Yige Yan

Charline Soraru

Valérie Keller

Nicolas Keller

Lydie Ploux

Antibacterial and biofilm-preventive photocatalytic activity and mechanisms on P/F-modified TiO 2 coatings

Keywords: Antibacterial P/F-modified TiO2 coating, photocatalytic activity, immediate versus long-term biofilm-preventive activity, mechanism, surface topographic properties

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Introduction

Surface adhesion of bacteria and formation of biofilm [START_REF] Ploux | Bacteria/material interfaces: role of the material and cell wall properties[END_REF] present health threat in many instances such as biomedical field, 2 food industry, [START_REF] Capita | Antibiotic-resistant bacteria: a challenge for the food industry[END_REF] water distribution [START_REF] Liu | Understanding, monitoring, and controlling biofilm growth in drinking water distribution systems[END_REF] and wastewater treatment. [START_REF] Rizzo | Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: A review[END_REF] Apart from conventional physico-chemical surface treatments, an alternative method for reducing frequency and level of surface contamination has been proposed in the last two decades: the selfdisinfecting surfaces. [START_REF] Tsibouklis | Preventing bacterial adhesion onto surfaces: the low-surface-energy approach[END_REF][START_REF] Weber | Self-disinfecting surfaces: review of current methodologies and future prospects[END_REF][START_REF] Bakhsheshi-Rad | In vitro degradation behavior, antibacterial activity and cytotoxicity of TiO2-MAO/ZnHA composite coating on Mg alloy for orthopedic implants[END_REF] Nowadays, surfaces impregnated, functionalized or coated with silver or copper are the most advanced. 9 However, the development of surfaces with active biocidal agent but harmless doses for human health is delicate. 10 As a consequence of the surface redox properties developed by the TiO2 semiconductor under ultraviolet A irradiation (UVA, 320-400 nm), TiO2 mostly in its anatase form exhibits strong antibacterial effect owning to the generation of various reactive oxygen species (ROSs) from photogenerated charges. 11 Applying TiO2-incorporated surfaces for self-disinfection enabled bacterial inactivation, even up to complete mineralization 12- 13 on a wide range of Gram-negative and Gram-positive bacteria species.

Aside from irradiation source power, 14 treatment time 15 and matrix (or substrate)-related factors, [START_REF] Adamek | Immobilisation of TiO2-P25 on a glass fibre mat: Preparation, photocatalytic activity and stability[END_REF][START_REF] Barthomeuf | Effect of titanium dioxide film thickness on photocatalytic and bactericidal activities against Listeria monocytogenes[END_REF] the intrinsic physico-chemical properties of TiO2 greatly affect the photocatalytic oxidation performance of the coating by influencing the amount, recombination rate and diffusion length of the photogenerated charges. [START_REF] Ganguly | Antimicrobial activity of photocatalysts: Fundamentals, mechanisms, kinetics and recent advances[END_REF] The role of TiO2 itself was addressed in many studies by comparing the bactericidal performance of new TiO2 coatings [START_REF] Clemente | Staphylococcus aureus resists UVA at low irradiance but succumbs in the presence of TiO2 photocatalytic coatings[END_REF] to that made of P25 TiO2, [START_REF] Ohtani | What is Degussa (Evonik) P25? Crystalline composition analysis, reconstruction from isolated pure particles and photocatalytic activity test[END_REF] the commercial photocatalyst largely admitted as a reference. The self-disinfection properties were improved thanks to chemical modifications of TiO2, mainly involving Ag and Cu. However, Ag/Cu-modified TiO2 is expected to reveal high bactericidal properties directly due to the heavy metals. [START_REF] Van Grieken | Photocatalytic inactivation of bacteria in water using suspended and immobilized silver-TiO2[END_REF] Other studies focused on the building of TiO2-based heterojunctions [START_REF] Karunakaran | Preparation and characterization of ZnO-TiO2 nanocomposite for photocatalytic disinfection of bacteria and detoxification of cyanide under visible light[END_REF] or TiO2 doping, [START_REF] Zahid | Fabrication of visible light-induced antibacterial and self-cleaning cotton fabrics using manganese doped TiO2 nanoparticles[END_REF] to provide self-disinfection properties under visible light. Meanwhile, TiO2 coatings with extended bactericidal efficiency under UVA irradiation without releasing of environmental-unfriendly agents such as heavy metals are lacking.

Efficacy of TiO2-based self-disinfection coatings also depends on the surface-related properties, especially those resulting from the weight of TiO2 deposited per surface area as well as from the surface morphology and texture, closely related to the size of the particles or of their aggregates.

They directly impact the photocatalytic activity [START_REF] Adamek | Immobilisation of TiO2-P25 on a glass fibre mat: Preparation, photocatalytic activity and stability[END_REF] and also change the surface area of contact between TiO2 and the adhered bacteria, thus affecting the degree of ROSs attack on cells. Further, both chemical and topographical surface properties, which change with the photocatalyst density and chemistry, modulate the amount of adhered and hence targeted bacteria. [START_REF] Ploux | Opposite responses of cells and bacteria to micro/nanopatterned surfaces prepared by pulsed plasma polymerization and UV-irradiation[END_REF] If present, the matrix in which the particles are embedded also modulates the disinfection efficiency by influencing bacterial adhesion [START_REF] Yao | Investigation of the performance of TiO2 photocatalytic coatings[END_REF][START_REF] Zhang | Advanced titanium dioxidepolytetrafluorethylene (TiO2-PTFE) nanocomposite coatings on stainless steel surfaces with antibacterial and anti-corrosion properties[END_REF] and may create a synergetic effect with the photocatalyst. [START_REF] Ganguly | Antimicrobial activity of photocatalysts: Fundamentals, mechanisms, kinetics and recent advances[END_REF] Other factors like the presence of ions and the oxygen content in the liquid environment may also influence the photocatalytic activity, thus having dramatic consequences for the performance in real aqueous phase conditions. [START_REF] Rincon | Effect of pH, inorganic ions, organic matter and H2O2 on E. coli K12 photocatalytic inactivation by TiO2 Implications in solar water disinfection[END_REF] However, except regarding the matrix, these topics are usually omitted.

Besides, bacterial inactivation on TiO2-based coatings is usually addressed by counting viable or cell wall-damaged cells. 11 Rare studies also investigate the photocatalytic toxicity to intracellular functions such as enzymatic and respiratory activities. [START_REF] Kubacka | Understanding the antimicrobial mechanism of TiO2-based nanocomposite films in a pathogenic bacterium[END_REF][START_REF] De Dicatillo | Antimicrobial effect of titanium dioxide nanoparticles[END_REF] However, they only touch upon TiO2 nanoparticles or nanocomposites but fail for TiO2 coatings. Hence, actual impairment of vital intracellular functions in bacteria adhered and treated thanks to TiO2 coatings has still to be clarified. Besides, bacteria are able to overcome the photocatalytic treatment thanks to ROSs defense mechanisms involving catalase and superoxide dismutase (SOD). [START_REF] Bruno-Bárcena | Role of antioxidant enzymes in bacterial resistance to organic acids[END_REF][START_REF] Alhasawi | Glycine metabolism and anti-oxidative defence mechanisms in Pseudomonas fluorescens[END_REF] In the case of oxidative damages caused by TiO2, the immediate inactivation rate of bacteria was shown to be inversely proportional to SOD and catalase activities. [START_REF] Koizumi | Deactivation kinetics of Escherichia coli cells correlated with intracellular superoxide dismutase activity in photoreaction with titanium dioxide particles[END_REF] In addition, a SOD-and catalase-deficient mutant strain was reported to be much more sensitive to photocatalytic treatment than the wild strain. [START_REF] Gogniat | TiO2 photocatalysis causes DNA damage via Fenton reactiongenerated hydroxyl radicals during the recovery period[END_REF] Importantly, this capacity varies with species and strains [START_REF] Bruno-Bárcena | Role of antioxidant enzymes in bacterial resistance to organic acids[END_REF] and can enable bacterial repair during the post-irradiation period. [START_REF] Rincón | Photocatalytical inactivation of E. coli: effect of (continuousintermittent) light intensity and of (suspended-fixed) TiO2 concentration[END_REF] Accordingly, the high immediate antibacterial efficacy of a self-disinfection coating cannot ensure high post-photocatalytic longer-term performance. To our knowledge, this has been never investigated for immobilized TiO2 especially regarding variations according to bacterial species and their sensitivity to oxidative stress.

We report here the photocatalytic liquid-phase antibacterial activity of a novel phosphorus (P) and fluorine (F) -modified TiO2 photocatalyst immobilized as a coating, which is expected to provide higher bactericidal efficiency under UVA light than a reference coating made of TiO2 P25 catalyst without releasing any environmental-unfriendly agent. The P/F-modified TiO2 powder was developed in a previous work and revealed higher activity in the photocatalytic degradation of gaseous methylethylketone compared to TiO2 P25. [START_REF] Yan | On the role of BmimPF6 and P/F-containing additives in the sol-gel synthesis of TiO2 photocatalysts with enhanced activity in the gas phase degradation of methyl ethyl ketone[END_REF] Here, both immediate curative performance and the corresponding longer-term biofilm-preventive antibacterial efficacy of the TiO2 coatings were analyzed, for the first time, by in situ monitoring growth of bacteria adhered on such coatings.

Impact of the photocatalytic treatment on the cell wall and on the vital respiratory and enzymatic functions was also in situ studied by following cell wall permeability as well as redox and esterase activities. Potential effect of the cell wall type and ability to resist oxidative stress of bacteria was questioned by using three bacterial species (E. coli, Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens)) differing in these terms. Possible correlations between physical surface characteristics of the coating and chemical intrinsic properties of the TiO2 photocatalyst on one side, and the antibacterial efficacy on the other side were considered.

Effects of certain ions and richness in dissolved oxygen of the reaction medium were also explored.

Experimental

Sample preparation

Aeroxide® P25 TiO2 photocatalyst was purchased from Evonik® (Evonik Resource Efficiency GmbH, Hanau-Wolfgang, Germany) and is referred to as "P25".

The synthesis of the novel P/F-modified TiO2 photocatalyst was described elsewhere. [START_REF] Yan | On the role of BmimPF6 and P/F-containing additives in the sol-gel synthesis of TiO2 photocatalysts with enhanced activity in the gas phase degradation of methyl ethyl ketone[END_REF] Briefly, 5 g of titanium (IV) isopropoxide (Ti(O i Pr)4, 97%, Aldrich) was mixed in 10 g of propan-2-ol (AnalaR Normapur, >99.5%, VWR Chemicals), before 6.2 g of 2.2 mol/L acetic acid solution containing 21.7 mg of sodium fluoride (NaF, ACS reagent, ≥99%, Sigma-Aldrich) and 59 mg of phosphoric acid (H3PO4 (PA), analytical reagent, ≥85%, R.P.Normapur) was further added dropwise. NaF and PA additives provided the fluoride anions (F -) and the phosphate anions to the synthesis medium, respectively, with a F/Ti and P/Ti molar ratio of 0.03. A white milky precipitate was formed immediately, and the solution was kept under stirring for 1 h before being further sealed by parafilm and aged at room temperature for 6 days without any stirring to partially generate anatase nanoparticles by crystallization. After filtration of the powder from the rest of the solvent, the solid was dispersed in 50 mL distilled water under stirring for 10 min, before being separated from liquid by centrifuge. The washing-centrifuge cycle was repeated 2 times. After drying for 2 h at 100 o C, the so-obtained white powder was finally calcined in air at 550 o C for 2 h with a heating rate of 5 o C/min in attempt to increase crystallinity and to remove fluorine adsorbed on the sample surface. [START_REF] Jung | TiO2 nanotube fabrication with highly exposed (001) facets for enhanced conversion efficiency of solar cells[END_REF] This sample is referred to as "PANaF".

For the preparation of coatings, 484 mg of both TiO2 photocatalysts were separately dispersed in 1 ml of 96% ethanol (0.484 g/ml) followed by 5 min of ultrasonication in a 100 W ultrasonic cleaner. Then the TiO2 suspensions were evenly deposited on borosilicate glass cover slips (ROTH Karlsruhe Deckglaser 22 x 22 mm) by drop casting with a surface density of 1 mg/cm 2 , while the substrates were continuously dried from below with a hairdryer. Drying was completed in several minutes. Both TiO2 immobilized samples are referred to as "TiO2 coatings".

Characterization of photocatalysts

X-ray diffraction (XRD) patterns of TiO2 powders were recorded on a D8 Advance Brucker powder diffractometer in a / mode and using the K1 radiation of Cu at 1.5406 Å. Surface area of TiO2 powders were measured on an ASAP2010 Micromeritics Tristar 3000 analyzer using N2 as an adsorbent at 77K. Before N2 adsorption, samples were outgassed at 150 o C for 12 h. BET surface area was calculated from the N2 adsorption isotherm.

Transmission Electron Microscopy (TEM) analysis of TiO2 powders was performed using a Philips CM200 in standard mode observation, equipped with thermo-ionic LaB6 filament, operating at a 200 kV acceleration voltage. Samples were firstly grinded and sonicated in ethanol solution, before a drop of the solution was deposited onto a copper grid covered by holey carbon membrane for observation.

Surface topographical properties of TiO2 coatings were analyzed by AFM by using a NanoScope IIIa with the following parameter settings: Tapping mode; Tip thickness of 7 µl, length of 225 µm, width of 38 µm; Resonance frequency of 190 KHz; Force constant of 13-77 N/m; Image size of 1µm x 1µm for 5 different zones on each sample. Surface roughness was extracted from each micrograph after adequate image treatment and analysis by WSxM 9.1 (www.wsxm.es).

X-ray Photoelectron Spectroscopy (XPS) surface characterization was performed on an XPS spectrometer SES-2002 (VG SCIENTA) apparatus equipped with an Al K (1448.6 eV) source with a pass energy of 20 eV. The spectra were decomposed assuming contributions with Doniac-Sunjic shape [START_REF] Doniach | Many-electron singularity in X-ray photoemission and X-ray line spectra from metals[END_REF] and Shirley background subtraction. [START_REF] Shirley | High-resolution x-ray photoemission spectrum of the valence bands of gold[END_REF]

Bacteria strains and culture methods

-80 o C frozen bacterial cells were spread on LB (Lysogeny broth, Sigma-Aldrich) agar plate and cultured for two nights at 30°C for E. coli SCC1, [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context[END_REF] or 37 o C for S. epidermidis ATCC 35983 (CIP 106510) , or on BHI (Brain-Heart-Infusion, Sigma-Aldrich) agar plate at 30°C for P. fluorescens ATCC 13525 (CIP 69.13T). Then, precultures were prepared with one colony of E. coli or S. epidermidis in LB, or of P. fluorescens in BHI, before overnight incubation at 30°C, 37°C and 30°C, respectively. Cultures were prepared with 10% volume of the precultures in fresh LB for E. coli and S. epidermidis, and with 33.3% volume of the preculture in fresh BHI for P. fluorescens.

Cultures were then incubated for 4 h at 30°C or 37°C before bacteria were harvested by centrifugation. Harvested bacteria pellets were re-suspended either in physiological NaCl (9 g/L, pH 6.8) solution or in the so-called M63G, a E. coli-selective minimum medium (pH 6.8). [START_REF] Vidal | Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression[END_REF] The obtained bacterial suspensions were adjusted to an absorbance at 600 nm (A600 nm) of 0.1 (5 × 10 7 CFU mL -1 ) for being ready to inoculate the TiO2 coatings. As a reference, i.e. internal control of bacterial adhesion and sessile population growth, cleaned borosilicate glass cover slip surface was inoculated and further analyzed similarly to the TiO2 coatings.

Photocatalytic antibacterial activity of TiO2 coatings

Immediate antibacterial effect

Coatings were sterilized by UVC irradiation (=245 nm) for 7 min at a 2 cm distance before immersion in 3 ml of freshly-prepared M63G medium (exclusively for E. coli) or physiological NaCl (9 g/L) medium (for all three species) in Petri dishes (ROTH, standard petri dish, PS, 35 mm diameter, 10 mm height). After 3 h of incubation at 30°C or 37 o C, the coatings were washed three times by 2 ml physiological NaCl without creating air-surface interface and ended being immersed in the last washing solution. Two of the three identical coatings of each type were exposed for 10 min or 45 min (and 3 h if necessary i.e. only for P. fluorescens) to UVA irradiation. The third coating was maintained without irradiation as a non-illuminated reference. The UVA irradiation was provided by using a 8W UV-A blacklight lamp (Sylvania Blacklight Blue F8W/BLB T5) located at a 1.5 cm distance above the coating samples (light distribution centered on =365 nm with a received irradiance of 30 W/m 2 recorded using a RPS900-W ILT wideband spectroradiometer). For each illumination duration on control, P25 and PANaF coatings, experiments were repeated three times using three identical coating duplicates of each coating type. A last washing was performed after photocatalytic treatment to remove any planktonic bacteria. And in this last washing solution, Fluorescent Syto9® (Aldrich-Sigma) and propidium iodide (PI, Aldrich-Sigma) dyes were added (no Syto9® stain for E. coli SCC1) to stain viable or membrane-damaged bacteria respectively.

Observations of coating's colonization by bacteria were then conducted under confocal microscope (LSM700, Carl ZEISS) equipped with a X63 water immersion objective (W Plan Apochromat X63/1.0, 2.0 mm) and working in fluorescence mode with the excitation laser at 488 nm and with acquisition wavelength at 528 nm and 645 nm for Syto9® (GFP for E. coli SCC1) and PI emission, respectively. Bacteria with active green GFP metabolism (for E. coli) or stained in green by Syto9® (for S. epidermidis and P. fluorescens) are called "Live" in the following text, while those with damaged membrane as demonstrated by the red staining of PI (for all bacteria species), are called "Damaged" in the following text. The obtained double-channel images in RGB form were analyzed via ImageJ 1.47V® software to access quantity of stained bacteria in terms of surface coverage fraction. Three different locations of each surface sample were observed, located on the diagonal line (Figure S1).

Longer-term biofilm-preventive antibacterial effect

The longer-term antibacterial efficiencies of the coatings, i.e. post photocatalytic action, were determined for different UVA illumination durations. The general procedure is similar to that conducted for immediate antibacterial effect analysis, except that the coatings were put back into an adequate nutritive culture medium immediately after UVA illumination. They were kept overnight at 30°C or 37°C for E. coli or P. fluorescens, and S. epidermidis respectively, before being washed and stained for the final confocal microscopy observation. For the P. fluorescens strain, a 3 h UVA illumination experiment was also performed.

Photocatalytic action mechanism

The experimental procedure was similar to that used for the analysis of immediate antibacterial photocatalytic effect but conducted on E. coli PHL 628, a biofilm-making K12 MG1655 derivate strain [START_REF] Junker | Transcription in Escherichia coli PHL628 biofilms[END_REF] without fluorescence properties like that of E. coli SCC1. After UVA illumination, Calcein AM (CAM, Aldrich-Sigma) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Aldrich-Sigma) fluorescent dyes were simultaneously used to stain bacteria with intact endogenous intracellular esterase activities and redox respiratory activities respectively. Staining was performed in LB medium to reach a detectable level of bacterial respiratory activity. [START_REF] Schaule | Use of 5-cyano-2, 3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water[END_REF] On each surface sample, NaCl 9 g/L solution was thus replaced by 2 ml of solutions of CAM (3 mmol/L) and CTC (1 mmol/L) dyes in LB before 20 min incubation. Surface samples were then washed once by physiological NaCl 9 g/L medium without creating air-surface interface and finally observed under confocal microscope as described above. Excitation laser was at 488 nm and the emission was observed at 520 nm for CAM and 630 nm for CTC.

Photocatalytic bactericidal action in oxygen-deficient condition

Immediate antibacterial effect of photocatalysis in oxygen-deficient condition has been evaluated on the E. coli SCC1 strain cultured in a confined environment (Figure S2). Briefly, 20 μl of bacterial suspension prepared with freshly-autoclaved medium (therefore with oxygen deficiency) was dropped on each coating, which was immediately covered by a glass cover slip. The air-liquid exchange interface area was thus limited at the outer edge of the double glass cover layer. Then UVA illumination was performed as described above and prolonged up to 3 h since the photocatalytic activity was expected to drastically decrease in confined environment. In addition to the confocal microscopy analysis performed as described above, quantity of sessile bacteria was also assessed by using a classical plate counting method. [START_REF] Lechevallier | Enumeration and characterization of standard plate count bacteria in chlorinated and raw water supplies[END_REF] Glass cover slip was confirmed not to absorb neither UVA nor the fluorescence emitted by bacteria or bacterial staining.

Besides, the variation of the oxygen content during the photocatalysis action in confined environment was monitored by using an oxygen sensor patch (HIOXY Oxygen Sensor patch, IDIL). In the above described confined environment setup, the patch was stuck on the interior side of the top cover slip, sandwiching liquid along with the TiO2 coating (Figure S3). The liquid used in this experiment was either distilled water, fresh bacteria-free physiological NaCl 9 g/L medium or bacteria suspension adjusted to an absorbance A600 nm of 0.1 or 1.0. Oxygen dissolved in liquid quenches the fluorescence of the sensor, allowing a modulation of the fluorescence intensity level vs. the dissolved oxygen concentration by observation under an up-right epifluorescence microscope (Olympus BX51). Excitation laser was at 405 nm and emission was observed at 630 nm. UVA illumination was applied on the bottom side of the coatings with the same irradiance as before. To compare the oxygen content with and without UVA illumination, micrographs were taken with both non-illuminated and illuminated coatings.

Data statistics

Significance of the two-by-two differences between averages of bacteria's quantity on the diverse coatings was evaluated by bilateral Student's t tests with significance thresholds of 0.05.

According to Scherrer, 44 the alternative hypothesis (H1: μP25 ≠ μPANaF) was assumed to be true when the main hypothesis (H0: μP25 = μPANaF) was rejected.

Results and discussion

Immediate photocatalytic antibacterial effect on E. coli SCC1

Surface fraction covered by E. coli SCC1 before and after illumination on the reference surface, P/F-modified and P25 TiO2 coatings are shown in Figure 1 and Figure S4. Colonization was similar before illumination on all the three types of surface (TiO2 coatings and reference), which indicates that the TiO2 coatings do not present any noticeable attractive or repellent properties for bacteria compared to the reference. After surface inoculation and subsequent bacterial adhesion, no significant bacterial growth was observed neither on the reference surface nor on the TiO2 coatings, as expected in NaCl 9 g/L medium. Regarding immediate photocatalytic effect, a 10 min UVA treatment resulted in a strong reduction of bacterial activity on P/F-modified TiO2 ("PANaF") coating, with almost complete elimination of the sessile "Live" population. In contrast, a 10 min UVA treatment on P25 coating only provided about 50% of "Live" population decrease, with high variability of the results with samples and locations on samples. This is illustrated by the confocal micrographs in Figure S5 B,C, which display very different ratios of "Live" to "Damaged" bacteria numbers depending on the analyzed region on P25 coatings. After 45 min of UVA illumination however, a similar strong reduction of the quantity of "Live" bacteria was observed on PANaF and P25 coatings. Besides, the quantity of "Damaged" bacteria increased with the irradiation time up to the colonization level by "Live" bacteria measured before illumination.

The average "Damaged" gain-to-"Live" loss ratios for 10 min and 45 min illumination are 1.71 and 0.84 on P25 coatings, and 1.09 and 1.15 on PANaF coating respectively. In general, good correlation was found between loss in "Live" bacteria and gain in "Damaged" bacteria for all the coatings and all illumination time. This clearly indicates that bacteria were not eradicated; rather their cell wall was more or less strongly degraded according to coating type and illumination time.

Immediately after 10 min of UVA illumination, enzymatic and respiratory activities of bacteria on TiO2 coatings were not affected (Figure 2, Figure S6, Figure S7 and Figure S8), as shown by the absence of any significant difference in the quantities of CAM-or CTC-positive bacteria respectively. In contrast, 45 min illumination resulted in a strong reduction in the quantity of CTCpositive bacteria on both coating types, which was more significant on the PANaF coating.

Reduction in respiratory activity is in accordance with the increase in physical damage of the cell wall, as shown by PI staining. A 45 min photocatalytic treatment also resulted in a loss in the quantity of CAM-positive bacteria, which was also more marked on PANaF coating than on P25 coating. This is indicative that bacteria have been hit at a sufficient level to inhibit any enzymatic activity in a large part of the bacterial population (80% and 60% on PANaF and P25 coatings, respectively), even though cells were not completely degraded.

Relation between the physico-chemical properties of the coating and the antibacterial activity on E. coli SCC1

PANaF TiO2 coatings present higher photocatalytic activity than the P25 reference coating in terms of immediate cell wall damage and reduction in vital metabolic activities of E. coli cell.

Experiments conducted without illumination or without TiO2 coatings demonstrated that neither UVA illumination nor TiO2 coatings were able by them-selves to induce these antibacterial effects.

Absence of toxicity of TiO2 coatings was made sure by the preservation of very high "Live" and very low "Damaged" bacteria's quantity on TiO2 coating samples in the absence of illumination (Figure S4, Figure S9). In addition, neither 10 min (data not shown) nor 45 min of UVA illumination (Figure S9) resulted in any loss of "Live" bacteria and any gain of "Damaged" ones on reference surfaces. The applied irradiance was thus unable to induce any detectable damage of bacterial cells. 3 h of UVA illumination had to be reached to achieve a slight killing effect on E. coli SCC1, with non-significant variation of the "Live" bacteria population but high increase in the "Damaged" population (Figure S10). In addition, the electrostatic interactions between the charged bacterial outer surface (negative for all known bacterial species at physiological pH, about 7) and the material surface, which are known to impact bacterial adhesion, 1 are expected to have been similar on TiO2 P25 and PANaF materials. [START_REF] Li | Bacterial adhesion to glass and metal-oxide surfaces. Colloids and surfaces[END_REF] Both materials exhibit isoelectric point (IEP) of 5.6  0.5 and 3.4  0.2, respectively, ie. lower than the physiological pH, so that both surfaces are negatively charged in the most common bacterial culture media (pH = 6.8-7.0). [START_REF] Suttiponparnit | Role of surface area, primary particle size, and crystal phase on titanium dioxide nanoparticle dispersion properties[END_REF] This was confirmed by the experiment that showed that sessile E. coli bacteria were in similar amount on P25 and PANaF coatings before any photocatalytic treatment. Damage and inactivation of bacteria observed with up to 45 min of treatment should be thus attributed to photocatalysis, i.e. the physico-chemical properties of the TiO2 photocatalysts or of the coatings. PANaF TiO2 is expected to perform better than the P25 TiO2 reference for many different aspects.

XRD patterns revealed that the PANaF TiO2 powders had a smaller mean crystallite size than the P25 TiO2 reference (about 10-11 nm vs. 22 nm for the anatase phase in P25) (Figure 3), associated to its higher specific surface area (130 m 2 /g vs. 55 m 2 /g). The results were confirmed by TEM analysis, for PANaF and P25 TiO2 powders (with a 12-15 nm mean crystallite size vs. 22-25 nm, respectively). It was shown that this fine-tuned material morphology directly results from the use of phosphoric acid (PA) and sodium fluoride (NaF) additives in a P/F-assisted sol-gel synthesis of TiO2. Thanks to the use of both additives, a much higher photocatalytic activity in the gas-phase methyl ethyl ketone degradation was obtained with PANaF TiO2 in comparison to P25. Indeed, the number of adsorption and reaction surface sites was increased and an improvement of the photogenerated electron-hole charge flow separation was proposed, which reduces the charge recombination rate. [START_REF] Yan | On the role of BmimPF6 and P/F-containing additives in the sol-gel synthesis of TiO2 photocatalysts with enhanced activity in the gas phase degradation of methyl ethyl ketone[END_REF] Here as well, the increased number of surface sites favors the production of ROS, and consequently the subsequent antibacterial effect in aqueous phase.

Besides, the surface topographic properties are likely to provide some advantage to the PANaF coating over its P25 counterpart. As measured by AFM and reported in Figure 4, strong differences in surface roughness and morphology are noticed: the surface of the P25 coating is composed of large grain-like convex structures with size of 180 ± 35 nm, while the PANaF coating surface is smoother (mean lateral particle size of 75 nm vs. 120 nm for PANaF and P25, respectively) and the grain size is smaller (42 ± 16 nm). This is consistent with the smaller TiO2 crystal size of PANaF compared to P25. Indeed, even though the observed grains are probably aggregates rather than single crystals, smaller crystals are expected to form smaller aggregates than larger crystals do, if the form of primary particles is non-anisotropic. This has been especially shown for aggregates of P25 and of smaller size sol-gel TiO2. [START_REF] Maira | Size effects in gas-phase photooxidation of trichloroethylene using nanometer-sized TiO2 catalysts[END_REF] This difference in topography does not result in different surface coverage by bacteria of the coatings as shown before UV-A illumination (Figure S4). However, the photocatalytic effect on adhered bacteria might be impacted. Due to their large size (1-3 µm) compared to the TiO2 aggregates, bacterial cells lie on the coating's surface with only a limited numbers of contact points. [START_REF] Anselme | The interaction of cells and bacteria with surfaces structured at the nanometre scale[END_REF] On PANaF coatings, the smoother topography results in a higher number of contact points with shallower valleys in between. This is beneficial to the photocatalytic activity, [START_REF] Tong | Effects of material morphology on the phototoxicity of nano-TiO2 to bacteria[END_REF] since the reactive generated ROSs need to diffuse for a shorter distance to reach the target i.e. the bacterium at the surface of the PANaF coating compared to the P25 coating (Figure 4), which has been reported to prevent dramatic loss of the ROSs. [START_REF] Pryor | Oxy-radicals and related species: their formation, lifetimes, and reactions[END_REF]

Longer-term, biofilm-preventive antibacterial effect

Bacteria's fate on the coatings after treatment by photocatalysis has been investigated. Adhered E. coli were allowed to recover for 16 h in favorable growth conditions after different photocatalytic treatment times. Their amounts after the recovery period are depicted in Figure 5 and Figure S11, and some typical micrographs are shown in Figure S12. As expected, the surface coverage by "Live" bacteria without photocatalytic treatment increased from 15-20 % to up to 40-50 % on the TiO2 coatings after overnight culture in nutrition-rich conditions. In contrast, the surface coverage increased from nearly 0% when a prior UV-A treatment was applied for 10 min or 45 min to 15-20 % on both P25 and PANaF coatings after overnight culture. Overall, the longer-term antibacterial effect of the TiO2 coating is able to limit the bacterial overnight regrowth to roughly 60% of surface coverage relative to that on a non-treated coating (Figure 5 B). This demonstrates that the few "Live" bacterial cells still adhered on the coatings after photocatalytic treatment (Figure 1C) were able to grow if placed in favorable conditions. However, only few bacteria that were damaged by the treatment were able to recover, as suggested by the similar fraction of bacteria remaining "Damaged" (i.e. red) after the overnight culture (10%) (Figure 5 A) compared to the fraction measured directly after the photocatalytic treatment (10-20%) (Figure 1 C).

Variations of the photocatalytic antibacterial performance with bacterial species

The photocatalytic antibacterial performance, regarding both immediate and longer-term effects, was expected not to depend only on the material and the coating properties, but also on the bacterial species mainly due to strong differences in resistance to oxidative stress. Investigation conducted with S. epidermidis ATCC 35983 (CIP 106510) and P. fluorescens ATCC 13525 (CIP 69.13T) strains on the PANaF and P25 coatings revealed significant differences in the photocatalytic effect compared to the results observed with E. coli.

With S. epidermidis ATCC 35983 (CIP 106510), the results reveal a strong reduction of the "Live" bacteria quantity on both the coatings after 45 min UVA illumination (Figure 6 A, Figure S13 and Figure S14 for micrograph examples). However, 10 min UVA treatment failed to cause any antibacterial effect on P25 coating and only a slight advantage of PANaF coating was evidenced with "Live" bacteria quantity being reduced to about 80% of the initial population. 10 min UVA treatment also failed to provide any antibacterial effect on P. fluorescens ATCC 13525 (CIP 69.13T) strain on the P25 coating, but also on the PANaF coating (Figure 6 B and Figure S15). Nevertheless, 45 min UVA illumination led to a significant reduction of the "Live" bacteria quantity on both P25 and PANaF coatings, but the reduction was only of about 70% on the PANaF coating and 50% on the P25 coating, thus not as efficient as with E. coli. In addition, as illustrated in confocal micrographs (Figure S16), some bacterial cells were stained as dark orange instead of red, which is the result from double cell staining by red PI and green Syto9®. This indicates that the cell wall integrity was affected, even though damages were not strong enough to allow PI to completely replace Syto9® in DNA. Longer UVA treatment, tested for up to 3 h illumination, did not result in any further improvement of the antibacterial effect as seen in Figure 6 B.

After overnight growth in nutrient-rich culture medium without any previous treatment, S. epidermidis population that initially covered about 10% surface of the coatings grew to about 50% coverage in absence of photocatalytic treatment (comparison between Figure S15 and Figure S17).

With a previous 10 min UVA treatment, the bacterial population was unchanged (49% and 50% on P25 and PANaF coatings respectively) (Figure 6 C, Figure S17 and Figure S18). However, with 45 min UVA illumination, longer-term growth was significantly reduced, the coverage by "Live" bacteria being lowered to 40% of the P25 coating and about 30% of the PANaF coating. "Damaged" bacteria quantity was almost unchanged after the overnight growth in favorable conditions compared to immediately after the photocatalytic treatment, which suggests that damaged bacteria were mainly unable to recover, similarly to that observed with the E. coli SCC1 strain. Thus, photocatalytic treatment of S. epidermidis on both P25 and PANaF TiO2 coatings was able to inhibit the growth of a significant part of the sessile bacterial population even after a long recovery time in favorable conditions. In contrast, 45 min of treatment failed to avoid the formation of a thick P. fluorescens biofilm after post-treatment growth in nutritive medium (Figure S19) even though the micrographs suggest some moderate inhibition on both TiO2 coatings (Figure S20). A more pronounced inhibition was achieved after 3 h illumination as evidenced by the much less dense "Live" bacterial biomass observed in the biofilm. In addition, "damaged" bacteria were in higher quantity on the PANaF coating than on the P25 one.

Finally, bacterial species are ranked as following in terms of their rate of inactivation after the same photocatalytic treatment on the same TiO2 coating: E. coli > S. epidermidis > P. fluorescens, from the most to the least sensitive. Indeed, the possible effect of differences in adhesion rate between species is rejected since the inactivation rate relates to the number of adhered bacteria of each species measured without treatment. Therefore, three species-related factors may be involved in this variation in sensitivity: 1) the production of extracellular matrix (ECM), 2) the structure properties of the bacteria cell wall and 3) the defense mechanism countering ROSs.

ECM is expected to form a screen that partially hides the TiO2 nanocrystals from the irradiation beam. [START_REF] Takeuchi | Neutrophil elastase contributes to extracellular matrix damage induced by chronic low-dose UV irradiation in a hairless mouse photoaging model[END_REF] It also probably acts as a diffusion barrier for ROSs that have been produced. [START_REF] D'haeze | Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata[END_REF] However, illumination treatments were all carried out on bacteria just adhered (3 h of incubation) in physiologic saline solution, which is not expected to conduct to the formation of a significant biofilm. [START_REF] Costerton | Introduction to biofilm[END_REF] Hence, ECM is here expected not to have been produced in a sufficient quantity to compromise the photocatalysis effect. Bacteria's cell wall type might also vary the bacterial susceptibility to ROSs. Indeed, both outer membrane and peptidoglycan (PGN) layers, which are the main barriers for ROSs before damaging the cytoplasmic membrane, differ with Gram-negative / Gram-positive class of bacteria (Figure 7). [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF] Lipopolysaccharide and lipid layers of the outer membrane of Gram-negative bacteria have been demonstrated to be easily degraded through photocatalysis. [START_REF] Sunada | Studies on photokilling of bacteria on TiO2 thin film[END_REF][START_REF] Maness | Bactericidal activity of photocatalytic TiO2 reaction toward an understanding of its killing mechanism[END_REF] In contrast, the PGN layer, that is much thicker in Gram-positive bacteria, was shown to be the most resistant component of the cell wall. [START_REF] Kiwi | Evidence for the mechanism of photocatalytic degradation of the bacterial wall membrane at the TiO2 interface by ATR-FTIR and laser kinetic spectroscopy[END_REF] However, the natural pores of the PGN layer are large enough for the diffusion (bypass) of ROSs (if live long enough) in the absence of any PGN degradation. [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF] Therefore, variation of sensitivity based on the typical cell wall composition associated to the Gram-positive and versus Gram-negative classification is not obvious. This is in agreement with the literature that failed to report direct correlation between photocatalytic antibacterial efficacy and the cell wall type so far. [START_REF] Pal | Photocatalytic inactivation of Gram-positive and Gram-negative bacteria using fluorescent light[END_REF] Whatever the cell wall type, ROSs finally reach the cytoplasmic membrane. There, vital metabolic functions such as enzymatic and respiratory activities may be affected. [START_REF] Schaule | Use of 5-cyano-2, 3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water[END_REF][START_REF] Kashket | Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance[END_REF] This is therefore the ultimate and the most vital defensive line against many environmental stresses 60 . In our study, adhered E. coli cells with active enzymatic and respiratory activities were still in high number on both TiO2 coatings after 45 min of UVA treatment, although their GFP production, which signs the metabolic activity, was shown to be almost completely inactivated. Their cell wall was also almost completely damaged. In addition, the enzymatic activity was less reduced than the respiratory activity. This confirms that ROSs attacked the cytoplasmic membrane as first, which is the location of E. coli respiratory, while the remaining quantity of ROSs reached then the cytoplasm (Figure 7). In the bacterial cell cytoplasm, all three bacteria used in the present work had catalase and SOD, two important anti-oxidative enzymes providing defenses against ROSs. However, their capacity to inhibit ROSs varies with species. [START_REF] Zeller | Detoxification of hydrogen peroxide and expression of catalase genes in Rhodobacter[END_REF] Facultative anaerobic S.

epidermidis and E. coli bacteria are more sensitive to oxygen exposure than aerobic organisms that have moved towards intricate mechanisms to neutralize ROSs. [START_REF] Groves | Adaptation to oxidative stress by Gram-positive bacteria: the redox sensing system HbpS-SenS-SenR from Streptomyces reticuli[END_REF][START_REF] Hassan | Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia coli[END_REF][START_REF] Jacobs | Effect of hemin on the formation of the cytochrome system of anaerobically grown Staphylococcus epidermidis[END_REF] Thus, Alhasawi et al. have demonstrated that an obligate aerobic wild P. fluorescens strain can survive in media with 500 μM H2O2 by generating NADPH, ATP and glyoxylate in an effort to fend off ROSs. [START_REF] Alhasawi | Glycine metabolism and anti-oxidative defence mechanisms in Pseudomonas fluorescens[END_REF] This pronounced difference in fighting against ROSs is the probable main cause of the difference in sensitivity to the photocatalytic treatment observed between E. coli and S. epidermidis on the one side and P. fluorescens on the other side. In addition, E. coli K12 strains such as E. coli SCC1 lack one of the SODs compared to environmental E. coli B strain. This allows us to suggest that E. coli SCC1 bacteria exhibit less resistance to ROSs in comparison to S. epidermidis and P. fluorescens bacteria, P. fluorescens being the most resistant due to adequate adaptation to aerobic conditions.

Influence of oxygen and ions on the photocatalytic antibacterial performance

Environmental factors such as the oxygen level and the presence of ions in the reaction medium are prone to strongly affect the photocatalytic antibacterial effects. We therefore investigated the immediate photocatalytic antibacterial effect in a confined, i.e. O2-poor, environment (Figure 8) for E. coli SCC1 strain on both P25 and PANaF coatings. The surface fraction covered by bacteria without treatment was low (2%-5%) in agreement with the low bacteria's quantity inoculated on the surface in the confined environment (only 20 μl of bacteria suspension). After a 45 min UVA treatment, neither P25 nor PANaF coating revealed any significant reduction in the quantity of "Live" bacteria, which is in a great contrast with the results obtained in the "open" aerated system used for the above reported results (Figure 1 B). The lack of significant photocatalytic activity on the confined TiO2 coatings was confirmed after 3 h UVA illumination (data not shown).

Noticeably, the lack of antibacterial effect was associated with a color change of the TiO2 coatings (Figure S21). Typically, the coating revealed white color in aerated conditions, which was unchanged after illumination. Placed in the confined environment, the coating turned from white to a bluish color, regardless of the concentration of bacteria used for the inoculation, which is attributed to the formation of reduced TiO2 (Ti 3+ ) with the formation of oxygen vacancies. [START_REF] Qiu | Hydrogenation synthesis of blue TiO2 for high-performance lithium-ion batteries[END_REF][START_REF] Valentin | Reduced and n-type doped TiO2: nature of Ti 3+ species[END_REF] By using an oxygen sensor patch, an immediate and strong decrease in the level of dissolved oxygen was actually revealed by a sharp boost of fluorescence intensity upon UVA illumination in the confined condition (Figure 9). In contrast, the red fluorescence intensity was much lower upon illumination in aerated conditions, as well as in the absence of illumination in both aerated or confined environments, as expected for oxygen-rich media. The low level of oxygen content in the confined environment might lead to the impossibility for photogenerated electrons to be evacuated through the usual reduction of adsorbed O2 molecules. [START_REF] Linsebigler | Photocatalysis on TiO2 surfaces: principles, mechanisms, and selected results[END_REF] Hence, the photogenerated electrons accumulate in the TiO2 material, thus reducing Ti 4+ species into Ti 3+ centers as expected from the redox potential level of Ti 3+ lower than that of anatase TiO2. [START_REF] Valentin | Reduced and n-type doped TiO2: nature of Ti 3+ species[END_REF] . The resulting TiO2 structural changes alter the photocatalytic properties. [START_REF] Valentin | Reduced and n-type doped TiO2: nature of Ti 3+ species[END_REF] Besides, the lack of oxygen prevents any freeradical-induced chain scission of (bio)polymers, [START_REF] Singh | Mechanistic implications of plastic degradation[END_REF] which results in the preservation of biopolymers and therefore survival of bacteria upon photocatalytic treatment. Importantly, the presence of bacteria did not enhance the color change despite the O2 uptake by bacteria for their respiratory activity, probably due to their low consumption of O2 especially in a non-nutritive medium (NaCl 9 g/L). [START_REF] Schaule | Use of 5-cyano-2, 3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water[END_REF] It should be noticed that the blue color was more intense in the liquid freshly sterilized by autoclave just before use (e.g. physiological saline water), which is attributed to the removal of the dissolved oxygen. Finally, reduction of Ti 4+ into colored Ti 3+ is a nonmaterial-dependent phenomenon that occurred similarly on both TiO2 coatings. Some ions in the reaction medium were also shown to influence the photocatalytic antibacterial effect. It was especially noticed that 1 h of photocatalytic treatment in M63G medium failed to result in any significant reduction in the "Live" bacteria's quantity on both TiO2 coatings (Figure 10), which is very different from the effect in physiological saline medium (Figure 1 B).

Complementary experiment of the liquid phase photocatalytic degradation under UVA of methylene blue on the PANaF coatings in the presence or absence of M63G medium confirmed that the M63G medium inhibited the photocatalytic reaction (cf. Figure S22 and its description), even with a M63G medium diluted by 100 times with saline solution. XPS surface analysis (Table S1, Figure S23) revealed the characteristic doublet of P 2p3/2-1/2 orbitals for non-polymerized phosphate species (133.6 eV and 134.5 eV) in the case of the PANaF coating, as the expected consequence of using phosphoric acid as additive for the TiO2 synthesis [START_REF] Yan | On the role of BmimPF6 and P/F-containing additives in the sol-gel synthesis of TiO2 photocatalysts with enhanced activity in the gas phase degradation of methyl ethyl ketone[END_REF] , while logically no phosphorous was observed in the case of P25 coating (data not shown). After immersion in M63G medium, the P/Ti surface atomic ratio strongly increased for both TiO2 coatings (Table S1), with the appearance of an additional P 2p3/2-1/2 orbital doublet contribution (Figure S23 A,C) similar to that observed on TiO2-based composites used to adsorb phosphate from wastewater. [START_REF] Wang | Adsorption mechanism of phosphate by polyaniline/TiO2 composite from wastewater[END_REF] The presence of phosphate deposit was here attributed to phosphate adsorbed from the medium to the coating's surface. It was consequently the probable cause for the suppression of the photocatalytic antibacterial effect. Indeed, Rincon et al. reported that phosphate ion is the most detrimental compound among other common ions for the TiO2 photocatalytic antibacterial effect on E. coli. It acts as a poison for the photocatalyst by competing with the oxidizing radicals, by blocking the surface active sites of the photocatalyst [START_REF] Rincon | Effect of pH, inorganic ions, organic matter and H2O2 on E. coli K12 photocatalytic inactivation by TiO2 Implications in solar water disinfection[END_REF] , and by further serving as a charge recombination center.

Conclusion

Photocatalytic antibacterial investigation was conducted under UVA irradiation on a heavymetals-free P/F-modified TiO2 coating against several bacterial species in comparison to a reference TiO2-P25 coating. In general, near-total inactivation of E. coli and S. epidermidis was obtained, with a clear of both nature and topography of the coating, and of illumination time.

Significantly higher efficiency was achieved on the PANaF coating compared to its TiO2-P25 counterpart. Especially, the smaller size of the PANaF TiO2 crystals lead to a smoother topography of the coating surface, which may reduce the diffusion length that the photogenerated ROSs must travel to attack the bacterial cell wall. For the first time on TiO2 coatings, in-situ investigation of the cell wall integrity as well as of the respiratory and intracellular esterase activities showed that the photocatalytic treatment acts on the bacterial cells by attacking the successive barriers of the cell structure, with a decreasing photocatalytic effect as ROSs progress through the cell. Besides, we have evidenced a longer-term, biofilm-preventive effect on E. coli and S. epidermidis species in good correlation with the immediate photocatalytic antibacterial effect, while complete inactivation was not achieved with P. fluorescens, probably due to the capacity of P. fluorescens to fight the ROS-induced oxygen stress. In addition, the adsorption of phosphate ions on the TiO2 surface and the lack of dissolved oxygen in the reaction medium are detrimental to the photocatalytic activity.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. (A) Example of fluorescent images of E. coli SCC1 bacteria on P25 and PANaF coating

Figure 4 .

 4 Figure 4. (A) Topological micrographs and (C) height profiles of the TiO2 P25 coating, and (B,

Figure 5 .

 5 Figure 5. (A) Surface fraction covered by "Live" (green) and "Damaged (marked as Dmg)" (red)

Figure 6 .Figure 7 .

 67 Figure 6. Fraction of "Live" bacteria after immediate photocatalytic treatment in the population

Figure 8 .Figure 9 .

 89 Figure 8. Immediate photocatalytic antibacterial effect on "Live" E. coli SCC1 bacteria adhered

Figure 10 .

 10 Figure 10. Immediate photocatalytic antibacterial effect on E. coli SCC1 species cultivated in M63G medium 70 on P25 and PANaF coatings and with or without 1 h of illumination. No

Acknowledgment

Dr A. Airoudj, Dr L. Vidal and Dr S. Garreau (IS2M) are thanked for the AFM, TEM and XPS measurements respectively. We acknowledge CRT Aérial (Illkirch, France) for its assistance in complementary microbiological tests.

Funding Sources

This work is part of CLEANCOAT project funded by the MICA CARNOT (Materials Institute Carnot Alsace).

Supporting Information. Examples of fluorescent images of bacteria, surface coverage by staining-positive bacteria, supplementary experimental information on studying the effect of phosphate ions adsorption and the lack of oxygen dissolved in the reaction medium on the photocatalytic bactericidal activity of TiO2 coating.

with and without 10 min of illumination. (B) Surface fraction covered by "Live" bacteria with and without 10 min of illumination on P25 and PANaF coating. (C) Surface fraction covered by "Live" (green) and "Damaged" (marked as "Dmg") (red) bacteria and (D) fraction of "Live" bacteria in the population of E. coli SCC1 as a function of the illumination time on reference, P25 and PANaF coatings. Differences compared to t=0 are significant (p-value < 0.05) at all illumination length on P25 and PANaF coatings (cf. also in Figure S6). The following graphic will be used for the TOC:


Vorakit Vorakitphan
email: vorakit.vorakitphan@inria.fr

Marco Guerini
email: guerini@fbk.eu

Elena Cabrio

Serena Villata

Regrexit or not Regrexit: Aspect-based Sentiment Analysis in Polarized Contexts

Emotion analysis in polarized contexts represents a challenge for Natural Language Processing modeling. As a step in the aforementioned direction, we present a methodology to extend the task of Aspect-based Sentiment Analysis (ABSA) toward the affect and emotion representation in polarized settings. In particular, we adopt the three-dimensional model of affect based on Valence, Arousal, and Dominance (VAD). We then present a Brexit scenario that proves how affect varies toward the same aspect when politically polarized stances are presented. Our approach captures aspect-based polarization from newspapers regarding the Brexit scenario of 1.2m entities at sentence-level. We demonstrate how basic constituents of emotions can be mapped to the VAD model, along with their interactions respecting the polarized context in ABSA settings using biased key-concepts (e.g., "stop Brexit" vs. "support Brexit"). Quite intriguingly, the framework achieves to produce coherent aspect evidences of Brexit's stance from key-concepts, showing that VAD influence the support and opposition aspects.

Introduction

Aspect-based Sentiment Analysis (ABSA) aims at capturing sentiment (i.e., positive, negative or neutral) expressed toward each aspect (i.e., attribute) of a target entity. The main interest is to capture sentiment nuances about different entities. However, in a context of opinion polarization, different groups of people can form strong convictions of competing opinions on such target entities, resulting in different (often opposite) evaluations of the same aspect. Compare, for example, the differences in the pro-and anti-Brexit discourses concerning the withdrawal of the United Kingdom from the European Union (EU), aligning with contrasting attitudes toward the EU, the immigration and the country's culture. In fact, while in standard scenarios of sentiment analysis about specific entities and their aspects it is assumed that sentiment is consistent (e.g., a big screen is a desirable characteristics for a TV), this is not the case for polarized contexts. Hence, for example, a "clean Brexit" might be desirable to some, but not to others.

Some previous work has showed that sentiment alone (i.e., positive, negative, neutral) is not able to grasp complex emotion phenomena in information diffusion scenarios [START_REF] Guerini | Deep feelings: A massive cross-lingual study on the relation between emotions and virality[END_REF][START_REF] Berger | What makes online content viral[END_REF]. Following the suggestion of [START_REF] Guerini | Deep feelings: A massive cross-lingual study on the relation between emotions and virality[END_REF], to analyse in a fine-grained manner our scenario where the same topic can appear in different news outlets that have opposed opinions, we decide to make resort to the model presented in [START_REF] Russell | A circumplex model of affect[END_REF][START_REF] Bradley | Measuring emotion: the self-assessment manikin and the semantic differential[END_REF]. This work developed a circumplex model, with a three dimensional space of affect along the Valence, Arousal, and Dominance coordinates, where all emotions can be mapped in the same dimensional space. In our work, we therefore hypothesize that VAD plays a significant role in the interaction with linguistic elements in ABSA settings where polarized opinions are at play.

More specifically, we answer the following research questions: how basic constituents of emotions, such as VAD, interact with aspect-based polarization? How polarized settings differ from standard SA scenarios? How aspects of Brexit and related key-concepts are perceived by opposite parties?

To answer these research questions, we propose a comprehensive framework for studying the interaction of ABSA with opinion polarization in newspapers and social media. We first trained an RNN that detects the emotions and their intensities at sentence-level, and then we map emotion intensities into the VAD circumplex model. Later, we build a framework to assess whether and how VAD are connected to polarized contexts, by computing the VAD scores of a set of key-concepts that can be found on newspapers with opposite views. These key-concepts (e.g., "stop immigration") are built from a set of aspects ("immigration" in our example) combined with relevant verbs or adjectives that represent a clear polarized opinion toward the aspect (e.g., "stop").

To experiment with the proposed approach, we focus on the Brexit scenario, whose unique circumstances have boosted opinion polarization because of the extremely rapid and volatile changes in the political panorama as the results of the EU referendum were announced (political leaders were quick to say that the outcome had "revealed a divided Britain")1 . While an analysis of this situation from the viewpoint of political sciences is not the focus of our work, the Brexit scenario as discussed on newspapers and on social media provides us with the required elements to carry out our study, because of the opinion divisions formed around one or more political positions or issues. Moreover, to be meaningfully polarizing, issues need to be important (or "salient") to a large section of the public, not just a minority of people with strongly held views, and in the Brexit scenario this is actually the case. In our experimental setting, we select two British newspapers known to be polarized, i.e., either for or against Brexit. Results show that VAD are not absolute, but relative to the newspaper's viewpoint on the key-concept. Our approach highlights that using the proposed key-concepts gives us fine-grained details about VAD elements that strongly interact with the polarized context. We show that standard SA approaches can be deceptive in such polarized setting (considering only the word "Brexit" on both newspapers, the valence is almost identical), while our ABSA approach shows a clear-cut polarization.

Related Work

ABSA approaches aim not only at capturing linguistic relations, but also at ascribing polarity stances of textual entities. We concentrate our attention to this second aspect being it closer to the purpose of our work. [START_REF] Demszky | Analyzing polarization in social media: Method and application to tweets on 21 mass shootings[END_REF] apply ABSA methods to pioneer tweets on the US mass shootings topic, where the topic was politically discussed from different viewpoints according to the locations of events with the contrasting use of the terms "terrorist" and "crazy", that contribute to polarization. Still, while in [START_REF] Demszky | Analyzing polarization in social media: Method and application to tweets on 21 mass shootings[END_REF] such terms where taken as evidence of polarization, in our case we want to understand how such concepts are evaluated -in polarized scenarios -by both parties (e.g., how people and journalists from each stance react when the same shooter is defined as "terrorist" or "crazy"). Other approaches in this direction employ NLP to show that certain topics are more polarizing than others [START_REF] Balasubramanyan | Modeling polarizing topics: When do different political communities respond differently to the same news[END_REF]. Moreover, [START_REF] Zhang | Aspect-based sentiment classification with aspect-specific graph convolutional networks[END_REF] and [START_REF] Chen | Aspect sentiment classification with document-level sentiment preference modeling[END_REF] show that most recent ABSA approaches rely on state-of-the-art supervised approaches combining complex layers of neural network models (e.g., transformers) to classify labels representing aspects from text elements with standard sentiment (i.e., positive, negative). This direction is computationally expensive and requires the annotation of the aspects. In contrast, our ABSA method consists in an unsupervised approach where aspects are conveyed with respect to fine-grained extraction of VAD affects in polarized situations.

Emotions Analysis

As the first step of the proposed framework, we focus on the task of detecting emotions and their intensities at sentence-level from the news context. We compare two standard emotion recognition approaches, i.e., a lexicon-based model called DepecheMood++ [START_REF] Araque | Depechemood++: a bilingual emotion lexicon built through simple yet powerful techniques[END_REF], and an RNN based approach that casts the task of multi-label ABSA as a regression problem. Both DepecheMood++ and the RNN model were built and trained on the same set of news extracted from the Rappler.com website. Rappler.com is an online news publisher, where each article is associated to a mood meter. Such mood meter allows each reader to vote the emotions evoked by the article after reading. We implemented a web crawler and harvested a total of 67,828 articles from January 2017 until April 2019 along with the 8 possible emotions voted by readers (i.e., happy, inspired, amused, afraid, angry, annoyed, don't care, sad). As we aim at detecting emotions at sentence-level, we pick all headlines of such articles and the associated emotions as our training set. For the RNN based approach, we use a deep contextualized word representation as our features, namely ELMo [START_REF] Peters | Deep contextualized word representations[END_REF], with a deep bidirectional language model (BiLSTM). We use BiLSTM of 128 dimensional layers with a Rectified Linear Unit (ReLU) activation function [START_REF] Fred | Deep Learning using Rectified Linear Units (ReLU)[END_REF]. Then, we apply Mean Squared Error (MSE) as a loss function with a standard adam optimizer. At the last layer, we use sigmoid function to gate our multi-label output neurons as the intensity of each emotion. To validate our model, we perform cross-dataset testing on the "Affective Text" dataset from SemEval2007-T14 [START_REF] Strapparava | SemEval-2007 task 14: Affective text[END_REF]) using Pearson's correlation (r) to evaluate the performances of our system on each individual emotion. In Table 1 2 , we report the results for each emotion, and the average of all emotions produced by the different models.

Given that the ELMo embeddings with BiLSTM model outperformed the other models, we selected the output of this model as input for the next step (i.e., to map the predicted emotions to the VAD model).

VAD Analysis

The VAD model of affects [START_REF] Russell | A circumplex model of affect[END_REF][START_REF] Bradley | Measuring emotion: the self-assessment manikin and the semantic differential[END_REF] has been extensively used, and relies on three dimensions: Valence, that can be positive/negative (e.g., FEAR has a negative valence, JOY has a positive valence) and corresponds to the standard dimension of sentiment analysis; Arousal, that can be low or high and ranges from "calm" to "excitement" (e.g., SADNESS has low arousal while ANGER has high arousal, even if they have the same sentiment); finally, Dominance, that can be low or high as well, and ranges from "controlled" to "in control" (e.g. FEAR refers to low dominance, INSPIRED to high dominance).

To map the emotions expressed in a sentence to the VAD model, we followed the approach of [START_REF] Guerini | Deep feelings: A massive cross-lingual study on the relation between emotions and virality[END_REF]. We use the VAD scores of emotion labels provided in [START_REF] Warriner | Norms of valence, arousal, and dominance for 13,915 english lemmas[END_REF] which serves as our gold standard to obtain VAD scores from emotion intensities. In particular, here below we report the procedure for Valence computation (Arousal and Dominance computation are akin): given a sentence S, to obtain its valence S v we multiply the intensity of each emotion I(e i ) -computed using the approach described in Section 3 -with the corresponding Valence score V (e i ) present in [START_REF] Warriner | Norms of valence, arousal, and dominance for 13,915 english lemmas[END_REF]. Then, we sum the n emotional dimensions to obtain the final Valence score, as expressed in Equation 1:

S v = n i=1 V (e i ) × I(e i ) (1)
After the conversion, VAD values are in absolute form (arbitrary range). Since newspapers could have different "affective styles" -that are prior to their stance on a specific topic -we apply standardization individually to each VAD dimension for each newspaper to make results better comparable across newspapers.

Key-concept

A Polarized Context Scenario: the Brexit

Dataset collection. To clearly define the use case, we conducted a survey by asking to 10 British participants currently living in England to agree on the stance of UK newspapers with respect to the Brexit, i.e., they were asked which newspapers are known to be pro-or anti-Brexit. As a result, we selected two newspapers that obtained the mutual agreement across all participants, i.e., The Sun (thesun.co.uk) as the pro-Brexit newspaper, and The Guardian (theguardian.com) as the anti-Brexit one. We built a web crawler to harvest the articles containing the word "brexit" from these two news websites from year 2017 to 2019. We obtained 28,212 articles in total. We segmented them at sentence-level resulting in 1.2 million sentences.

Key-concepts. For our analysis, we focused on a set of key-concepts relevant to the Brexit discussion (e.g., "brexit", "immigration", "EU"). We created two lists of words that could potentially trigger polarization occurring with the identified key-concepts in text (e.g., "stop", "promote"). These words have then been combined with each key-concept to obtain the "support" and "against" aspect clusters, e.g., "stop Brexit", "block Brexit" for the against aspect, and "support Brexit", "make Brexit" for the support aspect. We then computed the VAD scores for the sentences in our dataset containing an occurrence of the mentioned aspects. We used weighted average on all sentences collected for each aspect cluster to compute the final VAD scores for the support and against aspect clusters. For consistency reasons, we select key-concepts only when their frequency (in both support and against aspect clusters) > 1,000.

Results and discussion. Table 2 shows some clear-cut results for key-concepts "Brexit", "Immigration" and "Theresa May". Considering "Brexit" key-concept, we see that the valence of support aspect in pro-Brexit journal is positive, while it is negative in anti-Brexit (as expected). Turning to arousal, we see that the concept "Brexit" provokes a higher emotional activation in pro-Brexit journal, regardless of the aspect being support or against. "Immigration", instead, is the key-concept with the highest arousal in all the considered cases, both for pro-and for anti-Brexit in the support case, but with opposite valence (positive for anti and neutral/negative for pro). In the against aspect instead (e.g., "stop immigration"), pro-Brexit have an extreme arousal connected to positive sentiment (valence), while anti-Brexit have low arousal with slightly negative sentiment (i.e., a resignation attitude). Interestingly, dominance (the feeling of being in control of the situation) is consistent with the polarized sentiment in both aspects and journals, with the "resignation attitude" confirmed also in this case for the against aspect of anti-Brexit. Lastly, we consider a politician, "Theresa May", giving that at the time we crawled the articles she was the UK Prime Minister. Results show a convergence toward a neutral score on arousal for both pro-and anti-Brexit (both for support and against key-concepts). For what concerns valence and dominance, they are both in the negative area of the polarization: the anti-Brexit journal has a more negative valence in the cluster of the support aspect (i.e., they are against Theresa May), while the opposite holds for the against aspect cluster, where the pro-Brexit journal has, as expected, more negative valence. These results seem to imply that the pro-Brexit journal has a "lukewarm attitude" toward Theresa May (neutral arousal, almost neutral sentiment in support but negative sentiment when she is criticized).

Finally, in Table 3, we analysed some concepts for which we did not have enough occurrences to create the support and against aspect clusters, but are nonetheless important with respect to the journal stance. Referring to sub-concepts of the Brexit scenario, i.e., "clean Brexit", "soft Brexit" and "hard Brexit", we see a clear consistency on all VAD dimensions with the previous analysis. It is also interesting to note that without using the proposed aspects, we obtain a neutral position on the Brexit key-concept alone, and inexact positions on Theresa May as well (compare the two key-concepts in Tables 2 and3). This proves that in polarized settings, applying just standard SA to perform ABSA can be misleading for concepts with high occurrences, which is mostly the case in traditional sentiment extraction. Other sub-concepts of Brexit, such as "clean Brexit", "soft Brexit" and "hard Brexit", have highlighted the consistency of VAD trends such that the pro-Brexit journal reveals higher scores on all VAD elements for these sub-concepts, than the anti-Brexit one. The same holds for sub-concepts concerning "politician name" (i.e., pro-Brexit politicians have a positive valence in pro-Brexit journals and anti-Brexit politicians have better valence in anti-Brexit journals). Finally, also for "referendum" anti-Brexit journal shows a consistently higher valence (i.e., preference toward an additional referendum).

Conclusions and Future Work

In this work, we presented a methodology to extend the task of Aspect-based Sentiment Analysis so that to include affect and emotions representation in polarized settings. In particular, we adopted the three-dimensional model of affect based on Valence, Arousal, and Dominance. We tested the proposed framework on the Brexit scenario, showing how affect dimensions (VAD) vary on the same aspect when politically polarized stances are presented. Our approach was able to capture stereotypical aspect-based polarization from newspapers regarding the Brexit scenario using biased key-concepts (e.g., "stop Brexit" vs. "support Brexit").

As for future work, we plan to investigate the correlation of our results with virality indexes on social media (e.g., number of likes and retweets, number of replied messages). We also plan to have a comparative analysis with the textual material produced by the large audience that is supposed to share the same stance/polarization as the newspaper considering the comments to the articles.

Table 1 :

 1 Pearson's correlation (r) on Cross-dataset Testing of Emotion Recognition Models.

	Emotions	DepecheMood++ LSTM BiLSTM CNN
	ANGER	0.47	0.62	0.67	0.64
	FEAR	0.60	0.78	0.76	0.73
	JOY	0.38	0.64	0.63	0.51
	SADNESS	0.46	0.75	0.75	0.7
	SURPRISE	0.21	0.43	0.52	0.48
	AVG All Emotions	0.43	0.64	0.66	0.61

Table 2 :

 2 Polarization on VAD Dimensions using Key-Concepts Approach.

	Sub-concept	Valence	Arousal	Dominance
	Brexit
	Politician
	Name
	Referendum

Table 3 :

 3 Polarization on VAD Dimensions on Sub-concepts via Averaging Topic.

This work is licensed under a Creative Commons Attribution 4.0 International Licence. Licence details: http:// creativecommons.org/licenses/by/4.0/.

https://www.kcl.ac.uk/policy-institute/assets/divided-britain.pdf

The "DISGUST" label is excluded as there is no correspondence to Rappler emotions.

Acknowledgement

This work is partially supported by the ANSWER project PIA FSN2 n. P159564-2661789/DOS0060094 between Inria and Qwant. This work has been supported by the French government, through the 3IA Côte d'Azur Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-19-P3IA-0002.


Qiang Zhang

Dongxing Zheng

Yan Wen

Yuelei Zhao

Wenbo Mi

Aurelien Manchon

Olivier Boulle

Xixiang Zhang

Effect of surface roughness on the anomalous Hall effect in Fe thin films

Surface roughness plays an important role on the magnetotransport properties of thin films, especially in ultrathin films. In this work, we prepared Fe thin films with various surface roughness by using different seed layers and studied the electrical transport and anomalous Hall effect. By tuning surface roughness scattering, the longitudinal resistivity (ρ xx ) measured at 5 K increases by one order of magnitude and the corresponding anomalous Hall resistivity (ρ AHE ) increases by three times with increasing roughness. The intrinsic, skewscattering, and side-jump contributions to ρ AHE were separated from our data. The anomalous Hall angle depends on the surface roughness, which may be of importance to the material engineering for achieving large spin Hall angle.

I. INTRODUCTION

Anomalous Hall effect (AHE) in magnetic materials, since its discovery [1], has been extensively studied for the fundamental interests and potential applications. It is generally accepted that the intrinsic and extrinsic mechanisms, both of which are related to spin-orbit coupling, are responsible for the AHE. Microscopically, the intrinsic mechanism [2][3][4][5], independent of impurity scattering, originates from the Berry phase of the occupied Bloch states. The extrinsic mechanism arising from impurity scattering includes two contributions, skew scattering [6], and side jump [7]. Skew scattering comes from the asymmetric scattering at impurity sites and side jump emerges from transverse displacement of the wave function by impurity scattering.

Theories also suggested the scaling relations [2,6,7] between anomalous Hall resistivity (ρ AHE ) and longitudinal resistivity (ρ xx ) for each mechanism: ρ AHE ∝ ρ 2 xx for both the intrinsic and extrinsic side-jump mechanism, while ρ AHE ∝ ρ xx for extrinsic skew-scattering mechanism. The scaling law is usually applied to experimental data to explore the underlying mechanisms. Experimentally, both the ρ AHE and ρ xx should be tuned in some range to give a reliable scaling relation. To this end, one option is varying the film thickness where the ρ xx could be changed by surface scattering [8][9][10][11][12]. Films with different thickness are also eligible to study the surface scattering effect on the AHE [13,14]. However, surface scattering strength/potential is not only related to the thickness of the * Current address: Core Technology Platforms, New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, United Arab Emirates.

† Corresponding author: xixiang.zhang@kaust.edu.sa films but also to the surface roughness, especially in ultrathin 50 films. Furthermore, the studies of spintronic topics of spin 51 orbital torque (SOT), interfacial Dzyaloshinskii-Moriya Inter-52 action (DMI) and perpendicular magnetic anisotropy (PMA) 53 are all involved with ultrathin films. The surface/interface 54 roughness may play an important role on these topics (see 55 Sec. 1 in Ref. [15]), which has been omitted in these studies. 56 Few theoretical studies have suggested that surface roughness 57 scattering could lead to a completely different scaling relation 58 between ρ AHE and ρ xx for spin Hall effect [16] or enhance 59 the spin Hall angle [17] in ultrathin films. However, an 60 experimental study of surface roughness effect on magneto-61 transport properties, particularly AHE, in thin films is still 62 lacking.

63

In this study, we report the surface roughness effect on the 64 AHE in Fe thin films (see the definition of surface roughness 65 in Sec. 2 of Ref. [15]). The surface roughness could be 66 affected by several factors in deposited thin films: seed layer 67 on substrate [18], substrate temperature [19], thickness of 68 deposited films [20], argon pressure during deposition [21], 69 etc. To study the surface roughness effect, we must keep all 70 other parameters of the samples the same and only vary the 71 surface roughness. Therefore, we cannot use different deposi-72 tion temperature or thickness of films since it will change the 73 microstructure or surface scattering strength. We then used 74 different materials of seed layers to tune the surface rough-75 ness. This idea is utilizing the wetting property of materials, 76 which is similar to the chemistry concept of the wettability 77 of liquid contacting with a solid surface. By this method, we 78 may most probably be able to keep all parameters (such as 79 microstructure, thickness, etc.) the same but the roughness. 80 It is, therefore, feasible to the study of the impact of surface 81 roughness scattering on the AHE.

II. EXPERIMENTAL METHODS

The samples were deposited on substrates of oxidized silicon wafers by sputtering system (Rotaris, Singulus). The structure of the films is, from the substrate side, seed layer

III. RESULTS AND DISCUSSION

A. Structural characterizations

To demonstrate the surface roughness of the Fe layers, the samples was imaged by AFM. Due to the existence of the protection layers, the AFM images only give the surface roughness of the SiO 2 layers, which could indirectly reflect the surface roughness of Fe layers. As seen in Fig. S1 and the corresponding analysis in Supplemental Material [15], the roughness of sample Cu/Fe is higher than that of sample Ta/Fe, which indicates the larger roughness of Fe layer in Cu/Fe sample than that in Ta/Fe sample. To directly show the surface roughness of the Fe layers, we characterized the crosssections of the samples by HRTEM. The TEM specimens with cross-sections were extracted from the macroscopic samples using focus ion beam (FIB). Figures 1(a the wetting property of seed layers on SiO 2 . To examine the 140 microstructures of the samples, GID measurements were run 141 for all samples with grazing incident angle of 0.5°. Figure 1(c) 142 shows the GID spectra for all samples. The diffraction peaks 143 (110), (200), and (211) of bcc-Fe could be identified. For each 144 peak, the intensity and full width at half maximum (FWHM) 145 are quite identical for all samples, indicating that the samples 146 have similar crystallinity.

147

B. Longitudinal resistivity 148

To understand the surface roughness effect on the electrical 149 transport properties, we measured the temperature-dependent 150 longitudinal resistivity of the samples at zero magnetic field 151 in the temperature range of 5-300 K. Figure 2(a) presents 152 the ρ xx -T curves for all samples. It is evident that all 153 curves show an overall metallic behavior, i.e., all samples have 154 positive temperature coefficient of resistivity (TCR, dρ xx /dT ) 155 at high temperatures. The upturn of ρ xx (T ) curves below very 156 low temperature have a negative TCR and could be ascribed 157 to an electrical conduction dominated by two-dimensional 158 weak localization effect which was verified by the linear 159 dependence of ρ xx on lnT (see Fig. S2 in Ref. [15]). The 160 most important feature is that the ρ xx (T ) curves shift to higher 161 values over the whole temperature range from sample Ta/Fe 162 to Cu/Fe, although the thickness of Fe layers is identical 163 (5 nm) for all samples. At 5 K, the ρ xx increases by one order 164 of magnitude from sample Ta/Fe to Cu/Fe. The insulating 165 seed layers don't contribute any resistance to the measurement 166 results. For metal or alloy seed layers with thickness of 167 0.2 nm, they would have much higher resistance than that 168 of Fe layers due to the size effect [11]. Furthermore, the 169 0.2-nm-thick seed layers can hardly be continuous layers 170 but rather discontinuous clusters. Therefore, the seed layers important role in longitudinal resistivity although the samples have the same thickness and microstructure.

C. Anomalous Hall resistivity

Since we have observed that the surface roughness significantly affects the longitudinal resistivity, we now turn our attention to that how the surface roughness affects the anomalous Hall resistivity. The Hall resistivity (ρ xy ) for all samples was measured with a magnetic field applied perpendicularly to the plane of the films in a range of -50 kOe H 50 kOe and at temperatures ranging from 5 to 300 K. Figure 3(a) shows the field-dependent Hall resistivity of all samples measured at 5 K. As it is seen, the Hall resistivity follows a linear and strong dependence on applied magnetic field up to the magnetic saturation field (H sat ). At field higher than H sat , the Hall resistivity shows a linear and much weaker dependence on applied magnetic field. Zero coercivity has been observed in these curves. These are typical behaviors for magnetic thin films having in-plane magnetization at zero field. At high magnetic fields, the weakly field-dependent Hall resistivity should be ascribed to the ordinary Hall effect and the slight change of the AHE due to the improved alignment of spins. At this stage, the field-forced alignment of spins is a result of thermal agitation (except at 0 K) and nonferromagnetic coupling at Fe layer surface or defects inside Fe layers. Since Fe has high Curie temperature (1043 K), the thermal agitation

D. Anomalous Hall effect scaling analysis 233

To study the origin(s) of the AHE in these samples, the 234 conventional scaling relation,

235 ρ AHE /ρ xx = c + dρ xx , (1)
was usually employed, where c is the contribution of skew scattering, d is the contribution of intrinsic mechanism or side jump. This equation includes both the impurity scattering and phonon scattering effect on mechanisms of skew scattering or side jump. Recently, a new scaling relation [9],

ρ AHE = αρ xx0 + βρ 2 xx0 + bρ 2 xx , (2)
was proposed in Fe thin films, where α represents the contribution from the skew-scattering, β and b denote the sidejump and intrinsic anomalous Hall conductivity (AHC), respectively. The subscript "0" indicates that the data were obtained at low temperatures where thermal contribution is negligibly small. This scaling relation excluded the contribution of phonon scattering to skew scattering or side jump.

To demonstrate if the phonon scattering affects the extrinsic mechanisms, we plotted the relations, ρ AHE /ρ xx ∼ ρ xx and ρ AHE ∼ ρ 2 xx , for the data of each sample, as suggested in reference [9], to compare the linearity. We found that the relation ρ AHE ∼ ρ 2 xx shows better linearity for all samples (see Fig. S4 and the corresponding analysis in Ref. [15]). Therefore we could ignore the phonon scattering effect on skew scattering or side jump and use the new scaling, Eq. ( 2), to analyze our data.

To present the roughness effect on both the longitudinal resistivity and anomalous Hall resistivity, we plotted the relation ρ AHE0 ∼ ρ xx0 , obtained at 5 K, for all samples in Fig. 4(a). From sample Ta/Fe to Cu/Fe, the nonmonotonic dependence suggests very complicated roughness scattering effect on the AHE. One may ask if the ρ AHE0 could be affected by the saturation magnetization because anomalous Hall resistivity is not only related to the longitudinal resistivity but also the magnetization [START_REF] Zhang | [END_REF]24]. The saturation magnetization of the samples could be estimated by the magnetic saturation field in Hall resistivity curves, since the samples have in-plane magnetization (demagnetization factor N = 1 in out-of-plane direction), as shown in Fig. 3 would decrease, which has been reported in Fe ultrathin films [9,12]. For the flattest sample, Ta/Fe, the intrinsic anomalous

Hall conductivity is about 746.7 -1 cm -1 which is very close to the value obtained by first-principles calculation [5].

The intrinsic anomalous Hall resistivity could be subtracted from the total anomalous Hall resistivity and then the extrinsic contribution, including skew-scattering and sidejump, of anomalous Hall resistivity,

ρ AHE0(ex) = αρ xx0 + βρ 2 xx0
(3) could be obtained. The data of ρ AHE0(ex) versus ρ xx0 were plotted in Fig. 4(c). Obviously, this curve cannot be well fitted by Eq. ( 3). For the scattering-related transport properties in thin films, both the surface scattering and bulk scattering contribute. The longitudinal resistivity at 5 K could be expressed as

ρ xx0 = ρ xx0(s) + ρ xx0(b) . (4)
Accordingly, the scattering-related anomalous Hall resistivity at low temperatures is also expressed as

ρ AHE0(ex) = ρ AHE0(s) + ρ AHE0(b) . (5)
The subscripts (s) and (b) denote the contributions from the surface scattering and bulk scattering, respectively. In

instead of Eq. ( 3) is more appropriate to describe the behavior in Fig. 4(c). If we put Eqs. ( 4) and ( 5) into Eq. ( 6), Eq. ( 6) could be expressed as In this equation, ρ AHE0(ex) and ρ xx0 are variables and other 309 parameters are all constants. Using Eq. ( 7) to fit the 310 curve in Fig. 4(c), we can extract the parameters as β = 311 40.4 -1 cm -1 and α -2βρ xx0(b) = -1.59 × 10 -2 . Since 312 Eq. ( 7) is overparametrized, we cannot get the exact value 313 of α but roughly estimate it. For the sample Ta/Fe, ρ xx0 = 314 2.58×10 -5 cm and ρ xx0(b) should be lower than that. There-315 fore 2βρ xx0(b) ≪ 1.59 × 10 -2 and then α ≈ -1.59 × 10 -2 . 316 Comparing the ρ AHE0(ex) and intrinsic anomalous Hall resistiv-317 ity, the intrinsic one dominates the anomalous Hall resistivity 318 for all samples. Since we observed the variation of anomalous Hall resistivity for these samples with different seed layers, one may quest if the variation is caused by the interfacial spin-orbital coupling [25,26] or interfacial modification [27] other than the surface roughness. We then prepared several samples with the same roughness but different interfacial spin-orbital coupling. The sample structure is, from the substrate side, Fe(5 nm)/cover layer(0.2 nm)/SiO 2 (5 nm) with different cover layers (Ta, Ru, Cu). The sample, Fe(5 nm)/SiO 2 (5 nm), without metal cover layer was also prepared for comparison.

ρ AHE0(ex) = (α -2βρ xx0(b) )ρ xx0 + βρ 2 xx0 + βρ 2 xx0(b) -αρ xx0(b) + ρ AHE0(b) . (7)
The same substrate material (SiO 2 ) lead to the same roughness of Fe layers and different cover layers provide different interfacial spin-orbital coupling. We then measured the temperature-dependent longitudinal resistivity and anomalous Hall resistivity. Figure 5(a) shows the normalized longitudinal 358 resistivity curves for the four samples. As seen, the resistivity 359 ratio, ρ xx (T )/ρ xx (300 K), is quite identical, which indicates 360 the similar extent of surface scattering in these samples.

  (0.2 nm)/Fe (5 nm)/SiO 2 (5 nm) with different seed layers (Ta, Ir 20 Mn 80 , MgO, Ru, Co 60 Fe 20 B 20 , SiO 2 , Co, Ni, CuN, Cu). The samples were deposited, at room temperature, with the base pressure lower than 8.0 × 10 -9 mbar and the process pressure 3 × 10 -3 mbar. The deposition of SiO 2 with 5 nm thickness as a top layer for each sample is to prevent sample oxidization. The surface roughness of the samples was characterized by atomic force microscope (AFM) (Dimension Icon, Bruker). The cross-sections of the samples were imaged by high-resolution transmission electron microscopy (HRTEM) (Titan 80-300, FEI). Grazing incidence diffraction (GID) measurements were carried out using x-ray diffraction (XRD) (D8 ADVANCE DAVINCI design, Bruker) with Cu Kα radiation. The longitudinal resistivity and Hall resistivity were measured by a physical property measurement system (Dynacool 14T, Quantum Design). The Hall-bar samples with dimensions 1000 µm (length) × 50 µm (width) were patterned by photolithography and ion beam etching for transport measurements. A five-contact geometry was used for simultaneously measuring the Hall resistivity and longitudinal resistivity on the same piece of sample. A DC constant current of 20 µA was applied during all electrical transport measurements. The sample electrodes were connected to the sample holder of the PPMS by wire bonder using aluminum wires.

  FIG. 1. HRTEM images of cross-sections of the samples (a) Ta/Fe and (b) Cu/Fe. (c) GID spectra for all samples.

FIG. 2 .

 2 FIG. 2. (a) Temperature-dependent longitudinal resistivity (ρ xx ) curves for all samples. (b) Normalized ρ xx (T )/ρ xx (300 K) ∼ T curves for all samples.

  Fig.S3in Ref.[15], clearly demonstrating more frequent scat-186

FIG. 4 .

 4 FIG. 4. (a) Anomalous Hall resistivity (ρ AHE0 ) vs longitudinal resistivity (ρ xx0 ) at 5 K. The solid line is a guide to eyes. (b) Intrinsic anomalous Hall conductivity (b) as a function of ρ xx0 . The inset shows the linear dependence of b on σ xx0 . (c) Extrinsic anomalous Hall resistivity as a function of ρ xx0 . The solid line is a fitting curve by Eq. (7). The same set of data shown in the inset was fitted by Eq. (3). (d) The anomalous Hall angle (ρ AHE0 /ρ xx0 ) at 5 K vs ρ xx0 .effect could be ignored. For the nonultrathin Fe layers, the 213

  232

  (a). The variation of magnetization has minor effect on the ρ AHE0 . Since the linear relation, ρ AHE ∝ ρ 2 xx , has been observed for each sample, we could extract the intrinsic AHC which is independent of scattering by Eq. (2). The obtained values of b were plotted as a function of ρ xx0 in Fig. 4(b). The intrinsic AHC dramatically decreases with ρ xx0 increasing, which could be well described by the relation b ∝ 1/ρ xx0 or b ∝ σ xx0 . The inset shows the linear dependence of b on σ xx0 . Because the ρ xx0 was largely tuned by surface roughness scattering, the relation b ∝ 1/ρ xx0 clearly demonstrates the surface roughness effect on intrinsic AHC. In fact, the electrical transport properties of the samples are largely determined by the areas where the thickness is lower than the average thickness. With thickness decreasing, the intrinsic AHC

Fig. 4 (

 4 Fig. 4(c), the variation of ρ AHE0(ex) and ρ xx0 is only due to the surface scattering. The bulk scattering contribution, ρ AHE0(b) and ρ xx0(b) kept constant for these samples. Therefore using expression

FIG. 5 .

 5 FIG. 5. (a) Normalized ρ xx (T )/ρ xx (300 K) ∼ T curves for the samples listed in the legend. The legend is the same in (b) and (c). (b) Field-dependent Hall resistivity measured at 5 K. (c) Temperaturedependent ρ AHE for the samples.

Anomalous

  Hall angle characterized by the ratio of ρ AHE /ρ xx was also studied for these samples with various surface roughness. Figure4(d) shows the data, measured at 5 K, of ρ AHE0 /ρ xx0 versus ρ xx0 . The ρ AHE0 /ρ xx0 decreases with ρ xx0 increases at lower ρ xx0 and increases with ρ xx0 at higher ρ xx0 . Overall, the largest anomalous Hall angle was achieved in sample Ta/Fe, which is the flattest sample with minimum surface roughness. This finding opens a promising avenue for achieving large spin Hall angle by surface roughness engineering in heavy metals. The previous theoretical study on spin Hall effect has proposed surface roughness enhanced spin Hall angle in Cu and Al [17]. In this study, only surface roughness scattering related extrinsic mechanisms were considered and intrinsic mechanism was not discussed. It turns out that the side-jump mechanism contributes to the surface roughness induced spin Hall effect but screw scattering does not. In our work, both screw scattering and side jump contribute to the extrinsic mechanisms which may partially come from the surface scattering, because the roughness scattering and effective surface scattering cannot be separated experimentally. With the surface roughness increases, the side jump contributed anomalous Hall angle (βρ xx0(s) ) increases, which is consistent with the results of the theoretical work [17].

  Fig-361 ure 5(b) presents the field-dependent Hall resistivity measured 362 at 5 K for the four samples. The four curves show similar 363 behaviors and Hall resistivity. The values of ρ AHE , extracted 364 from the Hall resistivity curves at different temperatures, were 365 presented in Fig. 5(c). The ρ AHE gives identical values at 366 each temperature for different samples, which indicates that 367 the various interfacial spin-orbital coupling cannot explain the 368 large difference of ρ AHE in Fig. 3(b).

370

  We prepared Fe thin films with the same thickness but 371 various surface roughness by using different materials of seed 372 layers. The microstructure and magneto-transport properties 373 were studied systematically. The samples show similar crys-374 tallinity as seen in GID patterns and distinct surface roughness 375 confirmed by TEM images of cross-sections. The magnitude 376 of longitudinal resistivity measured at 5 K increases by one 377 order from sample Ta/Fe to Cu/Fe. The anomalous Hall 378 resistivity at 5 K vary with different roughness and shows 379 nonmonotonic dependence on ρ xx0 . The intrinsic contribution 380 dominated the anomalous Hall resistivity and shows linear 381 dependence on the longitudinal conductivity at 5 K. The 382 anomalous Hall angle depends on the surface roughness, 383 which provides an alternative to achieve large spin Hall effect 384 experimentally. 385 ACKNOWLEDGMENTS 386 The research reported in this publication was supported 387 by funding from King Abdullah University of Science and 388 Technology (KAUST), Office of Sponsored Research (OSR) 389 under Award No. CRF-2017-3427-CRG6. Q.Z. acknowledge 390 the financial support by KAUST sensor project (REP/1/2708-391 01).

  392