Aquatic Toxicology 144–145 (2013) 230–241

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Contrasting responses of marine bacterial strains exposed to carboxylated single-walled carbon nanotubes Lyria Berdjeb, Émilien Pelletier, Jocelyne Pellerin, Jean-Pierre Gagné, Karine Lemarchand ∗ Institut des sciences de la mer de Rimouski, Université du Québec à Rimouski, 310 allée des Ursulines, Rimouski, Québec G5L 3A1, Canada

a r t i c l e

i n f o

Article history: Received 18 June 2013 Received in revised form 5 October 2013 Accepted 8 October 2013 Keywords: Marine bacteria Carboxylated single-walled carbon nanotubes (SWNT-COOH) Toxicity effects Viability Stress-related gene expression

a b s t r a c t The potential toxic effects of carboxylated ( COOH) single-walled carbon nanotubes (SWNTs) were investigated on the cell growth and viability of two reference (Silicibacter pomeroyi, Oceanospirillum beijerinckii) and two environmental (Vibrio splendidus, Vibrio gigantis) Gram-negative marine bacterial strains. Bacterial cells were exposed to six concentrations of SWNT-COOH, during different incubation times. Our results revealed different sensitivity levels of marine bacterial strains toward SWNT-COOH exposure. A bactericidal effect of SWNT-COOH has been observed only for Vibrio species, with cell loss viability estimated to 86% for V. gigantis and 98% for V. splendidus exposed to 100 ␮g mL−1 of SWNT-COOH during 2 h. For both Vibrio strains, dead cells were well individualized and no aggregate formation was observed after SWNT-COOH treatment. The toxic effect of SWNT-COOH on O. beijerinckii cells displayed time dependence, with a longer exposure time reducing their specific growth rate by a factor of 1.2. No significant effect of SWNT-COOH concentration or incubation time had been demonstrated on both growth ability and viability of S. pomeroyi, suggesting a stronger resistance capacity of this strain to carbon nanotubes. The analysis of the relative expression of some functional genes involved in stress responses, using the real-time reverse transcriptase PCR, suggests that the cell membrane damage is not the main toxicity mechanism by which SWNT-COOH interacts with marine bacterial strains. Overall, our results show that SWNT-COOH present a strain dependent toxic effect to marine bacteria and that membrane damage is not the main toxicity mechanism of SWNT in these bacteria. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Over the last decade, the industrial production and commercial use of engineered nanomaterials (ENMs) increased significantly in industrialized country worldwide (for information concerning the presence of ENMs in commercial products see the Nanotechnology Consumer Product Inventory, 2011, http://www.nanotechproject.org/inventories/consumer). Due to this recent enthusiasm of the industry for ENMs, they are now considered as emerging contaminants in aquatic environments by the United States Environmental Protection Agency (2009). Among these ENMs, carbon nanotubes (CNTs) research and development projects are booming and flourished since their discovery in 1991 (Lijima, 1991). Due to their size (diameter = 10−9 m), CNTs display singular physical, chemical and electronic properties (Saifuddin et al., 2013), which make them a promising material for a wide range of applications in industrial sectors including energy

∗ Corresponding author. Tel.: +1 418 723 1986x1259; fax: +1 418 724 1842. E-mail address: karine [email protected] (K. Lemarchand). 0166-445X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.10.013

conversion and storage, wastewater treatment, green nanocomposite design and biomedical engineering (Tan et al., 2012; Bosi et al., 2013). As a consequence, their global production was increased from 340 tons (2008) to 2500 tons (2010), and is expected to exceed 12 800 tons in 2016 (Patel, 2011). CNTs can enter aquatic environments from their manufacturing processes to the disposal of CNTs-containing products. Released CNTs may originate from point sources such as production facilities, landfills or wastewater treatment plants or from nonpoint sources such as wear from materials containing CNTs (Nowack and Bucheli, 2007). As a consequence, such an increase in the production of use of CNTs by the industry could lead to their increasing transfer in natural ecosystems (soils, rivers and marine coastal environments), where their impacts on the indigenous microorganisms are still unknown. Contrary to metallic nanomaterials, CNTs are exclusively composed of carbon atoms a priori harmless for biological compartments. Surprisingly, numerous studies revealed high toxic effects of these nanomaterials on cells of different biological organisms (e.g. Murray et al., 2009; Umeda et al., 2013; Campagnolo et al., 2013), leading to their classification as “emerging pollutant” by the U.S. Environmental Protection Agency (U.S. EPA, 2009). Most

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of the literature dealing with CNT toxicity is focused on mammalian cells (e.g. Monteiro-Riviere et al., 2005; Bottini et al., 2006; Donaldson et al., 2006), and demonstrated strong respiratory toxicity and oxidative stress effects. However, studying the behavior and the becoming of CNTs in aquatic environments and analyzing their potential effects on key organisms (e.g. bacteria, phytoplankton) is also crucial to assess the impact of these nanomaterials on the functioning of ecosystems and make decisions to preserve their ecological integrity. To our knowledge, most of CNT studies were performed in freshwater ecosystems and highlighted a set of harmful effects on several fish and invertebrate species (Templeton et al., 2006; Smith et al., 2007; Cheng et al., 2007; Ghafari et al., 2008; Zhu et al., 2009). In marine environments, such studies remain scarce. Only two published papers reported on the effects of CNTs on marine organisms, revealing different types of impacts such as growth reduction, activity inhibition and behavior alteration (Kwok, 2010; Wei et al., 2010). Bacterial communities constitute a very interesting biological model to assess, in real time, the toxicological effects of CNTs in marine ecosystems. Due to their high growth rates, their short generation times (del Giorgio and Cole, 1998), and the vast diversity of their metabolic activities, these microorganisms are able to respond quickly and specifically to the presence of numerous contaminants. Furthermore, their large surface to volume ratio makes them highly sensitive to low concentrations of contaminants (Traas and Leeuwen, 2007). Moreover, from an ecological point of view, bacteria constitute a key component in the functioning of aquatic microbial food web and many biogeochemical processes in coastal marine waters are related to their activities (Pomeroy, 2007; Azam and Malfatti, 2007). Through its capacity to degrade organic matter and regenerate nutrients, this biological compartment plays an essential role in the transfer of matter and energy to higher trophic levels (Azam et al., 1983). Thereby, the potential impact of CNTs on bacteria might strongly affect their capacity to provide ecosystem services listed above. According to Arias and Yang (2009), single-walled carbon nanotubes (SWNTs) functionalized with carboxylic surface group ( COOH) are among CNTs that exhibit a very strong antimicrobial activity on several pathogenic bacterial strains (both Gram-negative and Gram-positive). Consequently, the aim of our study is to assess, for the first time, the potential toxic effects of SWNT-COOH on marine bacterial cells. We investigated the effects of both concentration and treatment time of COOH functionalized SWNT on the cell growth and the cell loss of viability of two reference (Silicibacter pomeroyi, Oceanospirillum beijerinckii) and two environmental (Vibrio splendidus, Vibrio gigantis) marine bacterial strains. The relative expression of some functional genes in response to SWNT-COOH exposure has been also analyzed.

2. Materials and methods 2.1. Preparation and characterization of SWNT-COOH High-purity carboxylated ( COOH) SWNTs were purchased from NanoAmor Inc. (Houston, TX, USA). According to the manufacturer, SWNTs were produced by a chemical vapor deposition. Because their low level of surface functional groups the purchased SWNTs were difficult to disperse in aqueous solution. We deliberately increased the percentage of the carboxyl functional group according to the protocol of Tasi et al. (2006) (Fig. 1A). Briefly, 100 mg of the commercial SWNTs were loaded in a round-bottom flask and 7 mL of 3:1 (by volume) mixture of concentrated sulphuric and nitric acid were added to oxidize SWNTs. The mixture was stirred at 80 ◦ C during 90 min, then diluted with 100 mL of

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deionized water and filtered on a 0.2 ␮m pore-sized polypropylene filter (GHP filter, Pall Corporation). The residual black solid was washed with nanopure water until the pH of filtered water was neutral. The solid was then dried in vacuum at 60 ◦ C overnight to yield an enriched carboxylated SWNT-COOH. In this work, the SWNT-COOH acronym designs the acid treated nanomaterial. The dry solid was suspended in 100 mL of nanopure water by ultrasonication for 1 h and the dispersion remained stable for months. The concentration of the aqueous SWNT-COOH solution was estimated by measuring the dry weight of 100 ␮L aliquots with a microbalance (Mettler Toledo MX5). Purity and physical properties of the enriched SWNT-COOH were examined by LC-ICP-MS, Raman spectroscopy and transmission electron microscopy (TEM). The elemental composition of SWNTs was determined by CHN analyzer. Quantitative analysis of trace metal impurities in SWNT-COOH was performed using the induced coupled plasma mass spectrometry (ICP-MS). Briefly, 50 mg of SWNT-COOH were completely digested in a mixture of concentrated nitric acid and hydrogen peroxide (ratio 4:1) at 80 ◦ C in microwave oven. The solution was then diluted with nanopure water and submitted to ICP-MS analysis for quantification following standard procedure (Pariseau et al., 2009). DXR Raman spectroscopy (Thermo Fisher Scientific, USA), equipped with Olympus microscope was used at an excitation wavelength of 532 nm. The ratio of G-band (1579 cm−1 ) to D-band (1349 cm−1 ) peaks in the Raman spectra provided qualitative information for comparing the structural imperfection and the content of amorphous carbon (Dresselhaus et al., 2005). The SWNT Raman radial breathing mode (RBM) (205 cm−1 ) was used to estimate the average tube diameter (Dresselhaus et al., 2005). The modification of the original carboxylic functionalized SWNTs under acid treatment was monitored with Fourier transform infrared spectroscopy (FTIR). Infrared measurements were carried out on a PerkinElmer Spectrum 400 Fourier transform spectrometer. Spectra were recorded in the 4000–650 cm−1 range at a spectral resolution of 4 cm−1 and by co-adding 200 scans and rationing the samples scans to the background scans. The samples were diluted in potassium bromide powder (KBr) at a concentration of 0.09 wt%. 100 mg of the mixtures were pressed for 1 min at 4000 psi under vacuum. This operation produced round clear pellets where black carbon nanotubes where visible but well distributed in pelletsTransmission Electron Microscopy (TEM) images of SWNT-COOH were captured using LVM 5 (Delong America, USA) with 5.2 keV accelerating voltage. SWNT-COOH were dried in 400 mesh cooper grids pre-coated with thin carbon film (Pacific Grid-Tech). 2.2. Bacterial strains and culture conditions S. pomeroyi DSS-3 and O. beijerinckii subsp beijerinckii were reference bacterial strains, obtained from the American Type Culture Collection (ATCC 700808 and ATCC 12754, respectively). V. splendidus 7SHRW (Mateo et al., 2009), and V. gigantis SL1 (GenBank accession number KC295715) were environmental strains, isolated from the Estuary and Gulf of St. Lawrence (Quebec, Canada), respectively. The classification of these strains is presented in Table S1. O. beijerinckii, V. splendidus and V. gigantis were grown in marine broth (Difco 2216) at 26 ◦ C for O. beijerinckii and 20 ◦ C for both Vibrio species. S. pomeroyi was grown in YTSS medium (ATCC medium 2138) at 28 ◦ C. Cells were harvested in the mid-exponential growth phase. Cultures were then centrifuged at 6000 rpm for 10 min. Marine cell pellets were washed three times with 0.2 ␮m-filtered and autoclaved natural seawater (32 PSU) to remove residual soluble macromolecules and other growth medium components. Finally, pellets were resuspended in 0.2 ␮m-filtered and autoclaved natural seawater. Bacterial suspensions were diluted in sterile seawater to obtain working bacterial suspensions containing 107 cell mL−1 .

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Fig. 1. (A) Qualitative assessment of the dispersion and settling characteristic of the SWCNTs functionalized with 3% (1) and 7.5% (2) of carboxyl group (COOH ). SWCNTs samples (260 ␮g mL−1 ) were sonicated in a sonication bath for 1 h and standing for still 2 h. (B) Mosaic TEM image of SWNT-COOH. (C) Raman spectra of SWNT-COOH at 532 nm.

Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aquatox.2013.10.013. The reference strain Escherichia coli (ATCC 25922) was also used in this study as a control due to its high sensibility to CNT exposure (e.g. Kang et al., 2009; Simon-Deckers et al., 2009; Pasquini et al., 2012). E. coli was grown in Trypticase Soy Broth (BBL Microbiology Systems, Cockeysville, MD) at 37 ◦ C and cell pellets were washed and resuspended in 0.9% saline solution, as described above.

suspensions were mixed, in triplicates, with 20 ␮L of each CNT solution, or 20 ␮L of 0.2 ␮m-filtered and autoclaved natural seawater for control, into 1.5 mL centrifuge tubes. Tubes were kept rotating on a shaker incubator at 100 rpm for 2 h for all bacterial strains. Additional incubation periods were tested for both Vibrio strains (5, 10 and 30 min) and O. beijerinckii and S. pomeroyi strains (6, 18 and 24 h) due to the response obtained after the 2 h incubation assay.

2.3. SWNT-COOH treatment to bacterial cells

After incubation with SWNT-COOH, 10 ␮L of each mixture (cell suspension + CNTs) were transferred into 96-well plates containing 200 ␮L of marine broth or YTSS medium, according to the strain used. Plates were incubated at the growth optimum temperature of each strain. Cell growth was estimated by measuring the optical density (OD) at 600 nm every 3–6 h during three days on a Multiskan Ascent Microplate Photometer (Thermo scientific, Vantaa, Finland). The growth curves were obtained by plotting the OD

Six different experimental concentrations of SWNT-COOH have been considered in this study (0, 10, 50, 100, 150 and 200 ␮g mL−1 ). Based on the preliminary tests performed on the control strain (E. coli), these concentrations corresponded to the lethal concentrations of SWNT-COOH generating between 50% and ∼100% of cell mortality after 2 h exposure. Aliquots of 200 ␮L of working bacterial

2.4. Cell growth

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values versus growth time. The specific growth rate (, h−1 ) of each strain was estimated from the slope regression of ln(OD600 nm ) versus exponential growth time (Fuchs and Kröger, 1999). 2.5. Viability assay Enumeration of cells with intact or damaged membrane, defined here as viable and dead cells, respectively, was performed using the LIVE/DEAD BacLightTM assay (Molecular probes, USA). This kit contains a red cell-impermeant nucleic acid stain (propidium iodide, PI, 20 mM) and a green cell-permeant nucleic acid stain (SYTO-9, 3.34 mM). The staining of bacterial cells was performed following manufacturer’s instructions. Briefly, after incubations with SWNTCOOH, 5 ␮L of diluted (1/5, v/v) bacterial suspension and 1.5 ␮L of each stain were added in 991 ␮L of 0.2 ␮m-filtered and autoclaved natural seawater and incubated for 15 min at room temperature in the dark. One microliter of fluorescent beads (Fluoresbrite yellowgreen microspheres 1 ␮m, Polysciences) was systematically added to each sample as an internal standard to normalize cell fluorescence emission and light scatter values. The distinction of viable and dead cells was based on their green (FL1, Em = 522 nm) and red (FL3, Em = 610 nm) fluorescence (Falcioni et al., 2008) detected by using EPICS ALTRA Flow Cytometer (Beckman coulter, USA), equipped with a 15 mW 488-nm air-cooled argon-ion laser and a standard filter set-up. The percentage of dead cells was determined from the ratio of the number of cells stained with PI divided by the sum of cells. 2.6. Fluorescence imaging The effect of SWNT-COOH on V. splendidus and V. gigantis, was examined visually, by epifluorescence microscopy, the uniform versus aggregate distribution of dead cells (red fluorescent cells), after 2 h of incubation with 50 ␮g mL−1 and 200 ␮g mL−1 of SWNTCOOH. Images were acquired using an epifluorescence microscope Olympus BX-51 equipped with an Olympus CCD camera DP 70 (Olympus Corporation, Japan), using a TRITC (tetramethylrhodamine isothiocyanate) filter (Em = 605 nm). One milliliter of Cell-SWNT suspensions was stained with propidium iodide (30 ␮M final concentration) and incubated in the dark during 30 min. After incubation, stained samples were gently filtered through a 0.2 ␮m Whatman Nuclepore black polycarbonate filter. Thirty representative pictures obtained from different fields on each filter were captured and edited using the Image-Pro Plus software (version 5.1 for windowsTM , Mediacybernetics, USA). 2.7. Gene expression of V. splendidus, a sensitive strain, in response to SWNT-COOH exposure Gene expression analyses were only performed on V. splendidus due to (1) its sensitivity to SWNT-COOH treatments and (2) the technical constraints relative to the design and validation of specific primer sets for real time PCR analyses of functional gene expression which have prevented the use the other studied strains. The relative expression of four functional genes coding for proteins or co-factors involved in the general stress responses (rpoS, dnaK and groEL genes) and membrane stress responses (rpoE gene) was examined (Table S2). Aliquots of 2 mL of bacterial cell suspensions were incubated with 0, 10 and 50 ␮g mL−1 of SWNT-COOH, at 100 rpm for 2 h. For each treatment, total RNA was isolated using the RNeasy Protect Bacteria Mini kit (Qiagen) according to the manufacturer’s instructions. RNA samples were treated with RNase-free DNase I (Qiagen) at room temperature during 15 min. RNA concentration was measured using a NanoVueTM spectrophotometer (GE Healthcare, Canada). The RNA extracts were considered pure for OD260 /OD280 ratio of 2.0 or higher. The integrity of RNA was

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checked by analyzing products on 1% agarose gels. Reverse transcription reactions were performed on 240 ng of total RNA using SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen) with random hexamer primers according to the supplier’s instructions. To determine if RNA samples are contaminated with genomic DNA, a no reverse transcriptase control was included during the reverse transcription step. All complementary DNA (cDNA) samples were kept at −20 ◦ C until further analysis. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aquatox.2013.10.013. Primers were designed using the Primer Express software version 3.0 (Applied Biosystems, Foster City, CA, USA) for amplification of gene fragments between 93 pb and 213 pb with an annealing temperature between 58 ◦ C and 60 ◦ C (Table S2). Primers were purchased from Integrate DNA Technologies (Coralville, IA, USA). The blast program was used to compare all primer sequences with V. splendidus LGP32 complete sequence (Genbank ref. NC 011753.2). The composition of each quantitative real-time PCR (qPCR) reaction was as follows in 25 ␮L final volume: 1 ␮L of cDNA (1/20), 1X RT2 SYBR Green qPCR Mastermix (SABiosciences, Frederick, USA) and 250 nM of each primer. Quantitative real-time PCR assay was performed in 96-well plates (Life Technologies) on a ABI 7900 HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA) with the following conditions: initial denaturation at 95 ◦ C for 15 min, 40 cycles of denaturation at 95 ◦ C for 30 s, annealing and extension at 60 ◦ C for 1 min. Each run included a no-reversetranscriptase control (NRTC) and a no-template control (NTC) and each PCR reaction was carried out in triplicates. To determine the amplification specificity, a melting curve analysis was performed at the end of each PCR by gradually increasing the temperature from 60 ◦ C to 95 ◦ C with continuous fluorescence acquisition. Specific gene amplification was confirmed by detection of a single curve in a melt curve analysis. No primer-dimer formation was detected. The PCR efficiencies were calculated using a relative standard curve derived from cDNA of sample control (without SWNT-COOH). The relative level of target gene expression was based on a comparative method (Livak and Schmittgen, 2001; Pfaffl, 2001). The threshold value (Ct) was determined for each target gene as the number of cycle at which the fluorescence curve entered exponential phase. The relative quantification value of a sample is expressed as 2−Ct , where Ct = Ct (target sample) − Ct (reference sample) and Ct = Ct (target gene) − Ct (housekeeping gene). The 16S rRNA was selected as the housekeeping gene. Samples without SWNT-COOH were used as reference. 2.8. Statistical analysis All the statistics were performed using XLSTAT ProTM (version 2012.01.02). Results were expressed as mean ± standard deviation of at least three replicates. One-way analysis of variance (ANOVA) was carried out to test the significant effect of SWNT-COOH on the cell growth and viability of marine bacterial strains. SWNT-COOH treatment effects on specific growth rate were compared by analysis of covariance (ANCOVA). For these both statistical tests, the p-values 2 (Table 1, Fig. 1C), indicating that SWNT-COOH are relatively free of defects and impurities. 3.2. Effect of SWNT-COOH on E. coli A delay in cell growth (around 3h30) was recorded for cells exposed to 10, 50, 100 and 150 ␮g mL−1 of SWNT-COOH compared to controls without SWNT-COOH (Fig. S2A). Delay time was proportional to the concentration of SWNT-COOH used. By contrast,

no cell growth was detected for cells exposed to 200 ␮g mL−1 of SWNT-COOH. This was confirmed by viability assays that demonstrated 50% (±5.50) to 90% (±3.70) of viability loss at concentrations ranging from 10 to 150 ␮g mL−1 and 99% (±2.10) of viability loss at 200 ␮g mL−1 (Fig. S2B). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aquatox.2013.10.013. 3.3. Effect of SWNT-COOH concentration on marine bacterial strains growth For both S. pomeroyi and O. beijerinckii (Fig. 2A and B), no significant differences (ANOVA one way, F = 1.95, p = 0.11) were observed in the temporal evolution of growth between cells exposed to SWNT-COOH and controls, independently from the concentration of SWNT-COOH. For each treatment, the growth phase started before 6 h for S. pomeroyi and after 6 h for O. beijerinckii (Fig. 2A and B, respectively). The mean (n = 6) specific growth rate () is estimated at 0.20 ± 0.00 h−1 and 0.27 ± 0.01 h−1 for S. pomeroyi and O. beijerinckii, respectively (Table 2). In contrast, variations in the temporal evolution of V. splendidus and V. gigantis cell growth were observed and related to the concentration of SWNT-COOH (ANOVA one way, p < 0.0001) (Fig. 2C and D). No significant differences were observed between treatments with 10 ␮g mL−1 of SWNT-COOH and controls, whereas an exposure to 50 ␮g mL−1 of SWNT-COOH delayed their growth for about 6 h compared to controls and a growth inhibition appeared for exposure ≥100 ␮g mL−1 (Fig. 2C and D). However, despite the growth delay observed at 50 ␮g mL−1 exposure, the specific growth rates were similar between cells exposed to 50 ␮g mL−1 of SWNT-COOH and controls (n = 6, mean  = 0.30 ± 0.07 h−1 for V. splendidus and mean  = 0.40 ± 0.04 h−1 for V. gigantis; Table 2). 3.4. Effect of the exposure time on marine cell growth Growth curves of V. splendidus and V. gigantis after 5, 10, 15 and 30 min of incubation with 100, 150 and 200 ␮g mL−1 of SWNTCOOH are presented in Fig. 3. No cell growth was observed after 10 min of exposure with 100 ␮g mL−1 of SWNT-COOH for V. gigantis (Fig. 3II-B) and after 15 min for V. splendidus (Fig. 3I-C). A lack of cell growth was also highlighted for cells exposed to 150 and 200 ␮g mL−1 of SWNT-COOH after only 5 min of exposure for both strains (Fig. 3I-A and II-A). Cell growth curves of S. pomeroyi and O. beijerinckii exposed to different concentrations of SWNT-COOH, during 6, 18 and 24 h are shown in Fig. 4. Although the SWNT-COOH concentration and the incubation time changed, no significant differences were observed in the temporal evolution of the cell growth of S. pomeroyi between cells exposed to SWNT-COOH and controls (Fig. 4I-A, I-B, I-C and Table 3). No delay in the cell growth of O. beijerinckii was recorded.

Table 2 Specific growth rate of S. pomeroyi, O. beijerinckii, V. splendidus and V. gigantis determined by the slope regression of ln(OD600 nm ) versus time, after 2 h of incubation with the different concentrations of SWNT-COOH. Treatment

Mean specific growth rate ± SD (h−1 ) S. pomeroyi

O. beijerinchii

SWNT-COOH concentration

0 ␮g mL−1 10 ␮g mL−1 50 ␮g mL−1 100 ␮g mL−1 150 ␮g mL−1 200 ␮g mL−1

0.20 ± 0.02 0.20 ± 0.03 0.20 ± 0.01 0.20 ± 0.00 0.20 ± 0.00 0.21 ± 0.01

0.27 ± 0.01 0.28 ± 0.01 0.26 ± 0.00 0.27 ± 0.01 0.26 ± 0.00 0.26 ± 0.02

ANCOVA test

F p-Value

1.02 0.42

0.23 0.92

V. splendidus 0.31 ± 0.04 0.30 ± 0.05 0.28 ± 0.03 0.27 ± 0.010 0.26 ± 0.00 0.26 ± 0.020 20.03 0.001

V. gigantis 0.40 ± 0.02 0.43 ± 0.01 0.40 ± 0.04 0.27 ± 0.010 0.26 ± 0.00 0.26 ± 0.020 9.50 0.001

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Fig. 2. Growth curves of S. pomeroyi (A), O. beijerinckii (B), V. splendidus (C) and V. gigantis (D) in marine broth or YTSS medium at optimal temperature, after 2 h of incubation with 0, 10, 50, 100, 150 and 200 ␮g mL−1 of SWNT-COOH. Optical density (OD) at 600 nm was used to monitor the bacterial growth.

Nevertheless, according to SWNT-COOH concentrations, a significant decrease in the specific growth rate was observed after 18 h incubation (Fig. 4II-B, II-C). Compared to controls, the specific growth rate of cells exposed to 150 and 200 ␮g mL−1 of SWNTCOOH was reduced on average by a factor 1.25 ± 0.17 after 18 h incubation (ANCOVA, F = 2.72, p = 0.04) (Table 3), whereas those exposed to more than 10 ␮g mL−1 of SWNT-COOH for 24 h displayed a decrease in their specific growth rate on average by a factor of 1.34 ± 0.09 (ANCOVA, F = 3.83, p = 0.01) (Table 3). 3.5. Effect of SWNT-COOH concentration on marine bacterial cell viability Proportions of both viable and dead cells for the four strains exposed to the different concentrations of SWNT-COOH are shown in Fig. 5. Proportions were similar from one treatment to another for

both S. Pomeroyi (ANOVA one way, F = 0.75, p = 0.60) and O. beijerincki (ANOVA one way, F = 0.70, p = 0.63) with a mean percentage of viable cells for S. pomeroyi and O. beijerincki estimated to 73% (±2.70) and 82% (±4.40), respectively (Fig. 5A and B). In contrast, proportions of viable cells for V. splendidus and V. gigantis exhibited a significant decline when exposed to 50 ␮g mL−1 SWNT-COOH (ANOVA one way, p < 0.0001, Fig. 5C and D). This result is consistent with the ones obtained during the growth tests (Fig. 2C and D). After 2 h, the exposure of bacterial cells to 50 ␮g mL−1 induced between 47% (±6.80) and 68% (±8.00) of cell death for both Vibrio strains. For the highest concentrations (i.e. ≥100 ␮g mL−1 ), these nanotubes were responsible for more than 86% (±1.50) and 98% (±0.30) in the viability loss of V. gigantis and V. splendidus, respectively (Fig. 5C and D). The uniform distribution of V. splendidus and V. gigantis dead cells without any SWNT-COOH exposure (control treatment) is

Table 3 Specific growth rate of S. pomeroyi and O. beijerinckii determined by the slope regression of ln(OD600 nm ) versus time, after 6, 18 and 24 h of incubation with the different concentrations of SWNT-COOH. Marine strain

Treatment

−1

Mean specific growth rate ± SD (h−1 ) Time of incubation 6h

18 h

24 h

S. pomeroyi

0 ␮g mL 10 ␮g mL−1 50 ␮g mL−1 100 ␮g mL−1 150 ␮g mL−1 200 ␮g mL−1

0.22 ± 0.00 0.21 ± 0.01 0.20 ± 0.00 0.20 ± 0.01 0.21 ± 0.01 0.22 ± 0.00

0.23 ± 0.02 0.22 ± 0.03 0.22 ± 0.02 0.22 ± 0.02 0.22 ± 0.03 0.21 ± 0.07

0.26 ± 0.00 0.25 ± 0.02 0.26 ± 0.02 0.26 ± 0.00 0.25 ± 0.01 0.25 ± 0.08

ANCOVA test

F p-Value

1.27 0.29

1.92 0.12

3.83 0.01

O. beijerinchii

0 ␮g mL−1 10 ␮g mL−1 50 ␮g mL−1 100 ␮g mL−1 150 ␮g mL−1 200 ␮g mL−1

0.15 ± 0.01 0.15 ± 0.00 0.15 ± 0.01 0.15 ± 0.01 0.15 ± 0.00 0.15 ± 0.02

0.23 ± 0.01 0.23 ± 0.01 0.22 ± 0.03 0.21 ± 0.01 0.19 ± 0.01 0.16 ± 0.01

0.22 ± 0.02 0.22 ± 0.01 0.18 ± 0.01 0.17 ± 0.04 0.16 ± 0.02 0.15 ± 0.01

ANCOVA test

F p-Value

0.15 0.96

2.72 0.04

3.83 0.01

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Fig. 3. Growth curves of V. splendidus (I) and V. gigantis (II) in marine broth medium at optimal temperature, after 5 (A), 10 (B), 15 (C) and 30 minutes (D) of incubation with 0, 100, 150 and 200 ␮g mL−1 of SWNT-COOH. Optical density (OD) at 600 nm was used to monitor the bacterial growth.

shown in Fig. 6I-A and 6II-A. V. splendidus cells exposed to 50 ␮g mL−1 and 200 ␮g mL−1 of SWNT-COOH displayed patterns similar to those observed in control treatments as no aggregate formation was observed in the different examined fields (Fig. 6I-B and I-C). For V. gigantis, the microscopic analysis also revealed a uniform distribution of cells, even if the presence of very small aggregates was observed in some cases (Fig. 6II-B and II-C). However, the number of these aggregates was relatively low (∼5) compared to the total of randomly distributed cells (such as observed in 30 examined fields). Overall, for both Vibrio strains, the dead cells did not display any aggregate formation and seemed to maintain rather an individualized distribution after incubation with SWNT-COOH. 3.6. Gene expression of V. splendidus, a sensitive strain, in response to SWNT-COOH exposure According to RT-qPCR analyses, the selected genes exhibited different patterns of expression according to the concentration of

SWNT-COOH tested (10 ␮g mL−1 versus 50 ␮g mL−1 ) (Fig. 7). At both concentrations, rpoS and groEL genes were up-regulated more than twice. The magnitude of expression was higher at 50 ␮g mL−1 than at 10 ␮g mL−1 , especially in the case of the rpoS gene (Fig. 7). The up-regulation of dnaK gene was only recorded after the exposition of V. splendidus to 50 ␮g mL−1 SWNT-COOH. In contrast to these three genes, no alteration of rpoE gene expression was monitored, after 2 h exposure to 10 and 50 ␮g mL−1 SWNT-COOH.

4. Discussion Most studies dealing with the toxicity of CNTs on bacteria focused on pathogenic strains in order to promote the potential and promising applications of CNTs as antimicrobial material (e.g. Brady-Estévez et al., 2010; Vecitis et al., 2011; Jain et al., 2012; Sweetman et al., 2013). The toxicity of CNTs on non-pathogenic environmental bacterial strains remains poorly understood,

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Fig. 4. Growth curves of S. pomeroyi (I) and O. beijerinckii (II) after 6 h (A), 18 h (B) and 24 h (C) of incubation with 0, 10, 50, 100, 150 and 200 ␮g mL−1 of SWNT-COOH. Optical density (OD) at 600 nm was used to monitor the bacterial growth.

particularly in coastal marine ecosystems. The present study thus contributes to overcome this lack of information. 4.1. Effects of SWNT-COOH on marine bacterial strains The first interesting element revealed in this study is the different levels of sensitivity of marine bacterial strains toward SWNT-COOH exposures, despite the fact that all studied strains are Gram negative. This would suggest that the cell wall structure alone could not explain for the contrasted bacterial responses observed in this work. According to previous studies (Kang et al., 2009; Liu et al., 2009), different levels of sensitivity to SWNTs toxicity have been highlighted between Gram negative and Gram positive bacteria. These studies underlined the difference in the peptidoglycan thickness and the mechanical properties between these strains to explain their specific response to the physical pressure of SWNT. Nevertheless, differences in the structural properties of the cell wall have also been reported between Gram-negative bacterial species, leading to strong variation in the mechanical behavior of their membranes (Vadillo-Rodriguez and Dutcher, 2009). These differences would be linked to different structural configurations of the cell wall such as the peptidoglycan layer thickness, the nature of bonding between the outer membrane and the peptidoglycan layer, the length of lipopolysaccharide molecules and proteins inserted into the lipid bilayer (Vadillo-Rodriguez and Dutcher, 2009). Based on these observations, cell wall properties may contribute to the different sensitivity levels of Gram-negative bacterial strains

toward SWNT-COOH exposures observed in our study. However, the ability of these strains to develop different stress response strategies could also explain the contrasting responses observed after SWNT-COOH exposure. Many studies that have highlighted the toxic effect of CNTs on bacteria have used E. coli as a reference microbial model (e.g. Kang et al., 2009; Simon-Deckers et al., 2009; Pasquini et al., 2012). The contrasted responses that we obtained from the four marine bacterial strains compared to those obtained from E. coli clearly indicated that E. coli could not be a good proxy of the toxic effect of CNTs on natural bacterial community. After 2 h of exposure to 10 and 50 ␮g mL−1 SWNT-COOH, our results showed that V. splendidus and V. gigantis cell growth rates reached the same optimal values even if a delay in the beginning of the growth phase was highlighted. This resilience in the cell growth after an exposure to 10 and 50 ␮g mL−1 SWNTCOOH indicates that Vibrio strains could fully recover, at optimal growth conditions, from the toxic effect of SWNT-COOH at this range of concentrations. Furthermore, the overexpression of identified stress-response genes (rpoS, dnak and groEL) recorded in V. splendidus cells indicates the ability of this strain to activate transcriptional responses related to the presence of SWNT-COOH at these concentrations. This genetic regulation may contribute to the relative tolerance of V. splendidus to CNT contamination. However, we observed that an exposure to concentrations higher than 100 ␮g mL−1 resulted in a total inhibition of cell growth, especially at very short time scales (i.e. during the first minutes of

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Fig. 5. Relative abundance (in %) of live and dead cells of S. pomeroyi (A), O. beijerinckii (B), V. splendidus (C) and V. gigantis (D), after 2 h of incubation with 0, 10, 50, 100, 150 and 200 ␮g mL−1 of SWNT-COOH.

exposure), revealing the high bactericidal effect of SWNT-COOH on Vibrio species at these high concentrations. According to previous studies, the toxic effect of CNTs on non-marine bacterial strains has been previously reported at concentrations between 1 and 100 ␮g mL−1 for short periods of time (less than 2 h) (Kang et al., 2007, 2008a; Arias and Yang, 2009; Liu et al., 2009; SimonDeckers et al., 2009; Akasaka and Watari, 2009; Vecitis et al., 2010). Compared to these results, the sensitivity of both Vibrio species was noted at a higher level of CNT concentrations (≥100 ␮g mL−1 ), suggesting a higher tolerance of these strains compared to those previously considered. Interestingly, no significant differences were observed in the cell growth abilities of O. Beijerinckii, after 2 h exposure to various concentrations of SWNT-COOH suggesting a higher tolerance of this marine bacterial strain to CNT action compared to Vibrio species. Nevertheless, increasing the exposure period from 2 h to 18 h and 24 h led to a significant reduction in the specific growth rate of this strain. This result indicates that, after a longer exposure period, SWNT-COOH could generate, for some marine bacterial strains, a bacteriostatic effect rather than a bactericidal one. To the best of our knowledge, it is the first time that such an effect of SWNT-COOH on the bacterial cell growth is reported. However, bacteriostatic properties have already been observed for some other nanomaterials, such as metallic nanoparticles (Cioffi et al., 2005; Petrus et al., 2011; Raghupathi et al., 2011; Yousef and Danial, 2012). The results presented in this work thus suggest that such an antibacterial property could be extended to CNTs. Another striking result in this study is the lack of sensitivity of S. pomeroyi toward various SWNT-COOH concentrations and incubation time, suggesting a stronger resistance capacity of this strain to SWNT-COOH contamination. The bacterial resistance to

CNTs has been previously reported for both Gram negative Cupriavidus metallidurans (Simon-Deckers et al., 2009) and Gram positive Bacillus subtilis (Kang et al., 2009). According to these authors, the highest tolerance of these strains might be related to their remarkable capacity to counteract the action of CNTs by an overexpression of protective components such as membrane restoration elements. The complete genome sequence analysis of S. pomeroyi revealed an abundant number of transport proteins and complex regulatory systems (Moran et al., 2004, 2007). This might give to these bacteria an important advantage to counteract the effect of SWNT-COOH especially by a quick activation of appropriate physiological mechanisms such as membrane restoration or oxidative stress counteraction. Furthermore, Zhang et al. (2009) observed a modification of the translational protein in S. pomeroyi which allowed gaining resistance to phage infection. However, it has not been yet established if such a protein modification is phage-specific or a general response to another stress. Consequently, exploring the potential protein modification of S. pomeroyi exposed to SWNT-COOH would deserve extensive studies which may help to understand the resistance mechanisms of this strain to the presence of carbon nanotubes. One of the most interesting phenotypic characteristics of S. pomeroyi is its ability to grow in multicellular rosette shape (Gonzalez et al., 2003; Bruhn et al., 2007). Reasons explaining this rosette formation are not well known, but some authors suggested that this could be a defence mechanism against grazing (Lebaron et al., 1999). In the same way, we might also assume that this structure could contribute to protect most of cells against the CNT action. Indeed, the S. pomeroyi cells located in the middle of this structure could be potentially protected by those located at the periphery which might prevent them from direct contact with CNTs.

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results are comparable to those obtained by Liu et al. (2009) using individually dispersed native SWNTs. According to these authors, the large length-to-diameter aspect ratio of SWNTs makes them behave like “nano darts”. Based on this hypothesis, the individually dispersed SWNTs could be visualized as numerous moving “nano darts”, constantly attacking bacteria to finally degrading the bacterial cell wall integrity and causing the cell death. However, no up-regulation of the rpoE gene has been detected in V. splendidus cells. Knowing that the rpoE gene encodes for the sigma E factor ( E ) that regulates gene expression to preserve the integrity of the membrane (Hayden and Ades, 2008; Bury-Moné et al., 2009), our results suggest that cell membrane damage is not the main toxic mechanism of SWNT-COOH toward V. splendidus cells. As previously mentioned in the literature (Kang et al., 2008a; Vecitis et al., 2010; Chae et al., 2011), the oxidative stress is the other possible action mechanism of CNTs toxicity on bacterial cells. According to Vecitis et al. (2010), SWNT could act as a conductive bridge over the insulating lipid layer releasing the cell energy into the external environment. The high observed up-regulation of rpoS supports the hypothesis that toxicity of SWNT-COOH toward Vibrio strains could be induced by an oxidative stress. The rpoS gene codes for the sigma S factor ( S ) which is involved in the regulation of general stress response and confers resistance against near-UV radiation, potential lethal heat-shock and hyperosmolarity (Hengge-Aronis, 2002). The  S factor is also essential for bacterial resistance to oxidative stress (Bang et al., 2005; Barth et al., 2009; Jangiam et al., 2010; Chiang and Schellhorn, 2012). During our experiments, temperature and pH were maintained at optimal levels in all treatments (including the control) and the cells were not exposed to radiations. As all the experimentations were performed under the same experimental conditions, we can assume that the observed up-regulation of rpoS in the treated cells is relative to an oxidative stress generated by the exposure to SWNT-COOH. 5. Conclusion Fig. 6. Epifluorescence microscope images representing the uniform distribution of dead cells (stained with PI) of V. splendidus (I) and V. gigantis (II), after 2 h of incubation with 0 (A), 50 (B) and 200 (C) ␮g mL−1 of SWNT-COOH.

Fig. 7. Relative expression of the four selected genes (rpoE, rpoS, dnaK and groEL) in V. splendidus after 2 h of incubation with 10 and 50 ␮g mL−1 of SWNT-COOH. *Significant difference (p < 0.05); **significant difference (p < 0.001).

4.2. Potential action mechanisms of SWNT on marine bacterial strains Previous studies have proposed that the antibacterial activity of CNTs was mainly associated with damages linked to a direct contact between CNT aggregates and bacterial cells (Kang et al., 2007, 2008a,b, 2009; Upadhyayula et al., 2009; Arias and Yang, 2009; Rodrigues and Elimelech, 2010; Vecitis et al., 2010; Chae et al., 2011; Pasquini et al., 2012). In the present study, the high majority of dead cells observed in V. splendidus and V. gigantis cultures after a 2 h exposure to SWNT-COOH were single cells (Fig. 6). These

Marine bacterial communities are key players in coastal marine ecosystems (Pomeroy, 2007; Azam and Malfatti, 2007) that are facing to multiple anthropogenic contaminants. Assessing the bacterial responses to NMs is essential to better understand the potential impact of CNTs on the natural microbial compartment. Our study demonstrates that the sensitivity of marine bacterial strains to SWNT-COOH exposure is strain-dependant, ranging for high sensitivity to resistance. On the four studied marine bacterial strains, the two Vibrio strains appeared sensitive to SWNT-COOH concentrations equal or higher than 50 mg mL−1 . The relative gene expression analysis indicated that SWNT-COOH affect these cells through oxidative stress rather than membrane damage. Furthermore, the toxicity of SWNT-COOH toward Vibrio strains does not seem to be through the formation of bacteria-SWNT-COOH aggregates. Our study highlights the complexity of dose–time–response relationships between marine bacterial strains and SWNT-COOH. Further studies are required to determine if the different responses of marine bacterial strains to SWNT-COOH exposure could lead to significant changing in natural bacterial community composition and functioning. Author’s contribution L.B. conceived and designed the study, carried out all the experiments and data analysis and wrote the manuscript. J.P. funded the study and contributed to the manuscript revision. E.P. prepared the working suspension of the COOH-SWNT, carried out both the TEM characterization and the elemental composition analysis of

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