Assessing the Effects of Silver Nanoparticles on Biological NutrientRemoval in Bench-Scale Activated Sludge Sequencing Batch ReactorsChristina L. Alito and Claudia K. Gunsch*

Department of Civil and Environmental Engineering, Duke University, Box 90287, Durham, North Carolina 27708, United States

Center for Environmental Implications of NanoTechnology (CEINT), Duke University, Durham, North Carolina 27708, UnitedStates

*S Supporting Information

ABSTRACT: Consumer products such as clothing and medicalproducts are increasingly integrating silver and silver nanoparticles(AgNPs) into base materials to serve as an antimicrobial agent.Thus, it is critical to assess the effects of AgNPs on wastewatermicroorganisms essential to biological nutrient removal. In thepresent study, pulse and continuous additions of 0.2 and 2 ppmgum arabic and citrate coated AgNPs as well as Ag as AgNO3 werefed into sequencing batch reactors (SBRs) inoculated withnitrifying sludge. Treatment efficiency (chemical oxygen demand(COD) and ammonia removal), Ag dissolution measurements, and16S rRNA bacterial community analyses (terminal restrictionfragment length polymorphism, T-RFLP) were performed toevaluate the response of the SBRs to Ag addition. Results suggestthat the AgNPs may have been precipitating in the SBRs. WhileCOD and ammonia removal decreased by as much as 30% or greater directly after spikes, SBRs were able to recover within 24 h(3 hydraulic retention times (HRTs)) and resume removal near 95%. T-RFLP results indicate Ag spiked SBRs were similar in a16s rRNA bacterial community. The results shown in this study indicate that wastewater treatment could be impacted by Ag andAgNPs in the short term but the amount of treatment disruption will depend on the magnitude of influent Ag.

■ INTRODUCTION

Ionic silver is an established antibacterial agent and has beenutilized for its antiseptic properties in consumer products suchas washing machines, clothing, and children’s toys.1 Morerecently, specially coated silver nanoparticles (AgNPs) havebeen designed to replace ionic silver in consumer productsbecause of their ability to slowly release ionic silver as theircoatings dissolve, making them better suited for long-termconsumer product usage. Because manufactured AgNPsgenerally range from 10 to 100 nm, researchers speculate thatthey may be small enough to pass through the cell membraneof a microorganism and, then, release ionic silver directly intothe microbe as their coatings dissolve.2 Since it is likely thatsome consumer products such as textiles will be washed, it iscritical that the concentration of AgNPs in wastewatertreatment plant (WWTP) influent be quantified and theirimpacts ascertained. Current reports show that there is a highvariability in silver concentration present in consumer productsand it is difficult to find estimations of the amount of total silverand AgNPs being added to consumer products. Benn et al.3

found that AgNP containing socks leached up to 68 μg silver/gsock in water with gentle agitation, while Geranio et al.4

measured up to 377 μg/g with the application of detergent.Since AgNP-containing consumer products are already in

use, several methods have been developed for estimating

speciation of AgNPs in complex media such as wastewater,where it will likely collect. Because one of the suggestedantimicrobial mechanisms of AgNPs is the release of ionic silverafter the particle coating dissociates, researchers are examiningnanoparticle coating interactions with solution properties suchas pH, ionic strength, and dissolved organic carbonconcentration.5 The characteristically high concentration ofthiols in wastewater sludge has also led to the discovery ofnaturally occurring silver sulfide nanoparticles in wastewater,which provides evidence that influent silver is likely to bebound up with wastewater sludge thiols.6 Nonetheless, becauseof their proven antimicrobial properties, even if AgNPs are onlypresent at very low concentrations in wastewater influent, it ispossible that AgNPs could alter important microbial functions.The disruption of biological nutrient removal, and especiallynitrification, in wastewater treatment is of particular concern.Nitrifying bacteria including Nitrosomonas spp. can be easilyinactivated by disruption of their membrane-bound enzymeammonia monooxygenase (AMO) which could ultimatelycause nitrification treatment failures.7

Received: August 15, 2013Revised: December 20, 2013Accepted: December 23, 2013Published: December 23, 2013

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The bactericidal nature of silver has been well established inprevious literature,8 but the toxicity of AgNPs appears to differgreatly depending on size and coating.9 Due to increasedutilization of AgNPs containing consumer products, furtherevaluation is needed to determine potential impacts of AgNPs.Although a handful of studies have examined the antimicrobialeffects of AgNPs on wastewater bacteria, none have examinedthe effects on wastewater bacterial diversity and overalltreatment efficiency by cycling pulse and continuous inputsof Ag. The objective of the present study was to determine theeffect of two AgNPs (citrate and gum arabic stabilized) oncommon treatment characteristics (namely, in terms ofchemical oxygen demand (COD) and ammonia removalefficiency) and microbial community structure in sequencingbatch reactors (SBRs) mimicking WWTP operation. Thesetypes of AgNPs were chosen to follow up our previousresearch, which studied the effects of AgNPs on the nitrifier,Nitrosomonas europaea.10 Gum arabic and citrate stabilizedAgNPs were found to be to most toxic to the sensitive nitrifiersresponsible for converting ammonia to nitrite.

■ MATERIALS AND METHODSAgNP Characterization. Gum arabic (GA) and citrate

stabilized AgNPs were used in the present study. Theseparticles were selected because they have been widely studiedand because their coatings are representative of AgNPs used inconsumer products, as previously described.10 Citrate AgNPswere produced by reducing Ag nitrate in water with sodiumcitrate.11 GA AgNPs were manufactured by reducing Ag nitritewith water and GA. The average particle sizes measured bytransmission electron microscopy (TEM) were 32.3 ± 0.5 and15.5 ± 0.5 nm for GA and citrate AgNPs, respectively. Moreinformation related to AgNP synthesis and characterization canbe found in Yin et al.12 and Meyer et al.13 In addition, the“AgNP Synthesis” section in the Supporting Information ofArnaout and Gunsch10 has extensive information on TEMimages, zeta sizing of AgNPs, and laboratory procedures.SBR Design. Eight 3-L bench-scale SBRs were constructed

out of square pieces of plexiglass (6 × 6 × 6 in, 1/8 in. thick),which were welded together using poly(methyl methacrylate).The peak volume in all reactors was 2.0 L, and the volume afterdecanting was approximately 1 L. SBRs were operated on an 8h cycle, starting with 30 min of influent synthetic wastewaterfeeding, 6 h of aerating and vigorous mixing, 1 h of settling, and30 min of decanting. A 13 d solids retention time (SRT) waschosen to ensure optimum conditions for nitrification, and thereactors were run with a 5.3 h hydraulic retention time (HRT),which falls in the typical range for contact stabilizationsystems.14 The SRT was maintained by wasting sludgeperiodically to retain 2000−2500 mg/L of mixed liquorsuspended solids (MLSS). The SBRs were fed syntheticwastewater (SWW) based on a previously published recipe,15

which had an average COD of 450 mg/L, average ammoniaconcentration of 40 mg/L, and pH of 7. All reactors werecovered with aluminum foil to prevent any photolysis. AgNPswere added in stock concentration either directly to thereactors (for pulse additions) or into the influent medium (forcontinuous additions).Figure S1, Supporting Information, shows the configuration

of SBRs built in this study. Air was fed into reactors via TetraWhisper small aquatic aerators (Madison, WI), and feedwaterwas pumped in via multihead pumps (Cole Parmer, MasterflexL/S, Vernon Hills, IL). The dissolved oxygen was maintained

between 6 and 6.5 mg/L. The reactors were continuouslymixed with large stir bars on stir plates at 700 rpm. The cyclingof air, feed, and pumping was controlled by IntermaticTN311C timer-controllers (Grove, IL). Reactors were kept atroom temperature (∼17−20 °C). The SBRs were initiallyspiked with 500 mL of nitrifying activated sludge taken fromthe North Durham Water Reclamation Facility (Durham, NC).This plant currently treats up to 20 MGD and utilizes biologicalnutrient removal systems to remove phosphorus, BOD, andammonia. Prior to beginning the AgNP spikes, the SBRs wereoperated for 120 d with no Ag additions. It took the reactors 90d to reach steady state. The reactors were allowed to perform atsteady state for 30 d prior to any AgNP addition; they weresteadily removing 90% or more of COD and ammonia duringthis period. Microbial community structure analysis was alsoperformed on steady state SBRs to determine the startingcommunity diversity. Reactors were divided into 2 replicategroups of 4 reactors based on the similarity of their startingmicrobial community profiles. In total, four SBR treatmentswere conducted in duplicate consisting of: (1) no Ag control,(2) citrate AgNP treatment, (3) GA AgNP treatment, and (4)Ag+ control. At the time of the initial Ag spike, the COD andammonia removal rates were not significantly different acrossall eight reactors (p > 0.05).

AgNP Addition. Concentrations of 0.2 and 2 ppm wereselected as the low and high Ag additions. These concentrationswere chosen on the basis of our previous study, which showedthat the effects of AgNPs on the model wastewater ammoniumoxidizing bacterium (AOB), Nitrosomonas europaea, becameapparent around 2 ppm total Ag concentration.10 Table 1

displays the chronology of Ag addition to the SBRs. Ag wasinitially added in pulses at 0.2 ppm followed by a continuousaddition. For the pulse input, either AgNPs or AgNO3 wasadded directly to the SBRs as a single pulse at the start of thecycles. Following each pulse input, the SBRs were allowed toadapt with no Ag addition for different lengths of time. The firstacclimation cycle was the longest (14 d) to assess the recoveryperiod needed by the SBRs, and then, shorter acclimationcycles followed (6 and 2 d, respectively). Following the pulseinputs, a continuous flow of Ag was added by spiking influentSWW with stock solutions of AgNPs and AgNO3. Theconcentration of Ag was increased to 2 ppm at the end ofthe 0.2 ppm continuous phase, and the pulse cycling wasrestarted (14 d pulse, 6 d pulse, 2 d pulse, continuous).

Analytical Methods. To monitor COD and N removalefficiency, influent and effluent samples were collectedapproximately every 3 d as well as immediately after each Agspike. Four parameters were measured using HACH reagents(Loveland, CO): COD (mercuric digestion method), ammonia

Table 1. Order of Ag Addition to SBRsa

order ofaddition

spike concentration,ppm type of spike

length of acclimationcycle

1 0.2 pulse 14 d, 6 d, 2 d2 0.2 continuous 3 SRTs3 2 pulse 14 d, 6 d, 2 d4 2 continuous 3 SRTs

aSBRs were first spiked with 14 d, 6 d, or 2 d pulse Ag additions,followed by a phase of continuous Ag addition. After the first 0.2 ppmAg cycle, the concentration was increased to 2 ppm and the cyclingwas restarted.

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(salicylate method), nitrite (diazotization method), and nitrate(cadmium reduction method). Samples were taken directlyfrom SBR effluent containers and tested within 30 min. Tomeasure the Ag concentration in the SBRs, samples werecollected directly from the SBRs during treatment as well asfrom the treated SBR effluent. Samples for Ag spikes weretaken at 1 min, 1 h, 6 h, 24 h, and at the end of the spike, whilesamples for continuous Ag spikes were taken at 24 h and every7 d following the continuous spike. Effluent treated watersamples were also collected for Ag analysis every 3 d. Aganalysis was performed as previously described in Arnaout andGunsch10 and in the Supporting Information section.DNA Extraction and PCR Conditions. Biomass samples

were taken periodically from the SBRs and microcentrifuged for1 min at 13 000g. Biomass samples were then immediatelystored at −20 °C until DNA extraction. Duplicate samples wereextracted for each treatment using the UltraClean DNAIsolation Kit (MoBio Laboratories, Solana Beach, CA). PCRof the bacterial 16S SSU rRNA gene region was carried out bythe methods described in Lukow et al.16 with small adjust-ments. 6-Carboxyfluorescein (6-FAM) was used to fluores-cently label the forward primer (27F), and 1392R was used asthe unlabeled reverse primer.17 Please refer to the SupportingInformation section for a further description of DNAextraction.Terminal Restriction Fragment Length Polymorphism

(T-RFLP) Analysis. Restriction enzyme digests were carriedout by following the protocol described in Lukow et al.16 MspIwas added to purified PCR product and incubated for 3 h at 37°C. Fragment analysis was carried out using POP6 polymer andROX-labeled MapMarker 1000 size standards. AppliedBiosystems GeneScan v3.7.1 software (Foster City, CA) wasused to interpret the raw data after electrophoresis. All sampleswere visually inspected for clean peaks, and raw data wastransferred into T-REX.18 T-REX aligned peaks and identifiedtrue peaks. Data analysis was performed in T-REX using thepeak area feature, so that abundance and diversity could beexamined. T-RFs smaller than 50 bp were not included inanalysis, as to eliminate primer dimer fragments. Data files werethen imported into PAST statistical software to create Bray−Curtis principle coordinate ordination and cluster similarity(Hammer & Harper, D.A.T. 2006. Paleontological DataAnalysis. Blackwell). Diversity indices were calculated bycomparing taxonomic species with the Simpson 1-D index.The greater the resulting value, the more diverse is the samplein species. Please refer to the Supporting Information sectionfor a detailed description of T-RFLP methodology.Statistical Analysis. The unpaired, two tailed student’s t

test was used to identify statistical differences between controlsamples and treated samples for all data analysis except T-RFLPmicrobial community statistical analysis. Bray−Curtis similarityand principle coordinate ordinations were utilized for T-RFLPdata interpretation.

■ RESULTS AND DISCUSSIONDissolution of Ag from AgNPs. The concentration of

total and dissolved Ag was measured in the SBRs to assess howquickly Ag dissolved from the AgNPs. Since Ag was measuredin the influent, effluent, and the supernatant of the SBRs, thepartition of the Ag in the SBRs could be deduced from Agconcentration measurements. In general, total Ag concen-trations during pulse inputs were near dosing concentration(0.2 and 2 ppm) immediately following spikes (1 min) and

gradually decreased after 24 h. Dissolved Ag remained below 4ppb for all 0.2 and 2 ppm total Ag spikes (Table S1, SupportingInformation). Figure 1 shows the drop in total Ag

concentration during the 14 d spike and the averageconcentration during the continuous input of Ag. For the 0.2ppm spike, the total Ag concentrations in the wastewater after 1h for the 14 d pulse spike were 96.8 ± 0.95, 70.8 ± 10.9, and134.2 ± 10.8 ppb Ag for citrate AgNPs, GA AgNPs, and Ag asAgNO3, respectively. The dissolved Ag concentrations were notstatistically significantly different when compared to the no Agcontrol concentrations (p > 0.05). Total and dissolved Agconcentrations in the no Ag control SBR were consistentlylower than ICP-MS detection limit (<100 ppt). After 24 h, thetotal Ag concentrations showed a 34.3 ± 4.6%, 87.3 ± 1.3%,and 41 ± 2.3% reduction in total Ag measured compared to the1 h time point for citrate AgNPs, GA AgNPs, and Ag asAgNO3, respectively. These results suggest that citrate AgNPswere the most persistent in the SBR supernatant. Dissolved Agwas undetectable for all types of Ag, which is attributed tocerargyrite (AgCl) formation in the SBRs. This hypothesis issupported by MINEQL speciation data created with our SWWmedium (Table S2, Supporting Information).The same trends were observed for the second and third 0.2

ppm pulse spikes; GA AgNPs had the greatest reduction intotal Ag concentration, while citrate AgNPs and Ag as AgNO3levels were higher (Figures S2 and S3, Supporting Informa-tion). Within 24 h after the second spike, total Agconcentrations had reduced by 28.4 ± 9.5%, 77.4 ± 0.4%,and 24.2 ± 2.6% for citrate AgNPs, GA AgNPs, and Ag asAgNO3, respectively. Levels in the citrate AgNPs and ionic Agcontrol were statistically indistinguishable (p > 0.05). Theaverage total concentrations over 30 d during the continuousaddition were 9.28 ± 10.9, 6.94 ± 4.2, and 9.37 ± 12.9 ppb forcitrate AgNPs, GA AgNPs, and Ag as AgNO3, respectively.Dissolved Ag concentrations were below detection. Similarly to

Figure 1. Total Ag concentration in SBRs during pulse and continuousinputs. (A) 2 ppm spikes and (B) 0.2 ppm spikes. Bar diagrams showthe rapid decrease in total Ag measured from the supernatantimmediately after pulse spikes and during the continuous phase of Agaddition.

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that observed during the pulse addition experiments, GAAgNPs had the lowest total Ag concentration in the SBRscompared to the other treatments, which may have been due toattachment with settling sludge flocs. Effluent total anddissolved Ag was also measured periodically to determinehow much Ag was flowing out of the reactors. The effluent totalAg concentration was very low and not statistically differentfrom the no Ag control (p < 0.05). Effluent dissolved Agconcentrations were below detection.After the 0.2 ppm spikes, the concentration of Ag was

increased to 2 ppm for citrate AgNPs, GA AgNPs, and AgNO3.Immediately following the first 14 d spike (1 min after Agaddition), the concentrations of total Ag were measured as 1.12± 0.06, 0.73 ± 0.15, and 0.77 ± 0.26 ppm for citrate AgNPs,GA AgNPs, and Ag as AgNO3, respectively. After 24 h, the totalAg concentration had reduced by 99.8 ± 0.05% in the SBRs forall Ag reactors. Effluent total and dissolved Ag measurementswere not statistically different from no Ag control SBRs (p <0.05), indicating that Ag was either precipitating andaggregating at the bottom of the reactor or sorbing to sludgebiomass. Sorption of Ag to biomass has been shown to beheavily prevalent in wastewater, with up to 98% removal offunctionalized AgNPs from supernatant.19 The same trends inAg concentrations were observed for the 2 and 6 d spikes. Thecontinuous phase followed the pulses, and GA AgNPs were stillconsistently lower in concentration than citrate AgNPs andAgNO3. Dissolved Ag samples were not statistically differentfrom no Ag controls (p < 0.05).Overall, total Ag concentration decreased most rapidly in

SBRs receiving GA AgNPs in their influent. This result issurprising since GA AgNPs are typically known to be verystable due to their steric and electrostatic stabilized coatingwhich keeps them well dispersed.20 In previous experiments, wefound that GA and citrate AgNPs had similar zeta potentials,

suggesting that their dispersion should have been similar.10 Thenanoparticles also have similar total organic carbon concen-trations, but GA AgNPs were nearly twice the size of the citrateAgNPs (Table S3, Supporting Information). GA AgNPs mayhave adsorbed to sludge biomass in the wastewater matrix morereadily than the citrate coating. Although GA AgNPs areclassified as hydrophobic, hydrophilic zones within the coatingmay be able to bind to sludge flocs.21 The hydrophobic andhydrophilic properties of the polymer coating may have boundto biomass more readily. Since effluent total Ag concentrationswere so low, it is likely that Ag and AgNPs precipitated out ofsolution as they bound with wastewater ligands. Similarly, Houet al.22 found that 90% of citrate AgNPs remained in SBRs andsubsequent treatment cycles would thus be exposed AgNPsattached to sludge flocs until sludge wasting occurred. Kiser etal.23 showed that AgNPs were removed not only by aggregationand sedimentation but also by biosorption onto heterotrophicsludge biomass. Dissolved Ag concentrations were minimal inthe SBRs suggesting that most of the Ag as AgNO3 or Ag+

released from the AgNPs may have been combined with theabundance of chloride or sulfide groups normally present insludge biomass.24

Reactor Treatment Efficiency. The presence of AgNPsand AgNO3 in wastewater SBRs was found to significantlyimpact heterotrophic and autotrophic activity in the SBRs (p <0.05). During the 0.2 ppm spikes, the removal efficiencies ofCOD and ammonia decreased rapidly immediately followingpulse additions but stabilized within 3−5 d (Figure 2). It isinteresting to note that the total Ag concentration decreased inall treatments by at least 40% in 24 h after the first pulseaddition. This rapid reduction in total Ag may be linked to therapid treatment recoveries observed at the 0.2 ppm exposures.During the 2 ppm spikes, the SBRs were not able to recover asquickly.

Figure 2. COD and ammonia removal percentages in SBRs during pulse and continuous inputs. Graphs show the capabilities of the SBRs to removenutrients and the upsets that occurred in each time period. 14 d, 6 d, and 2 d pulse spikes of Ag or AgNPs were followed by continuous Ag additionto investigate microbial resilience to Ag.

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COD removal was moderately affected by AgNPs, especiallyfollowing the first 0.2 ppm pulse addition. Removal percentagesdropped from 99% to 71%, 80%, and 90% for citrate AgNPs,GA AgNPs, and Ag as AgNO3, respectively, at the end of thefirst pulse addition treatment cycle. However, after 3 d, theCOD removal recovered significantly and efficiencies were92%, 95%, and 92% for citrate AgNPs, GA AgNPs, and Ag asAgNO3, respectively. The COD removal efficiencies were notstatistically different from the control (p > 0.05). The sametrend was observed for the second and third 0.2 ppm pulseadditions, with treatment efficiencies of 77%, 81%, and 79%following the third 0.2 ppm pulse addition. Overall, Ag fromAgNO3 did not affect COD removal significantly more thancitrate or GA AgNPs. This trend may be due to a handful ofreasons. Ag from AgNO3 may have bound quickly withchlorides and sulfides, reducing toxicity to heterotrophicbacteria that are mainly responsible for COD removal.25 It isalso possible that the heterotrophic bacterial community wasslowly adapting to the presence of Ag, similarly to their abilityof developing antibiotic resistance.26 The continuous spike of0.2 ppm Ag and AgNPs did cause a slight drop in treatmentefficiency after the first cycle from 96% to 77%, 81%, and 79%for citrate AgNPs, GA AgNPs, and Ag as AgNO3, respectively;however, the reactors recovered rapidly and removed COD by90% or more after 3 d. This trend was also observed with theaddition of the 2 ppm spikes. After the first 14 d spike, no largedecreases in COD efficiency were observed. This trendcontinued until the continuous spike, where removalefficiencies dropped slightly to 92%, 76%, and 82% for citrateAgNPs, GA AgNPs, and Ag as AgNO3, respectively, after 2 dbut the SBRs recovered to steady state efficiency after 4 d. Theimprovement in recovery may have been linked to a moredrastic shift in microbial community structure in response tothe constant flow of AgNPs. Please refer to the “Effects ofAgNPs on Microbial Community Structure” section for a moredetailed discussion on community dynamics.Ammonia removal was more strongly affected by Ag as

AgNO3 than AgNPs. In general, ammonia removal efficienciesdropped immediately after the pulse additions but were able torecover similarly to COD removal. After the first 0.2 ppm pulseaddition, ammonia removal decreased from 98% down to 97%,83%, and 32% for citrate AgNPs, GA AgNPs, and Ag asAgNO3, respectively. These results suggest that Ag as AgNO3caused a statistically greater drop in ammonia removal thanAgNPs (p < 0.05). This effect is congruent with our previousstudy that also found Ag as AgNO3 to be lethal to N. europaea,a model wastewater ammonium oxidizing bacterium, at 0.2ppm.10 GA and citrate coated AgNPs were found to havesimilar mortality effects at concentrations of 2 ppm total Ag.Treatment recovery of the Ag as AgNO3 spiked reactor tooklonger than the AgNP treated reactors. However, after 8 d, allreactors had recovered and were removing in excess of 88% ofthe influent ammonia. The second and third pulse additionshad virtually no effect on ammonia removal, indicating that thenitrifying microbial community in the SBRs likely adaptedfollowing the initial spike to maintain high levels of activityeven in the presence of AgNPs. This observation is possibly dueto the presence of functionally redundant bacteria in the SBRs.Additional information is presented later in the manuscriptdiscussing changes in microbial community structure. Thecontinuous spike of 0.2 ppm Ag did not significantly affectammonia removal, with the exception of the Ag as AgNO3

treatment that lowered removal to 64% on d 55, but overall, thereactors were able to recover.The 2 ppm spike of Ag was more disruptive and sustaining

than the previous lower concentration Ag addition. Similarly,Ag as AgNO3 had the most significant impact on ammoniaremoval and caused a decrease in removal from 95% to 64% inthe Ag as AgNO3 after 2 d. The reactor recovered after 14 d butconsistently underperformed with each additional spike andfinally showed constant poor performance in the continuousphase, with an average ammonia removal of 83%. On the otherhand, the other forms of Ag (i.e., AgNPs) did not appear tohave a strong impact on ammonia removal and were notstatistically different from the no Ag control during the 2 ppmspikes. Overall, these data suggest that AgNPs were less toxic toammonia oxidizing bacteria than AgNO3. One explanation forwhy AgNPs could be less toxic is the effects due to theircoating, which may have encouraged precipitation and limiteddissolution of toxic Ag species. Nitrate and nitrite concen-trations were consistently monitored but no strong inhibitorytrends were apparent in those results. Please refer toSupporting Information for more details on nitrate/nitritemeasurements (Figure S4).

Effects of AgNPs on Microbial Community Structure.T-RFLP was performed on DNA samples from the SBRs andanalyzed for clustering, diversity, and abundance. Microbialcommunities prior to the Ag spike were compared tocommunities after Ag was added. The universal 16S SSUrDNA T-RF chromatograms were compared, which provides arepresentation of bacterial communities in the SBRs. Principlecoordinate ordinations (Bray−Curtis similarity) help visualizethe clustering of different time points and treatments (Figure3). The SBR communities before Ag spiking all clearly clusteredbut, as Ag additions became more frequent, the Ag treatedcommunities became less similar from the control commun-ities. The first noticeable trend in clustering occurred followingthe first pulse addition (14 d). Grouping of CA14 and GA14and distancing of AG14 was observed. After the second andthird 0.2 ppm pulse additions (6 and 2 d), the GA and CAcommunities shifted more toward the AG14 community.During the continuous phase 0.2 ppm Ag addition, the AgNPcommunities heavily grouped with AG14 and shifted from thefirst spike to a tight cluster. The Ag as AgNO3 exhibited themost immediate effects on microbial community, while theAgNPs showed a more gradual shift.The control communities did not group with the Ag treated

communities, but there was some variation in their similarity toeach other. This could be due to the variability of the controls,which is common in wastewater sludge. The diversity wasrelatively higher than other Ag treated communities for sometime points such as day 32, shown by Simpson 1-D diversityindices. In general, the AgNP additions lowered diversity inmost treatments during the first 32 d of Ag addition (pulsesonly). The effects of individual AgNPs is not immediatelydiscernible from the ordination plots. However, diversityindices (Table S4, Supporting Information) suggest that GAAgNPs lowered diversity more than citrate AgNPs at severaltreatment time points. The citrate and GA AgNP treated SBRsnever reached the same diversity they had prior to Ag addition,but they did appear to improve in diversity by the last day ofthe continuous 0.2 ppm Ag addition. Most notably, diversitydropped dramatically immediately following the first 14 d pulseaddition of 2 ppm Ag. While Ag as AgNO3 decreased thediversity the most (i.e., 94.6%), GA AgNPs and citrate AgNPs

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also caused significant decreases (92.6% and 32.4% reduction,respectively). Zheng et al.27 observed a similar decrease in SBRstreated with TiO2 nanoparticles. However, to our knowledge,the present study is the first demonstration of a significantdecrease in microbial diversity in SBRs receiving AgNPs.Another study performed by Yang et al.28 investigated theaffected microbial genera through pyro-sequencing. Theirresults concluded that nitrifying bacteria were particularlysusceptible to upset by AgNPs and Ag as AgNO3, which issupported by the temporary loss of nitrification observed in thisstudy.Bray−Curtis similarity indices (Table S5, Supporting

Information) also confirmed that there was very little similaritybetween the microbial communities of the 0.2 ppm continuousAg addition and the communities characterized during the 2ppm 14 d pulse Ag addition. The 2 ppm pulse and continuousinputs of all Ag types drastically lowered diversity and causedmajor upsets in terms of microbial community. The similaritybetween the 0.2 ppm continuous addition communities and allof the first 2 ppm Ag spike communities approached zero,indicating a total shift in the bacterial community.Implications of AgNPs on Wastewater Treatment

Performance. These data suggest that AgNPs may disruptCOD and ammonia removal initially, but eventually, microbial

adaptation and Ag-ligand formation will reduce their overallimpacts. While there may be brief upsets in treatment at 0.2ppm Ag, it is likely that wastewater microbial communities haveenough functional redundancy to recover quickly. If concen-trations reach 2 ppm total Ag, major plant upsets could occurand biological nutrient removal could be disrupted. However,the likelihood of such a scenario is unknown currently since theconcentration of AgNPs from consumer products is not welldocumented. These results suggest that adsorption, aggrega-tion, and low dissolved Ag were important factors that dictatedhow AgNP could impact microbial community structure andfunction in wastewater sludge. In general, the microbialcommunity diversities decreased with Ag spikes but were ableto survive Ag addition more readily than nonacclimatedmicrobial communities. Our novel approach to dosing andspiking SBRs with Ag and AgNPs demonstrates thatnitrification can rebound rapidly, especially at the lower Agconcentrations. It should be noted that a microbialcommunity’s ability to survive under high metal concentrationswill be linked to the development of heavy metal resistance and,therefore, the effects of AgNPs on treatment efficiency couldvary between different nanoparticles. Despite this, our resultsclearly indicate that wastewater microbial communities are ableto recover from Ag and AgNP additions, depending on theconcentration, but their dynamics may shift. Studies areongoing to characterize more types of AgNPs, the developmentof microbial resistance to AgNPs, and the genera specific effectsof AgNPs.

■ ASSOCIATED CONTENT*S Supporting InformationDetailed information regarding experimental methods, nutrientand Ag measurements, Ag speciation, AgNP characterization,and microbial community diversity. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 919-660-5208; fax: 919-660-5219; e-mail: ckgunsch@duke.edu.NotesThis work has not been subjected to EPA review and no officialendorsement should be inferred.The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis material is based upon work supported by the NationalScience Foundation (NSF) and the Environmental ProtectionAgency (EPA) under NSF Cooperative Agreement EF-0830093, Center for the Environmental Implications ofNanoTechnology (CEINT). Any opinions, findings, conclu-sions or recommendations expressed in this material are thoseof the author(s) and do not necessarily reflect the views of theNSF or the EPA. We thank our collaborators at CEINT forproviding AgNPs and AgNP characterization to us.

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Figure 3. Bray−Curtis principal coordinate ordination diagrams of (A)0.2 ppm Ag additions and (B) 2 ppm Ag additions show clustering ofmicrobial communities. Each sample is denoted by its treatment (AG= Ag as AgNO3, GA = gum arabic AgNPs, CA = citrate AgNPs, C = noAg control) and the phase (0 = before Ag addition, 14 = 14 d pulse, 6= 6 d pulse, 2 = 2 d pulse, cont = continuous Ag addition).

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