Impact of Microbial Activities on the Mineralogy and Performance of

Mackenzie, P. D.; Horney, D. P.; Sivavec, T. M. Mineral precipitation and porosity losses in granular iron columns. J. Hazard. Mater. 1999, 68, 1−17...
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Environ. Sci. Technol. 2007, 41, 5724-5730

Impact of Microbial Activities on the Mineralogy and Performance of Column-Scale Permeable Reactive Iron Barriers Operated under Two Different Redox Conditions THOMAS VAN NOOTEN,† F R A N C¸ O I S L I E B E N , ‡ J A N D R I E S , † ERIC PIRARD,‡ DIRK SPRINGAEL,§ AND L E E N B A S T I A E N S * ,† Flemish Institute for Technological Research (VITO), Environmental and Process Technology, Boeretang 200, 2400 Mol, Belgium, GEMME Ge´nie Mine´ral, Mate´riaux et Environnement, Universite´ de Lie`ge, Sart Tilman B52, 4000 Lie`ge, Belgium, and Division Soil and Water Management, KULeuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.

The present study focuses on the impact of microbial activities on the performance of various long-term operated laboratory-scale permeable reactive barriers. The barriers contained both aquifer and Fe0 compartments and had received either sulfate or iron(III)-EDTA to promote sulfatereducing and iron(III)-reducing bacteria, respectively. After dismantlement of the compartments after almost 3 years of operation, DNA-based PCR-DGGE analysis revealed the presence of methanogenic, sulfate-reducing, metalreducing, and denitrifying bacteria within as well as up- and downgradient of the Fe0 matrix. Under all imposed conditions, the main secondary phases were vivianite, siderite, ferrous hydroxy carbonate, and carbonate green rust as found by scanning electron microscopy (SEM) combined with energy dispersive X-ray analysis (EDX), and X-ray diffraction (XRD). Under sulfate-reduction promoting conditions, iron sulfides were formed in addition, resulting in 7 and 10 times higher degradation rates for PCE and TCE, respectively, compared to unreacted iron. These results indicate that the presence of sulfate-reducing bacteria in or around iron barriers and the subsequent formation of iron sulfides might increase the barrier reactivity.

Introduction The use of zerovalent iron (Fe0) in permeable reactive barriers (PRBs) has been shown to be very effective for the passive, long-term treatment of groundwater contaminated with chlorinated organic compounds, radionuclides, and redoxsensitive metals (1, 2). Since the installation of the first Fe0PRB in the mid-1990s, more than 100 full-scale applications have been installed all over the world (3). A remaining issue, however, relates to the longevity of iron barriers. Considering the usually slow groundwater movement, PRBs have to * Corresponding author phone: +3214335179; fax: +3214580523; e-mail: [email protected]. † Flemish Institute for Technological Research (VITO). ‡ Universite ´ de Lie`ge. § Division Soil and Water Management. 5724

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function properly for decades. But, within time, the accumulation of mineral precipitates and hydrogen gas can reduce barrier reactivity (4-6) and permeability (7, 8). Only a few studies discuss the microbial community composition in and around iron barriers and its potential impact on barrier longevity. Wilkin et al. (9) used phospholipid fatty acids (PLFA) analysis to evaluate the community composition of two full-scale iron barrier applications. Gu et al. (2) studied the microbial community in a full-scale iron barrier by using both PLFA and DNA-analysis. The development of a specific microbial community is expected, due to specific conditions generated in the strongly reducing Fe0 environment (2). In addition to a direct contribution to contaminant degradation (10, 11), microorganisms can potentially positively affect barrier performance by consuming abiotically produced hydrogen, and by the reductive dissolution of passivating iron oxides (12, 13). In our laboratory, we have been operating, for 3 years, various column-scale PRB systems containing Fe0 and/or aquifer material aimed at the treatment of groundwater containing contaminant mixtures by a combination of physicochemical and microbiological processes (14). A mixture of PCE, TCE, BTmX, and metals was applied as a model contaminant mixture in which PCE and TCE are known to be easily degraded by Fe0, while BTEX can be degraded by microbial anaerobic oxidative metabolic pathways with sulfate and Fe(III) as ultimate electron acceptors. To promote BTmX degradation, some of the PRB systems included aquifer material enriched in either iron(III)-reducing or sulfatereducing oxidative BTEX degraders and were operated under either iron(III)-reduction promoting (IRP) conditions and sulfate-reduction promoting (SRP) conditions by addition of iron(III)-EDTA or sulfate to the feed, respectively. The aquifer material was either mixed with Fe0 or physically separated in a separate column in a series setup. Since those columns were operated for 3 years, we considered them as ideal objects to study the impact of the microbial community and its activity on PRB mineralogy and overall PRB activity for PCE/TCE-removal, and to examine the effect of the operating conditions on those factors. Therefore, the column systems were dismantled and a detailed microbial as well as mineralogical analysis was performed. DNA-based PCRDGGE analysis was used with the application of group specific primer sets, targeting the eubacterial and archaeal community, and different functional groups of bacteria expected to occur in a Fe0 environment. Iron corrosion products and other mineral precipitates were identified by optical microscopy, scanning electron microscopy (SEM) combined with energy dispersive X-ray analysis (EDX), and X-ray diffraction (XRD). The reactivity of the aged iron material was evaluated and compared with original unreacted Fe0 in a batch degradation experiment.

Experimental Section Laboratory PRB Systems and Medium Description. A comprehensive and detailed description of the column systems and operation modes is given by Dries (14). Briefly, two setups of four column systems were operated, by which one setup was operated under IRP conditions and the other under SRP conditions (Figure 1). Two reactive column filling materials were used, i.e., granular Fe0 (Gotthart Maier Metallpulver, 0.25-2 mm) and aquifer material. The latter was a homogenized mixture of 13 samples from various contaminated aquifers, which had been previously enriched for BTEX degraders either under SRP or IRP conditions (14). 10.1021/es070027j CCC: $37.00

 2007 American Chemical Society Published on Web 06/28/2007

FIGURE 1. Schematic overview of the laboratory PRB systems. Two setups of four column systems were operated, of which one setup was operated under IRP conditions and the other under SRP conditions. One setup consisted of (i) an aquifer-containing compartment [1, aquifer 1], (ii) a noninoculated iron compartment [2a, Fe0], (iii) a second aquifer compartment which was displaced as indicated after 400 days of operation [2b, aquifer 2], (iv) a second iron compartment inoculated by 2b [3, Fe0inoc], and (v) a mixed compartment with aquifer and Fe0 [4, Fe0 + aquifer]. To provide each column system with the same absolute amount of reactive material, both materials were mixed with filter sand (1-2 mm, considered nonreactive) to fill the columns completely. The first system consisted of a single compartment, filled with a mixture of aquifer material and filter sand without Fe0. Systems 2 and 3 were sequential configurations in which a compartment, containing a Fe0filter sand mixture, was positioned before or after a compartment, containing a mixture of aquifer material and filter sand. System 3 was initially used to investigate the effect of colonization of Fe0 by aquifer microorganisms, but the upgradient aquifer column was displaced after 400 days of operation and positioned after the iron compartment in system 2. The fourth system was a mixed configuration and consisted of a compartment filled with a mixture of sand, aquifer material, and Fe0. The column systems were fed with deoxygenated simulated groundwater, supplemented with an additional deoxygenated phosphate-bicarbonate buffered (KH2PO4, 0.15 mM; Na2HPO4, 0.15 mM; NaHCO3, 0.25 mM; KHCO3, 0.25 mM) minimal medium containing sulfate (Na2SO4, 0.5 mM) or iron(III)-EDTA (2.5 mM), promoting either sulfate-reducing or iron(III)-reducing conditions, respectively (14). After 375 days of operation, the phosphate buffer was replaced by an organic buffer (MOPS, 2.5 mM). The model pollutant mixture was composed of (i) zinc (5 mg L-1; as ZnCl2) and arsenate (0.2 mg L-1; as Na2HAsO4), (ii) tetrachloroethylene (PCE, 2 mg L-1) and trichloroethylene (TCE, 5 mg L-1), and (iii) the aromatic hydrocarbons benzene, toluene and m-xylene (BTmX, 2 mg L-1 each). Average total flow rate in the columns was 2.33 ( 0.48 mL h-1, corresponding to pore water velocities of 1.18 ( 0.25 cm h-1. During operation, influent and effluent samples were regularly taken from each compartment to determine Eh and pH with a PHM62 electrode (Radiometer Copenhagen) and a PH535 electrode (WTW), respectively,

and to measure contaminant concentrations. We focused on the removal of chlorinated ethenes and aromatic hydrocarbons as all metals were in all cases efficiently removed from the start of the experiment (14). Samples were prepared for analysis by adding ∼0.5 mL sample to a 12 mL glass vial, together with 4.5 mL H2O and 100 µL of concentrated H3PO4. Vials were capped immediately and headspace was analyzed using a Thermo Finnigan Trace GC-MS. Zn and As were not analyzed. Microbial and Mineralogical Characterization of the Laboratory PRB Systems. After 900 days of operation, core samples were taken. Therefore, all PRB systems were dismantled in an anaerobic glove box and the core material was vertically divided into 4 or 5 sections, with smaller sections at the entrance side of the compartments. Section dimensions are indicated in Figure 2. The samples were collected and homogenized in plastic 50 mL tubes, and stored anaerobically in anaerocult bags (Merck) at 4 °C. From each column section, subsamples of 2 g were taken in duplicate from which DNA was extracted as described by Hendrickx et al. (15). The microbial diversity of the samples was evaluated by PCR-DGGE, using group specific PCR primers for the amplification of 16S rRNA genes targeting eubacterial, archaeal or IRB (Geobacteraceae and Geothrix) communities as well as functional genes targeting specific functional groups including denitrifying bacteria (nirK and nirS genes), SRB (dsr gene), and methanogens (mcrA gene). Primer sets and PCR-DGGE conditions are reported in Table S1 (16-22). All primer sets were based on earlier publications but were tested against a wide range of bacteria to check their specificity (Table S2). For XRD analysis, 5 g subsamples were sonicated in 10 mL acetone for 30 min to detach precipitates from Fe0 filings. After filtration, the dry residue was manually divided into a fine fraction (precipitates) and a coarse fraction (Fe0 and sand). The fine fraction was stored anaerobically until analysis. Exposure to air was limited to a few hours maximum for all samples to minimize their oxidation. XRD analysis was performed using a Philips powder diffractometer (PWD3710). For microscopical analysis, approximately 1 g subsamples were taken and dried at room temperature in the anaerobic glove box. Fe0 filings were placed in epoxy, hardened for 12 h, and polished. Selected sections were silver coated and examined with an (ESEM XL-30) environmental scanning electron microscope (Philips). Energy dispersive X-ray analysis (EDX) of major phases and selected contaminants (Zn and As) was performed with a conventional scanning electron microscope (Hitachi), fitted with a Link detector. Moreover, some iron grains were directly secured to aluminum stubs and gold coated for direct observation in the scanning electron microscope. Reactivity of Fe0. The reactivity of original unreacted iron and iron from the columns, harvested after 900 days of operation, was compared via batch degradation experiments. The experiment was carried out in 120 mL glass vials, each containing 4.5 g of iron. Iron grains in the column samples were magnetically separated from sand grains and aquifer material in an anaerobic glovebox. The same deoxygenated simulated groundwater as used in the column experiment was stored in a separatory funnel and mixed with concentrated aqueous solutions of PCE and TCE, but no electron acceptors. Each vial was filled with 50 mL of the medium, immediately capped with 70 mL headspace, and placed on a rotary shaker at 100 rpm at 20 °C. Each condition was carried out in four vials of which two were poisoned with 0.175% formaldehyde to ensure that pollutants were abiotically degraded. Vials filled with medium but without iron were used as control. Vials were regularly placed on a Trace GC-FID (Thermoquest) and headspaces were analyzed for PCE, TCE, cis-DCE, 1,1-DCE, and VC with a 30 m Rt-U plot VOL. 41, NO. 16, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Schematic overview of the microbial and mineralogical characterization of samples taken from different horizontal sections of the PRB columns operated under either SRP or IRP conditions. PCR results obtained with the different primer sets and applied on duplicate samples are given for each section. The column sections that were mineralogically studied are indicated by the rectangular labels surrounding the columns. XRD results are indicated by the boxed labels at the left of each horizontal column section, whereas SEM-EDX results are indicated by the discontinuous labels at the right of each section. Section dimensions are indicated on the figure in cm. Column names are explained in Figure 1. column using helium as carrier gas at the flow rate of 6 mL min-1. After 17 and 44 days, vial headspaces were also analyzed with a Varian GC-FID (CP-3800) for the detection of acetylene, ethene, and ethane with a capillary DB-1 column. Every three headspace analyses, vials were refilled with 1.2 mL nitrogen gas. Initial pollutant concentrations, determined by measurement of the headspace concentration in the simulated groundwater medium alone (i.e., no iron), were 6.22 mg L-1 (PCE) and 3.76 mg L-1 (TCE).

Results Column System Operation. The degradation performance for the complete contaminant mixture and the conditions in the column systems during the first 450 days are described by Dries (14). Briefly, during this period TCE was almost completely removed by Fe0 in all column systems. The average PCE removal efficiency in the systems operated under SRP and IRP conditions was 78 ( 14% and 63 ( 16%, respectively. In all cases, the majority of PCE removal occurred in the Fe0 compartments. In the reference aquifer compartments without Fe0, less than 10% of the chlorinated ethenes was removed. Aromatic hydrocarbons toluene and benzene were removed by microbial degradation in the aquifer compartments but also in the Fe0 compartments. During this study, the column systems were monitored less intensively for another 475 days. In the course of the experiment, degradation of PCE appeared to decrease in time for both electron acceptor conditions, although degradation efficiencies of 75 ( 2% and 88 ( 1% were still observed during the latest samplings in the systems operated SRP and IRP conditions, respectively. Degradation of TCE remained high (∼98%) during the complete operation period, except for the noninoculated iron compartment operated under SRP conditions which showed a gradually decreasing degradation efficiency in time (to 76%). Benzene and toluene were still degraded (>60%) by the column systems operated under both electron acceptor conditions. The influent feed solution had a pH of 7.13 ( 0.28 and a Eh of 120 ( 67 mV. The effluent pH of the iron compart5726

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ments operated under SRP conditions was relatively elevated (8.0-8.4) during the first 375 days. To decrease pH and guarantee microbial degradation, the phosphate buffer was replaced by an organic buffer (MOPS) and pH was subsequently reduced to 7.4-7.6. Under IRP conditions, pH of the effluent of the iron compartments remained close to neutrality. Eh of the effluent of the systems with the single aquifer compartment ranged between 85 and -80 mV while the average Eh of the effluent of the iron compartments was significantly lower (-169 ( 80 mV). Microbiological Characterization of the Column Systems. Results of PCR analysis targeting the different bacterial groups in duplicate samples from different horizontal column sections are schematically presented in Figure 2. Generally, similar PCR results were obtained for the duplicate samples. Eubacteria could be detected in nearly all sections of the aquifer, iron, and mixed compartments. Strong PCR signals were observed for all sections of all systems operated under sulfate-reducing conditions. In the iron and mixed compartments operated under IRP conditions, Eubacteria were detected in a lower number of sections, with strongest PCRsignals in the bottom sections. In some sections, no Eubacteria could be detected, while other bacterial groups belonging to the Eubacteria could be detected. A possible reason for this could be a different PCR efficiency for the used PCR protocols. The presence of bacteria in the noninoculated iron compartments can be explained by the fact that the Fe0 and sand grains were not sterilized before use in the column systems, or by accidental bacterial contaminations of the column systems during the operation period. Compared to the Eubacteria, other bacterial groups were detected in a lower number of column sections, with detection of the highest number of groups in the bottom sections of the iron and mixed compartments. Archaea and methanogens were less-represented, especially in the systems operated under SRP conditions, with strong PCR-signals only in the system with the single aquifer compartment. As expected, there was a strong presence of SRB in systems operated under SRP conditions. Strong PCR-signals were

obtained in all compartments except the noninoculated iron compartment where SRB could only be detected in the bottom part. In systems operated under IRP conditions, SRB were detected in a lower number of sections, particularly in the inoculated iron compartment and the mixed compartment. Geobacteraceae were detected in all sections of the aquifer and inoculated iron compartments operated under SRP conditions, while they were detected in a lower number of sections of the iron compartments operated under IRP conditions. In contrast, Geothrix species were only detected in the compartments operated under IRP conditions. Finally, denitrifying bacteria were present in all compartments, with detection of both nitrite reductase genes. DGGE profiles of the amplification products with the eubacterial primer set GC-63-F/518-R are presented in Figure S1 for all section samples of the system with the single aquifer compartment and the sequential system, both operated under SRP conditions. Fingerprints of the duplicate samples were highly reproducible, with occasional differences in band intensity. In one and the same column, and particularly for the system with the single aquifer column, the banding patterns were very similar for the different section samples indicating a relatively stable microbial community in function of compartment height. Although a few similar bands can be observed, the banding patterns differ significantly between the system with the single aquifer compartment and the iron compartment of the sequential system, indicating different microbial communities for both compartments. Except for one strong common band with the iron compartment, the banding pattern of the downgradient aquifer compartment is also very different from the iron compartment, and particularly from the system with the single aquifer compartment, indicating that the iron compartment strongly influenced the community structure of the downgradient aquifer compartment, probably by inducing a lower redox potential. Mineralogical Characterization of Iron and Mixed Compartments. Upon dismantling the iron and mixed compartments, most of the iron material appeared to consist of loose and uncemented grains, except for the entrance part of the compartments which consisted of a layer of cemented material. The column systems operated under SRP conditions contained black iron grains and sand, while greenish iron grains and ochreous sand were observed in the column systems operated IRP conditions. In the bottom section of the compartments, the iron grains were covered with precipitate layers with a thickness ranging from 10 to 200 µm. In the more downstream located column sections precipitate layers had a thickness of less than 10 µm. Samples for XRD analysis were taken from different sections of iron and mixed compartments operated under both electron acceptor conditions. The main secondary phases that were detected included vivianite (Fe3(PO4)2‚ 8H2O), siderite (FeCO3), ferrous hydroxy carbonate (FHC: Fe2(OH)2CO3) and carbonate green rust (CGR: FeII4FeIII2(OH)12CO3‚2H2O). A semiquantitative overview of the results is shown in Table S3 where indicative phase proportions are reported, based on the height of their principal peak. This is only a rough quantitative estimation as the integrated surface of diffraction peaks is not only depending on the concentration, but also on instrumental factors, absorption coefficients, degrees of cristallinity, and an eventual preferential orientation of the grains (23). The four main secondary phases were present at variable intensities in the different compartments and at different compartment heights. Intense vivianite peaks were observed especially in the lower sections of the compartments operated under both SRP and IRP conditions. Siderite was detected more frequently under IRP conditions while the detection of carbonate green rust was more frequent under SRP conditions.

Using optical microscopy and SEM-EDX, the same secondary phases indicated by XRD were identified, along with iron sulfide (FeS). FeS, causing the macroscopically observed black color, was evidenced by EDX analyses in the compartments operated under SRP conditions. The absence of this phase in the diffractograms suggests an amorphous or poorly crystalline form. FeS was abundantly found in the bottom and second section of the inoculated iron compartment and in the bottom section of the mixed compartment. No FeS could be detected in the systems operated under IRP conditions. Vivianite was present in all compartments, particularly as 10-50 µm big prismatic crystals and radially or lancet-shaped aggregates, with a characteristic blue color. A thick coating of vivianite was present on most of the iron grains in the bottom sections, while in the downstream sections only finer coatings or isolated aggregates were observed on certain grains. CGR was observed as hexagonal platy crystals of 1-3 µm, more or less agglomerated in spherical aggregates in the order of 5 µm (Figure S2A). Macroscopically, the iron grains appeared to be covered by a very thin green-blue layer. Ferrous hydroxy carbonate is a mineral phase close to malachite that occurs as aggregates of lamellar crystals (24-26). This phase was commonly observed as a 20 µm thick coating constituted by an inner compact to spherulitic layer covered by a layer of larger lamellar crystals (Figure S2B). Finally, siderite was observed as 2-3 µm big rhombohedra. Oxides (e.g., magnetite, hematite, maghemite) were not identified among secondary minerals. In some cases a few magnetite spots were observed, in particular on iron grains which were relatively little covered with precipitates. These spots can be considered as primary minerals as these small quantities could also be observed on the original cast iron. XRD and SEM-EDX results are united and schematically represented in Figure 2. Reactivity of the Used Fe0 Filings. A batch experiment was conducted to compare the reactivity of original unreacted iron with the reactivity of iron material from the column systems. Values of pseudo-first-order rate constants (k) for PCE decay were determined by best fit of measured headspace concentrations as a function of time, using a pseudofirst-order rate law. Observed degradation products, mass recoveries after 44 days, and rate constants for transformation of PCE and TCE are reported in Table S4 and half-lifes are presented in Figure 3. Rate constants for the column iron samples were comparable or higher than rate constants for the original iron for both PCE and TCE degradation. Significantly higher degradation capacities were observed for the iron samples originating from the inoculated iron column and the mixed column operated under SRP conditions. Especially for the inoculated iron column, rate constants were 7 and 10 times higher than rate constants for the original unreacted iron, for PCE and TCE degradation, respectively (Figure 3 and S3). Although not for all conditions, higher rate constants could be observed for samples without formaldehyde in comparison with the poisoned samples, indicating a potential biological contribution to PCE and TCE degradation. Among degradation products, cis-DCE, VC, ethene, and ethane where formed together with the detection of acetylene, evidencing that both the sequential hydrogenolysis pathway and the β-elimination pathway were followed for PCE and/or TCE degradation (27). Incomplete mass recoveries (67-83%) may be due to adsorption of reactants and products to the iron surface, losses of volatile reactants and products during the sampling process, or formation of nondetectable products.

Discussion Microbiological Aspects. After ∼15 months of operation, Gu et al. (2) observed a microbial community that was substantially increased in biomass in and in the surroundings VOL. 41, NO. 16, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. PCE and TCE half-lifes for the original unreacted iron and the different column samples operated under SRP or IRP conditions. Column names are explained in Figure 1. of the Oak Ridge Y-12 reactive barrier (Tennessee), with sulfate-reducing and denitrifying bacteria among the most dominant groups, and relatively low levels of methanogens. It was suggested that the observed biomass increase might have been due to the utilization of residual byproducts of the Guar gum, such as glucose, which was used during trench excavation. A more comprehensive laboratory column-scale microbial characterization was performed in our study by also targeting iron(III)-reducing bacteria and using 16S rRNA gene PCR-DGGE fingerprinting to indicate different microbial communities between different iron and aquifer compartments. Gu et al. (2) observed relatively higher biomass levels in both the upgradient and downgradient soil cores than in the iron samples. This is in accordance with our findings, as we detected bacterial groups in a higher number of sections in both aquifer compartments, compared to the iron and mixed compartments. The presence of indigenous bacteria, nutrients and carbon sources, and the higher surface area and redox potential, probably make the aquifer material a more favorable environment for colonization and growth of microorganisms than the Fe0 grains. Despite the sufficient buffering capacity of the simulated groundwater medium and the near neutral pH of the bulk column fluid, pH might increase at microlocations in the iron pore space and inhibit biofilm growth at the iron surface. On the other hand, it has to be noted that the extracted DNA content of the iron compartments might have been underestimated (on a unit volume basis) due to the fact that Fe0 has a relatively high particle density and 2 g samples were taken for DNA extraction. In the iron and mixed compartments, the higher number of functional groups of bacteria at the entrance zone of the Fe0 matrix is consistent with the findings of Wilkin et al. (9) who reported the highest biomass levels in the upgradient aquifer/iron interface and a biomass decrease along the flow path of an iron barrier installed at Elizabeth City, North Carolina. The lower biomass levels in the downstream column sections might be explained by a decreasing redox potential and/or nutrient content along the flow path. A rising pH may also be inhibitory for microbial growth although in our columns pH was staying close to neutrality, due to the feeding of the phosphate and later on organic buffer. Mineralogical Aspects. Most of the secondary mineral phases identified in our iron column samples are more or less consistent with the findings described elsewhere (25, 28). However, highly oxidized species such as hematite and goethite found in many laboratory- and field-scale Fe0 barrier systems (8, 28), were not detected in our study. Furthermore, in our systems, vivianite was highly abundant while mineral phases like magnetite and calcium carbonates (calcite, aragonite) were scarce or not detected. Based on thermodynamics, the finding of vivianite was predictable as with 5728

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the addition of phosphate-bicarbonate buffered minimal medium vivianite will precipitate rather than siderite. In contrast, vivianite is unlikely to be formed under in situ conditions as phosphate concentrations are usually low. Among the green rusts, only carbonate green rust (FeII4FeIII2(OH)12CO3‚2H2O) could be detected in contrast to the sulfate-containing green rust (FeII4FeIII2(OH)12SO4. nH2O), which is only formed under highly elevated sulfate concentrations (29, 30). Ferrous hydroxy carbonate, initially observed as a corrosion product formed at elevated temperatures (24), has recently been found in several Fe0 barriers (28, 31) and can result from the remineralisation of fine-grained, biogenic magnetite from IRB under anoxic conditions (26). Ferrous hydroxy carbonate may act as a conductor for electrons between Fe0 and the contaminants although Kohn et al. (25) speculated that thick precipitate layers may adopt insulating capacities due to an excessive distance for efficient electron transfer. Impact on Barrier Performance. The obvious linkage of geochemistry to microbial activity in Fe0 barriers is H2, which is an excellent energy resource in anaerobic environments, and in high quantities generated by the anaerobic corrosion of Fe0 (6). Our results indicate that SRB might have indirectly improved the reactivity of Fe0 in the columns operated under SRP conditions. SRB can utilize hydrogen as a substrate for the reduction of sulfate to sulfide which can result in the precipitation of ferrous iron sulfide. Rates of chemical abiotic sulfate reduction at low temperatures are extremely slow (9). Our results suggest that the presence of sulfate-reducing bacteria resulted into the precipitation of FeS in the column systems operated under SRP conditions. The batch experiment indicated that after more than 900 days of operation, the Fe0 retained reactivity with possibly even improved ability to remove PCE and TCE, in comparison with original unreacted iron. Especially samples containing iron sulfides showed significantly higher degradation rates, with rate constants up to 10 times higher than rate constants for the original iron. FeS-coatings are reported to be significantly more reactive per unit surface area than iron metal in transformation of TCE and other halogenated aliphatic compounds (32, 33). Our results, in addition, suggest that iron metal, placed in subsurface reactive barriers, may gain reactivity by the formation of FeS coatings through the growth of SRB. However, caution is required when applying the results to field conditions. The use of buffers in this study resulted into a near-neutral pH which is more favorable to microbial activity in comparison with the elevated pH that can be observed in field-scale iron barriers, depending on the natural buffering capacity of the groundwater and the retention time in the barrier. On the other hand, SRB have also been detected in a field-scale iron barrier (2). It might be useful to stimulate the growth of sulfate-reducing bacteria

in the vicinity of Fe0 barriers, or to treat iron grains with sulfides prior to or after barrier installation to form FeS surface coatings. An increased reactivity for pollutants such as TCE might require less iron material to be installed, possibly resulting in cheaper barrier configurations. IRB may also have positively affected the reactivity of the Fe0 barrier matrix by reductively transforming passivating layers of secondary Fe3+-containing minerals into Fe2+ or mixed-valent phases such as magnetite, green rust, vivianite, and siderite (12, 13). It has been reported that green rust, a mixed FeII-FeIII hydroxide, and mixed iron oxides such as magnetite can reduce chlorinated organics (34). Although we detected green rust together with the presence of IRB, we cannot prove any positive impact on iron reactivity by using the column setup as described here. It is unlikely that microbial fouling by biomass accumulation will occur in the anaerobic iron barrier matrix itself. Biomass accumulation and subsequent bioclogging might cause permeability reductions in aerobic biological barriers or zones. However, it does not seem to cause problems in Fe0-PRBs where microorganisms have to deal with anaerobic, highly reducing conditions and an elevated pH (4, 8). In this study, no visible evidence of biomass buildup was noted after column dismantling.

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Acknowledgments This work was funded by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and the Flemish Institute for Technological Research (VITO). Mineralogical investigations were performed with the helpful contribution of the CAT-µ (Joint Center for Microscopy) at University of Lie`ge. Column systems were setup under the framework of the European MULTIBARRIER project (QLK3-CT-2000-00163). We thank M. Maesen, A. Hermans, and A. Bossus for their appreciated contribution to this study.

Supporting Information Available Tables of PCR-primers and PCR-conditions, primer specificity, semiquantitative XRD-results, and kinetic parameters; DGGE-gel picture, SEM photomicrographs of secondary precipitates; figures with degradation curves. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Received for review January 5, 2007. Revised manuscript received May 4, 2007. Accepted May 16, 2007. ES070027J