Influence of Bovine Serum Albumin and Alginate on Silver

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Influence of Bovine Serum Albumin and Alginate on Silver Nanoparticle Dissolution and Toxicity to Nitrosomonas europaea Ann-Kathrin Ostermeyer, Cameron Kostigen Mumper, Lewis Semprini, and Tyler Steven Radniecki Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 12 Nov 2013 Downloaded from http://pubs.acs.org on November 17, 2013

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Environmental Science & Technology

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Influence of Bovine Serum Albumin and Alginate on Silver Nanoparticle

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Dissolution and Toxicity to Nitrosomonas europaea

3 4

Ann-Kathrin Ostermeyer1, Cameron Kostigen Mumuper1, Lewis Semprini2, and Tyler

5

Radniecki1,2*

6 7

1

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University, 5500 Campanile Drive, San Diego, CA 92182-1324

Department of Civil, Construction and Environmental Engineering, San Diego State

9 10

2

11

Oregon State University, Corvallis, OR 97331

102 Gleeson Hall, School of Chemical, Biological and Environmental Engineering,

12 13

*

Corresponding author: [email protected], (541)737-2491

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Keywords: Silver nanoparticles; BSA; alginate; Nitrosomonas europaea; UV-vis

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spectra; toxicity

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Abstract Bovine serum albumin (BSA), a model protein, reduced the toxicity of 20 nm

33

citrate silver nanoparticles (AgNP) towards Nitrosomonas europaea, a model ammonia

34

oxidizing bacteria, through a dual-mode protection mechanism. BSA reduced AgNP

35

toxicity by chelating the silver ions (Ag+) released from the AgNPs. BSA further

36

reduced AgNP toxicity by binding to the AgNP surface thus preventing NH3-dependent

37

dissolution from occurring. Due to BSA’s affinity toward Ag+ chemisorbed on the AgNP

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surface, increased concentrations of BSA lead to increased AgNP dissolution rates. This,

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however, did not increase AgNP toxicity as the dissolved Ag+ were adsorbed onto the

40

BSA molecules. Alginate, a model extracellular polysaccharide (EPS), lacks strong Ag+

41

ligands and was unable to protect N. europaea from Ag+ toxicity. However, at high

42

concentrations, alginate reduced AgNP toxicity by binding to the AgNP surface and

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reducing AgNP dissolution rates. Unlike BSA, alginate only weakly interacted with the

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AgNP surface was unable to completely prevent NH3-dependent AgNP dissolution from

45

occurring. Based on these results, AgNP toxicity in high protein environments (e.g.

46

wastewater) is expected to be muted while the EPS layers of wastewater biofilms may

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provide additional protection from AgNPs but not from Ag+ that have already been

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released.

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TOC/Abstract Figure 2 ACS Paragon Plus Environment

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Introduction

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Due to their antibiotic properties, silver nanoparticles (AgNPs) are the fastest

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growing nanomaterials on the market with a steep increase in the number of products

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containing AgNPs from 25 in 2006 to 313 products in 2011 1. AgNP-containing products

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can release AgNPs after washing (e.g. textiles and food containers) or through direct use

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(e.g. toothpastes, shampoos and swimming pool disinfectants) and it is expected that a

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significant quantity of AgNPs will enter into the wastewater stream 2-4. The introduction

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of the antimicrobial AgNPs into the wastewater stream may adversely affect beneficial

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bacteria within wastewater treatment plants (WWTPs).

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Ammonia oxidizing bacteria (AOB) derive energy and reducing power through

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the oxidation of ammonia (NH3) to nitrite (NO2-), the first step in the removal of nitrogen

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from wastewater 5. AOB are widely considered to be the most sensitive microorganisms

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in WWTPs being inhibited by many compounds, including silver ions (Ag+) and AgNPs

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6, 7

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which has been found to be responsible for the majority of the toxicity exerted by AgNPs

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6, 8, 9

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. The toxicity of AgNPs towards AOB has been linked to the dissolution of Ag+,

. The interaction of AgNPs with constituents in its environment can have dramatic

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effects on the dissolution rates, stability and toxicity of the AgNPs 10, 11. Within

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wastewater, a wide range of environmental parameters exist that can influence the

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stability and dissolution of AgNPs, including Ag+-binding ligands (e.g. NH3, Cl-, S2- and

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thiols), AgNP destabilizing divalent cations (e.g. Mg2+, Ca2+) and AgNP stabilizing

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biological macromolecules (e.g. proteins and extracellular polysaccharides)11-16.

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Proteins comprise roughly 50% of the organics found in wastewater in WWTPs

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17

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influenced both their dissolution rates and their stability in the presence of destabilizing

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divalent cations 13, 18. These interactions may ultimately determine the toxicity of the

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AgNPs to wastewater biota, including AOB.

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. Model proteins have been shown to interact with AgNPs in test media conditions and

Extracellular polysaccharides (EPS) are a major component of wastewater

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biofilms comprising approximately 50-90% of the organic matter within a biofilm 19.

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EPS from wastewater biota can interact with AgNPs altering their stability 12, 20. These

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interactions have been suggested to influence AgNPs toxicity towards wastewater 3 ACS Paragon Plus Environment


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biofilms. Thus, EPS may play a significant role in influencing the toxicity of AgNP

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towards AOB 21.

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To understand how biological macromolecules may influence AgNP toxicity

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towards AOB, 3-h batch toxicity assays were conducted with Nitrosomonas europaea (a

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model AOB) in the presence of 20 nm citrate AgNPs, bovine serum albumin (BSA) and

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alginate. BSA is a model protein that has been successfully used in studies examining the

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fouling of reverse osmosis (RO) membranes during wastewater reclamation, the

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production of energy from wastewater proteins in microbial fuel cells and the degradation

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of wastewater proteins by microbial consortiums 22-25. Alginate is a model extracellular

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polysaccharide that has been successfully used in studies examining RO membrane

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fouling and is a naturally secreted lipopolysaccharide that plays a large role in biofilm

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formation 26, 27.

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This study utilized UV-vis spectroscopy to examine the abiotic interactions of

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AgNPs with BSA and alginate. Additionally, silver ion selective electrodes were used to

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quantify Ag+ interactions with BSA, alginate and N. europaea cells. Understanding how

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biological macromolecules influence AgNP behavior and their resulting toxicity towards

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N. europaea will provide important insights into the risks AgNPs pose toward nitrogen

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removal from WWTPs.

101 102

Materials and Methods

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AgNP and Ag+ inhibition studies with BSA and alginate

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Aliquots from stock solutions containing the 20 nm citrate BioPureTM AgNPs

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(nanoComposix Inc., San Diego, CA; 1000 ppm) or AgNO3 (10 ppm) were placed into

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155 mL batch bioreactors containing 35 mL of deionized (DI) water to a final

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concentration of 1 ppm and 0.125 ppm, respectively. The bottles were shaken at 250 rpm

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for 15 min to disperse the AgNPs or AgNO3. After 15 min of shaking, a concentrated

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(NH4)2SO4 and HEPES buffer (pH 7.8) solution was added to final concentrations of 2.5

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mM and 30 mM, respectively, as previously described 9.

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Simultaneous to the addition of (NH4)2SO4 and HEPES, either BSA or alginate

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was added to a final concentration ranging from 5 – 150 ppm or 25 ppm – 1000 ppm,

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respectively. The batch bioreactors were shaken for an additional 30 min, at which time 4 ACS Paragon Plus Environment

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N. europaea cells, in mid-exponential growth, were added to a final concentration of 5

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mg protein L-1 (OD600 ≈ 0.072).

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N. europaea containing batch bioreactors were shook in the dark at 250 rpm for 3-

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h at 30°C. Nitrite production, via colorimetric assay 28, was measured at 45 min intervals

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throughout the 3-h experiment, as previously described 9. The observed nitrite production

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was linear for all conditions tested over the course of 3 h. See Supporting Information

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for more details on N. europaea culturing conditions and AgNP inhibition assay

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procedures.

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AgNP characterization

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The average hydrodynamic diameter of the AgNP suspension in the test media was quantified

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using time-resolved dynamic light scattering (TR-DLS) by measuring the z-average diameter at

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15 s intervals for 180 s with a 90Plus particle size analyzer (Brookhaven Instruments, Holtsville,

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NY). To minimize background noise, the DDI water and HEPES buffer (pH 7.8)/(NH4)2SO4

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solution were filtered through a 20 nm syringe filter (Whatman International Ltd., Maidstone,

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England) to remove small ambient particles before the AgNPs were added to a final concentration

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of 1 ppm. The average hydraulic diameter of the AgNP suspensions was 25.5 + 0.1 nm.

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AgNP surface charge was measured using a Brookhaven ZetaPALS (Holtsville, NY), and

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the electrophoretic mobility was converted to zeta potential (ξ) using the Hückel

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equation. Electrophoretic mobility of each suspension, containing 10 ppm AgNPs, 2.5

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mM (NH4)2SO4 and 30 mM HEPES in the presence and absence of 600 ppm BSA or

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1000 ppm alginate, was measured for five runs consisting of 20 cycles per run.

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Measurements were made on solutions ranging from pH 3 to pH 11, with pH measured

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using a Thermo Scientific Orion micro pH electrode (Chelmsford, MA).

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AgNP dissolution studies Batch reactors used for AgNP dissolution experiments were prepared as described

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above except in a total volume of 15 mL and without N. europaea cells present, since

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their presence interfered with the absorption spectrum of the AgNPs. In all dissolution

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experiments, 30 mM HEPES buffer (pH 7.8) was present while the values of (NH4)2SO4,

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BSA and alginate varied from 0 – 25 mM, 0 – 600 ppm and 0 – 1000 ppm, respectively.

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The characteristic localized surface plasmon resonance (LSPR) absorption

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spectrum of the AgNP suspensions, with a peak absorbance (λmax) at 400 nm, were

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measured from 200-900 nm via UV-vis spectrophotometry (UV-Visible

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Spectrophotometer Biomate 3S, Thermo Scientific, Madison, WI) 13, 18. The LSPRs were

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measured at four time points; immediately before the addition of (NH4)2SO4/HEPES

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buffer/BSA/alginate to the batch reactor (t = -30 min), 30 minutes after the addition of

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(NH4)2SO4/HEPES buffer/BSA/alginate (t = 0 min) and 120 minutes and 210 minutes

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after the addition of the (NH4)2SO4/HEPES buffer/BSA/alginate (t = 90 min and 180 min,

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respectively). The nomenclature of t = 0, 90 and 180 was maintained in the AgNP

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dissolution studies to help compare to the toxicity data in which N. europaea cells are

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added to the batch bioreactors at t = 0.

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AgNP dissolution was quantified by measuring the intensity of λmax over time, as

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previously described 18, and comparing the sample’s λmax intensity against the λmax

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intensity of a AgNP standard curve ranging from 0 – 1 ppm (Figure S1). The percent

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dissolution was calculated using the following formula: % Dissolution = (1 – [AgNP]t=-

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30min/[AgNP]t=180 min).

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However, the UV-vis spectra were used to indicate if aggregation of the AgNPs was

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occurring.

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The UV-vis spectroscopy method was not used to size the AgNPs.

To ensure that changes in λmax were due to dissolution and not aggregation, the

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full width at half maximum (FWHM) values were measured. The FWHM is the distance

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(in nm) between the left and right hand sides of the spectroscopy adsorption curve at one

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half of the λmax intensity and was found at for each time point sampled 29, 30. Thus, a

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constant FWHM throughout the experiment indicates that the AgNP suspension not

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aggregating and that decreases in λmax are due to dissolution. An increase in FWHM

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during the experiment indicates aggregation of the AgNP suspension and the decrease in 6 ACS Paragon Plus Environment

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λmax may be due to the creation of AgNP aggregates as well as AgNP dissolution. See

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supporting information for more details on AgNP dissolution and stability procedures and

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FWHM determinations (Figure S2).

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Ag+ adsorption studies

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AgNO3 was added to test media containing 2.5 mM (NH4)2SO4 and 30 mM HEPES

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buffer (pH 7.8) to a final concentration of 10 ppm. The solution was rapidly mixed on a

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stir plate. A silver ion selective electrode (ISE) was used to measure the Ag+

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concentration per manufacturer’s instructions (Denver Instrument, Bohemia, NY). BSA,

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alginate or N. europaea cells were titrated into the solution until the measured Ag+

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concentration was below 2 ppm (160 ppm, 3200 ppm and 80 mg protein L-1,

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respectively).

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In the absence of BSA, alginate or N. europaea cells, the dominant species of Ag in the

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test media included Ag+, Ag(NH3)+ and Ag(NH3)2+

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free Ag+. To account for this limitation, per manufacturer’s instructions, the silver ISE

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standard curve was conducted in the test media (Figure S3). Thus, the output from the

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silver ISE includes the combined concentrations of Ag+, Ag(NH3)+ and Ag(NH3)2+.

31

. The silver ISE will only detect

186 187 188

Results and Discussion

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BSA reduced AgNP toxicity through a dual protection mechanism

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The majority of AgNP toxicity to AOB has been attributed to the release of Ag+

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from the AgNP surface 8, 9. To determine if BSA could prevent nitrification inhibition

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caused by the released Ag+, 3 h batch toxicity assays were conducted with Ag+ and

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various concentrations of BSA. The Ag+-induced inhibition studies demonstrated that

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nitrification activity increased nearly linearly with increasing BSA concentrations (Figure

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1). Additionally, ISE experiments showed that BSA extensively bound Ag+ (Figure 2A).

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These results indicated a direct interaction between Ag+ and the high number of Ag-

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ligands in BSA, including thiol groups 10, 32. Thus, the presence of BSA should greatly

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reduce the toxicity from released Ag+ from AgNPs.



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The presence of BSA also reduced the inhibition exerted by 1 ppm 20 nm citrate

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AgNPs with increased BSA concentrations resulting in increased N. europaea

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nitrification activity (Figure 1). However, unlike in the presence of Ag+, the protection

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that BSA provided against AgNP inhibition occurred in a non-linear fashion suggesting a

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more complex reaction than simply the binding of released Ag+. At low BSA

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concentrations, 5-60 ppm, nitrification activity increased near linearly with the

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concentration of BSA in the test medium. At concentrations greater than 60 ppm, the rate

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of increase in nitrification activity with increasing BSA concentration decreased, similar

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in form to a Freundlich type isotherm, which has previously been used to describe BSA-

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AgNP interactions 33.

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This non-linearity may be due to the reduced magnitude of the surface charge of

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the AgNPs in the presence of high concentrations of BSA, one of the key driving forces

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behind BSA-AgNP interactions 33, 34. The surface charge of AgNPs decreased from -

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13.99 + 1.68 mV in the absence of BSA to -10.99 + 0.68 mV in the presence of 600 ppm

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BSA suggesting that an interaction between the AgNP surface and BSA was occurring.

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BSA-AgNP interactions were also detected through the observed red-shift in the λmax

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from 401 nm, for AgNP suspensions without BSA present, to 405 nm in the presence of

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10 ppm BSA, 407 nm in the presence of 40 ppm BSA and 409 nm in the presence of 150

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ppm BSA (Figure S4 and Table S1). These red-shifts in λmax indicated that BSA directly

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interacted with the surface of the AgNPs 13, 34, 35.

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To determine if the observed protection provided by BSA was due to decreased

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AgNP dissolution to Ag+, 3 h abiotic dissolution experiments were conducted in the

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presence of various BSA concentrations. AgNP dissolution was quantified utilizing UV-

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vis spectroscopy to quantify the decrease in λmax intensity of the AgNP suspension. This

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method has been successfully utilized to quantify AgNP dissolution in a variety of

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biological media 31, 36, 37.

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Additionally, in the presence of 2.5 mM (NH4)2SO4 and 30 mM HEPES buffer

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(pH 7.8), the dissolution of 20 nm AgNPs over a 3 h period has been quantified to be

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either 30% or 34% utilizing either ultra-filtration/inductively coupled plasma optical

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emission spectroscopy (ICP-OES) or UV-vis spectroscopy, respectively 9, 31. Thus, the


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UV-vis spectroscopy method is a viable method for quantifying AgNP dissolution in the

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test media presented in this work.

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AgNPs in test media containing 0, 10, 40, 150 and 600 ppm BSA resulted in 31%,

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12%, 14%, 24% and 47% dissolution, respectively, after 3 h (Figure 3 and Table S2).

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Thus, the presence of low concentrations of BSA initially reduced the dissolution of the

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AgNP suspension compared to AgNP suspensions that did not include BSA. However,

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as BSA concentrations increased, AgNP dissolution was enhanced compared to AgNP

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suspensions that did not include BSA. The switch from NH3-dependent dissolution to

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BSA-dependent dissolution can explain this apparent contradiction.

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Previous studies have shown that proteins can substitute weakly bound capping

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agents (e.g. citrate) on noble metal NPs 13, 38, 39. Additionally, the lattice model of protein

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binding has been successfully applied to model the binding of BSA to colloidal

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aluminum oxide particles 40, 41. The lattice model predicts that under low BSA

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concentrations, BSA will bind side-on, forming many low-energy bonds with the surface

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across the length of the BSA molecule. Based on these observations, low concentrations

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of BSA likely reduced AgNP dissolution rates by adsorbing to the AgNP surface and

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reduced the interaction of the surface with NH3 in the test media, a silver ligand that has

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been previously shown to enhance AgNP dissolution 16.

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The increased AgNP dissolution with increased BSA concentrations might be

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attributed to the sequestration of surface chemisorbed Ag+ to thiol groups present in BSA,

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such as cysteine, which are known to have a high binding affinity for metal cations 13, 34,

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42

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equilibrium with each other 43, 44, it is possible that the binding of Ag+ to BSA caused

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instability within the NP structure and induced further AgNP dissolution. Thus, AgNP

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dissolution is no longer dependent on NH3 concentrations, which are blocked from the

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AgNP surface by BSA, but on BSA concentrations, which binds with chemisorbed Ag+.

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The catalytic role of BSA in AgNP dissolution is further supported by the BSA-

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dependent dissolution of AgNPs at various NH3 concentrations (Figure 4, Table S3). In

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the absence of BSA, AgNP dissolution rates were NH3-dependent with higher NH3

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concentrations leading to faster dissolution rates through the formation of sliver amines

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(Ag(NH3)+ and Ag(NH3)2+) with surface chemisorbed Ag+ 16. However, in the presence

. Since AgNPs consist of Ag0 nanoparticles and surface chemisorbed Ag+ in



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of 100 ppm and 600 ppm BSA, the AgNP dissolution rates remained constant and were

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independent of NH3 concentrations but were dependent on BSA concentrations with 600

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ppm BSA inducing more dissolution (~50%) than 100 ppm BSA (~34%).

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In the absence of NH3, the dissolution rate of AgNPs increased from 15% in the

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absence of BSA to 30% and 51% in the presence of 100 ppm and 600 ppm BSA,

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respectively. These dissolution rates were 2-3.4 times greater than would be expected

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based on NH3-based kinetics alone and suggests that the thiol groups within BSA are

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catalyzing the dissolution of the AgNPs. Therefore, higher BSA concentrations would

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provide more thiol groups and induce higher dissolution rates. Contrastingly, at 1.6 mM

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NH3, the dissolution rates of AgNPs decreased from 91% in the absence of BSA to 50%

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and 39% in the presence of 600 ppm and 100 ppm BSA, respectively. This decrease in

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AgNP dissolution rates suggests that the BSA molecules are coating the AgNP surface

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and preventing the interaction of the surface chemisorbed Ag+ with the NH3 in the test

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media. The BSA coating eliminated NH3-dependent dissolution, while simultaneously

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controlling the rate of AgNP dissolution through the binding of Ag+ to the thiol groups

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within BSA with higher BSA concentrations inducing higher dissolution rates.

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Thus, BSA protects N. europaea from AgNP inhibition through two potential

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mechanisms: 1) the chelation of released Ag+ by Ag-ligand groups within the BSA

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molecule, including thiol and cysteine; and 2) the coating of the AgNPs to prevent direct

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interactions with the bacteria (as indicated by red shifts in λmax with BSA present) and

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reduce the dissolution of Ag+ through limiting the interaction of NH3 in the test media

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with the surface chemisorbed Ag+.

282 283

Alginate reduced AgNP toxicity through a single protection mechanism

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Unlike BSA, 3 h batch toxicity assays revealed that alginate was unable to prevent

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Ag+ toxicity towards N. europaea, even at concentrations as high as 1000 ppm (Figure 5).

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This would suggest that alginate was unable to bind Ag+. Interestingly, ISE experiments

287

showed that alginate was able to bind to Ag+ and protection of N. europaea may be

288

expected (Figure 2B). However, ISE experiments also revealed that the affinity of Ag+

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for N. europaea was much higher than that for alginate and this could explain the lack of

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protection from Ag+ inhibition afforded by alginate to N. europaea (Figure 2C). 10 ACS Paragon Plus Environment

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Similar to BSA, alginate was able to prevent AgNP inhibition towards N.

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europaea in a non-linear fashion (Figure 5). It should be noted that the concentration

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necessary to achieve any given level of protection from alginate was about twice as high

294

as that required by BSA. This is primarily due to the lower affinity of alginate for Ag+

295

compared to that of BSA (Figure 2). Even with the lower affinity, alginate did interact

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with the AgNP surface as indicated by the observed redshifts of the λmax from 401 nm in

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the absence of alginate to 402 nm in the presence of 300 ppm alginate, to 407 nm in the

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presence of 600 ppm alginate and to 409 nm in the presence of 1000 ppm alginate (Figure

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S5 and Table S1). The presence of 1000 ppm alginate also increased the negativity of the

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surface charge of the AgNP suspensions from -13.99 + 1.68 mV in the absence of

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alginate to -29.23 + 3.63 mV in the presence of alginate further suggesting that

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interactions were occurring between the AgNP surface and alginate molecules. Alginate

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has previously been shown to replace citrate as capping agent on AgNP surfaces 45, 46.

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As alginate concentrations increased from 0 ppm, to 100 ppm, 300 ppm, 600

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ppm and 1,000 ppm, the 3 h AgNP dissolution percentage decreased from 31% to 19%,

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20%, 14% and 10%, respectively (Figure 6 and Table S4). This decrease in AgNP

307

dissolution directly correlated with the increase in nitrification activity observed with

308

increasing alginate concentrations (Figure 5). Therefore, the presence of alginate

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prevented AgNP dissolution in a similar fashion as BSA, by preventing interactions

310

between NH3 in the test media with surface chemisorbed Ag+ on the AgNP surface.

311

However, unlike BSA, alginate did not dominate the dissolution rates of the AgNPs.

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Increased NH3 concentration in the test media resulted in increased AgNP dissolution

313

rates, though they were still less than when alginate was absent (Figure 7, Table S5).

314

Thus, alginate protected N. europaea from AgNP inhibition through the singular

315

mechanism of coating the AgNP surface which prevented direct interactions with the

316

bacteria (as indicated by red shifts in λmax with alginate present) and reduced AgNP

317

dissolution of Ag+ through limiting the interaction of NH3 in the test media with the

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surface chemisorbed Ag+. Once Ag+ had been released from the AgNPs, alginate was

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unable to mitigate their toxicity.

320 321

During the lifecycle of AgNPs, wastewater may be the most complex environment that AgNPs will likely experience and potentially can be one of the most 11 ACS Paragon Plus Environment


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important in determining their ultimate fate. Understanding how wastewater constituents

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(e.g. ammonia, proteins and polysaccharides) interact with AgNPs will be critical in

324

predicting the fate and toxicity of AgNPs in these environments.

325

Proteins proved to be a dominant wastewater constituent in the fate and toxicity of

326

AgNPs. BSA protected N. europaea from AgNP inhibition utilizing a dual mechanism.

327

First, BSA prevented Ag+-induced nitrification inhibition of N. europaea through the

328

binding of the Ag+ with thiol groups located within the protein. Second, the high affinity

329

of thiols for Ag+ resulted in the binding of the BSA to the AgNP surface thus reducing

330

the interactions between N. europaea and the AgNPs. The binding of BSA to the AgNP

331

surface was strong and prevented the NH3-induced dissolution of the AgNPs, thus

332

lowering the AgNP toxicity to N. europaea.

333

Interestingly, while BSA prevented NH3-induced dissolution, BSA itself

334

catalyzed the dissolution of the AgNPs. The increased dissolution of the AgNPs in the

335

presence of the BSA is proposed to be due to the extraction of chemisorbed Ag+ on the

336

AgNP surface by the thiols located within BSA. An increase in nitrification inhibition,

337

however, was not observed due to the tight binding of Ag+ to the BSA molecules. Thus,

338

it is expected that AgNPs will have limited toxicity in high protein environments but

339

these same environments may lead to the dissolution of the AgNPs via protein-induced

340

dissolution.

341

In contrast to BSA, alginate did not prevent Ag+-induced nitrification inhibition of

342

N. europaea due to its low affinity towards Ag+. However, alginate did interact with the

343

surface of the AgNPs, slowing AgNP dissolution and preventing AgNP-induced

344

nitrification inhibition. Alginate could only slow and not completely prevent NH3-

345

induced AgNP dissolution suggesting that the interaction between the alginate and the

346

AgNP surface was not as strong as that between the AgNP surface and BSA.

347

Thus, in environments with high polysaccharide content (e.g. biofilms), the

348

toxicity of AgNPs would be expected to be reduced due to a decrease in dissolution of

349

polysaccharide-coated AgNPs. However, toxicity from Ag+ that have been released from

350

the AgNPs before coming into contact with the polysaccharides would not be expected to

351

be reduced since most polysaccharides lack Ag+ binding ligands. Future experiments will

352

examine how the coating of AgNP with proteins or polysaccharides influences their 12 ACS Paragon Plus Environment

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adsorption to bacterial biomass, a critically important process in the fate and transport of

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AgNPs in wastewater treatment plants and the environment.

355 356 357

Acknowledgements We would like to thank the Dan Arp lab at Oregon State University for kindly

358

supplying the N. europaea culture. We would like to thank Jeff Nason and Daniel Pike at

359

Oregon State University for providing assistance with DLS and zeta-potential

360

measurements. We would like to thank Danielle Webb, Joe Anderson, Margaret

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Schneider, Kelsey Beyer, Issa El Haddad, Robert Godfrey, Tanner Houston, Rachelle

362

Huber, Tyler Kirkendall, Mark Rein and Matthew Tallone for assisting with this work as

363

undergraduate research assistants. Funding was provided by a grant from the National

364

Science Foundation’s Division of Chemical, Bioengineering, Environmental and

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Transport Systems – Environmental Health and Safety of Nanotechnology program (#

366

1067572).

367 368 369

Supporting Information Available Supporting information contains additional details on N. europaea culturing, UV-

370

vis spectroscopy interpretation, Ag+ ISE standard curves and AgNP dissolution values.

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This information is available free of charge via the Internet at http://pubs.acs.org.

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References

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1. Trust, Woodrow Wilson Institute Center for Scholars and the Pew Charitable The Project on Emerging Nanotechnologies. http://www.nanotechproject.org

375 376 377

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Figure Captions

525 526

Figure 1. Percent nitrification activity of N. europaea cells in test media containing 2.5

527

mM (NH4)2SO4, 30 mM HEPES (pH 7.8), various concentrations of BSA and either

528

0.115 ppm Ag+ (○) or 1 ppm 20 nm citrate AgNPs (●) concentrations during a 3-hour

529

batch bioreactor experiment. Bioreactors without AgNPs and Ag+ were run as controls.

530

Error bars represent 95 % confidence intervals.

531 532

Figure 2. The measured unbound Ag+ concentration remaining in solution after (A) BSA,

533

(B) alginate or (C) N. europaea cells were titrated into the test media.

534 535

Figure 3. Average UV-vis absorption spectra of triplicate 1 ppm 20 nm citrate AgNP

536

suspensions in test media consisting of 2.5 mM (NH4)2SO4, 30 mM HEPES buffer (pH

537

7.8) and (A) 0 ppm BSA, (B) 10 ppm BSA, (C) 40 ppm BSA, (D) 150 ppm BSA and (E)

538

600 ppm BSA.

539 540

Figure 4. Percent dissolution of 1 ppm 20 nm citrate AgNP after 3 h of exposure to test

541

media containing 30 mM HEPES (pH 7.8), 0-1.6 mM NH3 and 0 ppm BSA (■), 100 ppm

542

BSA (∆) and 600 ppm BSA (○). Error bars represent 95% confidence intervals.

543 544


545

mM (NH4)2SO4, 30 mM HEPES (pH 7.8), various concentrations of alginate and either

546

0.115 ppm Ag+ (□) and 20 nm citrate AgNPs (■) concentrations during a 3-hour batch

547

bioreactor experiment. Bioreactors without AgNPs and Ag+ were run as controls. Error

548

bars represent 95 % confidence intervals.

549 550


551


552

7.8), and (A) 0 ppm alginate, (B) 100 ppm alginate, (C) 300 ppm alginate, (D) 600 ppm

553

alginate and (E) 1,000 ppm alginate.

554 18 ACS Paragon Plus Environment

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555


556

media containing 30 mM HEPES (pH 7.8), 0-1.6 mM NH3 and 0 ppm alginate (■) and

557

600 ppm alginate (∆). Error bars represent 95% confidence intervals.

558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576



577 578


579

mM (NH4)2SO4, 30 mM HEPES (pH 7.8), various concentrations of BSA and either

580

0.115 ppm Ag+ (○) or 1 ppm 20 nm citrate AgNPs (●) concentrations during a 3-hour

581

batch bioreactor experiment. Bioreactors without AgNPs and Ag+ were run as controls.

582

Error bars represent 95 % confidence intervals.

Page 20 of 25

583


Page 21 of 25


584 585

Figure 2. The measured unbound Ag+ concentration remaining in solution after (A) BSA,

586

(B) alginate or (C) N. europaea cells were titrated into the test media.

587



Page 22 of 25

588 589


590


591

7.8) and (A) 0 ppm BSA, (B) 10 ppm BSA, (C) 40 ppm BSA, (D) 150 ppm BSA and (E)

592

600 ppm BSA.

593 594


Page 23 of 25


595 596


597

media containing 30 mM HEPES (pH 7.8), 0-1.6 mM NH3 and 0 ppm BSA (■), 100 ppm

598

BSA (∆) and 600 ppm BSA (○). Error bars represent 95% confidence intervals.

599 600 601

602 603


604

mM (NH4)2SO4, 30 mM HEPES (pH 7.8), various concentrations of alginate and either

605

0.115 ppm Ag+ (□) and 20 nm citrate AgNPs (■) concentrations during a 3-hour batch

606

bioreactor experiment. Bioreactors without AgNPs and Ag+ were run as controls. Error

607

bars represent 95 % confidence intervals.

608 23 ACS Paragon Plus Environment


Page 24 of 25

609 610


611


612

7.8), and (A) 0 ppm alginate, (B) 100 ppm alginate, (C) 300 ppm alginate, (D) 600 ppm

613

alginate and (E) 1,000 ppm alginate.

614


Page 25 of 25


615 616


617

media containing 30 mM HEPES (pH 7.8), 0-1.6 mM NH3 and 0 ppm alginate (■) and

618

600 ppm alginate (∆). Error bars represent 95% confidence intervals.


Influence of Bovine Serum Albumin and Alginate on Silver

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