impact of microbial activities on the mineralogy and performance of column-scale permeable reactive...

Impact of Microbial Activities onthe Mineralogy and Performance ofColumn-Scale Permeable ReactiveIron Barriers Operated under TwoDifferent Redox ConditionsT H O M A S V A N N O O T E N , †

F R A N C O I S L I E B E N , ‡ J A N D R I E S , †

E R I C P I R A R D , ‡ D I R K S P R I N G A E L , § A N DL 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 Genie Mineral, Materiaux etEnvironnement, Universite de Liege, Sart Tilman B52,4000 Liege, Belgium, and Division Soil and WaterManagement, KULeuven, Kasteelpark Arenberg 20,3001 Heverlee, Belgium.

The present study focuses on the impact of microbialactivities on the performance of various long-term operatedlaboratory-scale permeable reactive barriers. The barrierscontained both aquifer and Fe0 compartments and hadreceived either sulfate or iron(III)-EDTA to promote sulfate-reducing and iron(III)-reducing bacteria, respectively.After dismantlement of the compartments after almost 3years of operation, DNA-based PCR-DGGE analysis revealedthe presence of methanogenic, sulfate-reducing, metal-reducing, and denitrifying bacteria within as well as up- anddowngradient of the Fe0 matrix. Under all imposedconditions, the main secondary phases were vivianite,siderite, ferrous hydroxy carbonate, and carbonate greenrust as found by scanning electron microscopy (SEM)combined with energy dispersive X-ray analysis (EDX), andX-ray diffraction (XRD). Under sulfate-reduction promotingconditions, iron sulfides were formed in addition, resultingin 7 and 10 times higher degradation rates for PCE and TCE,respectively, compared to unreacted iron. These resultsindicate that the presence of sulfate-reducing bacteria inor around iron barriers and the subsequent formation ofiron sulfides might increase the barrier reactivity.

IntroductionThe 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 withchlorinated organic compounds, radionuclides, and redox-sensitive metals (1, 2). Since the installation of the first Fe0-PRB in the mid-1990s, more than 100 full-scale applicationshave been installed all over the world (3). A remaining issue,however, relates to the longevity of iron barriers. Consideringthe usually slow groundwater movement, PRBs have to

function properly for decades. But, within time, the ac-cumulation of mineral precipitates and hydrogen gas canreduce barrier reactivity (4-6) and permeability (7, 8).

Only a few studies discuss the microbial communitycomposition in and around iron barriers and its potentialimpact on barrier longevity. Wilkin et al. (9) used phospholipidfatty acids (PLFA) analysis to evaluate the communitycomposition of two full-scale iron barrier applications. Guet al. (2) studied the microbial community in a full-scale ironbarrier by using both PLFA and DNA-analysis. The develop-ment of a specific microbial community is expected, due tospecific conditions generated in the strongly reducing Fe0

environment (2). In addition to a direct contribution tocontaminant degradation (10, 11), microorganisms canpotentially positively affect barrier performance by consum-ing abiotically produced hydrogen, and by the reductivedissolution of passivating iron oxides (12, 13).

In our laboratory, we have been operating, for 3 years,various column-scale PRB systems containing Fe0 and/oraquifer material aimed at the treatment of groundwatercontaining contaminant mixtures by a combination ofphysicochemical and microbiological processes (14). Amixture of PCE, TCE, BTmX, and metals was applied as amodel contaminant mixture in which PCE and TCE are knownto be easily degraded by Fe0, while BTEX can be degradedby microbial anaerobic oxidative metabolic pathways withsulfate and Fe(III) as ultimate electron acceptors. To promoteBTmX degradation, some of the PRB systems included aquifermaterial enriched in either iron(III)-reducing or sulfate-reducing oxidative BTEX degraders and were operated undereither iron(III)-reduction promoting (IRP) conditions andsulfate-reduction promoting (SRP) conditions by additionof iron(III)-EDTA or sulfate to the feed, respectively. Theaquifer material was either mixed with Fe0 or physicallyseparated in a separate column in a series setup. Since thosecolumns were operated for 3 years, we considered them asideal objects to study the impact of the microbial communityand its activity on PRB mineralogy and overall PRB activityfor PCE/TCE-removal, and to examine the effect of theoperating conditions on those factors. Therefore, the columnsystems were dismantled and a detailed microbial as well asmineralogical analysis was performed. DNA-based PCR-DGGE analysis was used with the application of group specificprimer sets, targeting the eubacterial and archaeal com-munity, and different functional groups of bacteria expectedto occur in a Fe0 environment. Iron corrosion products andother mineral precipitates were identified by optical mi-croscopy, scanning electron microscopy (SEM) combinedwith energy dispersive X-ray analysis (EDX), and X-raydiffraction (XRD). The reactivity of the aged iron materialwas evaluated and compared with original unreacted Fe0 ina batch degradation experiment.

Experimental SectionLaboratory PRB Systems and Medium Description. Acomprehensive and detailed description of the columnsystems and operation modes is given by Dries (14). Briefly,two setups of four column systems were operated, by whichone setup was operated under IRP conditions and the otherunder SRP conditions (Figure 1). Two reactive column fillingmaterials were used, i.e., granular Fe0 (Gotthart MaierMetallpulver, 0.25-2 mm) and aquifer material. The latterwas a homogenized mixture of 13 samples from variouscontaminated aquifers, which had been previously enrichedfor BTEX degraders either under SRP or IRP conditions (14).

* Corresponding author phone: +3214335179; fax: +3214580523;e-mail: [email protected].

† Flemish Institute for Technological Research (VITO).‡ Universite de Liege.§ Division Soil and Water Management.

Environ. Sci. Technol. 2007, 41, 5724-5730

5724 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 16, 2007 10.1021/es070027j CCC: $37.00 2007 American Chemical SocietyPublished on Web 06/28/2007

To provide each column system with the same absoluteamount of reactive material, both materials were mixed withfilter sand (1-2 mm, considered nonreactive) to fill thecolumns completely. The first system consisted of a singlecompartment, filled with a mixture of aquifer material andfilter sand without Fe0. Systems 2 and 3 were sequentialconfigurations in which a compartment, containing a Fe0-filter sand mixture, was positioned before or after a com-partment, containing a mixture of aquifer material and filtersand. System 3 was initially used to investigate the effect ofcolonization of Fe0 by aquifer microorganisms, but theupgradient aquifer column was displaced after 400 days ofoperation and positioned after the iron compartment insystem 2. The fourth system was a mixed configuration andconsisted of a compartment filled with a mixture of sand,aquifer material, and Fe0.

The column systems were fed with deoxygenated simu-lated groundwater, supplemented with an additional deoxy-genated 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) oriron(III)-EDTA (2.5 mM), promoting either sulfate-reducingor iron(III)-reducing conditions, respectively (14). After 375days of operation, the phosphate buffer was replaced by anorganic buffer (MOPS, 2.5 mM). The model pollutant mixturewas 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) thearomatic hydrocarbons benzene, toluene and m-xylene(BTmX, 2 mg L-1 each). Average total flow rate in the columnswas 2.33 ( 0.48 mL h-1, corresponding to pore water velocitiesof 1.18 ( 0.25 cm h-1. During operation, influent and effluentsamples were regularly taken from each compartment todetermine Eh and pH with a PHM62 electrode (RadiometerCopenhagen) and a PH535 electrode (WTW), respectively,

and to measure contaminant concentrations. We focusedon the removal of chlorinated ethenes and aromatic hydro-carbons as all metals were in all cases efficiently removedfrom the start of the experiment (14). Samples were preparedfor 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 analyzedusing a Thermo Finnigan Trace GC-MS. Zn and As were notanalyzed.

Microbial and Mineralogical Characterization of theLaboratory PRB Systems. After 900 days of operation, coresamples were taken. Therefore, all PRB systems weredismantled in an anaerobic glove box and the core materialwas vertically divided into 4 or 5 sections, with smallersections at the entrance side of the compartments. Sectiondimensions are indicated in Figure 2. The samples werecollected and homogenized in plastic 50 mL tubes, and storedanaerobically in anaerocult bags (Merck) at 4 °C. From eachcolumn section, subsamples of 2 g were taken in duplicatefrom which DNA was extracted as described by Hendrickxet al. (15). The microbial diversity of the samples wasevaluated by PCR-DGGE, using group specific PCR primersfor the amplification of 16S rRNA genes targeting eubacterial,archaeal or IRB (Geobacteraceae and Geothrix) communitiesas well as functional genes targeting specific functional groupsincluding denitrifying bacteria (nirK and nirS genes), SRB(dsr gene), and methanogens (mcrA gene). Primer sets andPCR-DGGE conditions are reported in Table S1 (16-22). Allprimer sets were based on earlier publications but were testedagainst a wide range of bacteria to check their specificity(Table S2).

For XRD analysis, 5 g subsamples were sonicated in10 mL acetone for 30 min to detach precipitates from Fe0

filings. After filtration, the dry residue was manually dividedinto a fine fraction (precipitates) and a coarse fraction (Fe0

and sand). The fine fraction was stored anaerobically untilanalysis. Exposure to air was limited to a few hours maximumfor all samples to minimize their oxidation. XRD analysiswas performed using a Philips powder diffractometer (PWD-3710). For microscopical analysis, approximately 1 g sub-samples were taken and dried at room temperature in theanaerobic glove box. Fe0 filings were placed in epoxy,hardened for 12 h, and polished. Selected sections were silvercoated and examined with an (ESEM XL-30) environmentalscanning electron microscope (Philips). Energy dispersiveX-ray analysis (EDX) of major phases and selected contami-nants (Zn and As) was performed with a conventionalscanning electron microscope (Hitachi), fitted with a Linkdetector. Moreover, some iron grains were directly securedto aluminum stubs and gold coated for direct observation inthe scanning electron microscope.

Reactivity of Fe0. The reactivity of original unreacted ironand iron from the columns, harvested after 900 days ofoperation, was compared via batch degradation experiments.The experiment was carried out in 120 mL glass vials, eachcontaining 4.5 g of iron. Iron grains in the column sampleswere magnetically separated from sand grains and aquifermaterial in an anaerobic glovebox. The same deoxygenatedsimulated groundwater as used in the column experimentwas stored in a separatory funnel and mixed with concen-trated aqueous solutions of PCE and TCE, but no electronacceptors. Each vial was filled with 50 mL of the medium,immediately capped with 70 mL headspace, and placed ona rotary shaker at 100 rpm at 20 °C. Each condition wascarried out in four vials of which two were poisoned with0.175% formaldehyde to ensure that pollutants were abi-otically degraded. Vials filled with medium but without ironwere used as control. Vials were regularly placed on a TraceGC-FID (Thermoquest) and headspaces were analyzed forPCE, TCE, cis-DCE, 1,1-DCE, and VC with a 30 m Rt-U plot

FIGURE 1. Schematic overview of the laboratory PRB systems.Two setups of four column systems were operated, of which onesetup was operated under IRP conditions and the other under SRPconditions. One setup consisted of (i) an aquifer-containingcompartment [1, aquifer 1], (ii) a noninoculated iron compartment[2a, Fe0], (iii) a second aquifer compartment which was displacedas indicated after 400 days of operation [2b, aquifer 2], (iv) a secondiron compartment inoculated by 2b [3, Fe0

inoc], and (v) a mixedcompartment with aquifer and Fe0 [4, Fe0 + aquifer].

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column using helium as carrier gas at the flow rate of 6 mLmin-1. After 17 and 44 days, vial headspaces were alsoanalyzed with a Varian GC-FID (CP-3800) for the detectionof acetylene, ethene, and ethane with a capillary DB-1column. Every three headspace analyses, vials were refilledwith 1.2 mL nitrogen gas. Initial pollutant concentrations,determined by measurement of the headspace concentrationin the simulated groundwater medium alone (i.e., no iron),were 6.22 mg L-1 (PCE) and 3.76 mg L-1 (TCE).

ResultsColumn System Operation. The degradation performancefor the complete contaminant mixture and the conditions inthe column systems during the first 450 days are describedby Dries (14). Briefly, during this period TCE was almostcompletely removed by Fe0 in all column systems. Theaverage PCE removal efficiency in the systems operated underSRP and IRP conditions was 78 ( 14% and 63 ( 16%,respectively. In all cases, the majority of PCE removaloccurred in the Fe0 compartments. In the reference aquifercompartments without Fe0, less than 10% of the chlorinatedethenes was removed. Aromatic hydrocarbons toluene andbenzene were removed by microbial degradation in theaquifer compartments but also in the Fe0 compartments.

During this study, the column systems were monitoredless intensively for another 475 days. In the course of theexperiment, degradation of PCE appeared to decrease in timefor both electron acceptor conditions, although degradationefficiencies of 75 ( 2% and 88 ( 1% were still observed duringthe latest samplings in the systems operated SRP and IRPconditions, respectively. Degradation of TCE remained high(∼98%) during the complete operation period, except forthe noninoculated iron compartment operated under SRPconditions which showed a gradually decreasing degradationefficiency in time (to 76%). Benzene and toluene were stilldegraded (>60%) by the column systems operated underboth electron acceptor conditions.

The influent feed solution had a pH of 7.13 ( 0.28 anda Eh of 120 ( 67 mV. The effluent pH of the iron compart-

ments operated under SRP conditions was relatively elevated(8.0-8.4) during the first 375 days. To decrease pH andguarantee microbial degradation, the phosphate buffer wasreplaced by an organic buffer (MOPS) and pH was subse-quently reduced to 7.4-7.6. Under IRP conditions, pH of theeffluent of the iron compartments remained close toneutrality. Eh of the effluent of the systems with the singleaquifer compartment ranged between 85 and -80 mV whilethe average Eh of the effluent of the iron compartments wassignificantly lower (-169 ( 80 mV).

Microbiological Characterization of the Column Sys-tems. Results of PCR analysis targeting the different bacterialgroups in duplicate samples from different horizontal columnsections 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 theaquifer, iron, and mixed compartments. Strong PCR signalswere observed for all sections of all systems operated undersulfate-reducing conditions. In the iron and mixed compart-ments operated under IRP conditions, Eubacteria weredetected in a lower number of sections, with strongest PCR-signals in the bottom sections. In some sections, no Eubac-teria could be detected, while other bacterial groups be-longing to the Eubacteria could be detected. A possible reasonfor this could be a different PCR efficiency for the used PCRprotocols. The presence of bacteria in the noninoculatediron compartments can be explained by the fact that the Fe0

and sand grains were not sterilized before use in the columnsystems, or by accidental bacterial contaminations of thecolumn systems during the operation period.

Compared to the Eubacteria, other bacterial groups weredetected in a lower number of column sections, withdetection of the highest number of groups in the bottomsections of the iron and mixed compartments. Archaea andmethanogens were less-represented, especially in the systemsoperated under SRP conditions, with strong PCR-signals onlyin the system with the single aquifer compartment. Asexpected, there was a strong presence of SRB in systemsoperated under SRP conditions. Strong PCR-signals were

FIGURE 2. Schematic overview of the microbial and mineralogical characterization of samples taken from different horizontal sectionsof the PRB columns operated under either SRP or IRP conditions. PCR results obtained with the different primer sets and applied onduplicate samples are given for each section. The column sections that were mineralogically studied are indicated by the rectangularlabels surrounding the columns. XRD results are indicated by the boxed labels at the left of each horizontal column section, whereasSEM-EDX results are indicated by the discontinuous labels at the right of each section. Section dimensions are indicated on the figurein cm. Column names are explained in Figure 1.

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obtained in all compartments except the noninoculated ironcompartment where SRB could only be detected in thebottom part. In systems operated under IRP conditions, SRBwere detected in a lower number of sections, particularly inthe inoculated iron compartment and the mixed compart-ment. Geobacteraceae were detected in all sections of theaquifer and inoculated iron compartments operated underSRP conditions, while they were detected in a lower numberof sections of the iron compartments operated under IRPconditions. In contrast, Geothrix species were only detectedin the compartments operated under IRP conditions. Finally,denitrifying bacteria were present in all compartments, withdetection of both nitrite reductase genes.

DGGE profiles of the amplification products with theeubacterial primer set GC-63-F/518-R are presented in FigureS1 for all section samples of the system with the single aquifercompartment and the sequential system, both operatedunder SRP conditions. Fingerprints of the duplicate sampleswere highly reproducible, with occasional differences in bandintensity. In one and the same column, and particularly forthe system with the single aquifer column, the bandingpatterns were very similar for the different section samplesindicating a relatively stable microbial community in functionof compartment height. Although a few similar bands can beobserved, the banding patterns differ significantly betweenthe system with the single aquifer compartment and the ironcompartment of the sequential system, indicating differentmicrobial communities for both compartments. Except forone strong common band with the iron compartment, thebanding pattern of the downgradient aquifer compartmentis also very different from the iron compartment, andparticularly from the system with the single aquifer com-partment, indicating that the iron compartment stronglyinfluenced the community structure of the downgradientaquifer compartment, probably by inducing a lower redoxpotential.

Mineralogical Characterization of Iron and MixedCompartments. Upon dismantling the iron and mixedcompartments, most of the iron material appeared to consistof loose and uncemented grains, except for the entrancepart of the compartments which consisted of a layer ofcemented material. The column systems operated under SRPconditions contained black iron grains and sand, whilegreenish iron grains and ochreous sand were observed inthe column systems operated IRP conditions. In the bottomsection of the compartments, the iron grains were coveredwith precipitate layers with a thickness ranging from 10 to200 µm. In the more downstream located column sectionsprecipitate layers had a thickness of less than 10 µm.

Samples for XRD analysis were taken from differentsections of iron and mixed compartments operated underboth electron acceptor conditions. The main secondaryphases that were detected included vivianite (Fe3(PO4)2‚8H2O), siderite (FeCO3), ferrous hydroxy carbonate (FHC:Fe2(OH)2CO3) and carbonate green rust (CGR: FeII

4FeIII2(OH)12-

CO3‚2H2O). A semiquantitative overview of the results isshown in Table S3 where indicative phase proportions arereported, based on the height of their principal peak. Thisis only a rough quantitative estimation as the integratedsurface of diffraction peaks is not only depending on theconcentration, but also on instrumental factors, absorptioncoefficients, degrees of cristallinity, and an eventual pref-erential orientation of the grains (23). The four mainsecondary phases were present at variable intensities in thedifferent compartments and at different compartmentheights. Intense vivianite peaks were observed especially inthe lower sections of the compartments operated under bothSRP and IRP conditions. Siderite was detected more fre-quently under IRP conditions while the detection of carbonategreen rust was more frequent under SRP conditions.

Using optical microscopy and SEM-EDX, the samesecondary phases indicated by XRD were identified, alongwith iron sulfide (FeS). FeS, causing the macroscopicallyobserved black color, was evidenced by EDX analyses in thecompartments operated under SRP conditions. The absenceof this phase in the diffractograms suggests an amorphousor poorly crystalline form. FeS was abundantly found in thebottom and second section of the inoculated iron compart-ment and in the bottom section of the mixed compartment.No FeS could be detected in the systems operated under IRPconditions. Vivianite was present in all compartments,particularly as 10-50 µm big prismatic crystals and radiallyor lancet-shaped aggregates, with a characteristic blue color.A thick coating of vivianite was present on most of the irongrains in the bottom sections, while in the downstreamsections only finer coatings or isolated aggregates wereobserved on certain grains. CGR was observed as hexagonalplaty crystals of 1-3 µm, more or less agglomerated inspherical aggregates in the order of 5 µm (Figure S2A).Macroscopically, the iron grains appeared to be covered bya very thin green-blue layer. Ferrous hydroxy carbonate isa mineral phase close to malachite that occurs as aggregatesof lamellar crystals (24-26). This phase was commonlyobserved as a 20 µm thick coating constituted by an innercompact to spherulitic layer covered by a layer of largerlamellar crystals (Figure S2B). Finally, siderite was observedas 2-3 µm big rhombohedra. Oxides (e.g., magnetite,hematite, maghemite) were not identified among secondaryminerals. In some cases a few magnetite spots were observed,in particular on iron grains which were relatively little coveredwith precipitates. These spots can be considered as primaryminerals as these small quantities could also be observed onthe original cast iron. XRD and SEM-EDX results are unitedand schematically represented in Figure 2.

Reactivity of the Used Fe0 Filings. A batch experimentwas conducted to compare the reactivity of original unreactediron with the reactivity of iron material from the columnsystems. Values of pseudo-first-order rate constants (k) forPCE decay were determined by best fit of measured head-space concentrations as a function of time, using a pseudo-first-order rate law. Observed degradation products, massrecoveries after 44 days, and rate constants for transformationof PCE and TCE are reported in Table S4 and half-lifes arepresented in Figure 3. Rate constants for the column ironsamples were comparable or higher than rate constants forthe original iron for both PCE and TCE degradation.Significantly higher degradation capacities were observedfor the iron samples originating from the inoculated ironcolumn and the mixed column operated under SRP condi-tions. Especially for the inoculated iron column, rateconstants were 7 and 10 times higher than rate constants forthe 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 withoutformaldehyde in comparison with the poisoned samples,indicating a potential biological contribution to PCE andTCE degradation. Among degradation products, cis-DCE, VC,ethene, and ethane where formed together with the detectionof acetylene, evidencing that both the sequential hydro-genolysis pathway and the â-elimination pathway werefollowed for PCE and/or TCE degradation (27). Incompletemass recoveries (67-83%) may be due to adsorption ofreactants and products to the iron surface, losses of volatilereactants and products during the sampling process, orformation of nondetectable products.

DiscussionMicrobiological Aspects. After ∼15 months of operation, Guet al. (2) observed a microbial community that was sub-stantially increased in biomass in and in the surroundings


of the Oak Ridge Y-12 reactive barrier (Tennessee), withsulfate-reducing and denitrifying bacteria among the mostdominant groups, and relatively low levels of methanogens.It was suggested that the observed biomass increase mighthave been due to the utilization of residual byproducts ofthe Guar gum, such as glucose, which was used during trenchexcavation. A more comprehensive laboratory column-scalemicrobial characterization was performed in our study byalso targeting iron(III)-reducing bacteria and using 16S rRNAgene PCR-DGGE fingerprinting to indicate different mi-crobial communities between different iron and aquifercompartments. Gu et al. (2) observed relatively higherbiomass levels in both the upgradient and downgradientsoil cores than in the iron samples. This is in accordancewith our findings, as we detected bacterial groups in a highernumber of sections in both aquifer compartments, comparedto the iron and mixed compartments. The presence ofindigenous bacteria, nutrients and carbon sources, and thehigher surface area and redox potential, probably make theaquifer material a more favorable environment for coloniza-tion and growth of microorganisms than the Fe0 grains.Despite the sufficient buffering capacity of the simulatedgroundwater medium and the near neutral pH of the bulkcolumn fluid, pH might increase at microlocations in theiron pore space and inhibit biofilm growth at the iron surface.On the other hand, it has to be noted that the extracted DNAcontent of the iron compartments might have been under-estimated (on a unit volume basis) due to the fact that Fe0

has a relatively high particle density and 2 g samples weretaken for DNA extraction. In the iron and mixed compart-ments, the higher number of functional groups of bacteriaat the entrance zone of the Fe0 matrix is consistent with thefindings of Wilkin et al. (9) who reported the highest biomasslevels in the upgradient aquifer/iron interface and a biomassdecrease along the flow path of an iron barrier installed atElizabeth City, North Carolina. The lower biomass levels inthe downstream column sections might be explained by adecreasing redox potential and/or nutrient content alongthe flow path. A rising pH may also be inhibitory for microbialgrowth although in our columns pH was staying close toneutrality, due to the feeding of the phosphate and later onorganic buffer.

Mineralogical Aspects. Most of the secondary mineralphases identified in our iron column samples are more orless consistent with the findings described elsewhere (25,28). However, highly oxidized species such as hematite andgoethite found in many laboratory- and field-scale Fe0 barriersystems (8, 28), were not detected in our study. Furthermore,in our systems, vivianite was highly abundant while mineralphases like magnetite and calcium carbonates (calcite,aragonite) were scarce or not detected. Based on thermo-dynamics, the finding of vivianite was predictable as with

the addition of phosphate-bicarbonate buffered minimalmedium vivianite will precipitate rather than siderite. Incontrast, vivianite is unlikely to be formed under in situconditions as phosphate concentrations are usually low.Among the green rusts, only carbonate green rust (FeII

4-FeIII

2(OH)12CO3‚2H2O) could be detected in contrast to thesulfate-containing green rust (FeII

4FeIII2(OH)12SO4. nH2O),

which is only formed under highly elevated sulfate concen-trations (29, 30). Ferrous hydroxy carbonate, initially observedas a corrosion product formed at elevated temperatures (24),has recently been found in several Fe0 barriers (28, 31) andcan result from the remineralisation of fine-grained, biogenicmagnetite from IRB under anoxic conditions (26). Ferroushydroxy carbonate may act as a conductor for electronsbetween Fe0 and the contaminants although Kohn et al. (25)speculated that thick precipitate layers may adopt insulatingcapacities due to an excessive distance for efficient electrontransfer.

Impact on Barrier Performance. The obvious linkage ofgeochemistry to microbial activity in Fe0 barriers is H2, whichis an excellent energy resource in anaerobic environments,and in high quantities generated by the anaerobic corrosionof Fe0 (6). Our results indicate that SRB might haveindirectly improved the reactivity of Fe0 in the columnsoperated under SRP conditions. SRB can utilize hydrogen asa substrate for the reduction of sulfate to sulfide which canresult in the precipitation of ferrous iron sulfide. Rates ofchemical abiotic sulfate reduction at low temperatures areextremely slow (9). Our results suggest that the presence ofsulfate-reducing bacteria resulted into the precipitation ofFeS in the column systems operated under SRP conditions.The batch experiment indicated that after more than 900days of operation, the Fe0 retained reactivity with possiblyeven improved ability to remove PCE and TCE, in comparisonwith original unreacted iron. Especially samples containingiron sulfides showed significantly higher degradation rates,with rate constants up to 10 times higher than rate constantsfor the original iron. FeS-coatings are reported to besignificantly more reactive per unit surface area than ironmetal in transformation of TCE and other halogenatedaliphatic compounds (32, 33). Our results, in addition, suggestthat iron metal, placed in subsurface reactive barriers, maygain reactivity by the formation of FeS coatings through thegrowth of SRB. However, caution is required when applyingthe results to field conditions. The use of buffers in this studyresulted into a near-neutral pH which is more favorable tomicrobial activity in comparison with the elevated pH thatcan be observed in field-scale iron barriers, depending onthe natural buffering capacity of the groundwater and theretention time in the barrier. On the other hand, SRB havealso been detected in a field-scale iron barrier (2). It mightbe useful to stimulate the growth of sulfate-reducing bacteria

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.


in the vicinity of Fe0 barriers, or to treat iron grains withsulfides prior to or after barrier installation to form FeS surfacecoatings. An increased reactivity for pollutants such as TCEmight require less iron material to be installed, possiblyresulting in cheaper barrier configurations. IRB may alsohave positively affected the reactivity of the Fe0 barrier matrixby reductively transforming passivating layers of secondaryFe3+-containing minerals into Fe2+ or mixed-valent phasessuch 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 canreduce chlorinated organics (34). Although we detected greenrust together with the presence of IRB, we cannot prove anypositive impact on iron reactivity by using the column setupas described here.

It is unlikely that microbial fouling by biomass ac-cumulation will occur in the anaerobic iron barrier matrixitself. Biomass accumulation and subsequent biocloggingmight cause permeability reductions in aerobic biologicalbarriers or zones. However, it does not seem to causeproblems in Fe0-PRBs where microorganisms have to dealwith anaerobic, highly reducing conditions and an elevatedpH (4, 8). In this study, no visible evidence of biomass build-up was noted after column dismantling.

AcknowledgmentsThis work was funded by the Institute for the Promotion ofInnovation through Science and Technology in Flanders(IWT-Vlaanderen) and the Flemish Institute for TechnologicalResearch (VITO). Mineralogical investigations were per-formed with the helpful contribution of the CAT-µ (JointCenter for Microscopy) at University of Liege. Columnsystems were setup under the framework of the EuropeanMULTIBARRIER project (QLK3-CT-2000-00163). We thankM. Maesen, A. Hermans, and A. Bossus for their appreciatedcontribution to this study.

Supporting Information AvailableTables of PCR-primers and PCR-conditions, primer specific-ity, semiquantitative XRD-results, and kinetic parameters;DGGE-gel picture, SEM photomicrographs of secondaryprecipitates; figures with degradation curves. This materialis available free of charge via the Internet at http://pubs.acs.org.

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Received for review January 5, 2007. Revised manuscriptreceived May 4, 2007. Accepted May 16, 2007.

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