Toxicity of titanium dioxide nanoparticles on Pseudomonas putida

Accepted Manuscript Toxicity of titanium dioxide nanoparticles on Pseudomonas putida R.G. Combarros, S. Collado, M. Díaz PII:

S0043-1354(15)30438-3

DOI:

10.1016/j.watres.2015.12.040

Reference:

WR 11738

To appear in:

Water Research

Received Date: 28 October 2015 Revised Date:

22 December 2015

Accepted Date: 23 December 2015

Please cite this article as: Combarros, R.G., Collado, S., Díaz, M., Toxicity of titanium dioxide nanoparticles on Pseudomonas putida, Water Research (2016), doi: 10.1016/j.watres.2015.12.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT 1

Toxicity of titanium dioxide nanoparticles on Pseudomonas putida

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Combarros, R.G. a, Collado, S. a, Díaz, M* a. a Department of Chemical and Environmental Technology. University of Oviedo, Spain. * Tel: 985 10 34 39. Fax: 985103434. E-mail: [email protected]

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The increasing use of engineered nanoparticles (NPs) in industrial and household

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applications will very likely lead to the release of such materials into the environment.

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As wastewater treatment plants (WWTPs) are usually the last barrier before the water is

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discharged into the environment, it is important to understand the effects of these

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materials in the biotreatment processes, since the results in the literature are usually

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contradictory. We proposed the use of flow cytometry (FC) technology to obtain

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conclusive results. Aqueous solutions of TiO2 (0-2 mgmL-1) were used to check its

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toxicity effect using Pseudomonas putida as simplified model of real sludge over room

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light. Physiological changes in P. putida from viable to viable but nonculturable cells

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were observed by flow cytometry in presence of TiO2. The damaged and dead cell

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concentrations were below 5% in all cases under study. Both FSC and SSC parameter

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increased with TiO2 dose dependent manner, indicating nanoparticles uptake by the

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bacteria. The biological removal of salicylic acid (SA) was also significantly impacted

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by the presence of TiO2 in the medium reducing the efficiency. The use of FC allows

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also to develop and fit segregated kinetic models, giving the impact of TiO2

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nanoparticles in the physiological subpopulations growth and implications for SA

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removal.

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Keywords: Antimicrobial activity; Flow cytometry; Metabolic activity; Pseudomonas

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putida; TiO2 nanoparticles; Wastewater treatment.

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1. Introduction

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Abstract

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ACCEPTED MANUSCRIPT TiO2 nanoparticles (TiO2-NPs) are effective opacifiers profusely used as pigments in

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paints, paper, inks, and plastics (Li et al. 2014). TiO2-NPs have chemical stability, high

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photocatalytic efficiency and low cost (Erdural et al. 2014). Apart from this, TiO2-NPs

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have recently become very important as catalyst in photodegradation processes aimed at

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removing biorecalcitrant and /or toxic pollutants from wastewaters or the inactivation of

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pathogens in water (Erdural et al. 2014; Ju-Nam and Lead 2008; Rawat et al. 2007).

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Due to this wide application in industries and human daily life, TiO2-NPs have been

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detected in soil, surface water, wastewater and sewage sludge samples, which raises

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concerns about their potential environmental impacts and their possible adverse effects

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on biological systems (Li et al. 2015; Li et al. 2014). For instance, TiO2-NPs were found

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to adsorb onto activated sludge in conventional biological nutrient removal systems of

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WWTPs (Li et al. 2015). Nevertheless, the bibliography reports that the factors which

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might have an impact on interactions between the different types of nanomaterials and

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biomolecules have remarkably increased, but the knowledge about them is not yet

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(Ghosh et al. 2012; Kumar et al. 2011).

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There have been many contradictory reports about the biocompatibility and

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antimicrobial activity of TiO2-NPs. Recently, Kapri et al. (2009) and Anil and Zaidi

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(2010) showed that a bacteria consortium grew faster and to a higher optical density

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when nanoparticles of nanobarium titanate were added to a bacterial consortium at 0.1

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mgmL-1. On the other hand, several researchers indicated a strong antibacterial activity

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of TiO2-film on E. coli either with irradiate light or sunlight (Bonnefond et al. 2015;

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Cho et al. 2004; Erdural et al. 2014; Gelover et al. 2006). Erdural et al. (2014)

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demonstrated that the survival rate of suspended E. coli decreased from 100% to 30% in

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just 60 minutes, when the bacteria were in contact with TiO2-film in an irradiated light

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ACCEPTED MANUSCRIPT process; nevertheless, no antibacterial activity was found in the dark process under the

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same conditions.

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The precise mechanism for bacterial toxicity of TiO2-NP is still being elucidated, but

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the possibilities include free metal ion toxicity and oxidative stress via generation of

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reactive oxygen species (ROS) (Besinis et al. 2014; Bonnefond et al. 2015; Cho et al.

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2004; Erdural et al. 2014). The cell inactivation has frequently been related to inhibition

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of cell respiration, decomposition of the lipopolysaccharide layer, DNA damage,

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genotoxicity, release of toxic constituents and structural disarrangement of the

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cytoplasmic membrane due to lipid per oxidation. (Besinis et al. 2014; Erdural et al.

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2014; Ju-Nam and Lead 2008; Li et al. 2014; Neal 2008).

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Many of these works about effects of TiO2-NPs on microbial activity almost always

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based their conclusions on microorganism death, which is usually measured by means

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of PI staining and confocal microscopy (Le et al. 2014), drop-test based on measuring

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the CFU/mL (Anil and Zaidi 2010; Bonnefond et al. 2015; Cho et al. 2004; Erdural et

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al. 2014; Le et al. 2014), or optical density (Anil and Zaidi 2010; Besinis et al. 2014).

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However, after an in-depth bibliographic review, new techniques, such as flow

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cytometry (FC), which can not only measure the bacterial death but also the reduction in

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the activity, have not been implemented for this purpose yet. The use of FC provides

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additional information to the studied so far, where only growth suppression and loss of

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bacterial viability as CFUmL-1 and OD were described (Bonnefond et al. 2015; Cho et

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al. 2004; Erdural et al. 2014; Fu et al. 2005; Kapri et al. 2009). In addition, most of

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these researches has been conducted with well-known model organisms, such as

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Escherichia coli (Bonnefond et al. 2015; Cho et al. 2004; Erdural et al. 2014; Fu et al.

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2005; Kumar et al. 2011; Rincón and Pulgarin 2007)), with TiO2-NPs usually forming

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ACCEPTED MANUSCRIPT part of thin film deposit over different surfaces (Bonnefond et al. 2015; Erdural et al.

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2014; Gelover et al. 2006).

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However, information about their impact on wastewater biological treatments is very

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scarce (Li et al. 2015; Li et al. 2014). In fact, as far is known, there are not previous

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studies dealing with the effect of TiO2-NPs on Pseudomonas putida, even when these

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bacteria are the predominant ones in activated sludge (Collado et al. 2014) and the

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results obtained can be employed as a reference during the comparison with those from

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other works using mixed cultures. Moreover, the use of a pure culture instead of a

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mixed sludge accelerates and amplifies the response of the system to perturbations

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(Collado et al. 2014).

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Taking into account the aforementioned considerations, the aim of the present work is

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to evaluate, for first time, the effects of TiO2-NPs on the viability and activity of P.

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putida. For this purpose, the evolution of the physiological status of P. putida when is

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exposed to different suspended TiO2-NPs concentrations was studied by means of flow

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cytometry, distinguishing three metabolic states: viable, damaged and dead cells.

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2. Materials and methods

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2.1. Preparation of TiO2 nanoparticle suspension

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The TiO2-NPs used in this study were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). According manufacturer data, mean nanoparticle size is less than 25 nm, and the

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purity is 99.5 %. Stock solution (50 gL-1) was prepared in sterile Milli-Q water and

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sonicated (35 kHz frequency, Fisherbrand FB 11010, Germany) for 1 h to disperse the

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materials (Besinis et al. 2014; Li et al. 2015; Li et al. 2014; Zucker et al. 2010).

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2.2. Cell preparation

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ACCEPTED MANUSCRIPT Pseudomonas putida (Leibniz Institute DSMZ-German Collection of Microorganism

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and Cells Cultures, Germany (DSM 4478)) was chosen. A detailed description of cell

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preparation procedure can be found in a previous work (Combarros et al. 2014). Stated

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briefly, P. putida colonies were inoculated in a growth medium and incubated at 30 ºC

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and 150 rpm for 16 h. Afterwards, an aliquot was centrifuged and washed at 10160 x g

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for 10 minutes, being then used as an inoculum for subsequent experiments.

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2.3. Cell viability test

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The effects of TiO2-NP on the activity and viability of P. putida were studied using a

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synthetic industrial wastewater, composed by a minimum mineral medium which was

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supplemented by 500 mgL-1 of salicylic acid (SA), in order to simulate an moderately

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biodegradable pharmaceutical wastewater. Additionally, some experiments were carried

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out in the growth medium recommended for P. putida by the supplier, with the aim of

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having a preliminary knowledge about TiO2-NPs toxicity on P. putida under optimal

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medium conditions. The detailed composition of synthetic industrial wastewater and

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growth medium can be consulted in table S1 (see supplementary data).

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For both media, TiO2 -NPs concentrations selected were 0, 0.1, 0.5, 1 and 2 mgmL-1.

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The stock dispersion diluted with the growth or the simulated industrial medium above

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at neutral pH, dispersions were sonicated for 1 h prior to assay to ensure dispersion of

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the particles (Besinis et al. 2014). Typical TiO2-NPs concentrations reported in

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bibliography for other toxicity studies usually range from several mgL-1 to 1 gL-1 (Cho

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et al. 2004; Le et al. 2014; Li et al. 2015; Rincón and Pulgarin 2007).

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For the growth medium recommended by the supplier (GM), the culture conditions

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were 200 rpm and 30 ºC in 250 mL Erlenmeyer flasks containing 100 mL of simulated

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medium (Combarros et al. 2014). While, for the simulated industrial wastewater (SIW),

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ACCEPTED MANUSCRIPT the culture conditions were 250 rpm and 30 ºC and 100 mL of simulated medium in 500

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mL Erlenmeyer flasks (Combarros et al. 2015a). The higher volume and stirring speed

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selected in the case of SIW were chosen in order to improve the adaptation and growth

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of bacteria in this medium. In all cases, initial P. putida concentration was around 107

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CFUmL-1. Once P. putida was inoculated in the medium, contact TiO2-NP-bacteria was

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maintained for 24 h in an incubator New Brunswick Scientific (Edison, New York) that

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kept constant the culture conditions, taking samples at different times. For each one of

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them, optical density (DO), CFUmL-1, pH, SA concentration and metabolic viability

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and activity were analysed.

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Additionally, experiments with different TiO2-NP concentrations (0, 0.1, 0.5, 1 and 2

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mgmL-1) but without bacteria were also carried out with the aim of discarding possible

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interactions between TiO2-NPs and SA.

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All material was autoclaved at 121ºC for 20 min in order to maintain the sterility of the

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system. The TiO2 NPs and SA stock solutions were sterilized separately.

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2.4. Flow cytometry analysis

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2.4.1. Staining procedures

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The stock solutions preparation and staining procedures have been described in

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(Combarros et al. 2015b). The samples were stained using SYBRgreen (SYBRgreen;

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Invitrogen) and Fluorescent microspheres (Perfect Count; Cytognos, Spain) to obtain

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the total count of cells. Additionally, a dual-staining procedure (cFDA/PI) was used to

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evaluate the bacterial physiological status: carboxyfluorescein diacetate (cFDA) and

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propidium iodide (PI) are indicators of esterase activity and membrane integrity,

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repectively (Amor et al. 2002; Díaz et al. 2010; Quirós et al. 2007; Quirós et al. 2009)

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ACCEPTED MANUSCRIPT 2.4.2. Multi-parameter flow cytometry

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Flow cytometry measurements were performed using a Cytomics FC 500 flow

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cytometer (Beckman Coulter) equipped with a 488 and 633 nm excitation light source

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from an argon ion laser. Green fluorescence from samples (corresponding to cFDA or

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SYBRgreen-stained cells) was collected on the FL1 channel (530 nm), whereas PI

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fluorescence was registered on the FL3 channel (610 nm). Each analysis was performed

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in duplicate at a low flow rate setting (4000 events s-1). Data acquisition was carried out

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using Cytomics RXP software (Beckman Coulter). Gates and quadrants were

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established according to staining controls. For cFDA/PI dual-parameter, data collected

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from 200000 events, were analysed using Kaluza® Flow Analysis Software, Beckman

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Coulter. The fluorescent microspheres presented two types, A microspheres, excitable at

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506 nm and B microspheres, excitable between 365 and 650 nm.

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2.5. Analytical methods

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Concentration of SA in the samples were measured by HPLC (Agilent 1200) using a

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Mediterranea Sea18 column (5 µm x 25 cm x 46 cm, plus a reverse-phase column from

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Waters) combined with a UV detector. The wavelength used for detection of SA was

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290 nm. The mobile phase consisted in a mixture of two solutions: acetonitrile and 0.4

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% phosphoric acid in water (Hsu et al. 2004), employing a binary gradient at a constant

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flow rate of 1 mLmin-1.

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Number of culturable bacteria (expressed as CFUmL-1) was determined by the standard

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counting method by plating, obtaining viable counts. The evolution of total biomass was

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determined by means of two techniques: by optical density measurement at 660 nm

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(Shimadzu, UV-vis 1203) and by SYBR staining- flow cytometry. In the first case,

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TiO2-NPs absorbance was corrected using control samples containing only NPs (Kuang

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ACCEPTED MANUSCRIPT et al. 2013). Viable and total cells were finally expressed in gL-1 taking into account the

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calibration curves previously obtained for viable or total cells stained by SYBRgreen

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(cellmL-1) versus dry weight (gL-1), respectively.

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Physiological status of bacteria were established by means of a multiparameter flow

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cytometry using a previous dual PI/cFDA staining differentiating metabolically active

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cells as cFDA stained, dead cells as PI stained and damage cells as PI-cFDA stained.

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Finally, the cell size and cell complexity were monitored by measuring parameters FSC

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and SSC respectively by FC.

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All experiments were carried out at least in duplicate and each sample was analysed in

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triplicate. Standard deviations (SD) obtained are shown as vertical lines in figures.

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3. Results and discussions

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3.1. Antibacterial activity of TiO2-NPs

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Before starting the experiments, it was observed that SA concentration did not change

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after 24 hours in contact with TiO2-NPs and in absence of P. putida, thus precluding

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any side reactions between TiO2-NPs and SA (data not shown). Antibacterial test were

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performed in room light with either growth medium or synthetic wastewater (Fu et al.

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2005). Bacterial viability experiments in dark were not carried out since previous

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studies demonstrate that TiO2-NPs have no antibacterial effect in dark (Erdural et al.

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2014; Fu et al. 2005; Rincón and Pulgarin 2007). Moreover, Li et al. (2014) discovered

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that the levels of TiO2-dependent inhibition of biological nitrogen removal were similar

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under both dark and light conditions.

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3.1.1. Growth medium (GM)

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ACCEPTED MANUSCRIPT Firstly, in order to get a preliminary estimation of the effect of TiO2-NPs on the growth

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and physiological status of P. putida, this bacterium has been exposed to different

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concentrations of nanoparticles in the growth medium recommended by the supplier.

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Before inoculation, the system TiO2-NPs-GM was sonicated to ensure de dispersion of

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nanoparticles. The prepared TiO2-NPs suspension (0, 0.1, 0.5, 1 or 2 mgmL-1) in GM

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were inoculated with around 107 CFUmL-1 of P. putida and incubated for 24 h at 200

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rpm and 30ºC. In all cases, the initial pH of the media was adjusted to a value of 7.

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Figure 1a shows the evolution of P. putida in presence of different concentrations of

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TiO2-NPs obtained by means flow cytometry.

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The results of total cells obtained by flow cytometry (Fig. 1a) reveal a clear negative

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effect of TiO2-NPs on the P. putida growth for concentrations higher than 0.5 mgmL-1.

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Therefore, for TiO2-NPs lower than 0.5 mgmL-1, P. putida showed a fast growth during

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the first five hours, until forty times the initial bacterial concentration, and then

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gradually decreases, probably due to the depletion of the GM. However, P. putida did

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not followed this trend for TiO2-NPs concentrations higher than 0.5 mgmL-1. In such

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cases, the higher the TiO2-NPs concentration, the lower the initial P. putida growth.

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Moreover, the final gradual decrease in bacterial population observed for low TiO2-NPs

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concentrations after the 5th hour of cultivation is hardly noticeable. Obviously, the lower

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the bacterial population, the higher the time required for the complete depletion of

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medium is.

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Specific growth rates (µ) for P. putida were calculated for every TiO2-NPs

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concentrations, getting a value of 0.67 ± 0.01 h-1 for TiO2-NPs concentration equal to or

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lower than 0.5 mgmL-1, whereas values of 0.501± 0.001 and 0.45± 0.01 h-1 were

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calculated for 1 and 2 mgmL-1 of TiO2-NPs, respectively. When comparing these results

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ACCEPTED MANUSCRIPT with those reported for E. coli, it can be concluded that P. putida shows a higher

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tolerance to TiO2-NPs than E. Coli. Thus, Fu et al. (2005) demonstrated that a 0.40

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mgmL-1 TiO2-NPs suspension inhibits effectively the E. coli growth.

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Figure 1b shows the FSC and SSC parameters depending on TiO2-NPs concentration

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present in the medium. The intensities of FSC and SSC are proportional to the size of

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cells and the intracellular density, respectively. Although both parameters increased

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with the TiO2-NPs concentration, this increase was more marked for SSC than for FSC.

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So the FSC or SSC signals for the culture with the TiO2-NPs highest concentration (2

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mgmL-1) were around 2 or 13 times higher than those values obtained for cultures

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without nanoparticles, respectively. Moreover, both parameters are dose-dependent but

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not time dependent, that is, the increases in their values occur during the first minutes of

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reaction, then remaining constant (see Fig S2). Others researches such as Kumar et al.

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(2011) and Zucker et al. (2010) have observed the SSC increased sequentially while the

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FSC decreased in response to TiO2-NPs concentration with E. Coli and ARPE-19 cells,

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respectively, presumably due to substantial light reflection by the TiO2 particles

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(Toduka et al. 2012; Zucker et al. 2010).

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Several authors reported that the changes in intensity of SSC signal are related to the

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internationalization of NPs by the bacterium (Ghosh et al. 2012; Kumar et al. 2011).

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Thus, it seems reasonable to conclude that P. putida shows a fast uptake of TiO2-NPs.

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Regarding the increase in FSC signal observed, it could be due to the agglomeration of

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NPs (Ghosh et al. 2012).

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3.1.2. Simulated industrial wastewater (SIW)

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The effects of different TiO2-NPs concentrations on the growth and activity of a P.

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putida population are also evaluated, but using in this case a SIW. This wastewater

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ACCEPTED MANUSCRIPT consists of a minimum mineral medium to which 500 mgL-1 of SA was added, in order

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to simulate a moderately biodegradable pharmaceutical wastewater. Figure 2 shows the

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evolution on the main parameters under these conditions, such as the bacterial growth or

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the specific SA removal rates.

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Although several studies have been focused on the toxicity of NPs on bacteria, the

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effect of NPs on their pollutant-degrading activity has hardly been studied. As can be

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seen in figure 2a, the presence of TiO2-NPs in the SIW involves a reduction in the SA

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removal rate of P. putida. For instance, reductions in SA concentration of 86%, 64%,

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46%, 43% and 27 % were observed after 11 hours of cultivation in presence of 0, 0.1,

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0.5 1 and 2 mgmL-1 of TiO2-NPs, respectively.

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The evolutions of SA concentrations were successfully fitted to a pseudo first reaction

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order model (C=C0·e(-k·t)), obtaining the pseudo first order kinetic constant (k) for each

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TiO2-NPs concentration. The relation between the k value and TiO2-NPs concentration

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in the medium is represented by the following equation: k h

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= 0.20 + 0.040 · TiO

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mgmL r = 0.95

Figures 2b and 2c shows the bacterial growth measured as optical density or as colony

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forming units, respectively. Both parameters followed the same trend, that is, a fast

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increase in their value during the first hours of cultivation, then remaining

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approximately constant. It can also been clearly seen in these figures that the presence

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of TiO2-NPs in the SIW reduced the P. putida growth, this effect being significant even

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for low concentrations of nanoparticles. So, the bacterial growth dropped by half with

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only 0.1 mgmL-1 and the growth of the bacteria was almost completely inhibited for

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TiO2-NPs concentrations in SIW higher than 0.5 mgmL-1. This trend is better observed

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when the specific growth rates (µ) are calculated for each concentration of

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ACCEPTED MANUSCRIPT nanoparticles. The µ values obtained from CFU data were 0.41 (r2=0.82), 0.26

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(r2=0.70), 0.08 (r2=0.99), 0.07 (r2=0.85), and 0.01 (r2=0.93) for 0, 0.05, 0.1, 0.25, 0.5

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and 1 mgmL-1 of TiO2-NPs, respectively. The relation between the µ value and TiO2-

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NPs concentration in the medium can be represented by the following equation: μ h

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mgmL r = 0.87

Comparing these results with those previously obtained in GM, it can also be pointed

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out that, as expected, the inhibitory effect of TiO2-NPs on the P. putida growth is more

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marked when the availability of carbon or nitrogen is limited. Finally, the specific

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degradation rates are shown in Fig. 2d; presented a maximum value at around 3 hours,

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this trend was similar with all TiO2-NPs concentrations.

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In regards to flow cytometry measurements in presence of different concentrations of

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TiO2-NPs, figure 3 plots the evolution of P. putida subpopulations, that are, total

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(SYBR+), viable (as CFU), viable but not culturable (VBNC) (cFDA+ less CFU), active

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(cFDA+), damaged (PI+ cFDA+) and dead (PI+ cFDA-) bacteria. A more detailed

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description about the gates selected for each subpopulation can be found in SI, Fig. S3.

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At this point, it is important to point out that a study about the fluorescence emitted by

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the TiO2 itself or by its interactions with the dyes used (SYBRgreen, PI, cFDA) was

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previously carried out (data no shown). Results indicated that the fluorescence emitted

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by TiO2-NPs either alone or in presence of fluorochromes, was always lower than 5%,

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so that, this fluorescence was not considered as significant. Therefore, interferences of

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TiO2-NPs with the flow cytometry procedure were discarded.

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Results of total cell concentration by flow cytometry are in accordance with those

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obtained by means of OD and plate counting. So, the presence of small amounts of

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TiO2-NPs in the SIW involves a significant reduction in the P. putida growth. So, flow

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ACCEPTED MANUSCRIPT cytometry

measurements

indicated

maximum

total

bacterial

populations

of

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(1.7·±0.4)·108 , (1.4 ±·0.1)·108 , (9.2 ± 2.8)·107 , (6.8 ±·0.2)·107 and (3.3 ±·0.5)·107

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cells mL-1 after 3 hours of cultivation with 0, 0.1, 0.5, 1 and 2 mgmL-1 of TiO2-NPs,

290

respectively (Fig. 3a). Total bacteria concentrations at zero time for all experiments

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were around 107 cellmL-1. Concentration of P. putida subpopulation in percentage and

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cells·mL-1 are shown in Figs. 3b-3f for 0, 0.1, 0.5, 1 and 2 mgmL-1 TiO2 concentration,

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respectively. In order to observe better the trends of the subpopulations, the figures S4

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and S5 show each subpopulation (non-damaged, damaged and dead cells (S4) and

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actives, viable and VBNC cells (S5)) with time under different TiO2-NPs

296

concentrations.

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The decrease in total cell concentrations observed in figure 3a for high times indicates

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that dead bacteria are rapidly lysed. This trend can be also observed in the evolution of

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non-damaged subpopulation (PI-) (see Fig. S4a). So, the higher the TiO2-NPs

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concentration, the lower the number of bacteria that have not lost their membrane

301

integrity. However, when non-damaged cells are expressed as a proportion of total cells

302

(see Fig. S4b), it is noteworthy that the higher percentages were obtained for the higher

303

TiO2-NPs concentrations. These results suggest that a high TiO2-NPs concentration

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decreases the number of total P. putida, but the surviving bacteria show a better

305

physiological status. In other words, the presence of TiO2-NPs in the SIW reduces the

306

number of total bacteria but also has an effect of screening, allowing obtaining a final

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population composed almost entirely for non-damaged bacteria (99% for 2 mgmL-1 of

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TiO2-NPs in front of 95% in absence of nanoparticles). These results are in accordance

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with the evolution of damaged and dead subpopulations (Figs. S4c-d and S4e-f,

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respectively). As can be seen in both figures, either the percentages of dead or damaged

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cells are lower when the highest concentrations of nanoparticles were used. It can be

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ACCEPTED MANUSCRIPT also pointed out that both subpopulations never exceeded the 5% of the total population,

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which corroborates the fast lysis of the dead cells observed in figure 3a. It is also

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remarkable that for TiO2-NPs concentrations lower than 0.1 mgmL-1, the presence of

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nanoparticles increases the percentage of damaged bacteria, whereas concentrations

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higher than this value has the opposite effect, that is, the percentage of cells that have

317

lost their membrane integrity decreased. In fact, the proportion of damaged cells in the

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SIW with 2 mgmL-1 of TiO2-NPs was one order of magnitude lower than in absence of

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nanoparticles. In the light of these results, it seems reasonable to propose that P. putida

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in presence of TiO2-NPs concentrations higher than 0.1 mgmL-1 are transformed into

321

dead cells, which are rapidly lysed, with a small population of non-damaged bacteria

322

remaining at the end of the experiment. PI cells stained discrimination in E. coli

323

exposed to TiO2 NPs using FC was carried out by Kumar et al. (2011), obtaining values

324

of dead cells between 3.5-10% with TiO2 concentrations of 10-80 µgmL-1. However,

325

Zucker et al. (2010) observed that TiO2-NPs showed a minimal decrease in cell viability

326

(2%) in human-derived retinal pigment epithelial cells, at the highest concentration

327

tested (30 µgmL-1), this being measured as PI membrane permeability

328

The impact of TiO2-NPs in activity and culturability of P. putida can be seen in Fig.

329

S5a-d. At this point, it is important to emphasize that active P. putida were defined as

330

those stained by cFDA, whereas viable cells were measured by plate counting (Quirós

331

et al. 2007; Quirós et al. 2009). Again, the higher concentration of nanoparticles, the

332

lower the active or culturable subpopulations. Nevertheless, when the viability results

333

are expressed as percentage of total cells, it should be noted that the lower proportion of

334

viable bacteria were obtained not with 2 mgmL-1 , but with concentrations between 0.1

335

and 0.5 mgmL-1, as in the case of the percentage of damaged cells. This behavior is not

336

unexpected because both parameters are related to one another: damaged bacteria have

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ACCEPTED MANUSCRIPT lost their membrane integrity and are supposed to be uncapable of showing reproductive

338

growth (Ziglio et al. 2002). The evolution of viable but non-culturable subpopulation,

339

calculated as the active minus the viable subpopulations, also corroborates this

340

assertation (Fig.S5e-f). It should be also noted that nearly all the total population is

341

active but only around one third is culturable when high concentrations of TiO2–NPs are

342

used. It is well known that plate-culturing techniques only reveal a small proportion of

343

the total microbial population (Giraffa 2004) For instance, several authors have reported

344

that it was only possible to recover 5-15% of the total bacteria in sludge, regardless the

345

optimization of the cultural medium (Hammes et al. 2008; Ziglio et al. 2002). Finally, it

346

was also observed that the addition of increasing amounts of TiO2-NPs did not really

347

correspond to an increased in cell death rate but accelerated the transition to VBNC

348

states. SA was consumed in all assays, showing the metabolic activity associated with

349

the VBNC state. As it was previously observed (Quirós et al. 2007; Quirós et al. 2009),

350

VBNC maintain the transport and biosynthesis systems, and are able to metabolize

351

substrates. In our experiments, the cFDA fluorescence intensity was maintained during

352

overall bacterial growth, so it can be considered that bacterial VBNC cells were able to

353

preserve the whole enzymatic activity (Quirós et al. 2007). This can be clearer observed

354

in supplementary material, Fig. S6, where the SA removal rate per cell is represented.

355

Therefore, the similar specific rates observed for all the TiO2-NPs concentrations

356

confirms that each non- dead bacterium maintained its level of activity.

357

FSC and SSC were monitored during all SA removal time; both parameters increased

358

with TiO2 concentration and remained constant with time as in the case of GM (data no

359

shown).

360

3.2. Segregated kinetic model : proposal and discussion.

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ACCEPTED MANUSCRIPT 361

In orden to quantify the SA removal of VBNC cells, experimental data for cell

362

subpopulations and SA uptake were fitted to a segregated kinetic model (Quirós et al.

363

2007; Quirós et al. 2009). For this model, the following assumptions were made: •

The total biomass, formed by viable (CV), viable but non-culturable plus

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damaged cells (CN) and dead cells (CD), were expressed as bacterial

366

concentrations (gL-1), using the corresponding calibration curves determined

367

previously (dry weight (g L-1) = 8.17·10-10·cells concentration (cells mL-1).. •

plates.

369

370

Viable cells are the cell subpopulation which shows the ability to grow on

•

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Both viable and nonculturable fractions can lose their integrity, which results in the cellular death. VBNC cells are either not able to recover their

372

“culturability”.

373

•

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The SA removal was conducted by CV and CN

Taking into account the following considerations, the segregated model showed in

375

Fig.4a can be proposed. Based on this scheme, equations 1 to 6 can easily be obtained,

376

where r1 and r5 are the cell growth rates of viable and VBNC cells, r2 is the

377

transformation rate from viable to VBNC state, r3 and r4 are cell death rates of viable

378

and VBNC cells, τ´ and τ´´ are the inverses of viable and VBNC cell concentrations in

379

stationary phase, and qV and qN are the specific SA uptake rates for viable and VBNC

380

subpopulations, respectively.

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r1 = k1·C V ·(1 - τ ´·C V

382

r2 = k 2 ·C V

)

(1) (2)

16

ACCEPTED MANUSCRIPT 383

r3 = k 3 ·C N

(3)

384

r4 = k 4 ·C V

(4)

385

r5 = k 5 ·C N ·(1 - τ ´´·C N

386

−

(5)

d SA = qV ·CV + q N ·C N dt

(6)

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Equations 1-6 were fitted to experimental data from cell counting by flow cytometry

388

and plate counting using MicroMath Scientist® version 2.0 . Fig. 4 shows experimental

389

and predicted data for P. putida growth curves and SA uptake with different

390

concentrations of TiO2-NPs. The value of the different kinetic parameters calculated for

391

the proposed segregated kinetic model are collected in Table 1.

392

The model considering the growth of both viable and VBNC subpopulations provided a

393

proper description of bacterial populations, as well as carbon source uptake (Quirós et

394

al. 2007).

395

As can be seen in table 1, the growth kinetic constant for viable bacteria is higher than

396

their transformation kinetic constant into non-culturable cells (k1>k2) for the experiment

397

without nanoparticles. In addition, these non-culturable bacteria are mainly generated

398

from division of others non-culturable bacteria, instead of from the viable cells which

399

has loosen their capacity to growth in plate (k5>k2). The increase in the TiO2-NPs

400

concentration leads to higher values of k5 and k2 and lower values of k1. These findings

401

indicate that nanoparticles had a negative effect on the viable subpopulation growth (k1)

402

but a positive effect on the viable but non culturable cells formation rate, increasing

403

their generation beginning from either the own multiplication (k5) or the culturability

404

loss of viable cells (k2). In addition, when are compared with the others constants, the

405

values of k3 and k4 were no significant, indicating that death reactions were a lot slower

406

than the rest of the proposed ones.

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ACCEPTED MANUSCRIPT Finally, qV and qN values can be used to discuss the effects of TiO2-NPs on the P. putida

408

capacity to degrade SA. In absence of nanoparticles, the specific degradative capacity

409

for the viable subpopulation was far higher than for the viable but non-culturable

410

subpopulation. This suggests that the entrance of viable cells into the VBNC state can

411

be also accompanied by a reduction in substrate transport and metabolic activity levels

412

in order to minimize cellular energetic requirements (Bogosian and Bourneuf 2001;

413

Quirós et al. 2007; Quirós et al. 2009). A similar behaviour has been observed for non-

414

culturable Lactobacillus higardii in response to SO2 presence (Quirós et al. 2009). The

415

presence of TiO2-NPs reduced appreciably specific SA uptake rates for viable and

416

VBNC subpopulations. So, qv in the SIW ranged from 0.9 gSA(h gCV)-1 in absence of

417

nanoparticles to 0.6 gSA(h gCV)-1 when the concentration was 2 mgmL-1. Interestingly,

418

the qV/qN ratio kept approximately constant, at a value close to 12.7, suggesting that the

419

inhibitory effect of the nanoparticles is similar for both subpopulations.

420

4. Conclusions

421

In this work, FC technique allows the study of the TiO2-NPs effect on the physiological

422

state of the P. putida. The increase of TiO2 concentrations meant a decrease of the total

423

cell concentrations in the medium, a change in the physiological state of the bacteria

424

was also observed since viable cells (CFUmL-1) became VBNC cells. However,

425

significant increases in the damaged or dead cells concentration were not produced.

426

Using FC technique, FSC and SSC parameters were also measured, obtaining, in this

427

case, an increase of both parameters with higher TiO2 concentration, which is due to

428

NPs uptake in bacteria

429

The viable cells transformation into VBNC cells affects the SA biodegradation since the

430

metabolic capacity of VBNC cells is generally lower than that of viable cells. Finally, a

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ACCEPTED MANUSCRIPT segregated model including viable, VBNC and dead bacteria was fitted to the

432

experimental data obtaining the growth and removal constants for each subpopulation

433

5. Acknowledgements

434

R. G. Combarros wishes to express gratitude for a research grant from the Government of the

435

Principality of Asturias (Severo Ochoa Programme).

436

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ACCEPTED MANUSCRIPT Zucker RM, Massaro EJ, Sanders KM, Degn LL, Boyes WK (2010) Detection of TiO2

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nanoparticles in cells by flow cytometry Cytometry Part A 77A:677-685

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doi:10.1002/cyto.a.20927

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ACCEPTED MANUSCRIPT Table 1: Kinetics parameters obtained for the segregated kinetic model proposed for P. putida growth and salicylic acid consumption in presence of TiO2-NPs concentrations ranging from 0 to

k3 (h-1) 1.2·10-6 1.4·10-6 3.3·10-6 3.7·10-6 1.5·10-6

k4 (h-1) 3.0·10-7 1.9·10-7 3.3·10-7 1.4·10-7 1.6·10-7

k5 qV (h-1) gSA(h gCV)-1 0.027 0.95 0.049 0.8 0.052 0.73 0.069 0.67 0.025 0.6

τ' qN gSA(h gCN)-1 (Lg-1) 0.070 14.5 0.065 22.5 0.061 35.7 0.054 50.6 0.045 40.9

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k2 (h-1) 0.009 0.035 0.043 0.054 0.032

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k1 (h-1) 0.047 0.045 0.037 0.031 0.041

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τ'' (Lg-1) 11.8 15.3 22.4 28.0 25.7

ACCEPTED MANUSCRIPT

a) 30 20 10

1.5

12

1

8

0.5

4

0

0 0

4

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12 16 Time (h)

20

24

0

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FSC·FSC0-1

40

0.5 1 1.5 TiO2 (mgmL-1)

0

2

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Fig. 1: a) Evolution of total P. putida bacteria in GM in presence of different concentrations of suspended TiO2- NPs: 0 mgmL-1 (▲), 0.1 mgmL-1 (■), 0.5 mgmL-1 (•),

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1 mgmL-1 ( ), 2 mgmL-1 ( ). b) Effect of suspended TiO2- NPs concentrations on the FSC (▲) and SSC (•) signals after 8 h of P. putida cultivation in GM. In all cases: initial SA concentration of 100 mgL-1, 200 rpm, 30ºC, P. putida inoculum of 107 CFUmL-1,

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ACCEPTED MANUSCRIPT 3.5

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2.5 0.6

OD·OD0-1

2

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0.4 0.2

1

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0

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24

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0 0

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Fig. 1: Evolution of salicylic acid concentration (a), growth of Pseudomonas putida as

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OD (b) or colony forming unit (c) and specific degradation rate of salicylic acid (d) under different TiO2-NPs concentrations in SIW (▲ 0 mgmL-1, ■ 0.1 mgmL-1, • 0.5 mgmL-1,

2 mgmL-1). In all cases: Initial SA concentration of 500 mgL-

, 250 rpm, 30ºC, P.putida inoculum of 107 CFUmL-1, 100 mL in 500 mL Erlenmeyer

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24

ACCEPTED MANUSCRIPT

A 8

100%

12 16 Time (h)

20

80% 1E+7 60% 1E+6

40%

C 0

2

11

24

1E+6

40%

1E+5

20% 0

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11

24

1E+8 1E+7 1E+6 1E+5

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D 2

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1E+4 11

24

100%

1E+8

80%

1E+7

60% 1E+6

40%

1E+5

20%

F

0% 0

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1E+4 24

Fig. 1: Total cells concentration (a) (▲ 0 mgmL-1, ■ 0.1 mgmL-1, • 0.5 mgmL-1, mgmL-1,

1E+4

24

40%

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60%

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5 8 Time (h)

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0%

B

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1E+5

20% 0%

X (cells·mL-1) % Cell subpopulation

0

1E+6

40%

1

2 mgmL-1) and evolution of different P. putida subpopulation during SA

removal with TiO2-NPs concentration of 0 mgmL-1 (b) 0.1 mgmL-1 (c), 0.5 mgmL-1 (d), 1 mgmL-1 (e) and 2 mgmL-1 (f). Subpopulation of VBNC cells, dead cells

X (cells·mL-1)

1

1E+7 60%

X (cells·mL-1)

2

80%

viable cells

and

are plotted as per centage of the total cells counts on the principal y-axis.

Metabolically active ( ), viable cells (■), dead cells (•) and damaged cells (▲) are expressed as cellsmL-1 on the secondary y-axis. In all cases: Initial SA concentration of

X (cells·mL-1)

3

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% Cell subpopulation

% Cell subpopulation

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X (cells·mL-1) % Cell subpopulation

Total cells·Total cells0-1

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ACCEPTED MANUSCRIPT

1E+6

CD

1E+5

r4

1E+7

1E+6

0.2

4

8 Time (h)

1E+8

12

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1E+5 0

4

EP

0.2

8 Time (h)

AC C

X (cell·mL-1)

1E+8

0 12

SA (g·L-1) X (cell·mL-1)

X (cells·mL-1)

0

c)

1E+8

0.2

1E+7

0.4

1E+6

0.2

1E+5

0 0

4

8 Time (h)

12

Fig. 1: Segregated kinetic model proposed (a) and evolution of VBNC (▲), viable (■) and dead cells (•)) and SA concentration ( ) during the growth of P putida in a SIW and in presence of different concentrations of TiO2-NPs 0 mgmL-1 (b) 0.1 mgmL-1 (c), 0.5 mgmL-1 (d), 1 mgmL-1 (e) and 2 mgmL-1 (f). In all the cases: Initial SA concentration of 500 mgL-1, 250 rpm, 30ºC, P.putida inoculum of 107 CFUmL-1, 100 mL in 500 mL Erlenmeyer flask.

SA (g·L-1)

r3

0.4

SA (g·L-1)

CN

1E+7

SA (g·L-1)

CV

0.6

RI PT

SA

r2

b)

1E+8

X (cells·mL-1)

r1

a)

SC

r5

ACCEPTED MANUSCRIPT Highlights SA degradation is reduced by 22% in presence of 0.1 mgmL-1 of TiO2-NPs Flow cytometry technique reveals TiO2 effects on bacterial viability and activity. TiO2-NPs uptake in bacteria, since FSC and SSC increased with the NPs concentration

RI PT

Bacterial growth completely inhibited for 0.5 mgmL-1 of TiO2-NPs in SIW

Change of physiological state of P. putida from VC into VBNC in presence of TiO2-

AC C

EP

TE D

M AN U

SC

NPs