Microbial Colonization of an In SituSediment Cap and Correlation toStratified Redox ZonesD A V I D W . H I M M E L H E B E R , † , ⊥

S A R A H . T H O M A S , † , ‡

F R A N K E . L O F F L E R , † , ‡

M A R T I A L T A I L L E F E R T , § A N DJ O S E P H B . H U G H E S * , † , |

School of Civil and Environmental Engineering, GeorgiaInstitute of Technology, Atlanta, Georgia 30332, School ofBiology, Georgia Institute of Technology, Atlanta, Georgia30332, School of Earth and Atmospheric Sciences, GeorgiaInstitute of Technology, Atlanta, Georgia 30322, and School ofMaterial Science and Engineering, Georgia Institute ofTechnology, Atlanta, Georgia 30332

Received July 2, 2008. Revised manuscript received October21, 2008. Accepted October 23, 2008.

In situ capping is a management technique for contaminatedsediments involving the placement of clean material at thesediment-water interface. This work combined porewatergeochemicalprofilingwithquantitativemicrobialdatato investigatethe intrinsic microbial colonization of a sand cap. Geochemicalcharacterization using voltammetric microelectrodes indicatedverticalstratificationofbiogeochemicalprocesseswithinacappedsediment column. Following dissection of the column, quantitativereal-time PCR (qPCR) enumerated microbial populationswithineachdiscreteredoxzoneandwasaccompaniedbyterminal-restriction fragment length polymorphism (T-RFLP) to elucidategeneral community shifts. Bacteria and Archaea were presentwithin the cap according to qPCR, with higher concentrationsgenerally observed in the underlying sediment. Iron-reducingpopulations were detected and quantified using newly designedqPCR primer pairs for Anaeromyxobacter spp. and Shewanellaspp. and published primer sets for δ-Proteobacteria andGeobacteracea. Results confirmed geochemical measurementsindicating that microbial Fe(III) reduction was a major processin the overlying cap. Genes encoding microbial sulfate reduction(dsrA) and methanogenesis (mcrA) were also present withinthe cap but were more prevalent in the sediment. Canonicalcorrespondence analysis of terminal-restriction fragment lengthpolymorphism (T-RFLP) patterns verified that spatial changesin bacterial community composition were significantly correlatedtodepthandFe2+andMn2+concentrationgradients.Cumulatively,results demonstrate that microorganisms indigenous to aquaticsedimentscolonizedtheoverlyingcaptoformcomplexcommunitiesmirroring redox stratification. Implications of capping forbiogeochemical cycling, contaminant fate and transport, andremedial design are discussed.

IntroductionContaminated aquatic sediments have adverse effects onaquatic life and limit recreational and economic uses,especially in urban and industrial settings (1). In situ cappinghas emerged as an option for managing contaminatedsediments and involves placing a layer of clean fill materialat the sediment-water interface to prevent contaminantrelease and contact with benthic macrofauna and overlyingsurface water. Traditionally, clean sand has been employedfor capping and can considerably delay contaminant break-through when diffusive transport processes dominate (2).Recent efforts have focused on active capping technologies,in which sediment caps are augmented with constituents tosequester or transform contaminants during transportthrough the cap (3-5). A recent study (6) demonstrated thatemplacing an in situ cap at the sediment-water interfaceinduces an upward, vertical shift of geochemical species intothe overlying cap while simultaneously conserving redoxstratification. One issue that has not been addressed,however, is if active microbial populations also shift fromthe sediment to colonize the overlying cap.

Intrinsic microbial colonization of an in situ sedimentcap will directly impact biogeochemical cycling and affectcontaminant fate and transport. Biogeochemical cycling atthe sediment-water interface of aquatic sediments can becomplex and subject to multiple site-specific variables (e.g.,organic carbon input, hydrodynamic flow patterns). Undis-turbed sediments below oxic surface waters typically possessan aerobic layer extending millimeters to centimeters intothe sediment bed (7) inhabited by an abundant and diversecommunity of heterotrophic microorganisms due to thepresence of oxygen and accessible organic matter (8). Beneaththis aerobic layer a gradient of anaerobic biogeochemicalprocesses exist, stratified according to the thermodynamicyields of their respective terminal electron accepting pro-cesses (9). Microbial composition and functionality con-tribute to this redox stratification and influence contaminantbioattenuation mechanisms within sediments. For example,metal sequestration (10) and the microbial transformationsof petroleum hydrocarbons (11), chlorinated solvents (12),polychlorinated biphenyls (PCBs) (13), and polycyclic aro-matic hydrocarbons (PAHs) (14) have all been documentedwithin aquatic sediments under specific redox conditions.

Studies described herein assess the ability and degree towhich microorganisms indigenous to sediments inhabit andcolonize an overlying sand cap. Spatial distributions of targetpopulations implicated in metal reduction, sulfate reduction,and methanogenesis are examined using quantitative real-time PCR (qPCR) and compared against porewater geochem-ical profiles. Bacterial community composition is monitoredwith terminal-restriction fragment length polymorphism (T-RFLP) analysis. Results from the qPCR and T-RFLP studies,coupled with high-resolution geochemical characterization,provide further insight into biogeochemical processes andcontaminant fate following in situ capping of aquaticsediments.

Materials and MethodsCapped Sediment Column. A laboratory-scale sedimentcolumn was constructed to monitor biogeochemical changesinduced by capping with a clean sand. Details of sedimentcolumn construction have been reported previously (6).Briefly, wet sediment from the Anacostia River, Washington,DC containing PCBs, PAHs, metals, and organic pesticides(3) was packed to a depth of 7 cm in a glass chromatography

* Corresponding author phone: (404) 894-2201; fax: (404) 894-2278; e-mail: joseph.hughes@ce.gatech.edu.

† School of Civil and Environmental Engineering.‡ School of Biology.§ School of Earth and Atmospheric Sciences.| School of Material Science and Engineering.⊥ Present Address: Geosyntec Consultants, Columbia, MD 21046.

Environ. Sci. Technol. 2009, 43, 66–74

66 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 1, 2009 10.1021/es801834e CCC: $40.75 2009 American Chemical SocietyPublished on Web 11/25/2008

column (20 cm long, 5.0 cm i.d.; Spectrum Chromatography)fitted with a Teflon bottom end plate. The sediment wasthen capped with 7 cm of dry, autoclaved ASTM C-33 concretesand (U.S. Silica). Overlying water was maintained at 4 cmabove the cap-water interface with a constant head reservoirand consisted of deionized water (>18.2 Ω) containing onlysodium chloride (1.26 mM) to match the salinity of theAnacostia sediment porewater. The capped sediment columnwas incubated under aerobic conditions in the dark at roomtemperature with static (diffusive) conditions for 13 weeks,then upflow (advective) conditions for 16 weeks. Duringupflow conditions, a syringe pump provided anoxic influentsolution to the bottom of the column at a flow rate of 2.0 cm3

hr-1 (seepage velocity ) 4.4 cm day-1; Darcy velocity ) 2.4cm day-1). The influent was equivalent to the overlying watersolution but sparged for 30 min with nitrogen gas (Airgas)to remove dissolved oxygen. No substrates or growth factorswere supplied with the influent solution.

Porewater Geochemical Profiles. Voltammetric micro-electrodes were used to analyze aqueous geochemical species,with details of the measurements provided elsewhere (6).Briefly, all voltammetric measurements were performed usinga platinum counter electrode, an Ag/AgCl reference electrode,and an Au/Hg solid-state microelectrode fabricated asdescribed by Brendel and Luther (15). The Au/Hg micro-electrodes were capable of simultaneously measuring O2(aq),H2O2, Fe2+, Mn2+, S2O3

2-, ΣH2S [)H2S + HS- + S2- + S0 +Sx

2-], organic-FeIII(aq), and FexSy(aq) species (15-17). Each Au/

Hg microelectrode was calibrated for dissolved oxygen bylinear sweep voltammetry then for manganese by cathodicsquare wave voltammetry (CSWV). Linear sweep voltammetry(LSV) was exclusively used to measure dissolved oxygen (MDL∼4 µM), while cathodic square wave voltammetry (CSWV)was used to quantify Mn2+ (MDL ∼15 µM), Fe2+ (MDL ∼25µM), and ΣH2S (MDL ∼0.2 µM) Concentrations of Fe2+andΣH2S were determined from manganese calibration curvesfollowing the pilot ion method (15). The sensitivities of theorganic-FeIII

(aq) complexes have yet to be determined due inpart to the number of natural ligands present in sediments,and their concentrations are therefore reported in units ofcurrent intensity (nA) (16). Similarly, the stoichiometry ofFexSy (aq) is unknown and current intensities (nA) are reportedfor these species (17). All voltammetric analyses wereperformed with a computer-operated DLK-100A potentiostatand a micromanipulator capable of vertical movements atthe submillimeter scale (Analytical Instrument Systems, Inc.).Voltammograms were processed with a Matlab-based pro-gram (18). Multiple porewater geochemical profiles wereobtained during static and upflow conditions, with a finalprofile obtained 16 weeks after the onset of upflow conditions(29 weeks of total incubation; 71 pore volumes flushed) (6).

Column Dissection. The capped sediment column wasdissected for microbiological analyses immediately after thefinal geochemical profiling at 16 weeks of upflow conditions.Overlying surface water was removed from the column witha needle and syringe under aseptic conditions. A core samplewas obtained from the center of the column using a zerocontamination soil sampler (JMC Soil Samplers), with thecore sample encased in a PETG copolyester liner (30 cmlength; 2.0 cm i.d.). The core was frozen at -20 °C thenextruded and dissected under aseptic conditions. DNA wasextracted from the solid and aqueous phases of eachdissection zone, with detailed methodology provided in theSupporting Information. Notably, the DNA extraction pro-cedure was also applied to autoclaved, dry ASTM C-33 sandand the extract accompanied all measurements as a controlto quantify microbial populations in the sand prior to capping.

The first column dissection was made 10 mm below thecap-water interface, with successive dissections at constantintervals until the sediment-cap interface was reached

(Figure 1). Seven samples were obtained from the sand capmaterial (y ) 0 to -70 mm) and four samples were obtainedfollowing dissection of the underlying sediment (y )-70 to-140 mm). The samples were immediately placed into sterile,15 mL centrifuge tubes and stored at-20 °C. Dissected zoneswere labeled sequentially beginning with Zone 1 at thecap-water interface and ending with Zone 11 in the sediment(Figure 1).

Primer Design. qPCR primer pairs for Shewanella spp.and Anaeromyxobacter spp. were designed with the OligoDesign and Analysis Tools (www.idtdna.com) based on thenearly complete 16S rRNA gene sequences available inGenBank. The 16S rRNA gene sequences were aligned usingthe MegAlign program of the Lasergene software package(DNA Star, Inc.). The sequences of the selected primers areprovided in Table 1. The reverse primer for Shewanella spp.,She220R, is the reverse complement of She211f, a traditionalPCR primer (19). The Anaeromyxobacter spp.-targeted prim-ers were obtained using Applied Biosystems Primer ExpressTM

software. A 200 bp section of the 16S rRNA gene sequencecontaining an Anaeromyxobacter-specific region was identi-fied by analysis of the alignment and entered into the Primer

FIGURE 1. Porewater geochemical characterization of AnacostiaRiver sediment capped with ASTM C-33 concrete sand after 16weeks of upflow conditions. Values for organic-FeIII

(aq) andFexSy (aq) complexes are reported as current intensity and plottedalong the top x-axis. The concentrations of O2, Fe2+, Mn2+, andΣH2S (µM) are referenced to the lower x-axis. Dissectionlocations are indicated by solid gray lines, with numberscorresponding to dissection zones. The cap-water interface islocated at y ) 0 mm. The sediment-cap interface is located at y )-70 mm.

VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 67

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68 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 1, 2009

ExpressTM program to find a short region with a consistentmelting temperature. BLAST analysis suggested primerspecificity to the target sequences.

Quantitative Real-Time PCR (qPCR) Analysis. Targetorganisms, reaction chemistries, primer sequences, andstandards for all qPCR analyses are summarized in Table 1.Total Bacteria were quantified as described by Ritalahti et al.(20). Total Archaea were quantified using primers andmethods described by Da Silva et al. (21), with genomic DNAextracted from Methanococcus maripaludis (ATCC 43000) asa standard. Quantification of δ-Proteobacteria was performedby Microbial Insights, Inc. using primers and conditionsdescribed by Stults et al. (22). The qPCR reaction mixturesfor Anaeromyxobacter spp.-, Geobacter spp.-, and Shewanellaspp.-targeted analysis contained 15 µL of Power SYBR GreenPCR master mix (Applied Biosystems), 300 nM of each primer,and 3 µL of template DNA (10.8-90.0 ng of DNA) in a totalreaction volume of 30 µL. The qPCR temperature programwas as follows: 2 min at 50 °C, 10 min at 95 °C, then 40 cyclesof denaturation at 95 °C for 15 s and annealing at 60 °C for1 min, with fluorescence measured after each annealing step.The dsrA gene was quantified according to Wilms et al. (23),with the exception that only 2.5 µL of template DNA wasutilized instead of 10 µL. The standard for dsrA calibrationwas genomic DNA from Desulfovibrio vulgaris (ATCC 29579;GenBank accession number NC_002937), whose genomecontains one copy of the dsrA gene. The mcrA gene wasquantified as described (23), except that the reverse primerwas modified such that a W (A or T) replaced S (C or G) atthe ninth position (Table 1) to fully complement the originalprimers designed by Hales et al. (24). The standard for mcrAcalibration was genomic DNA from Methanosarcina mazei(ATCC BAA-159; GenBank accession number NC_003901),whose genome contains one copy of the mcrA gene. Furtherdetails on qPCR procedures are provided in the SupportingInformation.

Terminal-Restriction Fragment Length Polymorphism(T-RFLP) Analysis. Dissection zones 1, 2, 3, 4, 6, 8, and 10exhibited unique geochemical characteristics and wereselected for T-RFLP analysis, following previously describedprocedures (25) described briefly in the Supporting Informa-tion. The restriction enzymes utilized were HhaI, MspI, andRsaI. Anaeromyxobacter dehalogenans strain 2CP-C DNA wasdigested with each digestion enzyme to ensure that experi-mentally derived T-RFLP patterns matched predicted in silicorestriction fragments (see the Supporting Information).

Data Analysis. T-RFLP profiles were analyzed to elucidatestatistically significant information regarding bacterial com-munity composition and diversity among dissection zones(26). Canonical correspondence analysis (CCA) was per-formed on Zones 1, 2, 3, 4, 6, 8, and 10 to examine potentialrelationships between bacterial community patterns andenvironmental variables (27) (CANOCO 4.5 software, Mi-crocomputer Power) with significance defined at p < 0.05.Quantitative environmental variables tested were averagedepth and concentrations of Mn2+, Fe2+, organic-FeIII

(aq), andFexSy(aq), measured during the voltammetric analysis. Resultswere summarized in ordination diagrams (CanoDraw 4.0).

Three additional, independent species diversity indiceswere calculated from normalized HhaI T-RFLP fragmentpatterns to analyze microbial diversity and composition. Thefirst indicator was species richness (S), equivalent to theamount of unique T-RFs in each zone (28). The secondindicator was Shannon’s diversity index (H), an estimate ofdiversity accounting for both species abundance and even-ness, calculated using StatsDirect Statistical Software (Stats-Direct, Ltd.). Typical values are between 1.5 and 3.5 withhigher values indicating a greater degree of diversity. Thethird indicator was the Smith and Wilson evenness value(Evar.), recommended by Blackwood et al. (29) to examine

species parity from T-RFLP data, ranging from 0 (skeweddistribution) to 1 (even distribution) (30). Phylogeneticanalysis using a web-based, in silico process was alsoperformed to identify dominant community members in eachdissection zone and is described in the Supporting Informa-tion.

ResultsPorewater Geochemical Profile. Voltammetric analysis ofporewaters indicate that multiple biogeochemical processeswere spatially distributed within the capped sediment column(Figure 1). Oxygen was present in the overlying water atrelatively constant concentrations before decreasing tonondetect concentrations only 2.8 mm below the cap-waterinterface. A region dominated by manganese reduction wasobserved below the aerobic portion of the column, evidencedby peaking Mn2+ concentrations (0 to -24.0 mm). This wasfollowed by a region dominated by Fe(III) reduction, indicatedby measured increases in Fe2+ and organic-FeIII

(aq) levels.Ferrous iron decreased to nondetect concentrations at-87.0mm due to the formation of dissolved iron sulfide complexeswithin the sediment. Sulfate reduction within the cap andsediment may have been utilized for FexSy(aq) formation (6).In general, the capped sediment column exhibited stratifiedredox zones distributed according to the thermodynamicyield of terminal electron accepting processes and encom-passed cap and sediment material. Spatial and temporaldevelopment of the stratified biogeochemical processes isthe focus of previous work (6) which includes more detaileddiscussions of the porewater profile.

Material from the dissection zones were characterized byvarious biogeochemical processes according to the porewatergeochemical profile (Figure 1). Zone 1 included oxygen andmanganese reduction; Zone 2 exhibited manganese reduc-tion; Zones 3 and 4 displayed iron and manganese reduction;Zone 5 included Fe(III) reduction; Zones 6 through 8displayed Fe(III) reduction and FexSy(aq) formation; and Zones9 through 11 included Fe(III) reduction and FexSy(aq) formationbut no dissolved, free ferrous iron.

Target Microbial Populations. qPCR analyses confirmedthat microbial populations were present and quantifiable ineach dissected zone of the sediment and overlying sand cap(Figure 2). Bacterial cell numbers in the cap generallydecreased in each subsequent zone (Figure 2A), from 5.2 ×108 cells per mL porewater in Zone 1 to 1.7 × 106 cells permL porewater in Zone 6, before an increase in Zone 7 directlyabove the sediment-cap interface. Bacterial cell titers wererelatively constant within the sediment (Zones 8-10), withobserved mean values between 5.9 × 108 and 6.8 × 108 cellsper mL porewater. Neither Bacteria nor Archaea weredetected in DNA extracted from dry, autoclaved ASTM C-33sand (Figure 2).

Bacteria associated with dissimilatory metal reductionand potential contaminant biotransformations were detectedin each zone of the sediment and the overlying sand cap(Figure 2A). δ-Proteobacteria were detected throughout thecolumn at levels similar to those of total Bacteria. Geobacterspp., Anaeromyxobacter spp., and Shewanella spp., were eachdetected in all zones. Geobacter cell titers in the cap rangedfrom 105 to 106 cells per mL porewater before peaking in thesediment (Zone 9) at 6.7 × 107 cells per mL porewater.Anaeromyxobacter numbers were most prominent in theupper cap layers (9.9 × 105 in Zone 1) and in the sediment(1.8 × 107 in Zone 9). The presence of Shewanella species,suspected to be present following detection of an organic-FeIII

(aq) complex observed previously in association with thisorganism (6, 31), were most abundant in the upper cap layers(1.0 × 106 in Zone 1), then stabilized to mean values between104 and 105 cell per mL of porewater before increasing slightlyin the sediment. In Zone 1, dissimilatory metal- and iron-

VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 69

reducing bacteria (Anaeromyxobacter, Geobacter, Shewanellapopulations) collectively accounted for only 0.4% of the totalBacteria (Table 2). Higher proportions of metal- and iron-reducers relative to total Bacteria were observed in theremainder of the cap and sediment, peaking at 8.7% in Zone6. Dehalococcoides strains were not detected in any regionof the column; however, the necessary electron acceptors(e.g., chlorinated ethenes) to enrich their population werenot provided.

The dsrA functional gene for sulfate reduction wasdetected in all sediment and cap zones; at 104 to 105 cells permL porewater in the cap before increasing 2-3 orders ofmagnitude in the sediment (Figure 2B). dsrA gene copieswere more comparable (i.e., within 1 order of magnitude) toδ-Proteobacteria levels in the deeper zones of the column

(Zones 7-10) compared to most cap layers (Zones 1-6). Theproportion of sulfate reducing prokaryotes (SRPs) to totalBacteria generally increased with depth, increasing from lessthan 0.1% in Zone 1, to 2.1% in Zone 5, and to 22.3% in Zone9 (Table 2). Archaeal cell numbers displayed slightly greaterabundance in the uppermost cap zone compared with thelower cap zones (Figure 2C). Archaeal cell numbers in Zone7 were 6.6 × 105 copies per mL porewater and increased inthe sediment to approximately 108 copies per mL porewater.The ratio of archaeal to bacterial 16S rRNA gene copies rangedfrom less than 0.1% in Zones 1-3 to 19.1% in Zone 9 (Table2). The mcrA gene was detected in the upper two cap zones,but was not detected in Zones 3 and 4, respectively. Copynumbers of mcrA in the remaining layers (Zones 5-10) werecomparable to those observed for Archaea, with the most

FIGURE 2. Quantitative real time PCR (qPCR) results for (A) total Bacteria and populations implicated in metal reduction andbioremediation, (B) total Bacteria and populations implicated in sulfate reduction, and (C) Total Archaea and mcrA gene copies. N/Dindicates results below detection limits.

70 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 1, 2009

mcrA gene copies detected in the sediment (2.1 × 108 inZone 9). The proportion of mcrA gene copies to Archaea 16SrRNA gene copies ranged from 12.5% in Zone 2 to greaterthan 100% in Zone 9 (Table 2). A modified mcr gene hasrecently been identified in enrichments of anaerobic methaneoxidizers (32) and may be involved in reverse methanogenesisunder anoxic conditions (33, 34). However, the mcrA geneis conserved among methanogenic Archaea, which are a morecommonly encountered environmental population.

Bacterial Community Analysis. Analysis of T-RFLP pat-terns via CCA testing revealed that changes in bacterialcommunity composition among dissection zones could besignificantly correlated to environmental variables. Averagedepth best explained changes in the T-RFLP fingerprints andwas the only variable independently related to the data (p) 0.015). The T-RFLP data were explained with the greatestdegree of confidence (p ) 0.008), when average depth, Fe2+,and Mn2+ concentrations (µM) were all considered, sug-gesting these three variables collectively influenced com-munity composition (Figure 3). Therefore, based on the CCAresults, the hypothesis of no relation between organism andenvironment is rejected due to statistically significant speciescomposition changes correlated to environmental variables.

Indices of bacterial richness and evenness were uniquebut generally constant across all zones analyzed (Table 3).Most species richness values ranged from 95 to 132, indicatinga complex community. Previous studies estimating richnessfrom T-RFLP data classified values between 45 and 72 ashaving a high degree of complexity in community structure(28). The maximum species richness was observed in Zone1 (S ) 132), followed by Zones 8 and 10 (S ) 122 for eachzone). A moderate, positive correlation between bacterialnumbers and species richness (R2 ) 0.75) was observed suchthat the zones with the highest richness also had the highestqPCR bacterial cell titers. Notably, Zone 6 had very low speciesrichness (S ) 21) and modifications to the T-RFLP experi-mental protocol and replicate T-RFLP analyses yielded similarresults. Shannon index values were between 3.45 and 4.00for most zones, with Zone 6 being the exception (H ) 2.13).A modest correlation was detected between qPCR bacterialnumbers and diversity (R2 ) 0.66). Smith-Wilson estimatesof evenness suggested that species abundances within mostzones were relatively similar, except for Zone 1 which showedskewed abundance values (Evar. ) 0.23) (i.e., certain popula-tions were dominant relative to the community). In silicophylogenetic assignment of T-RFLP peaks at the phylum-

class level provided additional insight into communitycomposition and shifts among dissection zones and can befound in the Supporting Information.

DiscussionThe microbial colonization of a laboratory-scale in situsediment cap was demonstrated using an integrated, culture-independent approach of voltammetry-based porewatergeochemical measurements, qPCR to enumerate targetmicrobial populations, and T-RFLP to examine changes inbacterial community structure. qPCR analysis confirmed thatBacteria and Archaea inhabited the cap in numbers ap-proaching those in the underlying sediment. Microorganismscolonizing the overlying cap originated from the sediment,since PCR analysis did not detect microorganisms associatedwith the sand prior to cap emplacement. Statistical analysisof T-RFLP data with CCA demonstrated that changes incommunity patterns were significantly related to environ-

TABLE 2. Proportion of Groups of Metal Reducers, SulfateReducers, And Methanogens to Total Microorganisms for EachDissection Zone, Calculated from qPCR Resultsa

dissectionzone

proportionof metal-reducersb

to Bacteria

proportionof dsrA toBacteria

proportionof Archaeato Bacteria

proportionof mcrA to

Archaea

1 0.4 <0.1 <0.1 23.42 4.0 0.3 <0.1 12.53 2.7 0.5 <0.1 N/A4 4.1 0.7 N/A c N/A5 6.3 2.1 0.2 >1006 8.7 2.9 0.3 92.57 1.2 1.6 2.2 57.98 3.8 22.2 18.5 75.99 6.9 22.3 19.1 >10010 0.2 6.7 4.7 52.2

a All values are reported in percentages of gene copiesto gene copies. The sediment-cap interface is locatedbetween Zones 7 and 8. b Metal- and iron- reducing bac-teria, defined as the cumulative gene copies of Anaeromyxo-bacter, Geobacter, and Shewanella. c Calculation not able tobe performed due to quantities below detection limits.

FIGURE 3. Ordination diagram based on canonical correspo-ndence analysis of T-RFLP community profiles from dissectionregions 1, 2, 3, 4, 8, and 10. The coordinates of the site points arethe scores of each individual site on the two synthetic axes.Arrows indicate quantitative environmental variables and point inthe direction of maximum change in the value of the associatedvariable. The eigenvalues of axis 1 (horizontal) and axis 2(vertical) are 0.696 and 0.497, respectively, and represent thepercentage of community variance explained by extracted,synthetic gradients.

TABLE 3. Indices of Bacterial Community Structure Calculatedfrom HhaI T-RFLP Fragment Patterns at Each DissectionRegion Analyzed

dissectionzone richness (S )

Shannondiversity (H )

Smith-Wilsonevenness

(Evar.)

1 132 3.87 ( 0.0023a 0.232 95 3.45 ( 0.0025 0.433 105 3.63 ( 0.0037 0.484 93 3.52 ( 0.0052 0.516 21 2.13 ( 0.0049 0.408 122 4.00 ( 0.0033 0.5310 122 3.73 ( 0.0029 0.45

a Estimated value ( one standard deviation.

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mental variables (depth, Fe2+, and Mn2+), suggesting thatmicrobial data collected in these studies did not reflect onlythe elution of planktonic microorganisms from the sediment.If this were the case, then environmental variables wouldnot adequately explain spatial changes in bacterial com-munities. Although the elution of organisms may havecontributed to the microbial numbers observed in the cap,the results demonstrate that microbial populations inhabitedthe overlying cap and formed distinct communities accordingto depth and redox gradients.

Metal- and iron- reducing populations (δ-Proteobacteria,Anaeromyxobacter spp., Geobacter spp., and Shewanella spp.)were detected in each zone of the cap and sediment,confirming geochemical data suggesting that microbial Fe(III)reduction was a major biogeochemical process (6). In Zone1, Anaeromyxobacter, Geobacter, Shewanella populationsaccounted for only 0.4% of the total Bacteria (Table 3),consistent with observed geochemical data indicating oxygenreduction was occurring. Zones 2-9 displayed higher pro-portions of Anaeromyxobacter, Geobacter, Shewanella popu-lations relative to total Bacteria, agreeing with the geochem-ical data that implied metal reduction in these zones.Geobacter consistently outnumbered Shewanella and Anaer-omyxobacter within the cap. Interestingly, Anaeromyxobacterlevels were low in the cap but exceeded Geobacter numbersin the sediment. Anaeromyxobacter and Shewanella cell titerswere higher in the upper zones relative to the rest of the cap,possibly due to their roles in oxygen and manganesereduction. Geobacter spp. were also detected in Zone 1 andcan remain viable under aerobic conditions (35) and/orparticipate in manganese reduction (36).

The proportion of SRPs compared to total Bacteriain-creased 3-4 orders of magnitude in the sediment comparedtothe cap (Table 2), complementing porewater geochemicalprofiles suggesting sulfate reduction in the sediment(Figure 1 (6)). The detection of the dsrA gene in the capregions means that microbial sulfate reduction may havesupplied the sulfide for FexSy(aq) formation in the cap, withsulfide possibly also contributing to manganese oxidereduction in the upper cap (y ) -20 mm (37)). Thedetection of dsrA in the more oxidized regions of the cap(Zones 1 and 2) is not unfounded since certain SRPswithstand exposure to oxygen (38). The detection of themcrA gene in the lower portion of the cap suggests thepresence of methanogenic Archaea, demonstrating thatthe entire range of microbial metabolic processes (oxygenreduction to methanogenesis) were spatially distributedwithin the cap. Thus, a diverse range of microorganisms,spanning the spectrum of microbially catalyzed redoxreactions, were colonized in the cap after 29 weeks ofemplacement. This colonization most probably contrib-uted to redox zone evolution within the system, in parallelwith abiotic reactions discussed in detail previously (6)(e.g., Mn(IV) reduction by Fe(II)). The microbial coloniza-tion and development of biogeochemical zones observedwithin the cap provides additional insight into microbialcommunity response following physical and geochemicalperturbations to the sedimentary environment. Microbialpopulations appear to sense, respond, and adapt to redoxchanges at the sediment-water interface to create strati-fied, functional communities in short time periods. Similarobservations have been made in natural systems such asflooded tropical soils (39).

Finally, intrinsic microbial colonization of an in situ capcan impact contaminant fate and transport within suchengineered systems. Organisms colonizing the overlying capduring this study included Anaeromyxobacter, Geobacter, andShewanella spp., with members of these groups capable oftransforming multiple classes of environmental contaminantsincluding radionuclides (40), petroleum hydrocarbons (41)

and chlorinated ethenes (42). In previous experiments,actively dechlorinating Dehalococcoides eluted from enrichedAnacostia River sediment under upflow conditions (43),suggesting they may also populate a sediment cap undersuitable conditions. This is significant considering Dehalo-coccoides have been linked to the reduction of chlorinatedethenes (44), PCBs (45), polybrominated diphenyl ethers (46),chlorophenols (47), and dioxins (48) among other chlorinatedorganics (48, 49). Despite previous detection and quantifica-tion of Dehalococcoides strains in Anacostia sediment (43),Dehalococcoides were not detected in this study due to a lackof chlorinated organics provided as electron acceptors. Inthe aerobic region of the cap, the presence of a diversebacterial community suggests that aerobic PAH mineraliza-tion (14) may occur if oxygen penetration is sufficient. Also,methanotrophic bacteria have been linked to the cometabolictransformation of organic contaminants (50) and weretentatively identified through T-RFLP analysis to be presentat the cap-water interface (Supporting Information TableS.2). Importantly, rapid microbial colonization providescapping with an intrinsic, secondary level of treatment (i.e.,biotransformations) in addition to physical isolation of thesediment contaminants. The natural colonization of the capmaterial demonstrated here suggests that enhanced microbialcontaminant degradation in the cap is feasible, thus openingup new avenues for innovative cap design (e.g., cappingcombined with biostimulation) and active capping tech-nologies. Alternatively, the presence of microorganismswithin subaqueous sediment caps can also have negativeimplications that should be considered. Biogenic gas pro-duction (e.g., methanogenesis) within the sediment and capcould lead to contaminant partitioning and transport in thegas phase (51), physical instability of the cap (3), andformation and release of greenhouse gases to the atmosphere.Additional research is needed to optimize cap design andemplacement to fully exploit microbial activity in caps andto ensure long-term cap stability and performance.

AcknowledgmentsWe thank Rebecca Daprato and Laura Guest for helpfuldiscussions and assistance with T-RFLP analysis, ClaribelCruz-Garcia for helpful discussions regarding qPCR analysis,and John Fortner for assistance and advice during columndissection. Funding for this research was provided by theHazardous Substance Research Center-South and Southwestand a fellowship to D.W.H from the Georgia Institute ofTechnology.

Supporting Information AvailableTabular qPCR data (Table S.1) and additional T-RFLP analysesincluding representative data (Figure S.1), positive controldata (Figure S.2), and in silico phylogenetic assignments(Table S.2). This material is available free of charge via theInternet at http://pubs.acs.org.

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