Christoforos Theodorou
email: christoforos.theodorou@grenoble-inp.fr

Michail Ziogas

A GENERAL USE CIRCUIT FOR AUDIO SIGNAL DISTORTION EXPLOTING ANY NON-LINEAR ELECTRON DEVICE

In this paper, we propose the use of the transimpedance amplifier configuration as a simple generic circuit for electron device-based audio distortion. The goal is to take advantage of the non-linearities in the transfer curves of any device, such as diode, JFET, MOSFET, and control the level and type of harmonic distortion only through bias voltages and signal amplitude. The case of a nMOSFET is taken as a case study, revealing a rich dependence of generated harmonics on the region of operation (linear to saturation), and from weak to strong inversion. A continuous and analytical Lambert-W based model was used for simulations of harmonic distortion, which were verified through measurements.

INTRODUCTION

Since the beginning of electrical musical instruments, musicians are trying to achieve a unique sound through their gear with all kinds of effect units. Modulation, reverb, overdrive and distortion effects are widely used for electric guitars but nowadays also by any electric string instrument, synthesizers, keyboards or even vocals. For example, a signal can be distorted through soft or hard clipping, or through any non-linear transfer function, symmetrical or asymmetrical. To achieve all these cases of signal distortion, the typical circuits used are single or multiple gain stages with or without diode clipping. For the gain stages there are hundreds of different combinations that can be used: from vacuum tubes [START_REF] Hamasaki | Learn about harmonics by means of vacuum tube guitar amplifier[END_REF], [START_REF] Takemoto | Supply voltage scaling technique of triode tube based on harmonic distortion characteristics[END_REF] to opamps [START_REF] Paiva | Emulation of operational amplifiers and diodes in audio distortion circuits[END_REF], from silicon to germanium diodes [START_REF] Reiss | Overdrive, Distortion, and Fuzz[END_REF], from BJTs to JFETs and MOSFETs [START_REF] Réveillac | Musical Sound Effects[END_REF], [START_REF] Dailey | Electronics for Guitarists[END_REF], each circuit has a different transfer curve and as a result a unique tone character, usually thanks to the devices' non-linear response. Diodes are used for both soft and hard or shunt clipping, in different configurations: in amplifier feedback loop or shunt to ground, respectively. The clipping of the signal can also be symmetrical or asymmetrical, leading to purely odd or odd/even harmonics.

To our knowledge, in all above cases, the non-linear devices are either used as non-linear resistors in an OPAMP's feedback loop or as shunting elements. As a result, only specific amplitudedependent regions of non-linear response are being exploited. In this work, we propose a method with which one can make use of any non-linear I-V characteristic exploiting various voltage bias regions, while having complete control of the generated harmonics. Despite the fact that there has been an in-depth analysis of harmonic distortion in MOSFETs [START_REF] Sansen | Distortion in elementary transistor circuits[END_REF] and even more advanced FET structures [START_REF] Paz | Non-linearity analysis of triple gate SOI nanowires MOSFETS[END_REF], [START_REF] Cerdeira | New method for determination of harmonic distortion in SOI FD transistors[END_REF], it has not yet been used in the configuration that we propose, therefore we chose the MOSFET device as a case study to demonstrate the applicability and advantages of our method.

DEVELOPMENT OF THE PROPOSED METHOD

In this section, we present the step-by-step development of the proposed method, presenting the case of a n-channel MOSFET as an example for electron device-based harmonic distortion.

The transimpedance amplifier as an electron device characterization instrument

The Transimpedance amplifiers (TIA) are widely used to translate the current output of sensors like photodiode-to-voltage signals [START_REF] Beguni | Photodiode Amplifier with Transimpedance and Differential Stages for Automotive Visible Light Applications[END_REF], since many circuits and instruments can only accept voltage input. Moreover, they are used as current preamplifiers in precision measurement instruments such as in current DC and noise characterization [START_REF] Chroboczek | Low Frequency Noise Point Probe Measurements On A Wafer Level Using A Novel Programmable Current Amplifier[END_REF]. for any device under test (DUT) connected at its input and controlled by various bias voltages (V, Vbias). Since the current flowing in the input of the OPAMP can be considered zero, and the negative feedback resistor guarantees the OPAMP's linear regime, the DUT current has to be equal to the current flowing in the feedback loop. Therefore IDUT = (Vo-V)/Rf, because the OPAMP functions in linear mode thus V+ = V-. So, the output voltage of a TIA would be:

𝑉 𝑜 = 𝑉 + 𝐼 𝐷𝑈𝑇 𝑅 𝑓 (1)
It becomes obvious that the DC voltage output is actually independent of any other device bias voltages other than the one connected to the OPAMP. Of course, it has to be noted that we consider an ideal OPAMP with linear response for a large range of voltage swings and with no input voltage offsets. In a real scenario, one needs to correct the input offset and make sure the output voltage does not exceed the linear response region of the OPAMP, which is directly limited by the supply voltage bias. Fortunately, this can be easily regulated with the value of Rf, so that the output signal is never higher than the maximum allowed limit for linear OPAMP operation. Moreover, a real OPAMP has also a maximum output current above which it also saturates. The latter issue cannot be avoided by adjusting Rf, but only by choosing carefully the OPAMP model so that it can provide the maximum current of the connected DUT. Now if we consider an AC output after a high-pass filter (HPF) that cuts frequency content below 10 Hz for example, we could approximate the output voltage signal as:

𝑣 𝑜 = 𝑖 𝐷𝑈𝑇 𝑅 𝑓 (2)
where iDUT are the current variations of the DUT due to variations in one (or more) of the bias voltages. Therefore the voltage signal of the output is a direct linear function of solely the device AC current, with a gain equal to the feedback resistor value, Rf.

An alternative way to obtain (2) without the use of a filter, which could impact the signal's phase at low frequencies, is to connect a voltage subtractor circuit in series with the output, to remove the same DC voltage V that we apply at the OPAMP's (+) input. Finally, as happens with all analog audio circuits, since both the DUT and the OPAMP have DC/AC characteristics and responses that vary for each copy of the device, some manual tweaking of supply bias and DUT bias may be needed to obtain exactly the same behaviour from one circuit to another identical one.

Controlling the harmonic distortion through voltage bias and signal amplitude

In the example of Fig. 1, the current flowing through the device under test can be expressed as IDUT = I(V, Vbias1, Vbias2). Therefore, an AC component, vin, is added to one of the bias voltages, and the rest are constant with time, the small-signal AC output of the OPAMP would be:

v 0 = 𝜕I 𝐷𝑈𝑇 𝜕V in 𝑣 in 𝑅 𝑓 = f(V in , v in , V bias,1-2 , … ) (3)
where

𝜕𝑖 𝐷𝑈𝑇
𝜕V in is a function of the DC bias voltages and corresponds to the 1 st order sensitivity of IDUT to Vin. This means that the dynamic output response is unique and fully controlled by the choice of signal input and the bias voltages. Now, if we also account for higher order components [START_REF] Sansen | Distortion in elementary transistor circuits[END_REF] introduced by a non-linear relation of IDUT with Vbias, we obtain:

v 0 = 𝑅 𝑓 ( 𝜕I 𝐷𝑈𝑇 𝜕V in 𝑣 in + 1 2 𝜕 2 I 𝐷𝑈𝑇 𝜕V in 2 𝑣 in 2 + ⋯ + 1 n! 𝜕 n I 𝐷𝑈𝑇 𝜕V in n 𝑣 in n ) (4)
We can therefore see that with the configuration of Fig. 1, even the generated harmonics that define the distortion levels only depend on the relation between IDUT and the voltage bias where the signal is applied, for the given DC biases.

CASE STUDY: THE MOSFET

TIA-based MOSFET current converter

A simple example of a TIA-based circuit is the current measurement schematic for a n-channel MOSFET (Fig. 2). The output signal's (Vo) linearity is directly dependent on the region of operation (linear/saturation, weak/strong inversion) and the varying voltage. In case the signal is applied on the gate, G, only the input characteristics (Id-Vg) should be accounted for, whereas only the output ones (Id-Vd) if the drain, D, carries the signal. To understand the potential advantages for distortion, compared to a MOSFET amplifier, we underline that for the latter, the signal gain is also a function of the region of operation (linear/saturation) which affects the DC operating point and the load line. In fact, in order for the gain to be independent from the drain bias, the MOSFET in linear amplifiers is always biased in saturation, where -supposing a perfectly saturated current-the current is no more dependent on the drain voltage. In conclusion, for each DC bias and signal amplitude, one would obtain a specific set of harmonics, which only depends on the I-V transfer curves of the DUT and not in any other circuit elements, such as bias resistances as is the case in MOSFET amplifiers. This way, a high level of control and predictability over the generated distortion can be achieved, only by knowing the static (DC) behaviour of the electron device.

MOSFET modelling for simulations

In order to simulate the precise response of such a circuit, we need MOSFET models that are continuous and analytical from weak to strong inversion and from linear to saturation regimes. Thankfully, such a modelling approach exists and utilizes the Lambert-W (LW) function [START_REF] Papathanasiou | Symmetrical unified compact model of short-channel double-gate MOSFETs[END_REF]- [START_REF] Karatsori | Full gate voltage range Lambertfunction based methodology for FDSOI MOSFET parameter extraction[END_REF], which provides a very good description of the inversion charge behavior in all bias regions. This modelling approach is described by equations (5): where W, L the width and length of the channel, μeff the effective mobility, Vt the threshold voltage, η the sub-threshold ideality factor (=1 for 60mV/dec), kT the thermal voltage (≅26mV at room temperature), and Cox the oxide capacitance per unit area.

I d = W L μ eff [(𝑞 𝑠 -𝑞 𝑑 ) + 1 2𝜂𝑘𝑇C ox (𝑞 𝑠 2 -𝑞 𝑑 2 )] (5a
The quantities qs and qd represent the carrier charge densities near the source, S, and drain, D, of the transistor, respectively. Fig. 3 shows an example of input and output MOSFET transfer curves as calculated by ( 5), with W = 1 μm, L = 0.2 μm, Vt = 0.5 V, μeff = 100cm 2 /Vs, Cox = 1.2μF/cm 2 , and η = 1.

The typical behavior of a MOSFET's drain current, Id, with gate and drain bias voltages can be seen:

1) exponential increase with Vg below Vt (weak inversion), whereas linear above Vt (strong inversion) for low Vd values and quadratic for Vd>Vg-Vt, whereas 2) linear increase with Vd for Vd<<Vg-Vt (linear regime), constant current (saturation) for Vd>Vg-Vt, and logarithmic behavior between the two (triode region).

Figure 3: Typical DC transfer characteristics for varying gate voltage (top/lin-log Y-scale) and drain voltage (bottom)

Thanks to the simplicity of the proposed TIA-based circuit (Fig. 2), these curves are actually translated into voltage transfer functions (Fig. 4), because V0 = Vd + Id Rf. The Rf value is adapted so that the maximum voltage does not surpass the power supply of the OPAMP (+15V). The offset in high Vd values can be corrected by subtracting the DC bias of the drain from the output signal.

Consequently, depending on the DC bias around which we apply our signal, as well as the signal amplitude itself, we can expect very different results in terms of distortion. This is graphically demonstrated in Fig. 5, where an AC signal is applied at the MOSFET's gate, around 2 different DC bias (moderate and strong inversion) and with two different amplitudes. The first signal, oscillating around moderate inversion, is objected to the exponential dependence of Id(Vg), resulting in a signal with almost clipped bottom half and an exponentially distorted top half. On the other hand, if we apply a signal around a Vg well above Vt, the signal has almost no distortion in linear region, while following a quadratic distortion in saturation (high Vd). It should be noted here that the signal amplitude was deliberately chosen to be significantly high, in order to capture the passage from weak to strong inversion and the quadratic dependence. If the signal amplitude is very small (mV range) and the DC bias is well above Vt, the distortion will be negligible.

Figure 5: Graphic demonstration of the distortion effect of the proposed circuit (Fig. 2), depending on the DC bias and AC amplitude of a signal applied at the transistor gate (TIA: Transimpedance amplifier, HPF: high-pass filter).

Harmonic distortion analysis

Following the same simulation method, we examined many different scenarios of gate and drain DC bias and AC signal amplitudes, to reveal the variety of different harmonic content. Fig. 6 and Fig. 7 show two examples where both DC biases are kept constant at Vg = Vd = 1 V, but the signal (f = 1 kHz) is applied at the gate (Fig. 6) or at the drain (Fig. 7). The power spectra shown are obtained using the Welch periodogram method in SciPy (Python), whereas the signals have been normalized in amplitude so that they can be easily plotted and visualized together in the same graph; in any case, large amplitude differences can be compensated at the output thanks to Rf.

It becomes evident from Fig. 6 that when the signal amplitude is large enough to cover two different transistor operation regimes, it can dramatically affect the harmonic distortion. That's because in the case of varying Vg in saturation (Fig. 6), the signal is prone to the ~(Vg-Vt) 2 behavior of Id, whereas varying Vd in strong inversion around 1 V (Fig. 7) can actually cause asymmetrical hard clipping of the signal due to the alternating between linear and triode regimes. As a result, the harmonic distortion in the latter case is much more severe, which is directly visible in the high amplitude of high-order harmonics.

We repeated this type of simulations for many combinations of DC biases Vg and Vd, from 0 up to 2 V, and various signal amplitudes, vamp, from 50mV to 0.5V, and we extracted the power values of each harmonic in order to visualize their dependence with the applied voltages. Fig. 8 shows the relative (with regard to the fundamental's power) power of four harmonics (2 nd to 5 th ) for vamp = 0.5 V applied at the gate and Fig. 9 at the drain. For the case where the signal is around Vg (Fig. 8), a clear reduction of harmonic distortion with Vg is visible, whereas this reduction for the 2 nd and 3 rd harmonic can be cancelled out by high Vd bias. The 4 th and 5 th harmonic seem to have a negligible amplitude, except for the case of very low Vg values (weak inversion).

Concerning the case where the signal is applied at the drain (V+ input of OPAMP), high Vd values can make the harmonic distortion immune to variations in Vg bias, and it is worth noting that here, even the 4 th and 5 th harmonics have very significant contributions.

By using formula (6), we also calculated the total harmonic distortion (THD) for various combinations of Vg and Vd, accounting up to the 5 th harmonic.

THD(%) = 100√

HD2+HD3+HD4+HD5 HD1 [START_REF] Dailey | Electronics for Guitarists[END_REF] where HDx the power of the x th harmonic. The results are plotted in Fig. 10, for vamp = 0.2 V applied at the MOSFET's gate, and in Fig. 11 for the case where the signal is applied at the (+) input.

It is worth noting that in both cases (AC around Vg and around Vd), the THD reaches 100% for certain DC bias combinations, while it can also be decreased down to 0.1% for others. This is a direct confirmation of our hypothesis that with the circuit architecture of Fig. 2, any amount of THD can be achieved, provided that one examines all the possible bias conditions.

Moreover, the THD has a characteristic value for each combination of Vg, Vd and vamp, making it easy to use this concept as the basis for a variety of audio distortion (or even tone control) applications. For example, as far as vamp is concerned, Fig. 12 DAFx.5

shows how it can impact THD when the signal is at the gate, for both linear (Vd = 0.1 V) and saturation (Vd = 2 V) regions. Similarly, in Fig. 13 is plotted the THD versus vamp when the signal is at the (+) OPAMP's input, for both weak (Vg = 0.5 V) and strong (Vg = 1.5 V) inversion regions. As it can be seen, the THD is always increasing with vamp, except when the DC bias is at weak inversion (Vg = 0.5 V), where THD reaches a plateau around vamp = 0.2 V. Note also how there is no signal for Vd = 1.5 V in weak inversion, because the signal amplitude needed to reach triode region is higher than the maximum value of vd,amp. Therefore the drain current is constant whatever the fluctuation of Vd.

MEASUREMENT RESULTS

We constructed the circuit shown in Fig. 2, using the Onsemi BS170 enhancement mode n-channel MOSFET as a device under test, which has a Vt around 2 V. Following the same methodology as in the simulations, we tested the output response for various Vg and Vd DC bias and signal amplitudes, with the signal applied at the MOSFET's gate. Some examples are shown in Fig. 14 (linear regime) and Fig. 15 (saturation regime). The value of Rf was modified for every case, in order to avoid OPAMP saturation or negligible output amplitude.

In Fig. 14 we note a verification of the bottom half soft clipping due to alternating from strong to weak inversion in linear region (as in Fig. 5), while in Fig. 15 the quadratic dependence of Id(Vg) in saturation regime gives a smooth distortion to the signal. For higher signal amplitudes, a more severe low-half clipping is visible, due to the passage to weak inversion (as in Fig. 6).

CONCLUSIONS

We have presented an analog circuit that can be used to exploit the non-linearities in any electron device, aiming the harmonic audio distortion. The n-channel MOSFET was shown and studied as an example, revealing a high level of control of the output signal harmonics through the DC gate and drain bias voltages, the signal's amplitude, as well as its input position (gate or drain). The effect was studied both through simulations and measurements. The proposed circuit configuration could be potentially used in guitar effect pedals or analog synthesizers.

  Copyright: © 2023 Christoforos Theodorou and Michail Ziogas. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, adaptation, and reproduction in any medium, provided the original author and source are credited.

Figure 1 :

 1 Figure 1: Typical current-to-voltage converter configuration with transimpedance amplifier for electron device characterization (DC and noise)As shown in Fig.1, the (TIAs) configuration of an operational amplifier (OPAMP) can be used as a current-to-voltage converter

Figure 2 :

 2 Figure 2: Current-to-voltage converter configuration for MOSFET characterization (DC and noise)

)

  𝑞 𝑠 = 𝜂𝑘𝑇𝐶 𝑜𝑥 𝐿𝑊(𝑒 (𝑉 𝑔 -𝑉 𝑡 )/𝜂𝑘𝑇 ) (5b) Proceedings of the 26th International Conference on Digital Audio Effects (DAFx23), Copenhagen, Denmark, 4 -7 September 2023 DAFx.3 𝑞 d = 𝜂𝑘𝑇𝐶 𝑜𝑥 𝐿𝑊(𝑒 (𝑉 𝑔 -𝑉 𝑡 -𝑉 d )/𝜂𝑘𝑇 ) (5c)

Figure 4 :

 4 Figure 4: Transfer characteristics of Fig.2's circuit with the I-V curves of Fig. 3.

Figure 6 :

 6 Figure 6: Power spectrum calculations for the case where a signal is applied at the MOSFET gate for 3 different AC amplitudes. DC bias: Vg = Vd = 1 V.

Figure 7 :

 7 Figure 7: Power spectrum calculations for the case where a signal is applied at the OPAMP's (+) input for 3 different AC amplitudes. DC bias: Vg = Vd = 1 V.

Figure 8 :

 8 Figure 8: Relative power levels of the 4 first harmonics for a signal of vamp = 0.5 V amplitude applied at the gate, and various combinations of Vg and Vd DC bias.

Figure 9 :

 9 Figure 9: Relative power levels of the 4 first harmonics for a signal of vd,amp = 0.5 V amplitude applied at (+), and various combinations of Vg and Vd DC bias.

Figure 10 :

 10 Figure 10: THD for a signal of vamp = 0.2 V amplitude applied at the gate, and various combinations of Vg and Vd DC bias.

Figure 11 :

 11 Figure 11: THD for a signal of vamp = 0.2 V amplitude applied at (+), and various combinations of Vg and Vd DC bias.

Figure 12 :

 12 Figure 12: THD versus varying signal amplitude vamp applied at the MOSFET's gate, for linear (left) and saturation (right) regimes.

Figure 13 :

 13 Figure 13: THD versus varying signal amplitude vamp applied at the OPAMP's (+) input, for weak (left) and strong (right) inversion regions.

Figure 14 :

 14 Figure 14: Measured input (yellow) and output (blue) signals for Vd = 50 mV, Vg = 3 V, vamp = 1 V (top) and 2 V (bottom) around gate voltage.

Figure 15 :

 15 Figure 15: Measured input (yellow) and output (blue) signals for Vd = 0.5 V, Vg = 2 V, vamp = 0.1 V (top) and 0.2 V (bottom) around gate voltage.


Cécile Blanchon

Eve Toulza

Christophe Calvayrac

Stanislawa Eichendorff

Marie-Agnes Travers

Jeremie Vidal-Dupiol

Caroline Montagnani

Jean-Michel Escoubas

Christophe Stavrakakis

Gaël Plantard

Marie- Agnès Travers

Gael Plantard

Inactivation of two oyster pathogens by photocatalysis and monitoring of changes in the microbiota of seawater: a case study on

Keywords: OsHV-1 µVar, Vibrio harveyi, seawater microbiota, photocatalysis, oyster pathogens, inactivation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Contaminated seawater is one of the biggest sources of economic losses in aquaculture (pisciculture, shellfish farming,…) and fishing activity with approximately 60 millions euros losses in 2014 (SE Tunisia) (El Zrelli et al., 2020). Those losses are related to a fish density decrease in the fishing areas due to ecosystem changes [START_REF] Hernández-Terrones | Water quality assessment in the Mexican Caribbean: Impacts on the coastal ecosystem[END_REF], and especially the contamination of seawater which can negatively affect fish, shellfish, molluscs and algae [START_REF] Bado-Nilles | Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg)[END_REF][START_REF] Bayen | Exposure and response of aquacultured oysters, Crassostrea gigas, to marine contaminants[END_REF][START_REF] Gagnaire | Combination of a pesticide exposure and a bacterial challenge: In vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg)[END_REF][START_REF] Gagnaire | Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: Screening of 23 molecules using flow cytometry[END_REF] or the rights of to harvest these organisms. At the same time, more intensive farms have been created but, in those places, the higher animal density enhance disease development. In seafood production, diseases are often caused by bacterial and/or virus infections and various microorganisms (algae, fungi, bacteria), and can induce production losses but sometimes those losses can be attributed to biomolecules as toxins or mycotoxins produced by fungi or algae [START_REF] Gholami | Environmental risk assessment of harmful algal blooms case study: Persian Gulf and Oman Sea located at Hormozgan Province, Iran[END_REF]Radcliffe, 2017;[START_REF] Yanong | Fungal diseases of fish[END_REF]. Till date, antibiotics remain the most used method to indulge the bacterial infections in aquaculture but this treatment does not protect against virus or toxins and it enhances emergence of resistance to antibiotics [START_REF] Pérez-Sánchez | Biological Approaches for Disease Control in Aquaculture: Advantages, Limitations and Challenges[END_REF][START_REF] You | Tackling Vibrio parahaemolyticus in ready-to-eat raw fish flesh slices using lytic phage VPT02 isolated from market oyster[END_REF]. To eliminate potential pathogens, disinfection procedures are currently applied in closed-loop aquaculture systems, such as filtration of incoming seawater to 20 microns [START_REF] Cordier | Removal of pathogens by ultrafiltration from sea water[END_REF][START_REF] Whittington | Removal of oyster pathogens from seawater[END_REF], chlorination [START_REF] Ben-Asher | Chlorine-based disinfection for controlling horizontal transmission of VNN in a seawater recirculating aquaculture system growing European seabass[END_REF], UVC disinfection [START_REF] Aguilar | Inactivation of a wild isolated Klebsiella pneumoniae by photo-chemical processes: UV-C, UV-C/H2O2 and UV-C/H2O2/Fe3+[END_REF][START_REF] Sharrer | Inactivation of bacteria using ultraviolet irradiation in a recirculating salmonid culture system[END_REF], disinfection with peracetic acid [START_REF] Liu | Peracetic acid is a suitable disinfectant for recirculating fish-microalgae integrated multi-trophic aquaculture systems[END_REF] or hydrogen peroxide [START_REF] Blanchon | Comparison of Infectious Agents Susceptibility to Photocatalytic Effects of Nanosized Titanium and Zinc Oxides: A Practical Approach 15[END_REF][START_REF] Pedersen | Hydrogen peroxide application to a commercial recirculating aquaculture system[END_REF], or ozonation [START_REF] Davidson | The effects of ozone and water exchange rates on water quality and rainbow trout Oncorhynchus mykiss performance in replicated water recirculating systems[END_REF]. These methods can lead to the production of toxic by-products in the water [START_REF] Chhetri | Acute toxicity and risk evaluation of the CSO disinfectants performic acid, peracetic acid, chlorine dioxide and their by-products hydrogen peroxide and chlorite[END_REF], are expensive due to chemical and energy costs [START_REF] Badiola | Energy use in Recirculating Aquaculture Systems (RAS): A review[END_REF] or do not inactivate toxins of biological origin. Therefore, although these seawater treatments are now being applied in the aquaculture sector, there is still a need to develop alternative seawater disinfection methods. In order to inactivate microbial contaminants present in seawater, as well as toxins and other organic contaminants, advanced oxidation processes (AOPs) [START_REF] Foster | Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity[END_REF], such as heterogeneous photocatalyis, are one of the most promising solutions. As opposed to homogeneous photocatalyis (ozonation or fenton), these require the use of a solid catalyst, which can be reused, and no toxic by-products are produced during the reaction.

Nonetheless, while the use of the photocatalytic oxidation reaction of titanium dioxide (TiO2) for the elimination of organic biotic contamination in freshwater and wastewater has been widely documented [START_REF] De Pasquale | TiO2-based nanomaterials assisted photocatalytic treatment for virus inactivation: perspectives and applications[END_REF][START_REF] Kacem | Modeling heterogeneous photocatalytic inactivation of E . coli using suspended and immobilized TiO 2 reactors[END_REF][START_REF] Liga | Virus inactivation by silver doped titanium dioxide nanoparticles for drinking water treatment[END_REF][START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends[END_REF][START_REF] Mayer | The Impact of Capsid Proteins on Virus Removal and Inactivation during Water Treatment Processes[END_REF][START_REF] Yamada | Identification of Vibrio alginolyticus as a causative pathogen associated with mass summer mortality of the Pacific Oyster (Crassostrea gigas) in China[END_REF], it remains largely unexplored in the case of seawater, particularly for bacterial and/or viral inactivation [START_REF] García-Garay | Wild microorganism and plankton decay in ballast water treatments by solar disinfection (SODIS) and advanced oxidation processes[END_REF][START_REF] Levchuk | UVA and solar driven photocatalysis with rGO/TiO2/polysiloxane for inactivation of pathogens in recirculation aquaculture systems (RAS) streams[END_REF][START_REF] Levchuk | Solar photocatalytic disinfection using ink-jet printed composite TiO2/SiO2 thin films on flexible substrate: Applicability to drinking and marine water[END_REF]Rubio et al., 2013). In addition, it should be pointed out that titanium dioxide (TiO2) is one of the most interesting catalysts for photocatalyis water treatment applications [START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends[END_REF], as this compound is inert, can be converted into different forms and allows the production of radicals capable of treating chemical compounds as pharmaceutical or herbicide substances and biotic contaminants such as gram-positive and gram-negative bacteria [START_REF] Brienza | Use of solar advanced oxidation processes for wastewater treatment: Followup on degradation products, acute toxicity, genotoxicity and estrogenicity[END_REF][START_REF] Kacem | Kinetics and efficiency displayed by supported and suspended TiO2 catalysts applied to the disinfection of Escherichia coli[END_REF]. However, using photocatalysis to treat seawater raises a number of challenges, not least because of the presence of salt and other suspended residues that can slow down the inactivation of targeted pathogens [START_REF] Blanchon | Comparison of Infectious Agents Susceptibility to Photocatalytic Effects of Nanosized Titanium and Zinc Oxides: A Practical Approach 15[END_REF].

In seafood production, aquaculture represents one of the fastest-growing food industries with more than 50% of the worldwide production [START_REF] Ritchie | Improving UV seawater disinfection with immobilized TiO2: Study of the viability of photocatalysis (UV254/TiO2) as seawater disinfection technology[END_REF]. The mollusk aquaculture production is dominated by oyster production which is subject to mortality events at every life stage, induced by many Vibrio bacteria. During the larval stage oysters are subject to mortality induced by Vibrio coralliilyticus or Vibrio tubiashii [START_REF] Richards | Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii[END_REF].

Then, juvenils oysters can be subject to mortality induced by Vibrio alginolyticus [START_REF] Yamada | Identification of Vibrio alginolyticus as a causative pathogen associated with mass summer mortality of the Pacific Oyster (Crassostrea gigas) in China[END_REF], Vibrio tasmaniensis or Vibrio crassostreae. Once adult, oysters can be subject to mortality induce by Vibrio aestuarianus [START_REF] Mesnil | Emergence and clonal expansion of Vibrio aestuarianus lineages pathogenic for oysters in Europe[END_REF] or Vibrio harveyi [START_REF] Oksanen | Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus[END_REF][START_REF] Saulnier | A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity[END_REF]. Production losses in aquaculture can also be caused by the accumulation of human vibrio pathogens on oysters such as Vibrio vulnificus and Vibrio parahaemolyticus [START_REF] Campbell | Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus : A review of ecology and processing parameters[END_REF], which will result in a ban on sales. The Pacific Oyster Mortality Syndrome (POMS) is one of the most prevalent diseases which can kill up to 100% of oysters spat in France every year [START_REF] Ecoscopa ; Zrelli | Economic impact of human-induced shrinkage of Posidonia oceanica meadows on coastal fisheries in the Gabes Gulf (Tunisia, Southern Mediterranean Sea)[END_REF]. This disease specifically affecting larvae and juvenile oysters is dependent on multiple environmental factors and microbes [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF][START_REF] Petton | The Pacific Oyster Mortality Syndrome, a Polymicrobial and Multifactorial Disease: State of Knowledge and Future Directions[END_REF]. We recently evidenced the course of events leading to oyster death when facing POMS [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]. POMS is triggered by an early infection with OsHV-1 µVar herpes virus (Ostreid herpes virus) which impairs the oyster antibacterial defenses following the infection of oyster immune cells (hemocytes). This immune suppression induces a dysbiosis that enhances the proliferation of opportunistic bacteria, ultimately causing septicemia and the death of the oyster.

The aim of this study was to investigate the potential of UV365/TiO2 seawater treatment for microbial inactivation in the context of oyster mortality. This article attempts to provide new insight into the impact of heterogeneous photocatalytic treatment on two major classes of aquaculture pathogens, Vibrio bacteria and herpes virus considered separately (Vibrio harveyi isolate) and on a complex microbial community composed of herpes virus and bacteria released by diseased oysters. The impact of photocatalytic treatment was systematically compared with that of UV365 treatment alone (photolysis), in terms of pathogen survival (viable, cultivable Vibrio or virus infectivity during experimental infection) and DNA integrity (DNA quantification).

Materials & Methods

Catalyst and pilot used for seawater disinfection

In this study, TiO2 in its photosphere form was used [START_REF] Joannis-Cassan | Towards an innovative combined process coupling biodegradation and photo-oxidation for the removal of pharmaceutical residues[END_REF]. These are anatase TiO2-coated glass microspheres centered on a diameter of 45 µm (range of 5 µm to 85 µm) and with a specific surface of 11.5 m 2 .g -1 [START_REF] Heydari | Passive or Semi-Passive Photocatalytic Treatment of Organic Pollutants in Water[END_REF]. Due to their low density (ρ=0.22 g.L -1 ), they facilitate the separation of the liquid/solid phases by flotation at the end of the treatment process (Reoyo-Prats et al., 2022). Preliminary tests carried out in freshwater during previous work on the decontamination of pharmaceutical targets concluded that a concentration of 5 g.L -1 of photospheres was optimal to capture all the incident radiation. All photons are absorbed across the thickness of the reactor, so transmittance is zero at the bottom. As the presence of salt in the medium has no influence on photon absorption, this concentration was retained in this study [START_REF] Plantard | Correlations between optical, specific surface and photocatalytic properties of media integrated in a photo-reactor[END_REF]. The experiments were conducted in 200 mL beakers (60 cm 2 surface area) containing seawater and catalyst. The suspension was homogenized with a magnetic stirrer and maintained at 19 ± 1 °C by a water bath. During the experiment, the beakers were subjected to generated artificial UV radiation (Vilbert Lourmat lamp, model VL-300 with an emission spectrum from 300 nm to 400 nm centered at 365 nm). A flux density of 35 W.m -2 was applied for 2 hours and checked at the surface of the batch reactor using a UV sensor (UV Radiation, apogee instruments).

Experiments were conducted in parallel to evaluate the effects of photolysis. Photolysis experiments were conducted in a beaker with seawater without a catalyst, maintained at a flux density of 35 W.m -2 .

2.2. Disinfection of a bacterial pathogen 2.2.1. Bacterial pathogen: Vibrio harveyi An oyster pathogen, Vibrio harveyi Th15_O_G11 modified with a stable Green Fluorescent Protein (GFP) plasmid pFD086 (GFP, TrimR) [START_REF] Oksanen | Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus[END_REF] was used to study the disinfection of a single pathogen. This bacterium was cultured in Zobell medium (4 g.L-1 bactopeptone, 1 g.L-1 yeast extract in sterile seawater, pH 7.4) with 10 µg.mL -1 of trimethoprim (T7883, Sigma Aldrich, France) in order to maintain the GFP plasmid at 20 °C until the stationary phase was reached. Then filtered natural seawater (Banyuls, France, seawater filtered at 1 µm, pH 8 and salinity of 35 PSU) was inoculated to reach 10 7 CFU/100mL at the beginning of the photocatalysis and photolysis treatments (Supplementary 1). The inoculation was performed directly without separating bacterial cells from the culture media.

To avoid contamination of seawater by cultivable bacteria, negative control was carried out by spreading 200 µl on Marine Agar plate before spiking the seawater with Vibrio harveyi.

Monitoring

Disinfection was monitored by the enumeration of cultivable bacteria. The culture media was Marine Agar (Marine Broth medium with 15 g.L -1 of agar, 76448, Sigma Aldrich, France) with 10 µg.mL -1 of trimethoprim and all counting were performed in triplicate by spreading 200 µl of treated seawater. This method allows a detection limit of 10 4 cfu/100mL, corresponding to 99.99% of inactivation (i.e. 3 log). The Vibrio samples were incubated at 20 °C for 48 h until counting (Supplementary 1). In addition to the trimethoprim screen, the fluorescence of the colonies was checked at 395 nm (Lepro Lampe Torche UV LED) before cultivable bacteria enumeration. The bacterial inactivation curves connected to the seawater disinfection by photocatalysis and photolysis were compared using Pairwise-t-test with Benjamini & Hochberg adjusting method, and Tukey multiple pairwise-comparisons. Then the disinfection efficiency was estimated by fitting the experimental point to a first-order kinetic model (Equation 1). In this law, the UV dose (Equation 2) was expressed as the quantity of energy received corrected by volume of water in the reactor as for solar treatment [START_REF] Plantard | Experimental and numerical studies of a solar photocatalytic process in a dynamic mode applied to three catalyst media[END_REF]. It allows to consider the reactor geometry with the irradiated surface / volume of the reactor ratio.

Disinfection of a seawater microbial complex community

Production of contaminated seawater

The microbial community tested in this work was composed of seawater enriched in virus and bacteria obtained following the experimental infection of juvenile oysters by OsHV-1 μVar, the inducer of the POMS disease. Susceptible pathogen-free juvenile oysters F14V [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]) aged of 3.5 months were injected in the adductor muscle with 4.10 8 Genomic Unity (UG) of OsHV-1 μVar virus (injection of 50 µl of F14-TFF suspension at 7.10 6 UG.µL -1 ). 30

Crassostrea gigas oysters spats (mean weight 3 g) were put in a 1 L tank to reach an animal density of 90 g of biomass/L. Oysters were kept in oxygenated seawater during 24 h at 21 °C by controlling the room temperature. The contaminated seawater (CSW) produced was directly used for photocatalysis and photolysis treatments (Figure 1a). During experiments, virus inactivation and seawater bacterial microbiota inactivation were analyzed separately.

Oyster mortality monitoring

During the seawater disinfection process, seawater was sampled and filtered at 0.2 µm (to eliminate bacteria) before injection on oysters. Susceptible pathogen-free juvenile oysters (family F14V aged 3.5 months) initially pathogen-free were injected in the adductor muscle either with 50 µl of contaminated seawater treated using photocatalysis (UV365/TiO2) or photolysis (UV365 alone) or not treated (kept to obscurity) (figure 1a and figure 1b). In addition, non-injected oysters were used as infection control and no mortality occurred in this group during the trial. As previous experiments (Supplementary 2) did not show viral adhesion on TiO2 catalysis, no adhesion control was performed in this study. Four tank replicates were used with 10 oysters (mean weight of 3g) / 500 mL tanks to reach an animal density of 60 g of biomass/L. Then oysters were put in aerated seawater at 21 °C. Oyster mortality was monitored twice a day for 10 days. Oysters were not fed during the 10 days of monitoring and day/night (10h/14h) cycles were performed.

The nonparametric estimates of the survival function were computed according to Kaplan & Meier (survfit, {survival}) [START_REF] Therneau | Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas[END_REF]. Survival time started when oysters were injected with treated seawater and stopped after 10 days, when no additional mortality has been observed in the past 48h. The data was read as the number of dead animals within each condition on each day, the tanks replicated for each condition were considered as one tank for the survival analysis (i.e. n = 40 per condition). Survival curves of exposed oysters were plotted and compared among the treatments and their duration. The survival time curves of oysters connected to the seawater disinfection by photocatalysis and photolysis were compared using the Cox regression model (coxph and cox.zph, {survival}) [START_REF] Therneau | Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas[END_REF] and the Log-Rank regression model (survdiff, {survival}) [START_REF] Therneau | Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas[END_REF]. To evaluate viral inactivation, the time required to reach 50% oyster mortality for each treatment (TL50) was calculated from the survival curves. This corresponds to the post-injection time at which 50% of the oysters in the condition have died.

DNA extraction

All seawater samples were stored at -80 °C until the DNA was extracted. The total DNA of seawater samples was extracted using the blood protocol from Machrey-Nagel NucleoSpin tissue kit (reference 740952). The extractions were realized on 200 µl of seawater with a final elution volume of 30 µl. In order to improve DNA extractions, a crushing step of 12 min mechanical lysis with zirconium beads followed by an RNAse step was added before the DNA binding step. The DNA extracts were stored at -20°C before analysis.

Metabarcoding analysis

The 16S rRNA gene of bacterial communities was amplified using the variable V3-V4 loops (341F / 805R). Paired-end sequencing was performed on the MiSeq system (Illumina) at the University of Perpignan Via Domitia (Bio-Environment platform, Perpignan University, France). Sequencing data are available at the NCBI-SRA under the accession number PRJNA987558. The microbiome bioinformatics analyses were performed with QIIME 2 2022.8 pipeline to generate Amplicon Sequence Variant (ASV) (Bolyen et al., 2019). Sequence data was already demultiplexed so we directly denoised it with DADA2 using the ---p-poolingmethod and -p-chimera-method parameters to pool the sequence between sample for the chimers elimination and the ASV creation. Then the taxonomy affiliation was performed on ASV using a Silva 138 V3V4 classifier (classifier train with the used primers). The phylogenetic tree was created with FastTree, using the mafft program. After creating the phylogenetic tree, the taxonomic assignment and the ASVs, all the statistical analyses were done using R v4.2.1 (2022-06-23) (Supplementary 3). Samples were normalized using the rarefaction method before any statistical analyses at a sample depth of 10 301. Principal coordinate analysis (ordinate, {phyloseq}) (McMurdie and Holmes, 2013) was computed to describe compositions of microbial communities between samples using Bray-Curtis dissimilarities (distance, {phyloseq}) (McMurdie and Holmes, 2013).

The impact of seawater treatment on seawater microbiota was tested using ANOVA (anova, {stats}) (R Core Team, 2022) on the Constrained Analysis of Principal coordinates calculated with the Bray-Curtis distance (capscale, {vegan}) [START_REF] Oksanen | Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus[END_REF]. Log Fold Change analysis was performed to analyze changes on bacteria abundance between treatment and control (DESeq, {DESeq2}) [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]) using a adjusted p-value at 0.05. For all analysis, the threshold significance level was set at 0.05.

Quantification of bacteria and virus

Quantifications of OsHV-1 µVar, total bacteria 16S rDNA and total Vibrio 16S rDNA were performed using quantitative PCR (qPCR) with a Roche LightCycler 480 thermocycler (Bio-Environment platform, University of Perpignan, France). The total qPCR reaction volume was Green Takyon Master Mix at 2X (Eurogentec SA). OsHV-1 µVar specific primer pairs targeted a DNA polymerase catalytic subunit (named DP or ORF100): 5′-ATTGATGATGTGGATAATCTGTG-3′ and 5′-GGTAAATACCATTGGTCTTGTTCC-3′ [START_REF] Webb | Primers for PCR-based detection of ostreid herpes virus-1 (OsHV-1): Application in a survey of New Zealand molluscs[END_REF]. The total bacteria quantification used the same specific primer pairs as for the metabarcoding analysis (see above). The specific primers used for the total Vibrio quantifications targeted 567F/680R region of the 16S rDNA: 5′-GGCGTAAAGCGCATGCAGGT-3′ and 5′-GAAATTCTACCCCCCTCTACAG-3′ [START_REF] Mansergh | Vibrio diversity and dynamics in the Monterey Bay upwelling region[END_REF]. qPCR reactions were performed in triplicate with the following program: 95°C for 3min, followed by 40 cycles of denaturation (95 °C, 10 s), hybridation (60 °C, 20 s) and elongation (72 °C, 30 s). The absolute quantification of the OsHV-1 µVar DNA copies in the seawater was carried out by comparison of Cp value of sample to Cp value of a standard curve (obtained from a plasmid of the DNA polymerase catalytic subunit) according to [START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF][START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF]. The results were expressed as the number of OsHV-1 µVar DNA copies per mL of seawater (UG/mL). The absolute quantification of the total bacteria and total Vibrio in the seawater was carried out by comparison of Cp value of sample to Cp value of a standard curve obtained from Vibrio genomic DNA of known concentration. The results were expressed as the number of Vibrio DNA copies per mL of seawater (CP/mL). The DNA concentration connected to the seawater disinfection by photocatalysis and photolysis was compared using Pairwise-t-test with

Benjamini & Hochberg adjusting method, and Tukey multiple pairwise-comparisons.

Results and discussion

Inactivation of the oyster pathogen Vibrio harveyi

The efficiency of photocatalysis and photolysis treatments to inactivate simple and complex microbial communities was evaluated by testing in water either a bacterial pathogen alone or the µVar OsHV-1 virus present in a complex community composed of seawater microbiota.

We began by assessing the inactivation by photocatalysis and photolysis of V. harveyi, a bacterium pathogenic to oysters in seawater.

Briefly, sterile seawater was artificially contaminated with V. harveyi population in stationary phase before applying photocatalysis and photolysis treatments. showed that photocatalysis treatment was much more effective than photolysis in inactivating Vibrio cells. Where only 30% (or 0.2 log) of the bacterium were inactivated by 2 h of UV365 irradiation alone ( = 0.046 ± 0.004 L.kJ -1 ; R 2 = 0.98) corresponding to a UV365 dose of 8 kJ.L -1 (or 25 000 mJ.cm -2 ), almost 80% (or 0.7 log) were inactivated in the presence of TiO2 catalyst ( = 0.218 ± 0.017 L.kJ -1 ; R 2 = 0.97). Most of the photocatalysis disinfection experiments found in the literature ran UVC sources (254 nm) (Rubio et al., 2013;[START_REF] Wu | Ballast waters treatment using UV/Ag-TiO2+O3 advanced oxidation process with Escherichia coli and Vibrio alginolyticus as indicator microorganisms[END_REF] which have a higher bacteria inactivation power than UVA (365 nm) [START_REF] Kim | Bacterial inactivation in water, DNA strand breaking, and membrane damage induced by ultraviolet-assisted titanium dioxide photocatalysis[END_REF] so a higher inactivation rate. Most of those studies compare efficiency of treatment by UV alone or with a catalyst and always found that photocatalysis treatments are the most efficient processes. The UV dose required to obtain the desired level of disinfection has a direct impact on the cost of disinfection [START_REF] Blanchon | Comparison of Infectious Agents Susceptibility to Photocatalytic Effects of Nanosized Titanium and Zinc Oxides: A Practical Approach 15[END_REF]. It is therefore valuable to estimate the ratio between the UV dose required for photocatalysis and photolysis to obtain the same level of disinfection. Rubio et al. ( 2013) reported a value of 38.6 mJ.cm -2 to achieve 99.9% disinfection of Alteromonas sp. with UV254 alone against 25.9 mJ.cm -2 with UV254/TiO2 which reduces the amount of UV needed by 1.5 (Rubio et al., 2013). In our study, a UV365 dose of 25 000 mJ.cm -2 (8 kJ.L -1 ) was needed to achieve 30% of the Vibrio harveyi inactivation with UV365 alone against 3 000 mJ.cm -2 (1 kJ.L -1 ) with TiO2, allowing to drastically reduce the amount of UV365 needed (by 8). As shown for Vibrio alginolyticus which is highly resistant to UV365 photolysis (UV dose greater than 18 J.cm -2 for less than 0.5 log inactivation) (Moreno-Andrés et al., 2023), V. harveyi strain tested in our study does not appear to be UV365 sensitive as it was shown by the kinetic curves (Figure 2). Indeed, it has already been observed that autochthonous bacteria such as Vibrio spp. (Vibrio owensii, Vibrio alfacsensis and V. harveyi)

in seawater are more resistant to inactivation by SODIS than non-indigenous bacteria such as E. coli, T. coliforms and Enterococci in contaminated drinking water [START_REF] Levchuk | Solar photocatalytic disinfection using ink-jet printed composite TiO2/SiO2 thin films on flexible substrate: Applicability to drinking and marine water[END_REF]. It is difficult to compare these results with our study, especially when crucial parameters as experimental conditions, bacteria concentration, radiation type and photocatalyst are different. However, given that the light intensity was 48 W.m -2 , we can estimate that the UV dose required for Vibrio spp. inactivation obtained in the study by [START_REF] Levchuk | Solar photocatalytic disinfection using ink-jet printed composite TiO2/SiO2 thin films on flexible substrate: Applicability to drinking and marine water[END_REF] under UVA and SODIS was greater than that found in the present study.

Given the good efficiency of the photocatalytic treatment obtained under our experimental conditions, it may be considered that this type of process could be applied to the disinfection of aquaculture influents and effluents [START_REF] Levchuk | UVA and solar driven photocatalysis with rGO/TiO2/polysiloxane for inactivation of pathogens in recirculation aquaculture systems (RAS) streams[END_REF][START_REF] Levchuk | Solar photocatalytic disinfection using ink-jet printed composite TiO2/SiO2 thin films on flexible substrate: Applicability to drinking and marine water[END_REF][START_REF] Moreno-Andrés | A comparison of photolytic, photochemical and photocatalytic processes for disinfection of recirculation aquaculture systems (RAS) streams[END_REF][START_REF] Villar-Navarro | Combination of solar disinfection (SODIS) with H2O2 for enhanced disinfection of marine aquaculture effluents[END_REF] or ballast water [START_REF] Moreno-Andrés | UV-based technologies for marine water disinfection and the application to ballast water: Does salinity interfere with disinfection processes?[END_REF], provided that further studies on the inactivation of other aquaculture pathogens are carried out.

Inactivation of a complex microbial community

Effect on OsHV-1 µVar infectivity

Complex microbial community composed of OsHV-1 µVar virus associated with a marine bacterial community was treated by photocatalysis and photolysis processes. The treated or untreated seawater was filtered before injection into the oysters, to eliminate the bacterial fraction (Figure 1). Briefly, the contaminated seawater infectivity was followed by evaluating the mortality in oysters, and monitoring of the viral DNA in seawater was also assayed. Three disinfection experiments were carried out to obtain various levels of virus contaminated seawater. The first two experiments involved a 2-hour treatment, one by photocatalysis and the other by photolysis (Figure 1), to achieve a final UV dose of 8 kJ.L -1 . A control that had not been subjected to any treatment and had been kept protected from radiation for 2 hours was used. Next, virus-susceptible oysters were injected with the treated or not contaminated seawater from the 3 disinfection experiments. For each treatment, 40 oysters were injected with treated or not treated seawater for 0, 0.5, 1 or 2 hours (respectively 0, 2, 4 and 8 kJ.L -1 ) and then mortality was followed to assess the suitability of the treatments (Figure 1).

The Kaplan-Meier survival curves thus obtained (Figures 3) allowed us to visualize the survival rates and the efficiency of photocatalysis and photolysis treatment to inactivate OsHV-1 µVar virus. A better oyster survival rate was directly correlated with better virus inactivation (with a lower quantity of virus still infectious). The oyster mortality is caused directly by the injection of still-infectious virus as verified using qPCR on oysters. As showed by blue curve, mortality occurred during the first 80 h on oysters injected with non-treated seawater whereas it started later and to a less rapid rate for oysters injected with treated seawater until there was no mortality at all (see curves in red and green and purple). On one hand, the mortality rate of oysters for the control seawater (seawater without treatment at 0 h and after 2 h) reached 100% (Figure 3a). All the oysters died quickly after 80 hours of monitoring, and no oysters were left alive in the tanks. We also note that the presence of the TiO2 catalyst does not seem to have a quantifiable impact on the survival rate, indicating that the catalyst alone does not affect the virus and does not induce direct mortality of the oysters (figure 3a).

On the other hand, the survival rates of oysters injected with treated seawater was significantly improved (Figure 3b and Figure 3c) and the longer the treatment was, the better the survival rate was. As indicated by statistical analysis (Cox test, p-value = 6e-16 for UV365 treatment vs. control and p-value < 2e-16 for UV365/TiO2 treatment vs. control), both treatments are significantly different from the control condition. For both oysters injected with seawater treated for long time by photolysis and photocatalysis (1 or 2 hours) part of those oysters were still alive after 10 days (20% to 100% of survival proportion) (Figure 3b, Figure 3c). Photocatalysis (UV365/TiO2) was efficient to inactivate viruses, same as, photolysis (UV365 alone) alone also decreased virus infectivity but to a lesser extent. Calculating the time needed to achieve 50% oyster mortality for each treatment (TL50) shows that virus inactivation by photocatalysis is twice as effective as by photolysis. In fact, it takes half as long to achieve 50% mortality in oysters injected with CSW treated for 1 hour by photolysis (96h)

as by photocatalysis (192h). Indeed, 2 hours of treatment by photocatalysis of the contaminated seawater allows sufficient inactivation of OsHV-1 µVar to have 100% survival of the oysters whereas exposure to UV365 alone only allows a 50% reduction in final oyster mortality (Figure 3b and Figure 3c).

Viral inactivation by photocatalytic process consists in three steps: (1) the viral particle shape distortion, (2) the protein oxidation, and (3) the DNA leakage and damage to the genetic material [START_REF] Zhang | Progress and challenges in photocatalytic disinfection of waterborne Viruses: A review to fill current knowledge gaps[END_REF]. Quantification of viral DNA (Supplementary 5a) showed that DNA degradation by photocatalytic treatment was not essential to effectively reduce viral infectivity and halt oyster mortality. We observed that the amount of viral DNA present in UV365-treated seawater was quite similar to that measured in UV365/TiO2-treated seawater (respectively for UV365/TiO2 and UV365 2h-treatment, 4.23e+03 ± 1.35e+03 UG/µL and 1.11e+04 ± 4.47e+03 UG/µL). However, we also observed that oyster mortality was lower for oysters injected with UV365/TiO2-treated seawater than with UV365-treated seawater alone.

These results are consistent with the data of [START_REF] Bounty | Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation[END_REF] [START_REF] Bounty | Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation[END_REF] who showed that the increase in viral inactivation by photocatalysis versus photolysis was not due to an increase of DNA damages. The 2 hours treatment reduced the OsHV-1 µVar DNA concentration for photolysis treatment and photocatalysis by 95 to 98% respectively whereas in seawater kept out of any UV sources the OsHV-1 µVar DNA was only reduced by 30%

(degradation of free DNA during the 2h at obscurity). Several authors have already reported that virus sensibility to photo-oxidative treatment depends on the capsid composition and the nature of the genetic material [START_REF] Mayer | The Impact of Capsid Proteins on Virus Removal and Inactivation during Water Treatment Processes[END_REF]. The OsHV-1 µVar virus is an herpesvirus composed of double-stranded DNA (dsDNA) encapsulated in an enveloped icosahedral capsid of 116 nm diameter [START_REF] Adamson | Mapping the proteins of herpes simplex virus type 1 capsid[END_REF][START_REF] Davison | A novel class of herpesvirus with bivalve hosts[END_REF]. The virus size, both in terms of diameter and genome length, plays a role in the virus inactivation by photocatalysis [START_REF] Mayer | The Impact of Capsid Proteins on Virus Removal and Inactivation during Water Treatment Processes[END_REF], as well as the presence of an enveloped capsid [START_REF] Blanchon | Comparison of Infectious Agents Susceptibility to Photocatalytic Effects of Nanosized Titanium and Zinc Oxides: A Practical Approach 15[END_REF][START_REF] Mayer | The Impact of Capsid Proteins on Virus Removal and Inactivation during Water Treatment Processes[END_REF][START_REF] Nakano | Broad Spectrum Microbicidal Activity of Photocatalysis by TiO2[END_REF]. The addition of TiO2 catalyst (the photocatalysis treatment vs photolysis one) enhances the viral inactivation without inducing more DNA degradation but could have target the viral envelope, increasing the reduction of the infectivity.

Effect on a complex pathobiota (virus and bacteria)

Both experiments showed that microbial contaminants were efficiently inactivated by the treatment, especially the photocatalytic one. The DNA quantifications of both total Vibrio and total bacteria highlighted that DNA was not destroyed by the 2h of treatment while more than 95% of the OsHV-1 µVar DNA was destroyed by the same treatment (Supplementary 5a).

In freshwater some authors have already reported that viruses were the most susceptible organisms to Advanced Oxidative Processes (AOPs) such as photocatalysis (Laxma [START_REF] Reddy | TiO 2 -based photocatalytic disinfection of microbes in aqueous media: A review[END_REF][START_REF] Zheng | Photocatalytic disinfection performance in virus and virus/bacteria system by Cu-TiO2 nanofibers under visible light[END_REF]. This observation seems to be verified both in terms of DNA quantification and in terms of virus infectivity or cultivable GFP V. harveyi quantification. The

OsHV-1 µVar infectivity does not induce any oyster mortality after the 2h of photocatalytic treatment whereas only 80% of cultivable GFP V. harveyi were inactivated. In addition, the photolytic treatment was less efficient than the photocatalytic one both the OsHV-1 µVar inactivation and the V. harveyi inactivation. The use of TiO2 with the UV treatment enhanced the formation of OH radical which improved the seawater disinfection but that was not linked to a higher level of DNA damage since the DNA quantification was the same between photolysis and photocatalysis experiments. A better oyster survival rate is directly correlated with better virus inactivation (with a lower quantity of virus still infectious). However, this reduction in the number of infectious viruses was not correlated with the viral DNA load detected. In fact, the viral DNA load remain the same for photolysis-treated and photocatalysis-treated seawater samples, whereas the infectivity of photocatalysis-treated seawater was lower than that of photolysis-treated seawater. Photocatalytic treatment therefore appears to inactivate viral particles without destroying their DNA. The same observation was already done on virus by [START_REF] Bounty | Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation[END_REF] with adenovirus inactivation in freshwater by UV254/H2O2 advanced oxidation [START_REF] Bounty | Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation[END_REF] and by [START_REF] Park | Inactivation efficiency and mechanism of UV-TiO 2 photocatalysis against murine norovirus using a solidified agar matrix[END_REF] with murine norovirus in solidified agar matrix by UV/TiO2 processes [START_REF] Park | Inactivation efficiency and mechanism of UV-TiO 2 photocatalysis against murine norovirus using a solidified agar matrix[END_REF]. On bacteria, [START_REF] Kim | Bacterial inactivation in water, DNA strand breaking, and membrane damage induced by ultraviolet-assisted titanium dioxide photocatalysis[END_REF] showed that the use of TiO2 with UV352 or UV306 treatment induces more damage to DNA than the UV alone including the plasmid DNA linearization [START_REF] Kim | Bacterial inactivation in water, DNA strand breaking, and membrane damage induced by ultraviolet-assisted titanium dioxide photocatalysis[END_REF].

Effects of the photocatalysis process on the seawater bacterial community

Microbiota change was linked to seawater treatments

As the POMS is triggered by an OsHV-1 µVar oysters infection causing immune-suppression inducing a fatal bacteraemia [START_REF] Petton | The Pacific Oyster Mortality Syndrome, a Polymicrobial and Multifactorial Disease: State of Knowledge and Future Directions[END_REF], we also analyzed the effect of treatment on whole seawater bacterial communities using the 16S rRNA gene sequencing from samples taken before and during the contaminated seawater treatment (0, 0.5, 1 and 2h). In parallel with the analysis of the total bacteria microbiota analysis, the total bacteria and Vibrio loads were quantified using qPCR. All the analyses presented in this section were carried out on contaminated seawater without water filtration, containing both bacteria and viruses (Figure 1).

To investigate the effect of treatment on the dynamics of bacterial microbiota in contaminated seawater, we analyzed total bacterial communities using 16S rDNA metabarcoding during (0.5, 1 and 2 hours) and after the treatment (4, 7 and 10 days). The normalization to a depth of 10 301 sequences per sample allowed us to keep 1 014 ASVs.

Given that the total composition of the microbiota is likely to evolve between the end of treatment and the various post-treatment we decided to analyze photocatalysis and photolysis effects during and after exposition separately. The principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarities computed on samples obtained during the treatment highlighted that seawater microbiota changed between the two treatments and the control (p-value = 7e-04 for capscale ANOVA) (Figure 4) but no significant differences were observed on total bacteria loads between treatments and control (p-value > 0.05 for both Pairwise-t-test and Tukey multiple pairwise-comparisons) (Supplementary 5b).

Photocatalysis-treated samples cluster further apart on the figure 4, revealing that the microbiota composition of photocatalysis-treated CSWs is more affected than that of photolysis-treated CSWs. Furthermore, the longer the treatment, the more important the effect on photocatalysis-treated CSWs. The same taxa were found (Figure 5a), but the proportion of each bacterial genus changed between the UV365 treatment, the UV365/TiO2 treatment and the control (seawater kept in the dark during treatment). In the control and UV365-treated samples, the relative proportion of bacteria did not change over time, whereas treatment duration had an impact on the relative proportion of bacteria for the photocatalysis treatment (Figure 5a). The DESeq2 analysis allowed us to identify genus significantly modified by the treatment applied by calculating the log2 fold change value. In UV365/TiO2-treated seawater, ASVs affiliated to the genera Aesturiibacter and Vibrio were found to be more abundant than in control seawater (log2 fold change > 0 with adjusted p-value < 0.05), while Croceibacter and Pseudophaeobacter were less abundant (log2 fold change < 0 with adjusted p-value < 0.05). (Figure 5b). In seawater treated by UV365 alone, only

Pseudoalteromonas genus was modified and was more abundant in UV365 treated seawater than in control seawater (Figure 5b). Although the log2 fold change shows a variation in Vibrio bacteria in the UV365/TiO2 treated seawater compared to the untreated one, the quantification of total Vibrio did not show any significant changes (Supplementary 5c).

Bacteria belonging to Vibrio and Pseudoalteromonas genera have been shown to be the opportunistic strains associated with POMS disease [START_REF] Petton | The Pacific Oyster Mortality Syndrome, a Polymicrobial and Multifactorial Disease: State of Knowledge and Future Directions[END_REF]. More specifically, numerous Vibrio species are involved in oysters production losses both by inducing mortality in oysters [START_REF] Kim | Bacterial inactivation in water, DNA strand breaking, and membrane damage induced by ultraviolet-assisted titanium dioxide photocatalysis[END_REF][START_REF] Oksanen | Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus[END_REF] or by making them not consumable [START_REF] Froelich | Vibrio bacteria in raw oysters: managing risks to human health[END_REF]. These results are consistent with data found in the literature concerning Vibrios associated with POMS disease during oyster life stages and in particular i)

during the spat or larval stage with V. anguillarum, V. alginolyticus, V. tubiashii, V. ostreicida, V. coralliilyticus, V. neptunius, V. bivalvicida and other Vibrios of the Splendidus clade [START_REF] Richards | Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii[END_REF], ii) during the juvenile stage with Vibrio of the Splendidus clade such as V. splendidus, V. tasmaniensis, V. cyclitrophicus or V. crassostreae in POMS mortality events [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF][START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF][START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF] and V. harveyi and iii) once adult by V. aestuarianus [START_REF] Garnier | Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas[END_REF][START_REF] Therneau | Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas[END_REF].

In conclusion, the 16S rDNA barcoding analyses of the Vibrio genera did not allow to show any pattern in the change of the global proportion of the Vibrio affiliated ASVs according to the oxidative treatment type or to the treatment duration (data no show). Nevertheless, the taxonomic resolution of the marker used in this study did not provide a reliable identification of the different species within the Vibrio genus.

Assessment of microbiota resilience at the end of photocatalyis treatment

After the end of the treatment and once the TiO2 was fully removed by flotation from the seawater, the microbiota shifted towards that of the one of control seawater (Figure 6a). Some bacteria genus remains nevertheless with abundances significantly different from those of the control seawater (Figure 6b). After the catalysis removal, the BD7-11 and Roseibacillus bacteria remain more abundant in UV365/TiO2 treated seawater than in the control seawater (log2 fold change > 0) whereas VadinHA49 bacteria were less abundant. The same trend was observed for UV365 treated seawater where Roseibacillus bacteria were also more abundant than in the control. Even if some genus showed significant abundance differences, no correlation was observed between seawater bacterial composition and the treatment applied a few days after the end of photo-oxidative treatments (pvalue = 0.9725 > 0.05 for capscale ANOVA). The total 16S rDNA bacteria quantification was also the same after treatment between treated seawater and the reference seawater (Supplementary 5b).

Thus, 10 days after the treatment, the total bacterial community was resilient both in term of quantity and quality by comparison to the control. In the present study, disinfection was not total, and it is quite conceivable that the part of the microbiota still viable after treatment could grow again, restoring a bacterial microbiota closer to that of untreated water. As previously mentioned by Falet et al. ( 2022), the resilience observed here is an imperative requirement for aquaculture, as pathogen removal must not alter the entire seawater microbiota in order to limit the impact on shellfish and fish development [START_REF] Fallet | Early life microbial exposures shape the Crassostrea gigas immune system for lifelong and intergenerational disease protection[END_REF].

Conclusion

The aim of this study was to apply a photocatalyis process to seawater, either using UV365 treatment alone, or using a combination of UV365 and titanium dioxide photospheres, in order to assess the microbial inactivation. The feasibility of viral and bacterial inactivation in a context of aquaculture influent and effluent disinfection was investigated as well as the effect of such treatment on the global bacterial community. The main conclusion of this study is that photo-oxidizing treatments effectively inactivate the OsHV-1 µVar virus and the V. harveyi

  kinetic equation with C and C0 respectively the bacteria concentration at the sampling time and at the beginning of the experiment in UFC/100mL,Qt) the UV dose accumulated since the start of the experiment in kJ.L - 1 (equation 2), and  the first order kinetic constant. equation 2 UV dose accumulated since the start of the experiment in kJ.L -1 , with Sirradiated the irradiated surface of the reactor in m 2 , Vtotal the total volume of the reactor in L and I the flux density received in W.m -2 .

  Two disinfection experiments were carried out to achieve various levels of seawater decontamination. The first one was a 2 h photocatalytic treatment (UV365 associated with TiO2 catalyst) and the second one was a photolytic treatment (only UV365 irradiation without catalyst). The bacterial inactivation was followed by counting the cultivable bacteria during treatment. Preliminary work (Supplementary 4) on the adhesion of V. harveyi has shown that under our experimental conditions of pH and salinity, adhesion to the catalyst was negligible in comparison with the duration of the treatment experiments. The inactivation results of V. harveyi in seawater are presented as a function of the cumulative UV365 dose (Q(t)) versus the normalized survival ratio (Figure2). The raw data was fitted by a first-order model (with or without shoulder) to calculate the constant rate k. Only the UV365 treatment exhibited a shoulder in the first 15 minutes of treatment corresponding to a UV365 dose of 1 kJ.L -1 and even if authors have already reported evidence of a photoreactivation mechanism of Vibrio in freshwater[START_REF] Das | Repair of ultraviolet-light-induced DNA damage in Vibrio cholerae[END_REF] and in marine water[START_REF] García-Garay | Wild microorganism and plankton decay in ballast water treatments by solar disinfection (SODIS) and advanced oxidation processes[END_REF][START_REF] Moreno-Andrés | Inactivation of the waterborne marine pathogen Vibrio alginolyticus by photo-chemical processes driven by UV-A, UV-B, or UV-C LED combined with H2O2 or HSO5[END_REF], no such observation had been observed in our study. The inactivation experiment (figure2)

  photocatalysis treatment (UV365/TiO2) was more efficient in inactivating pathogens without

Figures

  Figures

Figure 1 :

 1 Figure 1: (a) experimental design for the OsHV-1 µVar virus and seawater total bacterial community inactivation with (b) the detail of oyster injections. DNA extracts were used for the barcoding analysis and also for the total bacteria, total Vibrio bacteria and OsHV-1 µVar quantification by qPCR.

Figure 2 :

 2 Figure 2 : Photocatalyis kinetics of Vibrio harveyi inactivated by UV365 alone () or inactivated by UV365/TiO2 ().C/C0 is the survival ratio along with the standard error of the means(n = 3)

Figure 3 :

 3 Figure 3 : Survival curves for oysters injected by (a) seawater without any treatment, (b) seawater disinfected by UV365 only, and (c) seawater disinfected by UV365/TiO2 treatment. -CSW without treatment, -CSW after

Figure 5 :

 5 Figure 5: Bacteria genus modified according to the seawater photo-oxidative treatment applied (a) genus composition of the 17 more abundant genus, (b) genus significantly modified, log2 fold change (with a p-value adjust < 0.05) of photo-oxidative treatment compared to untreated seawater (kept out of any UV365 irradiation)

Figure 6 :

 6 Figure 6: Bacteria genus modified according to the time after the photo-oxidative treatment was applied (a) PCoA representation of the Bray-Curtis dissimilarity (•) at the end of the treatment, (•) 4 days, (•) 7 days, and (•)

Acknowledgements

We would like to express our sincere gratitude to all the individuals and organizations that have contributed to the publication of this research paper. First, we would like to thanks Virginie Le Razavet and Bruno Petton, from the IFREMER Bouin Marine Mollusc Experimental


Romane Manguin

María Galiana-Cameo

Tanakorn Kittikool

Cécile Barthes

Jompol Thongpaen

Etienne Bancal

Sonia Mallet-Ladeira

Sirilata Yotphan

Ricardo Castarlenas

Marc Mauduit

Jean-Baptiste Sortais

Olivier Baslé
email: olivier.basle@lcc-toulouse.fr

Iridium(I) complexes with bidentate NHC ligands as catalysts

come

Organosilicon compounds exhibit remarkable physicochemical properties that are of interest for applications in organic synthesis, in chemical biology and in material science. 1 Therefore, the development of new efficient methods to access organosilanes from easily accessible raw materials is an important research focus in modern organic chemistry. As an alternative to traditional methods employing stoichiometric amount of metals and to cross-coupling approaches involving pre-functionalized precursors, transition metal-catalyzed direct cross-dehydrogenative coupling of C-H and Si-H bonds to selectively form new C-Si bonds constitutes an atom-and stepefficient approach. 2 The directing-group assisted dehydrogenative C-H bond silylation, which can be catalyzed by a variety of transition metal-based systems, has emerged in recent years as a robust strategy for regioselective functionalization. 3 Among transition metals, iridium is recognized for its high performance in catalyzing dehydrogenative C-H silylation transformations, especially when in association with nitrogen-based ligand. 4 Furthermore, and despite the established advantage of N-Heterocyclic Carbene (NHC) ligand in transition-metal catalysis, 5 including for C-H bond functionalization, 6 the use of such ligands in C-H silylation is very scarce and limited to two independent reports by Mashima and co-workers in 2013 on the one hand, 7 and Oro and co-workers in 2017 on the other (Figure 1). 8 Herein, we described the synthesis of new iridium(I) complexes with bidentate NHC ligands complexes and their application as efficient catalysts for the ortho-selective dehydrogenative directed C-H silylation of arenes with bulky aromatic silylating agents. Precursors of unsymmetrical bidentate NHC-carboxylate are accessible through a straightforward multicomponent synthesis which we recently developed. 9 Previous reports have evidenced that these bidentate LX type ligands could strongly bind transition metals such as iridium(III), 10 rhodium(I/III) 11 and ruthenium(II) 12 to form robust cyclometallated complexes that notably demonstrated efficient catalytic activities in C-H borylation under thermic and photocatalytic conditions. 11,12 In a continued effort to design novel NHC-based transition-metal catalysts for C-H bond functionalization reactions, we investigated the synthesis of well-defined iridium(I) complexes combining an electron rich bidentate NHC-carboxylate ancillary

Ir-cat

Previous work:

This work: bidentate NHC-Ir(I) catalyst with exclusive ortho-selectivity using bulky aromatic silanes ligand and the labile 1,5-cyclooctadiene (cod) ligand. To this end, a series of amino acid-based NHC ligand precursors 1 were converted to their corresponding silver-intermediates and subsequently transmetalated to [Ir(cod)Cl]2 to afford the desired (NHC)Ir(cod) complexes in good yields (Figure 2). Complexes 2a, 2b and 2c, respectively derived from L-leucine, Lvaline and L-alanine, were fully characterized and their structures could be confirmed by X-ray diffraction analysis. Selected geometrical parameters are shown in Table 1. These three complexes exhibit a slightly distorted square planar geometry without noticeable influence of the substituent at the stereogenic carbon center of the 5-membered cyclometallated NHC-carboxylate "LX type" ligands; as evidenced by the C1-Ir-O1 trigonal angles of 85.5(5)°, 86.27(18)° and 86.2(3)° for 2a, 2b and 2c, respectively. Moreover, the "X-type" ligand nature of the carboxylate arm is attested by the distance between the oxygen atom (O1) that binds to the iridium center and the carboxylate carbon atom (C5) being longer than the bond length of O2-C5, in each case. 10 With the new complexes in hand, we decided to investigate their catalytic activity in the dehydrogenative silylation of aromatic C-H bonds directed by a pyridine group using a hydrosilane as silylating reagent (Table 2). First, at 120 o C in ( o C) Yield (%) [b] 1 Et3SiH 2a norbornene toluene 120 49 [c] 2 Et3SiH 2a -toluene 120 nd [d] 3 Et3SiH -norbornene toluene 120 nd [d] 4 16 [f] MePh2SiH (IMes)Ir(cod)Cl norbornene toluene 120 16

[a] Reaction conditions: 2-phenylpyridine (3a, 0.2 mmol), catalyst (2, 0.01 mmol), HSiR3 (0.6 mmol), H2 acceptor (0.6 mmol), solvent (1 mL) in preheated oil bath at stated temperature for 16 hours under Ar. [b] %Yield was determined by 1 H NMR using 1,3,5-trimethylbenzene as an internal standard. [c] isolated yields of 4a, 4b, and 4d are 47, 59, and 68%, respectively (See SI). [d] not detected. toluene and using norbornene as hydrogen acceptor, complex 2a bearing the NHC ligand derived from L-leucine furnished 40% of the desired ortho triethylsilylated product 4a (entry 1). In the absence of either hydrogen acceptor (entry 2) or iridium catalyst (entry 3) no desired product was detected. When changing the reflux conditions from toluene to THF or 2-MeTHF a significant drop of catalytic activity was observed (entries 4 & 5). The reaction performed well under neat conditions but with reduced yield compared to the use of toluene as solvent (entry 6), whose potential competitive functionalization was not observed. [START_REF] Liu | Silylation of toluene could not detected by GC-MS or NMR methods. 14 When Oro and co-workers used an Iridium(III) catalyst with a sterically demanding monodentate 1,3-bis(2,6diisopropylphenyl)imidazol-2-ylidene (IPr) ligand (see reference 8), silylation of 2-(p-tolyl)pyridine with bulky aromatic silanes such as H-SiMePh2 showed a selectivity shift towards exclusive C5-functionalization of the pyridine moiety. 15 In Ru(II)-catalyzed ortho C-H silylation of 2-phenyloxazoline, Et3SiH is the most efficient coupling reagent and other silanes such as Ph3Si-H or Me2PhSi-H were ineffective[END_REF] Replacement of norbornene by alternative hydrogen acceptor such as 1,5-cyclooctadiene, neohexene or norbornadiene did not lead to improved catalytic activities (entries 7-9). Interestingly, the use of dimethylphenylsilane as silylating reagent had a beneficial impact on the catalytic process affording 64% yield of the ortho-silylated product 4b (entry 10). A further improved of the silylation process could be achieved with the larger methyldiphenylsilane coupling partner to reach 82% yield of product 4c. However, a further increase of the bulkiness of the silylating agent had a detrimental effect on the dehydrogenative coupling process resulting in a reduced yield (entry 12). Under the optimal conditions using HSiR 3 (3 equiv.) H 2 acceptor (3 equiv.) 3a

N N SiR 3 4a (R 3 = Et 3 ), 4b (R 3 = Me 2 Ph), 4c (R 3 = MePh 2 ), 4d (R 3 = Ph 3 )
solvent, T °C methyldiphenylsilane, the use of complex 2b or 2c could not outperform the catalytic activities reached by complex 2a (entry 13 & 14). Finally, the ligand free [Ir(cod)Cl]2 and monodentate NHC-based (IMes)Ir(cod)Cl catalysts exhibited modest activities to afford product 4c in respectively 49% and 16% yield (entries 15 & 16), highlighting the beneficial contribution of the bidentate NHC ligand for the transformation.

To test the viability of this method, we first investigated the scope of the silylation reaction with respect to 2-arylpyridine based substrates (Table 3). Aryl groups bearing a substituent in para-position were well tolerated and both electron-poor and electron-rich substrates afforded the desired ortho-silylated products (4e-4h) in very good yields. Noteworthy is the exclusive ortho-selectivity observed in all cases, without detectable competitive C5-H bond silylation of the pyridine directing group. 14 Similar excellent results were also obtained when using 4-methylpyridine as directing group, with isolated yields of the ortho-silylated products varying from 81 to 92% independently of the electronic nature of the paraor metasubstituent aryl moiety (4i-4m). The isoquinoline-group was also amenable to direct the silylation in ortho-position of electronically different aryl fragments (4n-4o). Furthermore, excellent catalytic activities were achieved when using oxazole as a directing group, affording 93% isolating yield of the coupling product 4p. Interestingly, dehydrogenative silylation of 2-phenyloxazole with dimethylphenylsilane and triphenylsilane also performed well to produce 4q and 4r in 69 and 82% isolated yield, respectively. 15 On the other hand, while the use of 3,5dimethylpyrazole as a more sterically congested directing group led to a noticeable drop of efficiency providing 4s in low 27% isolated yield, almost full conversion of 2-phenylpyrimidine was achieved to obtain the corresponding silylated product 4t in 85% isolated yield. Pleasingly, the reaction also proved compatible with the use of aldimine as removal directing group affording the selective ortho-silylated aldehyde 4u in good 50% isolated yield. In addition, the late-stage silylation of pharmaceuticals attempted on the anxiolytic diazepam, 16 and on the gastroprotective drug zolimidine, 17 led to 72% and 31% isolated yield of the desired silylated drugs 4v and 4w, respectively. Finally, silylation of sp 3 C-H bond of 8methylquinoline could proceed quite satisfactorily with 45% yield to furnish 38% of the corresponding isolated product 4x.

To gain some insight into the mechanism of the present reaction, we performed a series of stoichiometric reactions in the presence of complex 2a in d8-toluene. First, when reacting 2a with 3a under increasing temperature (up to 120°C), no cyclometalated intermediate or noticeable decomposition of the NHC-iridium complex could be detected by 1 H NMR spectroscopy. 8 Moreover, the same observation was made in the presence of additional norbornene, demonstrating a total lack of reactivity of complex 2a in the absence of silylating agent. However, tentatives to isolate or characterize a hydridosilane based complex resulting from the reaction of 2a with HSiMePh2 were also unsuccessful, preventing from identifying transient iridium intermediate species that would be involved in the catalytic C-H silylation process. 7,18 On the other hand, the kinetic profiles for the silylation of 3a and its pentadeuterated Table 3. Scope of the reaction. [a] [a] Reaction conditions: 3 (0.2 mmol), 2a (0.01 mmol), Ph2MeSiH (0.6 mmol), norbornene (0.6 mmol), toluene (1 mL) in preheated oil bath at 120 o C for 16 hours under Ar. [b] Isolated yield (%). [c] yield (%) determined by 1 H NMR using 1,3,5trimethylbenzene as an internal standard. [d] PhMe2SiH (0.6 mmol). [e] Ph3SiH (0.6 mmol). [f] from N-(4-methoxybenzylidene)aniline and after hydrolysis (HCl, 3M).

analogue d5-3a were separately recorded in d8-toluene and a striking difference was observed. In fact, in the case of d5-3a, an induction period concomitant with a rapid deuterium-hydrogen exchange at ortho-positions was observed before the C-Si bond formation reaction takes place with a rate constant similar to that independently measured for 3a (Figures 3 & S6-9). 12,19,20 Moreover, we also compared the rates for the functionalization of 3a using H-SiMePh2 or D-SiMePh2 as silylating agent, and a KH/KD value of 2.0 was observed in this case (Figures 3 & S9-S10). Taken together, these results support the reversibility of the ortho C-H bond cleavage step and suggest that the Si-H bond breaking, and not the C-H bond, is involved in the ratedetermining step. 20,21 Finally, the catalytic silylation of substrate 3a conducted in the presence of access butylated hydroxytoluene (BHT), TEMPO [2,2,6,6-tetramethylpiperidine-1-oxyl], or mercury metal proceeded without noticeable decrease of activities (Figures S11-12), implying that both the involvement of free radical processes or heterogeneous catalysis are unlikely to be operative.

2a (5 mol%) 4 Yield [b] (NMR yield) [c] X [e] SiMe 2 Ph N O 4q, 69% (81%) [d] 4o Xray structure In summary, a series of three new chiral iridium(I) complexes bearing LX-type bidentate NHC-carboxylate ligands derived from amino-acids were synthesized in good yields and fully characterized, including their solid-state structures. These complexes demonstrated excellent catalytic activities in orthodirected dehydrogenative silylation of sp 2 C-H and sp 3 C-H bonds in the presence of norbornene as hydrogen acceptor. Despite the use of silylating agents, the developed catalytic system allowed for a complete control of the regioselectivity of the silylation reaction and prevents competitive functionalization of both the heterocyclic directing group and the aromatic solvent.

Figure 1 .

 1 Figure 1. State of the art of NHC-iridium catalysts in directed C-H silylation.

Figure 2 .

 2 Figure 2. Synthesis of (NHC)(cod)Ir(I) complexes (top). X-ray crystal structures of 2a (25% probability ellipsoids) and 2b-c (50% probability ellipsoids). Most hydrogen atoms are omitted for clarity.

  [e] 3,3dimethylbut-1-ene. [f] IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene.

Figure 3 .

 3 Figure 3. Kinetic isotope effects for parallel reactions

Table 1 .

 1 Selected bond lengths (Å) and angles (degree) for complexes 2a-c

	Bond length (Å)	(S)2a	(S)2b	(S)2c
	Ir-C1	2.052(13)	2.054(5)	2.059(10)
	Ir -O1	2.069(11)	2.073(4)	2.080(7)
	O1-C5	1.26(2)	1.291(7)	1.284(12)
	O2-C5	1.24(2)	1.221(7)	1.221(12)
	Angle ( o )	(S)2a	(S)2a	(S)2a
	O1-Ir-C1	85.5(5)	86.27(18)	86.2(3)

Table 2 .

 2 Optimization studies.[a]

					Temp.
	Entry	Silane	Catalyst	H2 -acceptor	Solvent

J. Name.,2013, 00, 1-3 | 3

Acknowledgements

We acknowledge the Centre National de la Recherche Scientifique (CNRS); the Ministère de l'Enseignement Supérieure, de la Recherche et de l'Innovation (MESRI, grant to RM), the Spanish Ministerio de Ciencia e Innovacion MCIN (PID2019-103965GB-I00 grant to MGC) and the Science Achievement Scholarship of Thailand (to T K). This work was supported by the Agence Nationale de la Recherche (ANR-15-CE07-0012-01 "CHADOC" grant to OB, JT),

Author Contributions

RM, MGC, TK, CB, JT, and EB performed experimental investigations; SML realized X-ray diffraction analysis; SY, RC, MM, JBS supervised; OB conceptualized and supervised this work and wrote the article.

Conflicts of interest

There are no conflicts to declare