Effect of Bare and Coated Nanoscale Zerovalent Iron on tceA and vcrA Gene Expression in Dehalococcoides spp. ZONG-MING XIU, †,‡ KELVIN B. GREGORY, § GREGORY V. LOWRY, § AND PEDRO J. J. ALVAREZ* ,‡ Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, Nankai University, Tianjin 300071, China, Department of Civil and Environmental Engineering, Rice University, Houston, Texas 77005, and Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 Received May 26, 2010. Revised manuscript received August 10, 2010. Accepted August 12, 2010. Nanoscale zerovalent iron (NZVI) can be used to dechlorinate trichloroethylene (TCE) in contaminated aquifers. Dehalo- coccoides spp. is the only microbial genus known to dechlorinate TCE to ethene as a respiratory process. However, little is known about how NZVI affects the expression of genes coding for reductive dechlorination. We examined a high-rate TCE- dechlorinating mixed culture which contains organisms similar to known Dehalococcoides to study the effects of NZVI on the expression of two model genes coding for reductive dehalogenases ( tceA and vcrA). A novel pretreatment approach, relying on magnetic separation of NZVI prior to reverse transcription qPCR (to avoid RNA adsorption by NZVI), was developed and used with relative quantification (relative to 16S rRNA as endogenous housekeeping gene) to quantify reductive dehalogenase gene expression. Both tceA and vcrA were significantly down-regulated (97- and 137-fold, respectively) relative to baseline (time 0) conditions after 72-h exposure to chlorinated ethenes (0.12 ( 0.03 mg/L cis-DCE, 0.69 ( 0.11 mg/L t-DCE, and 0.54 ( 0.16 mg/L VC) and bare-NZVI (1 g-NZVI/L). However, coating NZVI with an olefin maleic acid copolymer (a common approach to enhance its mobility in aquifers) overcame this significant inhibitory effect, and both tceA and vcrA were up-regulated (3.0- and 3.5-fold, respectively) after 48-h exposure. Thus, NZVI coating might enhance the expression of dechlorinating genes and the concurrent or sequential participation of Dehalococcoides spp. in the remediation process. Introduction Nanoscale zerovalent iron (NZVI) may be used as a reducing agent for in situ remediation of chlorinated ethene con- taminated sites (1-4). NZVI has a relatively high specific surface area and reactivity, and may be deployed in situ by slurry injection (5). Various types of NZVI, including surface- coated and bimetallic NZVI (1, 3, 6-8), have been extensively studied over the past decade and used to promote reductive dechlorination of chlorinated-solvent plumes (1) and DNAPL source zones (3, 4). Although NZVI is an effective bulk reductant for dechlorination, recent findings suggest that its use in situ may have unintended and detrimental environ- mental impacts. For example, bare (uncoated) NZVI can be toxic to bacteria (9-11). The mode of this toxicity is unknown but some evidence points toward the generation of reactive oxygen species by NZVI (9, 10). Recent studies demonstrate that the bacterial toxicity of NZVI is mitigated by coating the particles with engineered polymer or natural organic matter (11, 12). Li et al. (11) suggested that direct contact between bacteria and NZVI may be important for toxicity and that coatings on NZVI limit contact with the cells via electrosteric and/or electro- static repulsion. The emerging complexity of NZVI toxicity underscores the need to advance a fundamental under- standing of how NZVI interacts with bacteria to better predict the potential for detrimental environmental impacts and perhaps optimize NZVI-based strategies for chlorinated ethenes remediation. Dehalococcoides is the only known genus that completely dechlorinates trichloroethylene (TCE) to ethene, and mem- bers of this phylotype are commonly found in subsurface environments contaminated with chlorinated ethenes (13). These indigenous bacteria are important during stimulated bioremediation and natural attenuation of chlorinated solvents (14). Additionally, Dehalococcoides may serve as polishing agents following partial NZVI-based reductive dechlorination in a permeable reactive barrier or reactive zones created by NZVI injection. Previous work in our laboratory demonstrated that NZVI stimulated methanogenic activity (15, 16) while inhibiting biological dechlorination in a mixed culture containing Dehalococcoides spp. The inhibition of dechlorination suggests that Dehalo- coccoides spp. may be particularly sensitive to NZVI. However, the mechanism of inhibition of dechlorination has not been explored further and it is unknown whether bacterial toxicity may be associated with inhibition of dechlorinating gene expression or associated enzyme activity. Dehalococcoides spp. harbor reductive dehalogenase (RDase) genes, such as tceA, vcrA, and bvcA, which are responsible for their dechlorinating activity (17, 18). The product of the tceA gene in Dehalococcoides strains 195 and FL2 is thought to be responsible for the transformation of TCE to cis-dichloroethylene (cis-DCE) and vinyl chloride (VC) and cometabolism of VC to ethene (19-21). The vcrA gene in Dehalococcoides strains VS and GT codes for the enzyme that catalyzes the reduction of DCE and VC to ethene (22, 23). These genes and their mRNA transcripts are important biomarkers for evaluating in situ reductive dechlorination as well as the physiological state of the Dehalococcoides populations (18, 24, 25), and may also serve as important biomarkers to assess the impact of NZVI on dechlorinating activity in mixed cultures containing Dehalococcoides. This paper describes the effect of NZVI on the transcription of two functional genes that code for the dechlorination activity of Dehalococcoides spp. Employing a novel pretreat- ment approach based on magnetic separation of NZVI from cells (to avoid RNA adsorption by NZVI and associated interference from dissolved iron on qPCR), we quantified relative transcription levels of tceA and vcrA genes in a mixed dechlorinating culture in the presence and absence of NZVI. * Corresponding author e-mail: [email protected]; phone: (713)- 348-5903. † Nankai University. ‡ Rice University. § Carnegie Mellon University. Environ. Sci. Technol. 2010, 44, 7647–7651 10.1021/es101786y 2010 American Chemical Society VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7647 Published on Web 08/30/2010
Transcript
Effect of Bare and CoatedNanoscale Zerovalent Iron on tceAand vcrA Gene Expression inDehalococcoides spp.Z O N G - M I N G X I U , † , ‡
K E L V I N B . G R E G O R Y , §
G R E G O R Y V . L O W R Y , § A N DP E D R O J . J . A L V A R E Z * , ‡
Key Laboratory of Pollution Processes and EnvironmentalCriteria, Ministry of Education, Nankai University,Tianjin 300071, China, Department of Civil andEnvironmental Engineering, Rice University,Houston, Texas 77005, and Department of Civil andEnvironmental Engineering, Carnegie Mellon University,Pittsburgh, Pennsylvania 15213
Received May 26, 2010. Revised manuscript receivedAugust 10, 2010. Accepted August 12, 2010.
Nanoscale zerovalent iron (NZVI) can be used to dechlorinatetrichloroethylene (TCE) in contaminated aquifers. Dehalo-coccoides spp. is the only microbial genus known to dechlorinateTCE to ethene as a respiratory process. However, little isknown about how NZVI affects the expression of genes codingfor reductive dechlorination. We examined a high-rate TCE-dechlorinating mixed culture which contains organisms similarto known Dehalococcoides to study the effects of NZVI onthe expression of two model genes coding for reductivedehalogenases (tceA and vcrA). A novel pretreatment approach,relying on magnetic separation of NZVI prior to reversetranscription qPCR (to avoid RNA adsorption by NZVI), wasdeveloped and used with relative quantification (relative to 16SrRNA as endogenous housekeeping gene) to quantify reductivedehalogenase gene expression. Both tceA and vcrA weresignificantly down-regulated (97- and 137-fold, respectively)relative to baseline (time 0) conditions after 72-h exposure tochlorinated ethenes (0.12 ( 0.03 mg/L cis-DCE, 0.69 ( 0.11 mg/Lt-DCE, and 0.54 ( 0.16 mg/L VC) and bare-NZVI (1 g-NZVI/L).However, coating NZVI with an olefin maleic acid copolymer (acommon approach to enhance its mobility in aquifers)overcame this significant inhibitory effect, and both tceA andvcrA were up-regulated (3.0- and 3.5-fold, respectively)after 48-h exposure. Thus, NZVI coating might enhance theexpression of dechlorinating genes and the concurrent orsequential participation of Dehalococcoides spp. in theremediation process.
IntroductionNanoscale zerovalent iron (NZVI) may be used as a reducingagent for in situ remediation of chlorinated ethene con-taminated sites (1-4). NZVI has a relatively high specific
surface area and reactivity, and may be deployed in situ byslurry injection (5). Various types of NZVI, including surface-coated and bimetallic NZVI (1, 3, 6-8), have been extensivelystudied over the past decade and used to promote reductivedechlorination of chlorinated-solvent plumes (1) and DNAPLsource zones (3, 4). Although NZVI is an effective bulkreductant for dechlorination, recent findings suggest that itsuse in situ may have unintended and detrimental environ-mental impacts. For example, bare (uncoated) NZVI can betoxic to bacteria (9-11). The mode of this toxicity is unknownbut some evidence points toward the generation of reactiveoxygen species by NZVI (9, 10).
Recent studies demonstrate that the bacterial toxicity ofNZVI is mitigated by coating the particles with engineeredpolymer or natural organic matter (11, 12). Li et al. (11)suggested that direct contact between bacteria and NZVImay be important for toxicity and that coatings on NZVIlimit contact with the cells via electrosteric and/or electro-static repulsion. The emerging complexity of NZVI toxicityunderscores the need to advance a fundamental under-standing of how NZVI interacts with bacteria to better predictthe potential for detrimental environmental impacts andperhaps optimize NZVI-based strategies for chlorinatedethenes remediation.
Dehalococcoides is the only known genus that completelydechlorinates trichloroethylene (TCE) to ethene, and mem-bers of this phylotype are commonly found in subsurfaceenvironments contaminated with chlorinated ethenes (13).These indigenous bacteria are important during stimulatedbioremediation and natural attenuation of chlorinatedsolvents (14). Additionally, Dehalococcoides may serve aspolishing agents following partial NZVI-based reductivedechlorination in a permeable reactive barrier or reactivezones created by NZVI injection. Previous work in ourlaboratory demonstrated that NZVI stimulated methanogenicactivity (15, 16) while inhibiting biological dechlorination ina mixed culture containing Dehalococcoides spp.
The inhibition of dechlorination suggests that Dehalo-coccoides spp. may be particularly sensitive to NZVI. However,the mechanism of inhibition of dechlorination has not beenexplored further and it is unknown whether bacterial toxicitymay be associated with inhibition of dechlorinating geneexpression or associated enzyme activity.
Dehalococcoides spp. harbor reductive dehalogenase(RDase) genes, such as tceA, vcrA, and bvcA, which areresponsible for their dechlorinating activity (17, 18). Theproduct of the tceA gene in Dehalococcoides strains 195 andFL2 is thought to be responsible for the transformation ofTCE to cis-dichloroethylene (cis-DCE) and vinyl chloride (VC)and cometabolism of VC to ethene (19-21). The vcrA genein Dehalococcoides strains VS and GT codes for the enzymethat catalyzes the reduction of DCE and VC to ethene (22, 23).These genes and their mRNA transcripts are importantbiomarkers for evaluating in situ reductive dechlorinationas well as the physiological state of the Dehalococcoidespopulations (18, 24, 25), and may also serve as importantbiomarkers to assess the impact of NZVI on dechlorinatingactivity in mixed cultures containing Dehalococcoides.
This paper describes the effect of NZVI on the transcriptionof two functional genes that code for the dechlorinationactivity of Dehalococcoides spp. Employing a novel pretreat-ment approach based on magnetic separation of NZVI fromcells (to avoid RNA adsorption by NZVI and associatedinterference from dissolved iron on qPCR), we quantifiedrelative transcription levels of tceA and vcrA genes in a mixeddechlorinating culture in the presence and absence of NZVI.
10.1021/es101786y 2010 American Chemical Society VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7647
Published on Web 08/30/2010
Parallel studies with coated and bare NZVI provide insightinto the nature of the inhibitory mechanisms and thepotential for coatings to reduce or eliminate the inhibitoryeffect of NZVI on biological dechlorination activity.
Materials and MethodsChemicals and NZVI. Trichloroethylene, cis-dichloroethene(cis-DCE), trans-dichloroethene (trans-DCE), and vinyl chlo-ride (VC) were purchased from Sigma-Aldrich (St. Louis, MO).Molecular biology grade �-mercaptoethanol was purchasedfrom Research Organics (Cleveland, OH). NZVI particles wereobtained from Toda Kogyo Corporation (Onoda, Japan). Thephysical and chemical properties of this NZVI can be foundin Liu et al., 2005 (8). Briefly, the NZVI suspension consistedof irregularly shaped particles ranging in size from 5 to 70nm with a median radius of 20 nm. Its surface is negativelycharged. Coated NZVI (coated by a negatively chargedpolyelectrolyte-olefin maleic acid copolymer, MW 16,000g/mol) was also supplied by Toda Kogyo Corporation. Olefinmaleic acid is not toxic to bacteria and is a representative ofthe carboxylated organic macromolecules which are oftenused to prevent NZVI aggregation during emplacement(26-28). The slurry was prepared as described earlier (29).
Cultures and Medium Preparation. A Dehalococcoides-containing (∼109 gene copies/mL) TCE enrichment culturewas developed from an anaerobic methanogenic consortiumthat had been functionally stable in our laboratory for morethan 10 years (30, 31). An inoculum of this culture was createdby anaerobically transferring 800 mL to a 1-L serum bottlewith a Teflon-lined stopper and aluminum crimp seal cappedport on the side to add TCE as electron acceptor.
Analytical Procedures. TCE, cis-DCE, t-DCE, and VC wereanalyzed using a packed column (6 ft. × 1/8 in. o.d. 60/80carbopack B/1% SP-1000, Supelco, Bellefonte, PA) on a GC(HP5890, Ramsey, MN) equipped with a flame ionizeddetector (FID) as described previously (15).
Transmission Electron Microscopy (TEM). E. coli (ATCCstrain KW12) was grown in LB Miller broth medium at 37 °Cfor 12 h. The bacteria were harvested by centrifugation at5000g for 1 min. E. coli stock was prepared by resuspendingbacteria pellets in 10 mL of 2 mM sodium bicarbonate. NZVI/coated-NZVI (100 mg-NZVI/L) were added to the bacteriastock and incubated for 1 h. To examine the interaction ofbacteria with coated or bare NZVI, a 10-µL aliquot of theNZVI-bacteria mixture was placed on 400-mesh copper grids(ultrathin carbon type-A, Ted Pella, Redding, CA) and driedovernight. The NZVI-bacteria samples were examined byTEM performed with a JEOL 1230 operated at 120 kV (JEOL,Tokyo, Japan).
NZVI and Coated-NZVI Exposure Experiment. TheDehalococcoides-containing culture was activated by feedingwith TCE (228 mg/L, dissolved in methanol-electron donor)for three cycles. Residual chlorinated ethenes present at thebeginning of the experiment were cis-DCE (0.12 ( 0.03 mg/L), t-DCE (0.69( 0.11 mg/L), and VC (0.54( 0.16 mg/L). TCEwas not added during these experiments to avoid confound-ing effects on RDase gene expression associated withdifferences in TCE concentrations between treatments,resulting from differences in biological, abiotic, and combineddechlorination rates.
After the culture was activated, two treatments were setup in triplicate containing 100 mL of culture per serum bottle(Wheaton, NJ, USA) with/without NZVI (1 g/L, Fe0 ∼40%).This concentration is within the range (1-10 g/L) commonlyused when NZVI is injected into the subsurface for reme-diation purposes (32). The experiment with coated NZVI wasset up in the same manner. Specifically, 833 µL of coated-NZVI solution (120 g-NZVI/L in deionized water) was addedto one serum bottle, resulting in an iron concentration (1g/L, Fe0 ∼40%) equal to that of the bare NZVI treatment
group. The other bottle (without coated-NZVI addition)served as a control. Both treatments were placed in ananaerobic chamber at 25 °C and samples (1.8 mL) were takenperiodically. RNA protective reagent (3.6 mL) (Ambion,Austin, TX) was added simultaneously to avoid RNA deg-radation. All experiments were repeated for verificationpurposes.
RNA Extraction, DNA Removal, and cDNA Synthesis.Total RNA was isolated from the 1.8-mL samples using theQiagen RNeasy Mini Kit (Qiagen, Valencia, CA). To preventadsorption of RNA onto NZVI (SI Figure S1), the NZVI particles(which are magnetic (33)) were removed prior to cell lysisusing an Alnico Horseshoe Magnet (Fisher Scientific) placedat the bottom of the sample tube. The supernatant wastransferred into another tube, and cells were then collectedby centrifugation (Beckman Coulter Inc., Fullerton, CA)(5000g at 4 °C for 10 min). RNA isolation was carried outaccording to the manufacturer’s instructions. RNA sampleswere suspended in 50 µL of RNase-free water (Qiagen,Valencia, CA). The final DNA removal and First Strand cDNAsynthesis were performed using a First Strand cDNA SynthesisKit (Fermentas, Burlington, Ontario) according to the manu-facturer’s instructions. Purified RNA and synthesized cDNAwere stored at -80 °C prior to analysis by reverse tran-scriptase, quantitative PCR (RT-qPCR).
RT-qPCR. RT-qPCR was used to quantify the cDNA copynumber of tceA, vcrA, and 16S rRNA gene in Dehalococcoidesspp., using previously reported primer sequences (17, 34).RT-qPCR was performed on an ABI Prism 7500 sequencedetection system (Applied Biosystems, Foster City, CA). Eachreaction volume contained 10 µL of TaqMan Universal PCRMaster Mix (Applied Biosystems); 0.7 µM (tceA, vcrA, or 16SrRNA) forward and reverse primer; and 0.2 µM (tceA, vcrA,or 16S rRNA) probe. Thermocycling conditions were asfollows: 2 min at 50 °C, 15 min at 95 °C, and 40 cycles of 15 sat 95 °C and 1 min at 60 °C (31).
Gene Expression Data Analysis. mRNA losses duringsample preparation and inefficiencies of reverse transcription
FIGURE 1. Relative tceA and vcrA expression fold changesafter exposure to (a) bare NZVI; and (b) coated NZVI (1 g NZVI/L). Fold changes of target genes for each time point werenormalized to initial conditions (time 0). All data pointsrepresent average values from triplicate samples, and errorbars represent one standard deviation.
often limit the accuracy of RT-qPCR (35-37). A useful methodto discern the mRNA losses is adding luciferase control RNAas internal standard (38). However, the luciferase RNA wasnot an appropriate internal standard because of significantadsorption by NZVI (SI Figure S2). As an alternative, targetmRNA quantities can be normalized to the quantity of mRNAof an endogenous housekeeping gene (36, 37). The ratiobetween target and housekeeping gene remains stable evenwhen some mRNA are lost, so the relative quantification oftarget over housekeeping gene expression can overcome mRNAlosses during sample preparation, including enzymatic orabiotic degradation and inefficiencies in reverse transcription.
The 16S rRNA gene of Dehalococcoides spp. was selectedas an endogenous housekeeping gene because its transcriptoccurs at much higher level than RDase mRNA, regardlessof growth stage and substrate availability (39). 16S rRNA geneexpression was tested in the presence of bare NZVI, coated-NZVI, and coatings separately. The 16S rRNA remained stableover 168 h (p > 0.05, determined by one way ANOVA, SIFigure S3), demonstrating its suitability as a housekeepinggene. The amplification efficiency of target (tceA and vcrA)and housekeeping (16S rRNA) genes was statistically un-distinguishable (SI Figure S4). Thus, tceA gene and vcrA mRNAwas normalized to 16S rRNA gene within the same RNAextract.
Relative gene expression levels were calculated using thecomparative CT method (2- ∆∆CT method (40), where ∆∆CT )
(CT · target-CT ·16S)treatment- (CT · target-CT ·16S)control. The expressionof target genes in bare and coated NZVI treatments wasnormalized to that in the unexposed control group (SIFormulas S1 and S2). As a positive control for the relativequantification and RT-qPCR method, tceA and vcrA expres-sion in the presence of TCE (20 mg/L, dissolved in methanol)was quantified (SI Figure S5). Both tceA and vcrA weresignificantly up-regulated by TCE exposure (p < 0.05),corroborating previous results obtained by absolute quan-tification and RT-qPCR (38, 41).
Whether up-regulation or down-regulation of gene ex-pression was statistically significant relative to baseline orcontrol conditions was determined using Student’s t test atthe 95% confidence level.
Results and DiscussiontceA and vcrA Expression after Exposure to NZVI. Magneticseparation of iron prior to Rt-qPCR precluded mRNAadsorption onto NZVI and interference on qPCR, whichenabled quantification of gene expression Thus, this novelpretreatment method could also benefit the study of othermicrobial-iron interactions relevant to corrosion and ironmineral cycling.
Significant down-regulation of tceA (p < 0.05) was observedfollowing exposure to NZVI in the presence of residualchlorinated ethenes (Figure 1a), despite the biostimulatoryeffect of the cathodic H2 (405 ( 10 µmol, SI Figure S6)generated by NZVI corrosion (42). After 24 h, tceA expressionrelative to the housekeeping (16S rRNA) gene decreased by1.6-fold compared to time 0, and reached the lowest levelafter 72 h with a dramatic 97-fold down-regulation. A higherdegree of down-regulation was observed for vcrA (Figure1a), with a 7.5-fold decrease in relative expression in 24 hand maximum 137-fold down-regulation after 72 h. Theseresults are consistent with a previous study showing thatTCE dechlorination by this mixed culture was inhibited byNZVI (1 g/L), with the overall first-order degradation ratecoefficient decreasing by 54% from 0.115 ( 0.005 to 0.053 (0.003 h-1 (15).
tceA and vcrA Expression after Exposure to Coated-NZVI.NZVI is commonly modified by surface coatings to preventaggregation and enhance its distribution in contaminatedaquifers (29, 43-46). Therefore, most NZVI that is used foraquifer remediation has engineered polymeric coatings.These coatings can significantly change the mobility andreactivity of NZVI in the subsurface, so understanding thetoxicity of coated NZVI is essential to determining itsenvironmental fate and impact.
FIGURE 2. Relative tceA and vcrA expression fold changesafter exposure to surface coatings alone. Fold changes oftarget genes for each time point were normalized to initialconditions (time 0). All data points represent average valuesfrom triplicate samples, and error bars represent one standarddeviation.
FIGURE 3. TEM images of E. coli incubated with (a) bare NZVI (∼40% Fe0) and (b) coated NZVI (coated with olefin maleic acidcopolymer, MW ) 16 000 g/mol).
Unlike the initial response to bare NZVI, both tceA andvcrA were significantly up-regulated in treatments with coatedNZVI (p < 0.05); tceA expression increased 3-fold while vcrAincreased 3.5-fold in 48 h compared to time 0 (Figure 1b).This up-regulation was likely due to the presence of theresidual chlorinated solvents (24, 47, 48): cis-DCE (0.12 (0.03 mg/L), t-DCE (0.69 ( 0.11 mg/L), and VC (0.54 ( 0.16mg/L). Accordingly, when olefin maleic acid copolymercoatings of NZVI were added alone, both tceA and vcrA weresimilarly up-regulated in the presence of these residualchlorinated solvents (Figure 2). Specifically, tceA expressionincreased 2.9-fold while vcrA increased 2.7-fold in 72 h, whichis similar to the up-regulation associated with coated-NZVIexposure. Thus, the coating had no inhibitory or stimulatoryeffect on RDase gene expression.
Coated-NZVI had a slight inhibitory effect after 48 h (p< 0.05). The expression of tceA was down-regulated 10.6-fold, while vcrA was down-regulated 6.9-fold at 160 hcompared to time 0. However, these levels of down-regulationwere relatively small compared to those observed with bareNZVI (96-fold and 137.1-fold for tceA and vcrA, respectively).Thus, coating of NZVI with olefin maleic acid copolymerreduced the inhibition of tceA and vcrA gene expression.Coatings on NZVI electrosterically prevent direct contactbetween NZVI and cells (11), thus mitigating toxicity (9, 10).The observation of slight down regulation of tceA and vcrAin the presence of coated NZVI may be due to the eventualadhesion of a small number of NZVI to cells over the courseof the experiment because coatings do not cover 100% of theNZVI surface.
The mechanism of tceA and vcrA gene-expression inhibi-tion by NZVI is unclear. Expressions may be lowered fromdecreased residual chlorinated ethene concentrations(24, 47, 48) (cis-DCE: from 0.12 to 0.01 mg/L; t-DCE: from0.69 to 0.51 mg/L; and VC: from 0.54 to 0.16 mg/L) or strongreducing conditions created at the membrane by directinteractions with NZVI that interfere with expression patternsof Dehalococcoides spp. More than 90 reductive dehaloge-nase-homologous genes have been identified in Dehalococ-coides spp. genus (22, 49, 50) and most of the RDases aremembrane-bound (22, 48, 51) and potentially susceptible todirect contact with bare NZVI. Previous studies with Deha-lococcoides show that strong reductants (e.g., S2-) greatlyreduce halorespiration activity (52).
Cultures exposed to coated or uncoated NZVI wereexamined using TEM to visualize the association betweenthe cells and nanoparticles. Because test conditions neces-sitate mixed cultures containing Dehalococcoides spp., it isdifficult to distinguish the cells and clearly show associationbetween NZVI and Dehalococcoides spp. To illustrate thepotential for cell association of NZVI with gram negativebacteria such as Dehalococcoides (which is extremely difficultto isolate and is not commercially available in pure culture),a pure culture of E. coli was examined. E. coli is a wellcharacterized bacterium that has been previously used as amodel to study microbial-NZVI interactions (9-11). Mi-crographs show that exposure of E. coli to bare NZVI for 1 hresulted in attachment of nanoparticles to cells (Figure 3a),which is conducive to toxicity (11), whereas coated NZVI didnot (Figure 3b). Moreover, some E. coli cells exposed tocoated-NZVI are shown reproducing by binary fission,indicating that some cells continued to grow after exposureto coated-NZVI. These observations were representative ofall observed fields on the copper grid.
Overall, these results indicate that appropriate NZVIcoatings may enable the concurrent or sequential participa-tion of Dehalococcoides spp. in the cleanup process. Fur-thermore, this study shows a potential for coatings to reduceor eliminate the inhibitory effect of NZVI on biologicaldechlorination activity, which would consequently make
coated-NZVI more favorable in remediation of chlorinatedethene contaminated sites.
AcknowledgmentsThis research was sponsored by U.S. EPA (R833326) and theNSF (EF-0830093) Center for Environmental Implications ofNanotechnology (CEINT). We thank Dr. Brian G. Rahm(Cornell University) for valuable instructions on housekeep-ing gene selection; Dr. Yu Yang (Rice University), Chun Chen(Nankai University), and Jun Cui (Baylor College of Medicine)for valuable discussion and assistance on relative quantifica-tion analysis; and Dr. Li Lin (Rice University) for importantdiscussions on possible interaction of RNA and NZVI.
Supporting Information AvailableData for RNA adsorption onto NZVI, 16S rRNA, tceA, andvcrA expression after exposure to TCE, amplification ef-ficiency of target and housekeeping genes, and H2 productionby NZVI (as well as details on gene expression analysismethodology). This material is available free of charge viathe Internet at http://pubs.acs.org.
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