + 500 + 50 mg kg-1 of anthracene, chrysene and dibenz(a,h)anthracene] soil demonstrated for the first time that dissipation of condensed PAHs may be enhanced in the presence of arbuscular mycorrhiza [66 and 42% reductions in chrysene and dibenz(a,h)anthracene, respectively, versus 56 and 20% reductions in nonmycorrhizal controls]. Addition of a surfactant accelerated initial PAH dissipation but did not attain final PAH concentrations below those obtained with nonmycorrhizal plants. Toxicity tests (earthworm survival and bioluminescence inhibition in Vibrio fischeri) indicated that mycorrhiza reduced the toxicity of PAHs and/or their metabolites and counteracted a temporally enhanced toxicity mediated by surfactant addition. Phospholipid fatty acid profiles demonstrated that the imposed treatments altered the microbial community structure and indicated that the mycorrhiza-associated microflora was responsible for the observed reductions in PAH concentrations in the presence of mycorrhiza.
Introduction Polycyclic aromatic hydrocarbons (PAH) in soil are pollutants of major concern due to their recalcitrance and mutagenic/ carcinogenic properties (1). Phytoremediation has long been recognized as a cost-effective method for removal of organic pollutants from soil (2), although data on the phytoreme* Corresponding author phone: +33383 510 465; fax: +33 383 576 523; e-mail:
[email protected]. † Centre de Pe ´ dologie Biologique, CNRS. ‡ Royal Veterinary and Agricultural University. § Institute for Agrobiotechnology. 10.1021/es000288s CCC: $20.00 Published on Web 06/02/2001
2001 American Chemical Society
diation of PAHs are less abundant and less unanimous than data on other organic pollutants. There are several advantages of including plants in bioremediation schemes. Plant cover reduces wind and water erosion and, thus, spreading of polluted soil. Plants improve soil structure and, thus, aeration and hydrological aspects that may impose limitations to biodegradation. Plants may exude oxidative enzymes that contribute to PAH degradation. Finally, roots furnish and constitute easily degradable carbon and energy that generally increase microbial activity in soil, which may ultimately lead to enhanced degradation of organic pollutants through direct metabolism (diauxic growth and related phenomena) or cometabolism. Plant-microbe symbioses are ubiquitous in natural and most anthropogenically influenced soils. Whereas phytoremediation of inorganic soil pollutants has considered various plant-microbe symbioses (e.g., Rhizobia, Frankia, ecto- and endomycorrhizas) for some time (3, 4), phytoremediation of organic pollutants has only started to consider N-fixing symbioses (5) as well as the extremely frequent symbiotic root-fungus associations called mycorrhiza (6, 7). Contrary to N-fixing symbioses that concern a limited number of plant families, the large majority of natural and cultivated plants form mycorrhiza (8). The most widespread type of mycorrhiza, the arbuscular mycorrhiza (AM), colonizes mainly herbaceous plants. It is thus the most relevant mycorrhiza for the plants commonly used for phytoremediation of organic pollutants. AM has a profound impact on growth (9), stress tolerance (10), and interspecies competition of plants (11), as well as composition and activity of soil microorganisms (12). Recently, we reported a positive effect of AM on plant establishment and survival in PAH-polluted soil (13) and obtained indications that AM may have a positive reciprocal effect on PAH dissipation during phytoremediation (14). These results need to be validated using a wider range of plants, AM fungi, and PAH compounds but suggest that AM has potential as a bioremediation agent that should be considered in phytoremediation schemes. The objectives of the present study were to assess the influence of AM on the dissipation of three common PAH compounds with different recalcitrances spiked into an agricultural soil. The time course of this dissipation was correlated with ecotoxicological tests and a characterization of the soil microbial community to evaluate whether degradation products of the imposed treatments differed with regard to toxicity and whether differential development of the soil microbial community could reveal differences other than the imposed biological treatments (plants/no plants; mycorrhiza/no mycorrhiza) to explain any observed differences in PAH degradation.
Materials and Methods The experiment had an unbalanced factorial design with four main treatments (unplanted soil, planted soil with and without mycorrhizal inoculation, and mycorrhizal inoculation plus surfactant addition, all with PAH spiked soil), two harvest times (8 and 16 weeks), and five replicate pots per treatment. In addition to this factorial part, three replicate pots were included to monitor mycorrhizal effects in soil without PAH and harvested after 8 weeks. A silty sand topsoil (11% clay; pHCaCl2 6.6; 2.5% organic matter; 6 mg kg-1 inorganic N, 26 mg kg-1 Olsen P) was sieved ( 0.05, n ) 5).
DBA in 1 L of dichloromethane, adding this in three portions to 2060 g of soil (3% water) in a desiccator, and evaporating the solvent under vacuum with heating (45 °C). When dry, the spiked soil was mixed with moist (10% water) nonspiked soil 1:3 to obtain the final PAH concentrations given above. Five batches of 8 kg of soil were prepared separately, thoroughly mixed, and stored for 4 weeks for aging of added PAHs. Soil microorganisms were reintroduced after aging by adding 50 mL kg-1 of a soil suspension [50 g of the original soil plus 1 L of water blended in a Waring blender for 30 s, sieved (
