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Impacts of pristine and transformed Ag and Cu engineered nanomaterials on surficial sediment microbial communities appear short-lived Joe D Moore, John P Stegemeier, Kyle Bibby, Stella M. Marinakos, Gregory V. Lowry, and Kelvin B. Gregory Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05054 • Publication Date (Web): 03 Feb 2016 Downloaded from http://pubs.acs.org on February 4, 2016
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Environmental Science & Technology
Impacts of pristine and transformed Ag and Cu engineered nanomaterials on surficial sediment microbial communities appear short-lived Joe D. Moore,1, 2 John P. Stegemeier,1, 2 Kyle Bibby,3, 4 Stella M. Marinakos,2 Gregory V. Lowry,1, 2 Kelvin B. Gregory1, 2* 1
Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA
2
Center for the Environmental Implications of NanoTechnology (CEINT), Durham, NC 27708, USA 3
Civil and Environmental Engineering, University of Pittsburgh, Pittsburgh, PA 15260, USA
4
Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
*Corresponding author: 5000 Forbes Avenue, 119 Porter Hall, Pittsburgh, PA 15213, USA. Phone: 412-268-9811. Fax: 412-268-7813. Email:
[email protected] 1
ABSTRACT
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Laboratory-based studies have shown that many soluble metal and metal oxide engineered
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nanomaterials (ENM) exert strong toxic effects on microorganisms. However, laboratory-based
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studies lack the complexity of natural systems and often use “as manufactured” ENMs rather than
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more environmentally relevant transformed ENMs, leaving open the question of whether natural
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ligands and seasonal variation will mitigate ENM impacts. Because ENMs will accumulate in
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subaquatic sediments, we examined effects of pristine and transformed Ag and Cu ENMs on
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surficial sediment microbial communities in simulated freshwater wetlands. Five identical
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mesocosms were dosed through the water column with either Ag0, Ag2S, CuO or CuS ENMs
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(nominal sizes of 4.67±1.4, 18.1±3.2, 31.1±12 and 12.4±4.1, respectively) or Cu2+. Microbial
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communities were examined at 0, 7, 30, 90, 180 and 300 d using qPCR and high-throughput 16S
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rRNA gene sequencing. Results suggest differential short-term impacts of Ag0 and Ag2S,
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similarities between CuO and CuS, and differences between Cu ENMs and Cu2+. PICRUSt-
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predicted metagenomes displayed differential effects of Ag treatments on photosynthesis and of
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Cu treatments on methane metabolism. By 300 d all metrics pointed to reconvergence of ENM-
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dosed mesocosm microbial community structure and composition, suggesting limited long-term
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microbial community impacts from a pulse of Ag or Cu ENMs.
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Environmental Science & Technology
INTRODUCTION
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Engineered nanomaterials (ENM) are increasingly incorporated into commercial and industrial
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products.1 ENMs have already been detected within wastewater streams,2 and, with increasing
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usage, their concentration in the environment and potential for environmental impact will rise.
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While it is known that Ag and Cu ENMs are toxic or inhibitory to pure cultures and communities
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of microorganisms in the laboratory,3–6 it is not clear whether, in natural environments, their effects
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will be mitigated. It is also known that ENMs transform.7 However, the degree to which
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transformation will contribute to mitigation of ENM impacts is also an open question. Here we
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report the findings of a long-term (300 d) study in which we evaluated the magnitude and longevity
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of the effects of Ag0 and CuO ENMs, and their sulfidized analogs, on sediment microbial
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communities in large-scale, outdoor wetland mesocosms.
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Metal nanoparticles (NP) are some of the most commonly incorporated ENMs, with silver (Ag)
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and copper (Cu) being two of the most frequently, and increasingly, used metals.1 These NPs are
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often added to commercial products specifically because of their antimicrobial and antifungal
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properties.8–10 Applying ENMs to agricultural land within fertilizers is also a developing area of
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research.11 With increasing usage, the number of ENM-contaminated environmental
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compartments and the concentrations of ENMs within those compartments will rise. Ag NPs have
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already been detected in wastewater treatment plants.2 Environmental fate models for ENMs
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predict accumulation of ENMs in subaquatic sediments. This is a result of ENMs in wastewater
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effluent and of runoff from agricultural lands that apply biosolids.12,13 With runoff from farmlands,
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ENMs are expected to enter surface water, and freshwater and estuarine sediments.14–16 In
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wastewater treatment plants, and freshwater sediments, on agricultural land and within commercial
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products, ENMs will encounter diverse microbial communities that perform services important to
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human and ecosystem health. Thus, both in terms of the effectiveness of ENM-enabled
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antimicrobial products and the indirect environmental impacts of ENMs, studies of ENMs’ impacts
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on microbial communities are warranted.
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Ag NPs are toxic to microorganisms.3,17 Though Cu-based NPs are less well studied,
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comparisons with other ENMs have suggested that Cu-based NPs also have considerable potential
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for environmental impact,4,18,19 which is in line with the well-documented antimicrobial nature of
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Cu.20 A recent study comparing the toxicity of a variety of ENMs to bacteria and other unicellular
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organisms found Ag and Cu NPs to be the most toxic to bacteria, yeast, and algae.21 By studying
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the impacts of Ag and Cu NPs, we intended to capture the impacts of a worst-case scenario of
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possible ENM exposures.
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Pristine metal and metal oxide ENMs are known to transform to different species under
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environmental conditions,7,22–24 e.g. Ag0 transforms to Ag2S 25 and metallic Cu transforms to CuO
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then CuS.24,26 Recent work comparing effects of pristine and transformed Ag NPs revealed that
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they disparately inhibit E. coli,27 as well as nematodes, aquatic plants, and several fish species.28
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Dissolved constituents and redox conditions affect ENM transformation, aggregation and
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bioavailability.29–31 Very recent work has begun to consider the effect of environmental
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transformations when assessing environmental impacts of ENMs, typically observing lesser
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impacts.32–34 Nevertheless, studies purporting to assess potential ENM environmental toxicity still
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perform experiments in biological growth media or deionized water using pristine ENMs.35–38 In
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this study, we dosed mesocosms with pristine and transformed ENMs to determine if
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transformation mitigates the impacts of ENMs.
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Of the relatively few studies that have investigated impacts of Ag NPs on complex
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environmental microbial communities, there is little consensus regarding the degree or even the
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presence of impacts. Bradford et al. did not detect impacts of Ag NPs on transplanted estuarine
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sediment microbial communities using denaturing gradient gel electrophoresis (DGGE).39
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Similarly Colman et al. did not observe impacts on terminal restriction fragment length
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polymorphism (T-RFLP) analysis following Ag NP dosing of sediment slurries.40 Conversely,
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very recent work using 16S rRNA gene sequencing to detect impacts of Ag NPs has detected
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changes in microbial community structure in wastewater sludge41 and nitrifying batch reactors.42
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Others have observed impacts on rhizosphere microbial communities43 and microbial
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photosynthesis and phototrophic bacteria,44,45 calling into question the effects of Ag NPs on C
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cycling. The shortest of these studies investigated impacts after only two hours, and the longest
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was less than two months. In the present study, we opted for high-throughput 16S rRNA gene
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sequencing to obtain a high resolution view of impacts on microbial communities and embarked
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on a nearly year-long study to assess the longevity of impacts.
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Freshwater wetland microbial communities provide numerous ecosystem services, e.g. primary
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production, carbon and phosphorus sequestration and water quality improvements through
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denitrification.46–48 Ag NPs are likely to accumulate within subaquatic sediments.39,49 In fact, very
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recent work tracking the fate of Ag NPs within freshwater lakes showed biofilms within the lake,
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along with the sediment, to be Ag NP sinks.50 These results raise concerns around Ag and other
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antimicrobial metal NPs, specifically whether they will harm extant freshwater wetland microbial
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communities and the ecosystem services they provide.
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The goal of this study was to assess whether Ag and Cu ENMs will have lasting impacts on
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microbial communities in freshwater wetlands and whether sulfidation and/or environmental
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resilience will mitigate these impacts. We used large-scale, open air, outdoor mesocosms to captur-
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e as much of the natural complexity as possible, e.g. natural ligands, temperature changes,
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precipitation influxes, and ecology. We dosed separate mesocosms with Ag0, Ag2S, CuO or CuS
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NPs or with a Cu ion (Cu2+) control. (In a previous study, we included a Ag ion control which was
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found to transform into Ag2S,51 so we did not include a Ag ion control in the present study.) We
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hypothesized that given our relatively high dosing regimen – representing a worst case release
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scenario – pristine Ag0 and CuO NPs would have more pronounced impacts than their sulfidized
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counterparts, Ag2S and CuS NPs. We further hypothesized that sulfidation and environmental
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factors would combine to mitigate impacts of the NPs. To the best of our knowledge, this is the
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first study to assess long-term (10 months) effects of pristine and transformed metal ENMs on
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microbial communities under realistic conditions.
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MATERIALS AND METHODS
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Nanomaterial synthesis, sulfidation and characterization. For synthesis of pristine, gum
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arabic (GA)-coated Ag0 NMs, 1.37 L of ultrapure water (Barnstead Nanopure Diamond, Thermo
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Fisher Scientific, Waltham, MA), 45 mL of 10 g/L gum Arabic (Thermo Fisher Scientific), and 45
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mL of 0.1 M silver nitrate (Sigma Aldrich, St. Louis, MO) were added to an Erlenmeyer flask. The
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solution was stirred for 5 min. Forty-five mL of 0.1 M ice-cold sodium borohydride (Sigma
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Aldrich) were added all at once, and stirring was continued for 10 min. Multiple batches were
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combined, and the NPs were purified and concentrated by dialysis (Optiflux F200NR Fresenius
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Polysulfone Dialyzer, Fresenius Medical Care, Bad Homburg, Germany). The suspension was
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diluted in water and concentrated two additional times in order to obtain the final product as has
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been previously described.52 To produce transformed, GA-coated Ag2S NMs, Ag0 NMs were
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sulfidized following a modified published procedure (see Supplemental Methods in Supporting
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Information for more details).53
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One gram of
