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Silver Sink Effect of Humic Acid on Bacterial Surface Colonization in the Presence of Silver Ions and Nanoparticles Alex Joseph Bertuccio, and Robert D. Tilton Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04957 • Publication Date (Web): 20 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Environmental Science & Technology

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Silver Sink Effect of Humic Acid on Bacterial Surface Colonization in the Presence of

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Silver Ions and Nanoparticles

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Alex J. Bertuccio1 and Robert D. Tilton1,2*

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Center for Complex Fluids Engineering and Center for Environmental Implications of Nanotechnology, 1Department of Chemical Engineering, and 2Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213

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*Corresponding author. Tel: 1-412-268-1159, email: [email protected]

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Abstract Silver nanoparticles (AgNPs) released from consumer products may enter the

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environment and possibly harm microbial communities. Prior research showed that surface

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adherent AgNPs inhibit bacterial surface colonization, a precursor to biofilm formation, only

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when planktonic bacterial inoculum concentrations are less than a threshold level (Wirth and

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coworkers, J. Colloid Interface Sci. 2016, 467, 17–27). This inoculum effect is due to a decrease

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in free silver ion concentration associated with sublethal binding to bacteria. Natural organic

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matter can be an additional silver sink in environmental systems. Using Pseudomonas

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fluorescens as a model biofilm-forming bacterium, we find significant increases in minimum

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bactericidal concentrations for AgNP suspensions and Ag+ in solution when adding humic acid

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(HA) to bacterial suspensions. When HA is present, planktonic bacteria survive and colonize

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AgNP-laden glass surfaces at lower bacterial inoculum concentrations than were needed for

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survival and colonization in its absence. This occurs despite the observed tendency of HA to

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inhibit colonization on bare glass surfaces when silver is absent. Results are interpreted through

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equilibrium Ag+ binding isotherms to HA and suspended bacteria. These results indicate that

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silver ion sinks may lessen AgNP impacts on natural microbial ecology relative to the disruption

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observed in pristine laboratory conditions.

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1. Introduction

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Recent decades have experienced a proliferation of products including fabrics, personal

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care products and catheters that incorporate antimicrobial silver nanoparticles (AgNPs),1–5 and

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the potential of these biocidal nanoparticles to enter the environment via waste streams

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represents an environmental risk.3,6,7 Silver is toxic to microorganisms either in the form of Ag+

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or AgNPs,1,8–17 and there is concern that AgNPs may interrupt bacterial lifecycles.18 Bacterial lifecycles typically encompass an existence in the planktonic state, surface

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colonization and biofilm formation.19,20 Previous research has thoroughly investigated AgNP

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toxicity to planktonic bacteria 8,10,21–23 as well as to bacteria in biofilms.24–27 Silver can

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significantly damage a bacterium through a variety of mechanisms including Ag+ binding to

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DNA and proteins in the cell, formation of cell wall pits or other cell wall damage that degrades

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its performance as a transport barrier, and production of reactive oxygen species (ROS).8,9,28–34

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Although bacteria in biofilms are still vulnerable to Ag+ and AgNP toxicity, bacteria in biofilms

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have significantly higher resistances to antimicrobials in general compared to their planktonic

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counterparts,20,35,36 and this includes increased tolerance of AgNP exposure.26,37

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In addition to studies of pre-established biofilm susceptibility to AgNPs, bacterial

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colonization on AgNP-decorated surfaces has also been investigated by a number of groups.4,5,38–

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plastics,4,5,38–41,44 these investigations address the question of whether or not bacteria are capable

Using glass42,43,45 and a variety of other types of surfaces, such as TiO2 or AgNP-impregnated


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of colonizing a surface in the presence of nanoparticles, but they do not indicate particular

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factors that promote or inhibit colonization. The effects of humic acid on bacterial colonization

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in the presence of AgNPs were not considered. Wirth and coworkers45 showed that Pseudomonas

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fluorescens freely colonize directly on AgNP-decorated surfaces. Surface colonization was

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shown to depend significantly on planktonic bacterial inoculum concentrations. This inoculum

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effect was attributed to the decreased availability of silver ion bound to cells at sublethal levels.

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In addition to bacterial cells serving as silver sinks, in the environment there are numerous

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materials such as natural organic matter (NOM) that can bind silver and reduce the amount that is

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free to interact with bacteria. Several forms of organic matter have been observed to increase

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bacterial tolerance of silver nanoparticles in the planktonic state.23,26,46,47 The critical aspect of a

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silver sink effect is how silver binding to that sink decreases the concentration of silver ions that

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are free to bind to bacteria. The prior literature concerning organic compound effects on silver

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tolerance does not quantitatively evaluate silver ion partitioning between solution, bacteria-

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bound and organic matter-bound states, and as a result it is not known whether there is a critical

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lethal threshold of silver binding to silver bacterial silver binding.

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The objective of the current research is to elucidate the combined effects of initial

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bacterial inoculum concentration and the presence of NOM on the ability of planktonic bacteria

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to colonize a surface that displays adherent AgNPs. The governing hypothesis is that NOM, as

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exemplified by humic acid (HA) in this investigation, will increase bacterial tolerance of AgNP

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and Ag+ by functioning as an Ag+-binding sink that decreases the free silver concentration and

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thus the amount of bacteria-bound Ag+, and thereby allows bacteria to proliferate and colonize

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AgNP-laden surfaces at lower inoculum concentrations than would be possible in the absence of

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NOM. We consider a Gram- negative model biofilm-forming species, P. fluorescens, suspended



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in minimal media over glass surfaces decorated with polyvinylpyrrolidone (PVP)-stabilized

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AgNPs that have been used in previous model system studies. Gram negative bacteria are more

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vulnerable to AgNP and Ag+ than Gram positive bacteria which have thicker cell walls that

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reduce the likelihood of silver uptake.48–51 Typical aqueous NOM concentrations in the

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environment range from 2 to 10 mg C/L in rivers and lakes and up to 60 mg C/L in bogs,

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marshes and swamps.52 We consider the higher end of the natural HA concentration range to

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capture the magnitude of NOM effects that may be expected in high organic carbon ecosystems.

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We will show that HA increases the minimum inhibitory concentration (MIC) and minimum

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bactericidal concentration (MBC) of silver in both AgNP and Ag+ form and allows bacteria to

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colonize the AgNP-laden surface at lower bacterial inoculum concentrations than would

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otherwise be possible. This study also reveals that the overall effect of HA on surface

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colonization is complex and involves several competing tendencies. We find that HA exerts two

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counteracting effects that impact bacterial survival: by competitively binding Ag+, HA decreases

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the amount of Ag+ bound to bacteria for a given total silver concentration, which favors survival,

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but HA also sensitizes P. fluorescens to bound Ag+ such that growth inhibition and loss of

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viability both occur at lower extents of Ag+ binding to bacteria when in the presence of HA than

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in its absence. The net outcome of these competing effects favors P. fluorescens survival, which

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allows them to withstand silver exposure and proceed to colonize AgNP-laden glass surfaces.

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That is colonization also occurs despite the observed tendency of HA to otherwise decrease the

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amount of P. fluorescens colonization of glass surfaces, an effect observed here in the absence of

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silver.

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2. Experimental

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2.1 Silver nanoparticle deposition on glass coverslips.

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The AgNPs used in this study were described previously.53 They are stabilized by PVP

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(MW 55,000) and have a diameter of 9.3 ± 2 nm based on TEM. Their zeta potential (Malvern

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Zetasizer Nano ZS) in minimal Davis media (MDM) (50 mM ionic strength) was -2.35 ± 4 mV.

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Additional details can be found in Supporting Information. Glass coverslips (22×22 mm, No. 1.5, Fisher Scientific) were used as substrates for

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bacterial colonization. Coverslips were thoroughly cleaned before each experiment as described

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in Supporting Information and sterilized under a UV light (Spectroline Model EN-180) for 15

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minutes prior to use.

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2.2 AgNP-Decorated Coverslips To deposit AgNPs onto coverslips, the AgNP stock suspension was diluted with MDM to

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a final concentration of 10 ppm. 5 mL aliquots were added to 50 mL polypropylene centrifuge

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tubes (Beckman Coulter). One clean, UV-sterilized slide was placed with flame sterilized forceps

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into each tube and incubated to allow particles to adhere in the dark for 1 h at 23 ±2°C.

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Afterwards coverslips were rinsed aseptically with 300 mL of sterilized ultrapure water and

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immediately added to new sterile 50 mL centrifuge tubes containing 15 mL of MDM to begin a

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colonization experiment. AgNP-decorated coverslips were analyzed with a Zeiss Axio Observer

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Z1 microscope and a 20 X LD plan-NEOFLUAR objective (Zeiss) using darkfield microscopy

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and following the protocol described by Wirth and coworkers.45 Figure 1 shows representative

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images of a three separate AgNP-decorated coverslips.

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Figure 1. Representative darkfield microscopy images of AgNP-decorated coverslips. AgNPs appear as bright spots due to resonance Rayleigh scattering associated with the localized surface plasmon resonance. The AgNP area densities on these images are 3.34 (A), 4.01 (B) and 3.39 (C) × 105 particles/mm2. Circular features are out of focus objects.

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2.3 Bacterial strain and culture conditions. Pseudomonas fluorescens (ATCC 13525) was grown in pH 7.2 MDM that contains 1 g/L

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glucose as the primary carbon source. A frozen stock of P. fluorescens stored at -80 °C was

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inoculated in MDM on an orbital shaker at 160 rpm until the bacteria reached early log-growth.

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(Media and growth information are provided in Supporting Information.)


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2.4 Humic acid preparation.

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Pahokee Peat reference humic acid was purchased from the International Humic

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Substances Society. A stock solution of ~200 mg C/L HA was prepared in ultrapure water and

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adjusted to pH 7.1 by adding 0.1 M KOH (≥ 99.99%, Sigma-Aldrich). Additional details are

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described in Supporting Information.

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2.5 Bacterial surface colonization. A 25 mL volume of bacterial suspension with an initial bacterial inoculum of OD600 =

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0.0001 (~1×105 CFU/mL), 0.001 (~1×106 CFU/mL), or 0.01 (~1×107 CFU/mL) was exposed to

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a vertically oriented, pristine or AgNP-decorated coverslip in MDM with 0, 20 or 60 mg C/L HA

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in 50 mL centrifuge tubes and orbitally shaken at 160 rpm at room temperature for 72 h with

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loose caps to maintain aerobic conditions. A similar separate set of experiments, described in

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Supporting Information, was conducted with bacteria exposed to Ag+ in solution.

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2.6 Microscopic analysis of bacterial surface colonization. A Zeiss Axio Observer Z1 inverted microscope equipped with HBO 100 illuminating

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system was used to image bacteria adhered on the coverslips. LIVE/DEAD® BacLight™

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Bacterial Viability staining (Life Technologies) was used to aid visualization and assess adherent

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bacteria viability based on membrane integrity. Previous work has demonstrated that BacLight

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staining intensities and percentage viability determinations are not affected by the presence of

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AgNPs.26 The field of view was 37,300 µm2. Coverslip preparation, imaging procedures and

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representative live/dead images are provided in Supporting Information.



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2.7 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration

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(MBC)

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P. fluorescens in early log-growth was diluted with MDM containing suspended AgNPs

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or dissolved AgNO3 in MDM to obtain OD600 = 0.01, 0.001, or 0.0001 in volumes of 200 µL in -

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black polystyrene clear-bottomed well plates (Corning Costar). The MIC was determined as the

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lowest concentration yielding no change in OD600 over 24 h. The MBC was determined as the

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lowest concentration yielding no colony formation on agar. Additional details are described in

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Supporting Information.

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2.8 Ag+ binding to bacteria and HA. To measure Ag+ binding to P. fluorescens, a bacterial suspension in early log-growth

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phase (OD600 = 0.07 to 0.10) was added to fresh MDM containing AgNO3 with total Ag+

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concentrations ranging from 0.25 to 50 ppb. Bacterial inocula ranged from OD600 = 0.001 to

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0.045. The suspensions were orbitally shaken for 1 h and the amount of Ag+ bound per CFU was

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analyzed using inductively coupled plasma – mass spectrometry (ICP-MS, Agilent 7700x)

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following the procedure described in Supporting Information. A similar procedure was used to

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measure Ag+ binding to HA in AgNO3 solutions containing HA at 1 to 60 g C/L.

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3. Results and Discussion

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3.1 AgNP-decorated surfaces.

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AgNP adhesion was measured on six coverslips; two sets of three coverslips were prepared from independent AgNP dispersions, with representative images in Figure 1. The


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average deposition density was (3.5 ± 0.8) × 105 particles/mm2. Clean coverslips were also

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analyzed and averaged 500 ± 150 “spots”/mm2 indicating that any extraneous scattering

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contributions from dust or surface defects were insignificant. Wirth and coworkers showed that

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AgNP detachment is not significant during subsequent colonization experiments.45

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3.2 Effect of initial inoculum and presence of humic acid on bacterial colonization

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Bacterial colonization on glass coverslips with or without adherent AgNPs, in MDM or

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MDM augmented with 60 mg C/L HA, after 72 hours of orbital shaking, is shown in Figure 2.

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In the absence of HA (Figure 2D) colonization was significant for each initial inoculum

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concentration on pristine AgNP-free coverslips and exceeded monolayer coverage, where the

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maximum countable amount of bacteria would be ~26,800 cells/mm2 for this microscope. When

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adherent AgNPs were present, colonization was greatly reduced to sub-monolayer coverages, by

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a factor of at least 360, except for the most concentrated initial inoculum, ~107 CFU/mL, which

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showed similarly extensive colonization as on the pristine coverslips.



Figure 2. Representative images with a false color overlay for P. fluorescens colonization on AgNP-decorated coverslips at > 26,800 (A), 60 (B) and 700 cells/mm2 (C). Bacterial colonization over 72 h on coverslips without and with adherent AgNPs in HA-free MDM (D) or in MDM with 60 mg C/L HA (E). Red, blue and green bars correspond to initial inoculum concentrations of 105, 106, and 107 CFU/mL respectively. Initial inoculum concentrations below a certain amount resulted in statistically significant reductions in colonization on AgNP-decorated coverslips compared to pristine ones (comparing 105 and 106 CFU/mL with AgNP in D against similar inocula with no AgNP in D, p < 0.001). P. fluorescens showed a statistically significant increase in colonization in MDM with HA on AgNP-decorated surfaces compared to HA-free MDM (comparing 105 and 106 CFU/mL with AgNP in E against the same inocula with AgNP in D, p < 0.001). Data points represent at least three replicate coverslips and 10 random locations measured per coverslip. 183 ACS Paragon Plus Environment

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During these colonization experiments OD600 was monitored to identify changes in

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planktonic bacterial concentration. In the absence of HA, the optical density did not change over

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72 h in the presence of adherent AgNPs, i.e. there was no growth, for 105 or 106 CFU/mL initial

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inocula, but OD600 increased 14-fold, from 0.010 ± 0.001 to 0.14 ± 0.1, for 107 CFU/mL. This

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strong inoculum concentration dependence in both planktonic growth and colonization of the

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AgNP-decorated surface corroborates the prior observation of an inoculum effect whereby silver

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toxicity is highly dependent on initial planktonic bacterial concentrations.45 High initial

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planktonic bacterial concentrations provide an excess of silver binding sites that decrease the

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amount of free silver in solution. By distributing the silver among a large number of binding sites

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among the bacterial population, most bacteria take up a sublethal amount of silver which allows

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the majority of the population to survive, proliferate and colonize the AgNP-decorated surface.

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Adherent cell viability on the AgNP-decorated coverslips in MDM and MDM augmented

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with HA was also measured. In MDM, the average percentages of viable cells on coverslips were

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84 ±16% (ntotal = 55 cells distributed over 30 fields of view) and 61 ± 22% (ntotal = 57 over 30

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fields of view) for initial inocula of 105 and 106 CFU/mL respectively. A thick biofilm was

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observed with an initial inoculum of 107 CFU/mL and no dead cells were observed by

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LIVE/DEAD staining with epifluorescence microscopy. When MDM was augmented with 60

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mg C/L HA, viability was 99± 2% (ntotal = 501 over 30 fields of view), 94± 2% (ntotal = 1414

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over 30 fields of view), and 91± 6% (ntotal = 2774 over 30 fields of view) for 105, 106, 107

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CFU/mL respectively. Viability from the low initial inoculum concentrations of 105 and 106

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CFU/mL was greater in the presence of HA.

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Comparing data for colonization of the AgNP-decorated surface in the presence of 60 mg C/L HA (Figure 2E) with the similar data in its absence (Figure 2D) indicates that HA allowed



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significantly greater surface colonization on AgNP-decorated surfaces for the 105 and 106

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CFU/mL inocula. Colonization increased by factors of 7.7 (p < 0.001) and 9.4 (p < 0.001)

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relative to the HA-free experiments for 105 and 106 CFU/mL inocula, respectively (based on the

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statistical sign test54). This supports the hypothesis that HA provides a protective effect that

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enables low concentrations of P. fluorescens to colonize AgNP-decorated surfaces to a

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significantly greater extent. This protective effect on the bacterial surface colonization phase is

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consistent with prior evidence that HA can increase bacterial tolerance to silver ions and

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nanoparticles for bacteria in either the planktonic or the biofilm state.23,26,46,47

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Colonization on the AgNP-decorated surface by the 107 CFU/mL inoculum was

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decreased in the presence of HA compared to its absence. This is not due to a failure of HA to

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provide a protective silver sink. Comparing colonization on AgNP-free surfaces with (Figure 2E)

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and without (Figure 2D) HA indicates that HA has an overall effect of significantly decreasing

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colonization, by 37-, 15- and 4-fold for initial inocula of 105, 106, and 107 CFU/mL respectively.

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Even though bacteria have a decreased overall tendency to colonize coverslips in media

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containing HA, the lower inoculum concentration data show that there is a greater extent of

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colonization on AgNP-decorated coverslips in media with HA than in media without HA.

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The greater colonization of AgNP-decorated surfaces in the presence of HA, despite the

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otherwise unfavorable effect of HA on colonization, is attributable to the preservation of

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planktonic growth in the presence of HA. The optical density of the planktonic suspension

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bathing the AgNP-decorated coverslips in the presence of 60 mg C/L HA increased by 286%

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from 0.294 ± 0.001 to 1.135 ± 0.009, by 272% from 0.295 ± 0.003 to 1.1 ± 0.04, and by 253%

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from 0.271 ± 0.002 to 0.96 ± 0.090 over 72 h for 105, 106 and 107 CFU/mL inocula respectively.


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For each inoculum planktonic growth was far more extensive in the presence of HA than in its

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absence; in fact, HA allowed growth and colonization on AgNP-decorated surfaces in the two

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most dilute inocula that exhibited no growth and minimal colonization in its absence.

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The decreased bacterial colonization on AgNP-free surfaces in the presence of HA is

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considerable. We considered the hypothesis that HA inhibits P. fluorescens colonization of silica

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glass surfaces by adsorbing to the surface and creating an electrosteric barrier to bacterial

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adhesion, but prior literature indicates that HA does not adsorb to silica.55 We verified that

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Pahokee peat HA does not adsorb to silica via a quartz crystal microbalance with dissipation

Figure 3. Representative growth curves for P. fluorescens in HA-free MDM (open blue triangles) and MDM with 60 mg C/L HA (open red circles) when subcultured to a starting OD600 = 0.001. These experiments have no silver. Error bars represent standard deviations based on triplicate samples.

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(QSense E4) experiment (SI Figure S3). The decreased colonization may be a result of HA making the bulk solution environment more favorable for P. fluorescens. Figure 3 shows that P. fluorescens growing in MDM has a



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greater growth rate in the presence of HA than in its absence. The growth rate constants were

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0.101 ± 0.001 h-1 in the absence of HA and 0.21 ± 0.06 h-1 in the presence of 60 mg C/L HA.

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The faster growth in media with HA may suggest that P. fluorescens are able to metabolize HA

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as a carbon source, but independent growth experiments conducted with P. fluorescens in MDM

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containing no carbon source other than HA exhibited no growth. P. fluorescens cannot

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metabolize HA alone. It was also determined that HA did not significantly affect media pH,

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ruling out a buffering effect on growth. The origins of the growth rate increase have yet to be

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established, but it is clear that HA makes the planktonic phase more suitable for P. fluorescens

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growth, which could decrease a biological driving force for bacteria to select the surface

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environment. There also may be a colloidal basis for the lessened colonization in the presence of

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HA. The electrophoretic mobility of P. fluorescens in 10-fold diluted MDM was -1.1 ± 0.25

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µmcm/Vs in the absence of HA, but increased in magnitude significantly (p = 0.016, using the

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rank-sum test54) to -1.48±0.08 µmcm/Vs with the addition of 60 mg C/L HA. The increased

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negative electrophoretic mobility is consistent with HA adsorption to bacteria, which would

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increase their negative surface charge and could inhibit deposition on negatively charged silica

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surfaces.

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3.3 Bacteria and humic acid Ag+ binding isotherms. Isotherms were measured for Ag+ binding to P. fluorescens and to HA. Figure 4 shows

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the normalized amounts of silver bound per unit amount of P. fluorescens (Nbac = number of Ag

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ions bound per CFU) or per unit amount of HA (NHA = number of Ag ions per mg C HA). The

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normalized isotherms collapse reasonably well onto single curves for 45-fold and 60-fold

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variations in bacteria or HA concentration respectively. Normalized isotherms were fitted to


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empirical equations (1) and (2) to analyze the silver material balance in cell growth experiments.

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Coefficients were determined by regression. These equations have the form of a two-site binding

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model for P. fluorescens and a one-site Langmuir model for HA, but we do not ascribe any

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physical significance to the coefficients beyond curve fitting. The fitted isotherms give the

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normalized number of silver ions bound as a function of the free silver concentration, Cfree for

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binding to P. fluorescens as =

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1.31 81.88 + 10 0.1 + 100.9 +

and for binding to Pahokee peat HA as


(1)


4.14 = 10 19.54 +

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(2)

Figure 4. Ag+ binding isotherms to P. fluorescens (A) and to Pahokee peat humic acid (B). Different symbols represent different bacterial inocula of OD600 = 0.001 (black circles), 0.01 (red squares) and 0.045 (blue triangles) (A) or HA concentrations of 1 (red diamonds), 5 (blue triangles), 10 (purple crosses), 20 (green squares) or 60 mg C/L HA (black circles) (B). Curves are fitting equations [1] and [2]. Error bars are included to represent instrumental uncertainty and are typically smaller than the markers. To aid comparison of bacteria or HA binding, assuming a dry mass of 0.33 pg per bacterium,58 with carbon content 47%59, 10 million Ag ions/CFU would correspond to 6.4 × 1016 Ag ions/mg C bacteria.

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3.4 P. fluorescens MIC and MBC for Ag+ and AgNP. P. fluorescens MIC and MBC were determined for Ag+ and AgNPs for 105, 106, and 107 CFU/mL in MDM and MDM with 20 or 60 mg C/L HA. All results are reported in


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Tables S1 and S2. Figure 5 uses some of the MIC and MBC data to highlight important trends

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observed. Figure 5A show Ag+ (as AgNO3) MIC (threshold concentration for complete growth

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inhibition) and MBC (threshold concentration for 100% viability loss) values at different initial

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bacterial concentrations. Increasing initial inoculum concentration increases both the MIC and

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MBC, consistent with the greater capacity for sublethal Ag+ binding. In Figure 5B, when the

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bacterial inoculum is held constant at OD600 = 0.0001, corresponding to 105 CFU/mL, as the HA

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concentration increases the MIC and MBC both increase. The MBC values were consistently

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50% greater than the respective MIC values. These same trends were established for 106 and 107

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CFU/mL inocula and when AgNPs were the silver source (Figure 5C and 5D). These results

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confirm that HA functions as a silver binding sink and that it significantly protects against silver

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toxicity.



Figure 5. MIC and MBC values for Ag+ (added as AgNO3) and AgNPs for varying initial bacterial concentrations without HA (A) and (C) and for varying concentrations of Pahokee peat HA with an initial bacterial inoculum of 105 CFU/mL (B) and (D). White bars represent MICs and blue bars represent MBCs.

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3.5 Silver material balance and silver binding toxicity thresholds The sink effect of HA decreases the free Ag+ concentration from any silver source in the

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system. The total silver material balance below incorporates the binding isotherms, equations (1-

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2) and allows a threshold amount of silver binding per bacterium to be determined for growth

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inhibition (Nbac at the MIC) or for 100% viability loss (Nbac at the MBC):


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= ( + + ) 289

where Ntot is the total amount of dissolved silver ions in the system, V is the media volume, Cbac

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is the bacterial concentration, and CHA is the HA concentration.

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(3)

Threshold degrees of silver binding to bacteria at the MIC and MBC were calculated via

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equations (1-3) from the MIC and MBC results obtained for Ag+ (added as AgNO3) and

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presented for various inoculum and HA concentrations in Figure 6. When HA is absent, the

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lethal threshold amount of Ag ions bound per bacterium, Nbac (MBC) is on the same order of

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magnitude for each inoculum, 2.5 ± 0.6, 4.5 ± 2.1 and 10.6 ± 6.2 million Ag ions bound per

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CFU for 105, 106 and 107 CFU/mL inocula respectively. The trend of observing similar threshold

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amounts of Ag ions bound per CFU for the different inocula is also observed with respect to the

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values of Nbac at the MIC (Table S3). The order of magnitude similarity for threshold Ag+

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binding across three orders of magnitude in inoculum concentration suggests a critical degree of

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silver binding needed for 100% lethality.

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Despite the overall protective effect of HA manifested as higher MIC and MBC values,

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the critical threshold for lethal binding is substantially lower in the presence of HA compared to

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when HA is absent. Nbac (MBC) decreased by two orders of magnitude relative to HA-free

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conditions (p < 0.001, based on the rank-sum test54). In the presence of 20 mg C/L HA the

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amount of Ag ions bound per bacterium at the MBC is 0.012 ± 0.001, 0.021± 0.002 and 0.052

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± 0.004 million Ag ions bound per CFU for bacterial concentrations of 105, 106, and 107

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CFU/mL, respectively. In the presence of 60 mg C/L HA the amount of Ag ions bound per

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bacterium at the MBC is 0.013 ± 0.001, 0.016 ± 0.003 and 0.114 ± 0.003 million Ag ions

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bound per CFU for bacterial concentrations of 105, 106, and 107CFU/mL, respectively.

310

Comparing Figure 6A and 6B also shows that Nbac (MIC) also decreased with the addition of



311

HA. The significant decrease in the amount of silver ions bound per bacterium at the MIC and

312

MBC when HA is present compared to when it is absent suggests that P. fluorescens is more

Page 20 of 35

Figure 6. Ag+ bound per bacterium at the MIC (A) or MBC (B) in 0, 20 and 60 mg C/L humic acid with minimal Davis media for bacterial inoculum concentrations of 105, 106, and 107 CFU/mL which are represented by the red, blue and green bars, respectively.

313 314

sensitive to silver in systems with HA. Since there is scatter in the Ag+ binding data, in order to validate the conclusions

315

regarding the MIC and MBC threshold levels of bacterial Ag+ binding in the presence of HA,

316

silver material balance calculations were repeated using binding isotherm equations that

317

represent the extrema of the binding data. For example, upper bound estimates of the amount of

318

Ag ions bound per bacterium at the MIC or MBC were determined by using a binding isotherm

319

that would overestimate the amount of bacterial Ag+ binding (see Figure S4A) and a binding

320

isotherm that would underestimate the HA binding data set (see Figure S4B). Even using binding


Page 21 of 35


321

extreme representations that de-emphasize HA binding, the threshold amounts of bacterial silver

322

binding without HA remain well over one order of magnitude greater than with HA (Supporting

323

Information Table S3). Uncertainty in the binding isotherms does not affect the conclusion that

324

P. fluorescens is more sensitive to the toxic effects of bacterial bound Ag+ in the presence of HA.

325

The overall protective effect of HA is due primarily to the decrease in the free silver

326

concentration, since P. fluorescens is apparently more intrinsically vulnerable to silver toxicity

327

(lower lethality threshold of silver bound per bacterium) in the presence of HA than its absence.

328

That is, HA has two opposing effects: it decreases free silver concentrations but makes P.

329

fluorescens more sensitive to each silver ion bound. Here the net effect is overall protective. The

330

mechanism for the apparent increase in vulnerability is as yet unknown. One possible

331

explanation for the increased sensitivity is that HA adsorbs and shuttles bound Ag+ to the cell

332

wall. This would be consistent with electrophoretic mobility results in Section 3.2.

333 334 335

3.6 Bacterial colonization in the presence of Ag+ and humic acid The main toxic agent associated with AgNPs is believed to be Ag+ released from the

336

nanoparticles.11,15,56,57 ICP-MS analysis of the bacteria suspensions bathing the AgNP-decorated

337

coverslips showed that the total silver ion concentration (in either free, bacteria-bound or HA-

338

bound states) released from the AgNPs after 3 h of exposure was 3.2 ±1.5 ppb in MDM, 5.8±

339 340 341 342 343

0.8 ppb in MDM augmented with 20 mg C/L HA, and 5.4 ± 0.4 ppb in MDM augmented with

60 mg C/L HA. For comparison, the MIC values for 105 CFU/mL are 1.2 ± 0.4 ppb in MDM, 4.7 ± 1.6 ppb in MDM with 20 mg C/L HA and 11.7 ± 1.2 ppb in MDM with 60 mg C/L HA. The

MIC values for 106 CFU/mL are 4.2 ± 3.7 ppb in MDM, 6.3 – 1.6 ppb in MDM with 20 mg C/L HA and 11.5 ± 2.4 ppb in MDM with 60 mg C/L HA. (Where the uncertainty is indicated only



344

by a minus sign, it means every serial dilution experiment yielded the same MIC, so the

345

uncertainty is estimated as half the difference between the observed MIC and the next lower

346

concentration in the serial dilution.)

347

For both 105 and 106 CFU/mL initial inocula in the absence of HA, the silver released

348

after 3 h exceeded the MIC. Accordingly, there was no detected growth and minimal

349

colonization of the AgNP-decorated coverslips. However, in the presence of 60 mg C/L HA, the

350

silver released after 3 h was less than the MIC for these initial inocula, and there was significant

351

planktonic growth and surface colonization of the AgNP-decorated coverslips for both initial

352

inocula.

353

After 72 h the total concentrations of released Ag+ had increased to 6.1± 2.4 ppb in

354

MDM, 16.9 ± 1.0 ppb in MDM with 20 mg C/L HA and 20.4 ± 4.2 ppb with 60 mg C/L HA.

355

These concentrations somewhat exceeded the MIC values for 105 and 106 CFU/mL initial

356

inocula. Yet, the results in Figure 2 showed that the AgNP-decorated coverslips were colonized

357

in the presence of 60 mg C/L HA for both these initial inocula. This is attributed to the growth

358

that occurs during the early stages of exposure to AgNP-decorated surfaces, when the released

359

Ag+ concentrations remain below the MIC. That growth produces higher planktonic

360

concentrations, and since the MIC increases with increasing bacterial concentration, the

361

planktonic growth and colonization evidently outpace the release of Ag+. The relative kinetics of

362

silver release and bacterial growth are important. Quantitative considerations relating to this

363

issue are presented in Supporting Information section S15 and Table S4.

364

Since released Ag+ may influence bacteria in the nearby vicinity of AgNP deposition

365

sites in the environment, it is also important to investigate the potential for HA to aid bacterial

366

colonization on bare surfaces in the presence of dissolved Ag+. 105 and 106 CFU/mL P.


Page 22 of 35

Page 23 of 35


367

fluorescens inocula were exposed to 5 ppb Ag+ in MDM and MDM augmented with 20 and 60

368

mg C/L HA. This concentration was selected because it approximated the 3 h release of Ag+

369

from surface-adherent AgNPs. Both inocula concentrations have MIC values near or below 5

370

ppb Ag+ for 0 or 20 mg C/L HA and well above 5 ppb Ag+ with 60 mg C/L HA. Thus, we

371

expected bacterial growth would be inhibited in all media except MDM containing 60 mg C/L

372

HA for both bacterial concentrations. Since hindered growth correlated with significantly

373

diminished colonization (with adherent AgNPs, Figure 2), we also anticipated diminished

374

colonization on bare glass with 0 or 20 mg C/L, but not with 60 mg C/L HA.

375

Compared to silver-free systems (Figure 2), the only appreciable amount of colonization

376

in the presence of 5 ppb AgNO3 (Figure 7) occurred in MDM with 60 mg C/L HA. Accordingly,

377

there was no detectable change in OD600 in the presence of MDM or MDM with 20 mg C/L HA,

378

while in the presence of 60 mg C/L HA, OD600 increased by 0.80 ± 0.05 and 0.84 ± 0.02 for 105

379

and 106 CFU/mL, respectively. HA had a concentration-dependent effect of preserving

380

colonization on bare glass surfaces when all silver was added in the form of dissolved silver, and

381

the effect was quantitatively consistent with the effect of HA addition on the MIC.



Page 24 of 35

382

Figure 7. Bacterial colonization on AgNP-free coverslips when exposed to 5 ppb Ag+ as AgNO3 in 0, 20 or 60 mg C/L HA in MDM. Initial inoculum concentrations are represented by the red and blue bars for 105 and 106 CFU/mL, respectively.

383 384

3.7 Environmental Implications Provided that inoculum concentrations exceed a threshold level, bacteria can directly

385

colonize surfaces displaying an abundance of silver nanoparticles. The key is whether the

386

population is large enough that a fraction of the original population survives and proliferates.

387

Surviving bacteria colonize the nanoparticle-decorated surface. Silver ion binding to humic acid

388

extends the range of this inoculum effect, allowing bacterial populations to proliferate and

389

colonize silver nanoparticle-decorated surfaces at inoculum concentrations much smaller than

390

would otherwise be required in the absence of humic acid. Silver sinks such as humic acid are

391

abundant in many environments and can be expected to offer significant bacterial protective

392

effects against silver and potentially other toxic metals. The increased tolerance provided by

393

these silver sinks may allow bacteria to proceed through normal lifecycle phases under


Page 25 of 35


394

conditions where AgNP disruption of surface colonization and biofilm formation may be

395

expected otherwise. A more subtle but still significant consequence of the presence of humic

396

acid is an apparent sensitization of P. fluorescens to bound silver ions; the amount of Ag ions

397

bound per bacterium at the MIC and MBC are significantly lower when humic acid is present

398

compared to when it is absent. This effect opposes the protective effect of humic acid binding to

399

Ag+ to decrease the free silver ion concentration. In the current system, the balance of these

400

competing effects favored P. fluorescens proliferation and colonization, but it cannot be certain

401

that the balance would be favorable for other species if this sensitization also exists for other

402

bacteria.

403 404

ASSOCIATED CONTENT

405

Supporting Information.

406

AgNP characterization, coverslip cleaning, MDM composition, bacterial growth

407

conditions, HA preparation, bacterial surface colonization when exposed to Ag+, image analysis

408

method for colonized coverslips, representative Live/Dead images of bacteria on AgNP-

409

decorated coverslips, MIC and MBC protocol, Ag+ binding, QCM-D data for HA adsorption,

410

MIC and MBC tabulation for AgNP and Ag+, sensitivity considerations for silver material

411

balance, and P. fluorescens growth data and considerations for near-MIC Ag+ concentrations,

412

and discussion about the Ag released from AgNP-decorated coverslips and Ag+-binding

413

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

414 415

AUTHOR INFORMATION

416

Corresponding Author



417

*Phone: (412) 268-1159; e-mail: [email protected].

418

Notes

419

The authors declare no competing financial interest.

Page 26 of 35

420 421

ACKNOWLEDGMENT

422

This material is based upon work supported by the National Science Foundation (NSF)

423

and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF-

424

0830093, Center for the Environmental Implications of NanoTechnology (CEINT). Any

425

opinions, findings, conclusions or recommendations expressed in this material are those of the

426

author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been

427

subjected to EPA review and no official endorsement should be inferred. The authors thank the

428

PPG Industries Colloids, Polymers and Surfaces Laboratory at Carnegie Mellon University,

429

Malvern Instruments for use of light scattering equipment, Stella Marinakos for AgNP

430

characterization, and John Riley for QCM-D measurements.

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