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Published online 31 August 2007
Published in J Environ Qual 36:1444-1451 (2007)
DOI: 10.2134/jeq2006.0516
© 2007 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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Integrating Biodegradation and Electroosmosis for the Enhanced Removal of Polycyclic Aromatic Hydrocarbons from Creosote-Polluted Soils

José-Luis Niqui-Arroyo and José-Julio Ortega-Calvo*

Instituto de Recursos Naturales y Agrobiología, C.S.I.C., Apartado 1052, E-41080-Seville, Spain

* Corresponding author (jjortega{at}irnase.csic.es).

Received for publication November 23, 2006.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results
 Discussion
 Conclusions
 REFERENCES
 
This paper presents a hybrid technology of soil remediation based on the integration of biodegradation and electroosmosis. We employed soils with different texture (clay soil and loamy sand) containing a mixture of polycyclic aromatic hydrocarbons (PAH) present in creosote, and inoculation with a representative soil bacterium able to degrade fluorene, phenanthrene, fluoranthene, pyrene, anthracene, and benzo[a]pyrene. Two different modes of treatment were prospected: (i) inducing in soil the simultaneous occurrence of biodegradation and electroosmosis in the presence of a biodegradable surfactant, and (ii) treating the soils sequentially with electrokinetics and bioremediation. Losses of PAH due to simultaneous biodegradation and electroosmosis (induced by a continuous electric field) were significantly higher than in control cells that contained the surfactant but no biological activity or no current. The method was especially successful with loamy sand. For example, benzo[a]pyrene decreased its concentration by 50% after 7 d, whereas 22 and 17% of the compound had disappeared as a result of electrokinetic flushing and bioremediation alone, respectively. The use of periodical changes in polarity and current pulses increased by 16% in the removal of total PAH and in up to 30% of specific compounds, including benzo[a]pyrene. With the aim of reaching lower residual levels through bioremediation, an electrokinetic pretreatment was also evaluated as a way to mobilize the less bioaccessible fraction of PAH. Residual concentrations of total biodegradable PAH, remaining after bioremediation in soil slurries, were twofold lower in electrokinetically pretreated soils than in untreated soils. The results indicate that biodegradation and electroosmosis can be successfully integrated to promote the removal of PAH from soil.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results
 Discussion
 Conclusions
 REFERENCES
 
BIOREMEDIATION is a reasonable alternative to treat soils and sediments polluted by polycyclic aromatic hydrocarbons (PAH) when the characteristics of the polluted site do not demand urgent and more expensive and aggressive remediation solutions, such as incineration. Indeed, the successful bioremediation of PAH pollution in soils and sediments is well documented (Mueller et al., 1989; Carriere and Mesania, 1995; Breedveld and Karlsen, 2000; Breedveld and Sparrevik, 2000; Eriksson et al., 2000). The process usually consists of sustainable agricultural methods (landfarming) that involve precise nutrient amendments and periodical substrate homogenization, what promotes the oxygenation and bioaccessibility of nutrients and PAH for native microbial populations (Harmsen, 2004). In spite of its success, bioremediation of PAH still faces a major limitation, which is the low bioaccessibility of the target compounds. This causes, sometimes, too slow pollutant removal and residual concentrations that are still above legal standards. Indeed, biphasic ("hockey stick") kinetics of biodegradation, consisting of an initial period of fast degradation, followed by a second, much slower phase, is commonly observed in soils and sediments during bioremediation. The exact magnitude of the risk associated with the residual pollutant concentrations remaining after bioremediation is still a matter of debate (Semple et al., 2004).

During recent years an emerging technology, based on the controlled application of low-power DC electric fields to polluted soils, has shown promising results at the laboratory scale with PAH and other hydrophobic chemicals (Saichek and Reddy, 2005a). The limited transport that PAH exhibit in soil during electrokinetic treatment can be promoted with the use of solubility-enhancing agents (surfactants, co-solvents, and cyclodextrins), leading to significant pollutant removal through in situ flushing. The technique has been successfully tested with added, individual PAH, mainly phenanthrene, alone (Ko et al., 2000; Saichek and Reddy, 2003a, 2003b, 2005b; Reddy and Saichek, 2004; Yang et al., 2005), or present along with heavy metals (Maturi and Reddy, 2006), and with PAH mixtures present in historically contaminated soils (Maini et al., 2000; Reddy et al., 2006). However, the technology still faces major limitations due to the complex nature of electrokinetic phenomena (electromigration, electroosmosis, and electrophoresis), what results in a poor prediction capacity for key operating aspects in PAH removal such as direction and duration of the electroosmotic flow (Saichek and Reddy, 2005a).

The integration of bioremediation and electrokinetics for the treatment of hydrophobic organic soil contaminants has recently been addressed as a relevant innovative step in soil remediation (Wick et al., 2007). However, to our knowledge, there have been only two documented attempts to connect these two technologies in PAH remediation. One study focused on electrokinetic influence on transport of specialized, PAH-degrading strains through alluvial sand, clayey soil, and glass beads (Wick et al., 2004). These authors showed that electroosmotic transport is the main mechanism by which PAH-degrading microorganisms can be electrokinetically dispersed in soil. The other study showed the electrokinetic enhancement of PAH biodegradation in creosote-polluted clay soil (Niqui-Arroyo et al., 2006). This work postulated, on the basis of experimental results with phenanthrene, that the mobilization of soil fluids by electroosmosis, in the absence of surfactants, can promote biodegradation by increasing mass transfer of PAH and nutrients toward bacterial cells.

This research constitutes a new attempt to integrate biodegradation and electroosmosis into an electrokinetic bioremediation procedure for the efficient PAH removal from soil. We employed soils with different textures containing a mixture of PAH present in creosote, and inoculation with a representative soil bacterium that was able to degrade six different PAH. Our main objectives were (i) to test the effect of simultaneous biodegradation and electroosmosis on PAH removal from soil in the presence of a nontoxic and biodegradable surfactant, and (ii) to determine if a pretreatment step with low-voltage DC current causes a change in soil in the bioaccessibility of PAH, which would improve bioremediation performance.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
 Discussion
 Conclusions
 REFERENCES
 
Soils
Three soils were used in this study: two creosote-polluted clay soils and an agricultural soil. The clay soils (samples I and E2), classified as a calcaric fluvisols, were provided by EMGRISA (Madrid, Spain) from a wood-treating facility in Andújar (Jaén, Spain) with a record of pollution by creosote exceeding 100 yr. Clay soil I was obtained from a site with heavy pollution (4501 mg kg–1 total PAH, sum of 16 EPA PAH), whereas clay soil E2 originated from a neighboring site with low PAH levels (approximately 50 mg kg–1 total PAH). Both samples had the same organic matter content and particle size distribution, namely 3% organic matter, 1% coarse-grained sand (2.0–0.2 mm), 2% fine-grained sand (0.20–0.05 mm), 37% silt (0.050–0.002 mm), and 60% clay (<0.002 mm). A detailed description of clay soil I characteristics is given elsewhere (Niqui-Arroyo et al., 2006). The agricultural soil was a loamy sand from Coria del Río, Seville, Spain (Typic Xerochrepts, 0.8% organic matter, pH 6.5). Its particle size distribution was 73.3% coarse-grained sand, 6.0% fine-grained sand, 14.0% silt, and 6.7% clay . The soils were air-dried and sieved (2 mm mesh). Clay soil I was used as an intact soil matrix or in 1:10 mixtures with clay soil E2 or loamy sand. These PAH-containing soil mixtures, obtained after homogenization in a glass vessel with a rotary shaker for 6 h, will be referred as "clay soil II" or "loamy sand," respectively. To account for possible variability of PAH concentrations among the batches of loamy sand prepared for each electrokinetic experiment, analysis of each batch is reported and labeled with different capital letters: I, II, and III. The content of target PAH in the soil batches used for electrokinetic experiments is shown in Table 1.


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  		Table 1. Content of target polycyclic aromatic hydrocarbons (PAH) in the different creosote-polluted soil batches used for electrokinetic experiments.

 
Biodegradation
Biodegradation was measured in the solid phase and in slurries. Solid-phase experiments were performed in duplicate in closed biometer flasks (Bellco Glass, NJ) with 15 g of dry soil, three times autoclaved, and adjusted to 80% of field capacity with an inorganic salts solution (pH 5.8) described elsewhere (Niqui-Arroyo et al., 2006). Preliminary experiments showed that this sterilization procedure was efficient to prevent any biodegradation activity during the experimental period in the absence of inoculation. Fifty microliters of an acetone solution, with approximately 100000 dpm of [9-14C]-phenanthrene (13.1 mCi mmol–1), [3-14C]-fluoranthene (45.0 mCi mmol–1), or [4,5,9,10-14C]-pyrene (58.7 mCi mmol–1), all of them with a radiochemical purity > 98%, and provided by Sigma Chemical Co., St. Louis, MO, were added to the soil. Once the solvent was evaporated, the soil was inoculated with the bacterium Mycobacterium gilvum VM552. It was cultured with phenanthrene and prepared for mineralization experiments as previously described (Gomez-Lahoz and Ortega-Calvo, 2005). Each flask received an inoculum of 1.4 x 107 cells g–1. The flasks were closed with Teflon-lined stoppers, and incubated at 23 ± 2°C. Production of 14CO2 was measured as radioactivity appearing in the alkali trap of the biometer flasks. The NaOH solution was mixed with 5 mL of liquid scintillation cocktail (Ready Safe, Beckman Instruments Inc., Fullerton, CA, USA), and the mixture kept in darkness for about 8 h for dissipation of chemiluminescence. Radioactivity was measured with a liquid scintillation counter (Beckman Instruments, Inc., Fullerton, CA, USA; model LS5000TD). Mineralization rates were calculated and compared as the slope of the regression lines drawn with the points belonging to the phase of maximum mineralization (Ortega-Calvo et al., 1995). Residual contents of native PAH were determined in separate flasks that were incubated under the same conditions but contained no 14C-labeled compound. After mineralization reached the plateau, duplicate flasks were sacrificed and kept frozen at –80°C until analysis for PAH content by high performance liquid chromatography (HPLC).

For experiments with soil slurries, 1 g of soil was placed (in duplicate) in 250-mL Erlenmeyer flasks, 1 mL of distilled water containing 100000 dpm of [9-14C]-phenanthrene was added, and the mixture was homogenized for a 72 h period. A sterile, inorganic salts solution (pH 5.8) was added to complete a final volume of 70 mL. To test possible effects on biodegradation of the chemicals used in electroremediation experiences, the solution contained, when appropriate, the desired concentration of the biodegradable surfactant Brij 35, from Sigma-Aldrich Chemie, GbmH, Steinheim, Germany, or Tris-acetate-buffer (TA) of pH 7 (Sambrook et al., 1989). Brij 35 is a nonionic alkylpoly (ethylene glycol) ether surfactant [C12H25O(CH2CH2O)23H] that was included in the experiments on the basis of previous work that showed limited losses of PAH after electrobioremediation in the absence of surfactants (Niqui-Arroyo et al., 2006). It was selected on the basis of an extensive literature review that focused on the desorption, solubilization, and/or biodegradation performance of various nonionic surfactants (Liu et al., 1991, 1992, 1995; Yeom et al., 1996; García et al., 2001). Moreover, Brij 35 was chosen because it has been found to be nontoxic to bacteria (Tiehm, 1994). The slurries were then inoculated with the bacterium M. gilvum VM552. Each flask received an inoculum of 3.6 x 109 cells g–1. The bacterium was able to use acetate but not Brij 35 as a carbon or energy source (M. Bueno, IRNASE, personal communication, 2007), and therefore no attempt was made to measure the concentration of the surfactant during the different experiments. The flasks were then closed with Teflon-lined stoppers, from which an 8-mL vial containing 1 mL of 0.5 M NaOH was suspended to trap 14CO2. The flasks were incubated at 23 ± 2°C on an orbital shaker operating at 150 rpm. Measurements of mineralization of radiolabeled phenanthrene and residual concentration of native PAH were performed as above.

Electrokinetic Treatment
The cell designed, shown in Fig. 1 , consisted of a polyethylene body protected with an inner layer of glass, to prevent PAH loss due to sorption. Two rod electrodes, made of a titanium base and coated with a mix of platinum metal group oxides (METAKEM GmbH, Usingen, Germany), were inserted in the soil inside cylindrical glass filtercandles (Robuglasfilter-Geräte GMBH, Hattert, Germany) which served as electrode reservoirs. The filtercandles had porous walls (160–250 µm pore size), allowing the exchange of electrode solution into and out of the reservoirs. The separation distance between the electrodes was 16 cm. The test soil (200 g) was packed in the cell as a 5-cm-wide zone perpendicular to the line between the electrodes. The PAH-polluted soil was between two zones each containing 300 g of uncontaminated, agricultural soil, which was in contact with electrode reservoirs (Fig. 1). The soil was packed in layers and saturated with 150 mL of inorganic salts solution (Niqui-Arroyo et al., 2006) that contained, when appropriate, Brij 35 at a concentration of 36 mg mL–1 (or 35 mg g–1 soil). This concentration was well above the critical micelle concentration (CMC) of this surfactant in aqueous solution, 0.078 mg mL–1, and in aqueous suspensions of clay soil I, 0.817 mg mL–1 (M. Bueno, personal communication, 2007). During packing, the soil was inoculated with M. gilvum VM552, at a cell density of approximately 107 to 109 cells g–1. The voltage applied to the test cell ranged from 0.9 to 1.1 V cm–1. The DC power supply used was a Freak HY3005D-3 model unit, 30 V, 5 A (Instrumentación y Componentes, Zaragoza, Spain). Voltage measurements along the anode-cathode axis at points located in the same sections of the cells showed a linear relationship to the distance from the electrodes, and confirmed the monodimensional distribution of voltage within the cell. In some experiences, separate cells were maintained under exactly the same conditions, but without an electric field, to determine compound losses due to bioremediation. Abiotic treatments were also run with soil autoclaved three times, which received no inoculum, and was treated with electric field as described above, to assess possible effects due to electro-osmotic flushing.


Figure 1
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  		Fig. 1. Design of electroremediation cell used to treat polycyclic aromatic hydrocarbon (PAH)-polluted soils.

 
Preliminary tests showed that for an appropriate performance of the electrokinetic cell it was crucial to avoid the generation of extreme pH values in the electrode reservoirs (acidic pH at the anode and basic pH at the cathode) due to electrolytic reactions. This was achieved by continuous renewal of electrode reservoir solution, maintaining its pH at a constant value, by constant re-circulation at a flow rate of 10 mL min–1. Experiments that involved an electrokinetic pretreatment used, as electrode solutions, phosphate buffer (0.1 M). To exclude any indirect effect on biodegradation from the slight increases in temperature observed during electrokinetic treatments with phosphate buffer (approximately 8–9°C), experiments designed to integrate simultaneous biodegradation and electroosmosis were performed with 0.05 M TA of pH 7, which minimizes heating in electrobioremediation systems (Wick et al., 2004). After treatment, test soil cores (25 g dry soil) were taken from the cells and analyzed in duplicate for residual PAH content. Statistical comparisons between separate determinations were performed with analysis of variance and Duncan post-hoc test at P = 0.05. At the end of some experiments, the number of PAH-degrading microorganisms present in soil were estimated in duplicate as colony forming units (CFU), by plating soil suspensions on solid mineral medium (Gomez-Lahoz and Ortega-Calvo, 2005) supplemented with solid phenanthrene. The PAH was added to the sterile medium at 45°C in acetone solution (0.033 g mL–1), to give a final concentration of 0.10 g L–1. This procedure resulted in the formation of fine crystals whose dissolution through the agar allowed the growth of phenanthrene-degrading microorganisms.

Percolation columns were used, in a similar way to that one previously described for the study of bacterial transport through clay-rich porous media (Ortega-Calvo et al., 1999; Lahlou et al., 2000), to assess the electroosmotic mobilization of PAH. A detailed description of the method used is given elsewhere (Niqui-Arroyo et al., 2006). Briefly, clay soil II or loamy sand I was wet-packed with Brij 35 (35 mg g–1 soil) and TA buffer in glass columns with an inside diameter of 0.9 cm (cross-sectional area = 0.125 cm2), and a length of 10 cm. A portion of soil (1 cm) next to the cathode was spiked during packing with 100000 dpm of 14C-phenanthrene. Hollow, stainless steel cylindrical electrodes were connected to each side of the columns, and a constant electric field of 1 V cm–1 was applied for 5 h. Radioactivity present in the column effluent was then measured with a liquid scintillation counter.

Polycyclic Aromatic Hydrocarbon Analysis
Soil samples were defrosted, previously dried at 40°C, and finally dried completely using anhydrous sodium sulfate to ground the mixture in a mortar and pestle. Samples were extracted in a Soxhlet with 100 mL dichloromethane for 8 h. Then, the organic solvent was evaporated to near dryness, and the residue was re-dissolved in 4 to 5 mL of dichloromethane. Cleanup of the samples were then performed using Sep-Pak Plus Florisil cartridges (WATERS Corp., Milford, MA). Once this cleanup was performed, the organic phase was evaporated in a gentle stream of nitrogen and residue was dissolved in a known volume of acetonitrile (between 8 and 10 mL). Then, the sample was filtered through a syringe filter of nylon (0.45 µm, 13 mm Ø, Teknokroma, Barcelona, Spain). Analysis of PAH was performed using a Waters HPLC system (2690 separations module and 474 scanning fluorescence detector. Column: Nova-Pak C18 Waters, 5 µm, 4.6 x 250 mm; flow: 1 mL min–1; mobile phase: 45% acetonitrile, 55% water). The mobile phase used was an acetonitrile-water gradient comprising 45% (v/v) acetonitrile from 0 to 5 min, 95% (v/v) acetonitrile from 5 to 25 min, and programmed to 100% acetonitrile between 25 and 38 min. The initial composition was maintained for a further 13 min. Analysis of phenanthrene content of a reference material was in good agreement with the certified value (Niqui-Arroyo et al., 2006). Soil samples containing surfactant Brij 35 were extracted in Soxhlet with 100 mL acetone, instead of dichloromethane, as described above. Preliminary experiences showed no significant effect of Brij 35 on the recovery of PAH with this extraction method.


   Results
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
Discussion
 Conclusions
 REFERENCES
 
Bioremediation Potential
Parallel measurements of 14CO2 production from radiolabeled phenanthrene, fluoranthene, and pyrene, and residual contents of the same native compounds by HPLC analysis showed that these PAH were simultaneously metabolized in inoculated clay soil II (Fig. 2 ). It was expected that native PAH contained an aged fraction, with a probably higher persistence than the freshly added 14C-PAH. However, a significant compound disappearance of the native PAH occurred together with an active release of 14CO2, suggesting that the added 14C-PAH were homogeneously mixed with most of the native chemicals. The residual concentration of these native PAH remaining at the end of the mineralization plateau was likely due to the presence of a slowly desorbing fraction of limited bioaccessibility (Gomez-Lahoz and Ortega-Calvo, 2005; Niqui-Arroyo et al., 2006). Furthermore, analysis of the residual contents of other native PAH (Fig. 2B) revealed that the bacterium used to inoculate the soils, M. gilvum VM552, was also capable to degrade, through growth linked or cometabolic reactions, fluorene, anthracene, and benzo[a]pyrene. However, other high molecular weight (HMW)-PAH like chrysene (which is shown in this study as internal reference), benzo[a]anthracene, and benzo[b]fluoranthene were not degraded. This metabolic versatility has been previously reported in other PAH-degrading strains from Mycobacterium genus, which seems to be specialized in the biodegradation of HMW-PAH (Kanaly and Harayama, 2000; Wick et al., 2001; McLellan et al., 2002).


Figure 2
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  		Fig. 2. (A) Mineralization of 14C-polycyclic aromatic hydrocarbons (PAH) and (B) disappearance of native PAH in solid-phase treatments of clay soil II. (A) Mineralization of 14C-phenanthrene (circles), 14C-fluoranthene (triangles) and 14C-pyrene (squares). (B) Mean concentration of native PAH in soil before (black bars) and after (gray bars) treatment. Error bars correspond to one standard deviation of measurements in duplicate flasks. The detection limit for fluorene was 96 µg kg–1. Abbreviations: Flne, Fluorene; Phe, phenanthrene; Ant, anthracene; Ftne, Fluoranthene; Pyr, pyrene; B(a)pyr, benzo(a)pyrene; Crys, chrysene.

 
The possible effects on PAH biodegradation of the surfactant and TA buffer used in electrokinetic experiments was tested in the absence of electric current through 14C-phenanthrene mineralization assays. Biodegradation was determined at optimal conditions for microbial activity (inoculation, slurrying, shaking, and nutrient addition), and at concentrations of Brij 35 and TA buffer similar to those used in the electrokinetic experiments. Far from inhibiting biodegradation, the surfactant caused, in clay soil II and loamy sand I, slight increases in maximum mineralization rates, as compared with the controls without surfactant (data not shown). Mineralization of phenanthrene in soil slurries was also not affected by TA buffer at a concentration of 1 mL buffer g–1 soil. The rates of phenanthrene mineralization in clay soil II slurries with and without TA buffer were, respectively, 4.72 ± 0.24 and 4.37 ± 0.27 mg kg–1 h–1.

Current Reversal and Pulses to Improve Electrobioremediation
Electrokinetic experiments were designed to test in situ the effect of electric fields coupled to PAH biodegradation in soil, in the presence of the surfactant Brij 35. Figure 3 shows the time evolution during a representative experiment, performed without changes in current regime (i.e., continuous application of current and no changes in polarity), of the three cell parameters determined during each treatment (potential drop, current density, and temperature). The figure shows that highly stable conditions could be set during the electrokinetic experiments, and that no significant differences in temperature were observed between the test cells and the corresponding controls without current.


Figure 3
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  		Fig. 3. Time-course evolution of cell operating parameters (potential drop [circles], current density [triangles], and temperature [squares]) during electrokinetic treatment of clay soil II in the presence of Brij 35. Temperature evolution of control cell without current (dashed line) is shown for reference.

 
Table 2 shows the PAH losses detected after electrokinetic bioremediation under continuous current application in clay soil and loamy sand. Compound disappearances were more evident with loamy sand than with clay soil. For example, average losses of total PAH (i.e., sum of PAH potentially degradable by M. gilvum VM552) were 23% for clay soil and 53% for loamy sand after 14 and 7 d, respectively (Table 2). To assess the relative contributions of biodegradation and electrokinetic flushing to these losses, Table 2 compares them with those observed in control treatments where the soils were maintained under identical conditions, including surfactant concentration, but without electric field (to test bioremediation) and with current only (i.e., abiotic controls to test electrokinetic flushing). Losses in total PAH and many individual compounds were significantly higher in loamy sand treated with electrokinetic bioremediation, as compared with control cells, evidencing the benefit of the simultaneous occurrence of both processes. The differences were especially significant with benzo[a]pyrene. Whereas 49% of the compound had disappeared from loamy sand after electrokinetic bioremediation (from 3.54 ± 0.01 to 1.79 ± 0.16 mg kg–1), a significantly (P = 0.05) smaller fraction of the compound disappeared in the cells from the other two treatments (17 and 22% after bioremediation and electrokinetic flushing, respectively, resulting in 2.92 ± 0.29 and 2.76 ± 0.01 mg kg–1). Treatment of clay soil with electrokinetic bioremediation also resulted in higher losses compared with the respective controls, although the differences were not significant.


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  		Table 2. Effect of electrokinetic bioremediation with continuous application of electric field on percentages of removal of polycyclic aromatic hydrocarbons (PAH) in clay soil and loamy sand, as compared with cells with sterilized soil under continuous application of current and cells without current.{dagger}{ddagger}

 
The design of the electroremediation cell (Fig. 1) did not allow measurements of the mobilized water and PAH due to electroosmotic flow. Therefore, to test whether the applied electric field induced the electro-osmotic flow of water through soil and the mobilization of PAH dissolved in the soil fluid, a different experimental setup was used. The system was a column-type cell in which the electric field was applied between two hollow, cylindrical electrodes. The portion of the soil close to the cathode was spiked with radiolabeled phenanthrene. In this way, we could estimate electroosmotic flow rates of approximately 0.08 and 0.06 mL h–1 in clay soil II and loamy sand I, respectively, whereas 14C-phenanthrene elution rates were 1.05 and 2.68 {propto}g h–1. It is therefore probable that the higher disappearance rates observed in loamy sand during electrokinetic treatments were related to a higher mobilization rate of PAH.

Extending the treatment period to 18 d did not result, with loamy sand, in further decreases in PAH concentration, as compared with the 7-d treatment (Tables 2 and 3). This may have been caused by the interruption of the electroosmotic flow, what has often been observed in electrokinetic studies that have investigated the continuous application of the electric field (Reddy and Saichek, 2004; Maturi and Reddy, 2006). These studies have shown that flow interruption can be avoided by periodical changes in polarity and/or current application period. Therefore, electrokinetic experiments were performed with loamy sand to test the effects of periodical changes in current application on process performance. Three different regimes were used: inversion of polarity every 24 h, inversion every 72 h, and current pulses of 96 h alternated with 72 h without current application. The results, obtained with loamy sand III after 18 d of treatment are shown, for comparative purposes, in Table 3 together with those obtained after treatment for the same period of loamy sand II with continuous current. In all cases a clear improvement of electrokinetic bioremediation was observed by these changes. For example, in the three current regimes used, losses of total PAH were 63 to 64%, significantly higher than the losses observed after continuous application of current without changes in polarity (48%, respectively). No significant differences were observed between these different periodical regimes.


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  		Table 3. Effect of different current application regimes on percentages of removal of polycyclic aromatic hydrocarbons (PAH) after 18 d in loamy sand treated with electrokinetic bioremediation, as compared with control cells without current.{dagger}{ddagger}

 
To test if bacterial growth or die-off occurred in the system during electroremediation, the number of phenanthrene-degrading microorganisms was estimated at the end of the experiments with current inversions and pulses in loamy sand III (shown in Table 3). The resulting average numbers (2.8, 2.1, and 3.0 x 108 CFU g–1 with 24 h inversion, 72 h inversion, and pulses treatments, respectively) indicate that PAH biodegradation activity was still present. Indeed, these numbers were not very different to the initial bacterial densities used for inoculation in these specific experiments (1.7 x 109 CFU g–1).

Electrokinetic Pretreatment to Increase Bioaccessibility
Further research was performed on the effects of an electric field on the mobilization and mineralization of PAH in contaminated soils. A set of electrokinetic experiments were performed, in the absence of a surfactant, with the heavily contaminated clay soil I, with the goal of determining possible effects of electrokinetic pretreatment on biodegradation kinetics. After 20 d of exposure to an electric field, phenanthrene concentration in this soil was 1100.0 ± 39.9 mg kg–1 (the original content was 1321.4 ± 24.1 mg kg–1). Mineralization experiments in soil slurries, constituted with original and electrokinetically treated soil, and inoculated with M. gilvum VM552, showed that bioaccessibility of PAH was increased after these 20 d of electrokinetic treatment (Fig. 4 ). The maximum rate of conversion to 14CO2 of 14C-labeled phenanthrene was approximately three times higher in the soil samples obtained from the electrokinetic cells than in the soil with no treatment. This suggests a higher bioaccessibility of the native phenanthrene that eventually was homogenized with (and therefore represented by) 14C-labeled phenanthrene. Furthermore, residual levels of native PAH were approximately twofold lower in pretreated soils than in the control (Fig. 4B). The residual content in total PAH after bioremediation were 477.37 ± 35.80 and 932.56 mg kg–1 in the treated and control soils, respectively.


Figure 4
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  		Fig. 4. (A) Mineralization of 14C-phenanthrene in inoculated clay soil I slurries before (circles) and after (triangles) exposure to an electric field. The numbers denote maximum mineralization rates in mg kg–1 h–1. (B) Mean concentration of native polycyclic aromatic hydrocarbons (PAH) in soil after bioavailability assay without (grey bars) and with (black bars) electrokinetic pretreatment. Error bars correspond to one standard deviation of measurements in duplicate flasks. No error bars are shown in (B) for results without pretreatment because the results correspond to one single flask. The detection limit for fluorene was 54 µg kg–1. Abbreviations: Flne, Fluorene; Phe, phenanthrene; Ant, anthracene; Ftne, Fluoranthene; Pyr, pyrene; B(a)pyr, benzo(a)pyrene; Crys, chrysene.

 

   Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
Conclusions
 REFERENCES
 
The coexisting capacities in our system for PAH removal from soil through biodegradation and electroosmosis provided us with a new scenario to investigate the electrokinetic remediation of PAH-polluted soils. Previous studies have reported the electrokinetic removal of PAH from soil in the presence of solubility-enhancing agents such as surfactants, co-solvents, and cyclodextrins (Saichek and Reddy, 2005a). However, the aim of our study was not to confirm previous findings, but to determine whether bioremediation and electrokinetic flushing could be integrated into a new hybrid technology for the efficient removal of PAH from soil.

Separate experiments that included biodegradation assays in flasks (Fig. 2) and incubations in electrokinetic cells performed without electric current (Table 2) showed that up to six different PAH were simultaneously degraded in soil, through growth-linked or cometabolic reactions, during electrokinetic bioremediation treatments. In addition, the mobilization of PAH due to electroosmotic flow was supported by (i) the losses in chrysene, which was not degraded by M. gilvum VM552, in most electrokinetic bioremediation treatments (Table 3), (ii) the losses in most individual PAH during abiotic (flushing) electrokinetic experiments (Table 2), and (iii) separate estimations of electroosmotic flow and 14C-phenanthrene elution from percolation columns. It is difficult, however, from the data presented, to determine the exact contribution of biodegradation and transport to the overall PAH losses after electrokinetic bioremediation. The possible influences of one of these two processes on the other should be the subject of further investigation. In any case, the comparison with control experiments, performed under similar conditions, evidenced higher losses when these two processes acted simultaneously, thus showing the benefit of this hybrid technology. The new remediation method often resulted in significant reductions in total PAH levels as well as individual compounds, including benzo[a]pyrene, with special relevance for soil remediation projects due to its carcinogenicity. Indeed, benzo[a]pyrene is often used as an indicator of remediation performance in PAH-polluted soils (Juhasz and Naidu, 2000).

While the continuous use of current led to an apparent stop of the removal process after 1 wk of treatment, the use of periodical changes in polarity and current application period resulted in a significant improvement of process performance (Table 3). These results agree well with studies that showed an enhancement effect of changes in polarity and current pulses on PAH removal by electrokinetic flushing with surfactants (Reddy and Saichek, 2004) and cyclodextrins (Maturi and Reddy, 2006). These authors have postulated that applying the electrical potential in a periodic manner, or disconnecting the voltage periodically, increases micellar solubilization, and allows a maintained electroosmotic flow due to the depolarization of the diffuse double layer around soil particles. It is possible that, in our study, these processes contributed positively to PAH losses, both through enhancement of biodegradation of PAH desorbed into the pore fluid and PAH transport out from the soil.

Electrokinetic pretreatment also resulted in lower residual PAH concentrations when followed by biodegradation in soil slurries (Fig. 4). It is conceivable that physicochemical changes produced in polluted soil particles that were exposed to electric fields may promote bioaccessibility, thus improving bioremediation performance. These changes may have mobilized pollutant fractions present in the soil nanoporosity, which are often not accessible to soil microorganisms (Nam and Alexander, 1998). Thus, the occurrence of electroosmosis inside soil aggregates may have caused the mobilization of slowly desorbing PAH into the fast-desorbing pool, which possesses a higher bioaccessibility (Gomez-Lahoz and Ortega-Calvo, 2005). It should be noted that, during the experimental procedure, autoclaving of the soil may have induced possible modifications of the desorption pattern of PAH. However, in spite of this limitation, and given that all treatments (including the controls) included autoclaving, the observed relative differences still suggest an effect of electrokinetic pretreatment on PAH bioaccessibility.


   Conclusions
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 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
 Conclusions
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These results indicate that biodegradation and electroosmosis can be successfully integrated to enhance PAH removal from soil in two ways: (i) inducing their simultaneous occurrence in situ with the help of a non-toxic, biodegradable surfactant, and (ii) mobilizing the less bioaccessible fraction of PAH with an electrokinetic pretreatment to reach lower residual levels through bioremediation. The optimization of these processes for a cost-effective application of the technology in situ to meet remediation goals set by legislation will be the subject of future investigations.


   ACKNOWLEDGMENTS
 
We thank EMGRISA for the provision of the soil sample. Support for this research was provided by the European Union (contract QLRT-1999-00326), Spanish CICYT (BIO2000-1857-CE), and Spanish Ministry of Environment (057/2004/3, 1.2-270/2005/3-B and 392/2006/3-1.2).


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