domain: sdv sde

text:
Hélène Montanié
email: helene.montanie@univ-lr.fr

Margot De Crignis

Johann Lavaud

Viral impact on prokaryotic and microalgal activities in the microphytobenthic biofilm of an intertidal mudflat (French Atlantic coast)

Keywords: Virus, Prokaryotes, Microphytobenthos, Photosynthesis, Sediment, Mudflat

This is the first report on viriobenthos activity within the microbial biofilm located at the topsurface of the intertidal mudflat during emersion in Marennes-Oléron Bay (France). By combining in situ and ex situ approaches, the viral production (VP) was linked to the dynamics of prokaryotes and microphytobenthos (MPB). VP averaged 2-4 x 10 8 viruses ml -1 h -1 . VP correlated positively with the Virus to Prokaryote Ratio, and both were correlated negatively with the water content. The virus-induced mortality of prokaryotes was lower in winter than in summer (6.8 vs 39.7 % of the production) and the C-shunting may supply 2 to 12% of their Carbon Demand, respectively. VP accounted for 79% of loss in Prokaryotes but the response was delayed compared to the increase in VP suggesting a simultaneous release of viruses of MPB origin. This hypothesis is supported by capsid-sizing of virions by transmission electronic microscopy and bioassays. Harvesting and ex situ maintenance of topsurface sediments was carried out to monitor the dynamics of viruses, prokaryotes and MPB after inoculation with benthic or planktonic viruses. Benthic viruses modified the prokaryotic and MPB dynamics and decreased the photosynthesis efficiency in contrast to planktonic viruses that impacted MPB but not the prokaryotes.

INTRODUCTION

Microbial communities are structured by the intrinsic activities of viruses (Sime-Ngando, 2014) in terms of diversity and dynamics, directly through the process of virus-mediated cell lysis and changes in metabolic properties and/or indirectly by the bioavailability of significant amounts of viral lysates that may infer a reduction in competition pressure [START_REF] Suttle | Marine viruses -major players in the global ecosystem[END_REF]. The viral shunt of matter [START_REF] Wilhelm | Viruses and Nutrient Cycles in the Sea[END_REF] tends thus (i) to promote the recycling of carbon and nutrients by bacterial remineralizers [START_REF] Suttle | Marine viruses -major players in the global ecosystem[END_REF], (ii) to reduce the transfer of organic carbon to higher trophic levels [START_REF] Fuhrman | Marine viruses and their biogeochemical and ecological effects[END_REF] and (iii) overall to lubricate the microbial food-web on a short-time scale [START_REF] Weinbauer | Viral ecology of organic and inorganic particles in aquatic systems: avenues for further research[END_REF]. In the water column, up to 25% of the bacterial community [START_REF] Weinbauer | Ecology of prokaryotic viruses[END_REF]) may be infected and viruses are assumed to account for 20 to 40% of the daily mortality of the standing stock of planktonic bacteria [START_REF] Suttle | Marine viruses -major players in the global ecosystem[END_REF] and for 10 to 30% of the daily loss of bacterial production [START_REF] Fuhrman | Marine viruses and their biogeochemical and ecological effects[END_REF]. They affect both the biomass of phytoplankton (i.e: 10 to 50% of microalgae [START_REF] Gastrich | Viruses as potential regulators of regional brown tide blooms caused by the alga, Aureococcus anophagefferens[END_REF]) and the photosynthesis, probably through metabolic reprogramming [START_REF] Hurwitz | Metabolic reprogramming by viruses in the sunlit and dark ocean[END_REF], and reduce their primary productivity [START_REF] Suttle | Infection of Phytoplancton by viruses and reduction of primary productivity[END_REF][START_REF] Juneau | Effetcs of viral infection on photosynthetic processes in the bloom-forming alga Heterosigma akashiwo[END_REF]. Concerning the benthic deep-sea body, virus-induced mortality could globally represent as high as 80 % of the benthic prokaryotic heterotrophic production (Danovaro et al., 2008a).

The so-called phage kills the winner concept (KtW; Thingstad and Lignell, 1997) was tested on a panel of planktonic and benthic data sets [START_REF] Winter | Trade-offs between Competition and Defense Specialists among Unicellular Planktonic Organisms: the "Killing the Winner" Hypothesis Revisited[END_REF] and revealed a paradox in freshwater benthos where there is an apparent low infectivity of viruses together with an high abundance of bacterial hosts and viruses [START_REF] Filippini | Infection Paradox: High Abundance but low Impact of Freshwater Benthic Viruses[END_REF]. However, to question the universality of this paradox, the panel of environments considered needs to be enlarged, particularly in light of the fact that information on viriobenthos is scarce. Although analyses have been performed on viriobenthos from a variety of sediments (rewiewed by Danovaro et al., 2008b;[START_REF] Middelboe | Viral abundance and activity in the deep sub-seafloor biosphere[END_REF][START_REF] Helton | Interannual dynamics of viriobenthos abundance and morphological diversity in Chesapeake Bay sediments[END_REF] including surface layers in subtidal estuaries, coastal areas, continental lakes and deep-ocean sediments, there is only parcelled information of abundance concerning viriobenthos in the sediments of intertidal mudflats [START_REF] Montanié | Microbial interactions in marine water amended by eroded benthic biofilm: A case study from an intertidal mudflat[END_REF][START_REF] Careira | Counting viruses and bacteria in photosynthetic microbial mats[END_REF].

In Western European macrotidal estuaries and semi-enclosed bays, the primary productivity of intertidal mudflats is supported by motile microalgae (microphytobenthos, MPB) which are generally dominated by diatoms and form the main component of a dense biofilm at the surface of the sediment at low tide [START_REF] Pierre | Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France)[END_REF]. The MPB biofilm is stabilized by the exudation of Exocellular Polymers Substances (EPS) by both microalgae and prokaryotes (Orvain et al., 2014a). These epipelic diatoms were shown to be highly resistant to lighttemperature stress and its associated photooxidative stress, thanks to their motility and to the physiological non-photochemical quenching (NPQ) of chlorophyll a fluorescence [START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF].

The MPB biofilm is thus a unique transient biogeomorphological structure which constitutes a specific case study for in situ analyses of biological processes in surficial sediment. We investigated the dynamics and the activity of the viriobenthos associated with the MPB biofilm of the mudflat of Marennes-Oléron Bay (MOB; France) during the diurnal emersion period. The aims of our study were primarily, [START_REF] Auguet | Structure of Virioplankton in the Charente Estuary (France): transmission electron microscopy versus pulsed field gel electrophoresis[END_REF] to evaluate the temporal dynamics of viruses at the macro-(monthly) and at the micro-scale (hourly) and their horizontal distribution, and (2) to estimate the viral production and the virus-induced prokaryotic mortality. Secondly, we postulated that part of benthic viruses may also originate from microalgae and may interact with their dynamics. We confronted the in situ data with ex situ experimental values obtained from sediment surface layers containing motile MPB and inoculated with benthic and planktonic viruses in order to question the viral impact on both heterotrophic prokaryotes and microalgae with a focus on the photosynthetic productivity of MPB.

II MATERIALS AND METHODS 2.1 Study site and sampling stations

Sampling was conducted at diurnal low tide, during the emersion period (4 hours in length on average), on the mudflat located at the south end plume of the Charente estuary in Marennes-Oléron Bay (45°53'N 01°07'W, France). Intertidal mudflats represent 60% of the bay at low tide (Fig. S1). MPB can migrate vertically through the fine muddy sediment particles (median grain size around 11µm) and may rapidly cover between 80 and 90% of the top-surface of the sediment during the first half of the emersion. First, a 4 km cross-shore transect was surveyed at three stations ( In May 2009, the correspondence Weight/Volume was estimated as 1.29 ± 0.02 g per ml of fresh sediment (n=30), while the water content was 58.63% ± 1.55 (mean ± SD, n=10; range [START_REF] Middelboe | Spatial distribution and activity of viruses in the deep-sea sediments of Sagami Bay, Japan[END_REF][START_REF] Middelboe | Viral abundance and activity in the deep sub-seafloor biosphere[END_REF][START_REF] Montanié | Virus Like Particle analysis in a seston-rich coastal pond using Transmission Electron Microscopy[END_REF][START_REF] Montanié | Microbial interactions in marine water amended by eroded benthic biofilm: A case study from an intertidal mudflat[END_REF][START_REF] Nagasaki | Dinoflagellates, Diatoms, and their Viruses[END_REF][START_REF] Nagasaki | Previously unknown virus infects marine diatoms[END_REF][START_REF] Noble | Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria[END_REF][START_REF] Orvain | Tidal and seasonal effects on the short-term temporal patterns of bacteria, microphytobenthos and exopolymers in natural intertidal biofilms (Brouage, France)[END_REF][START_REF] Orvain | Relevance of macrozoobenthic grazers to understand the dynamic behaviour of sediment erodibility and microphytobenthos resuspension in sunny summer conditions[END_REF][START_REF] Ory | Pelagic food web patterns: Do they modulate virus and nanoflagellate effetcs on picoplankton during the phytoplankton spring bloom?[END_REF][START_REF] Pascal | Seasonal variation in consumption of benthic bacteria by meio-and macrofauna in an intertidal mudflat[END_REF][START_REF] Pérez-Bueno | Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus[END_REF][START_REF] Pérez-Clemente | Photosynthetic and antioxydant responses of Mexican lime (Citrus aurantifolia) plants to Citrus tristeza virus infection[END_REF][START_REF] Perkins | The application of variable chlorophyll fluorescence to microphytobenthic biofilms[END_REF]. Given this water content, 1 ml of fresh sediment weighed 0.53 g after desiccation.

In each case, the 1 cm top surface sediment of three independent cores were sliced, pooled and homogenized before sub-sampling in triplicate using 5 ml sterile syringe corers; they were then fixed with 4%-formaldehyde (V/V; 2% final concentration) and frozen (-20°C) one hour later until analysis within a week. In parallel, subsamples may serve to acquire environmental data: salinity, Chl a concentration measured using a Fluorometer Turner TD-700, water-content estimated by drying (60°C for 12h) and after a supplementary burning of 2 h at 490°C the concentration of organic matter (Table 1). Water column samples were taken at the sub-surface at high tide on the same day, either on the vertical of station 2 (2003-2004 survey) or at station E (mouth of the Charente estuary; [START_REF] Auguet | Dynamic of virioplankton abundance and its environmental control in the Charente Estuary (France)[END_REF]. Samples were fixed on board (36%-formaldehyde, 1% final concentration), stored at 4°C and analysed within 6 h in the laboratory.

Extraction of viruses

Benthic viruses were extracted, in triplicate. Briefly, 1.0 ml of tetrasodium pyrophosphate (10 mM final) and 3.6 ml of Milli-Q water were added to a slurry of 400 µl of fixed samples (i.e: 200µl of fresh sediment) defrosted at 37°C, followed by 30 min of gentle shaking at 4°C on a rocking table and one centrifugation for 30 min at 1000 g. Use of ultrasounds [START_REF] Danovaro | Determination of Virus Abundance in Marine Sediments[END_REF] have been discarded after a first test, confirmed then by a comparative test (July 2011) by which the accuracy of our method was analysed on three sediment samples, in triplicate, by comparison with the extraction method using probe sonication instead of shaking (Sonimasse S20, two periods of 30s at 60W separated by 30s of manual soaking). This surfactant-procedure can occasionally be performed two or three times more with the pellet of the remaining settled sediment to test the efficiency of virus extraction, notably in February (n=11) and July 2008 (n=23), July 2011 (n=9) and May 2013 (n=12) and at each new sampling period in triplicate. The different supernatants, stemming from successive S-steps, were separately quantified immediately after recovery. Briefly, 2 ml of a final dilution of 20, 200 and 400 times in MilliQ-water (from the first to the third supernatant, respectively) were filtered through a 0.02 µm Anodisc 25 membrane (Whatman) and stained with SYBRgreen I [START_REF] Noble | Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria[END_REF]. Slides were immediately enumerated for virus counts (15 fields) under a blue light (filter set 38, Zeiss) at x1000 magnification on a Zeiss Axioskop 2 Mot Plus epifluorescence microscope (Carl Zeiss, Inc.) with a 100x Plan APO oil objective lens. For the comparative test of method of extraction, the supernatants have been also quantified by flow cytometry according to the protocol of Brussaard et al. (2010): 10 -3 dilutions were stained by Sybr-green I and 80°C heated for 10 min before the analysis using a FACSCanto II cytometer, calibrated with 0.47 µm beads and the FACSDiva software.

Virus size and morphology

Viruses were first extracted in triplicate using pyrophosphate, a rocking shaker and centrifugation as described above for the epifluorescence counts. Supernatants were pooled and ultracentrifuged for 3.5 h at 150000 g (LE 70 Beckman ultracentrifuge, SW 28 rotor) and the pellet was resuspended in 100 µL of TN buffer (0.02 M Tris-HCl, 0.4 M NaCl, pH 7.4). The diversity in shape and size was analysed by TEM. Two carbon-colodion coated grids (Cu/Pd grid, 300 mesh) were prepared per sample by negative staining using 2% phosphotungstate [START_REF] Montanié | Virus Like Particle analysis in a seston-rich coastal pond using Transmission Electron Microscopy[END_REF]. Observations were performed with a Jeol 2011 transmission electron microscope operating at 200kV, calibrated with graphite grids, at a magnification of 50,000x to count at least 100 Virus Like Particles (VLP). Capsids were sized using Olympus analySIS software.

Prokaryotic enumeration

Triplicate samples (5 ml), defrosted at 37°C, were diluted 2000 times with 10 mM tetrasodium pyrophosphate at serial dilutions of 0.5, 10 -1 and 10 -2 (adapted from [START_REF] Pascal | Seasonal variation in consumption of benthic bacteria by meio-and macrofauna in an intertidal mudflat[END_REF] and validated (Fig. S2) by comparison with two other extraction methods using either cation-exchange resin [START_REF] Lucas | Bacteria of sediments: Extraction and distribution in a macrotidal mudflat (Bay of Saint-Michel)[END_REF] or methanol [START_REF] Lunau | An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy[END_REF]). A subsample was ultrasonicated for 30 s at 60 W (Sonimasse S20 sonicator), filtered onto a 0.2 µm black polycarbonate membrane, and the prokaryotic cells stained with DAPI [START_REF] Porter | The use of DAPI for identifying and counting aquatic microflora[END_REF] then enumerated under UV illumination (filter set 01, Zeiss) at 1000x magnification on a Zeiss Axioskop 2 Mot Plus epifluorescence microscope (100x Plan APO oil objective lens).

In the text, bacterial and archaeal cells are indifferently grouped as prokaryotes.

Microphytobenthos (MPB) counts

Homogenised mud (1 ml) was diluted ten-fold with <30-kDa ultrafiltered seawater (obtained by tangential flow through polysulfone cartridge [START_REF] Montanié | Microbial interactions in marine water amended by eroded benthic biofilm: A case study from an intertidal mudflat[END_REF]. Then 0.5 ml were diluted and fixed with 5 ml of alkaline lugol (1% final concentration). Algal cells were counted using a Nageotte chamber counter at x40 magnification on a Zeiss Axioskop microscope. MPB diatoms were divided into two groups: 'small cells' which were mainly Navicula sp. and 'large cells' which were mainly Pleurosigma and Gyrosigma spp., the socalled P-G taxon.

In situ viral production and virus-mediated mortality of prokaryotes

In situ viral production was monitored during the emersion period as the net in situ viral abundance change by sampling, in triplicate (three independent cores), three randomly chosen quadras to evaluate the initial abundance and three other quadras to determine the change in viral abundance after 3 h of emersion. Samples were V/V fixed with 4% formaldehyde (2% final concentration) and frozen at -20°C. The lytic viral production (VP) was calculated as described by [START_REF] Luef | Online program "Vipcal" for calculating lytic viral production and lysogenic cells based on a viral reduction approach[END_REF] as the maximum minus the minimum viral abundance divided by the time elapsed. Virus-mediated mortality of prokaryotes (% of cell loss per time unit; [START_REF] Weinbauer | Determining rates of virus production in aquatic systems by the virus reduction approach[END_REF] was deduced by dividing VP by the burst size (BS) as the number of lysed cells (VLC) and then by reporting it to prokaryotic standing stock (PSS), assuming a constant BS of 36 (mean value in [START_REF] Corinaldesi | Viral decay and viral production rates in continental-shelf and deep-sea sediments of the Mediterranean Sea[END_REF]. Virus-induced mortality of prokaryotes (VIM, %) has also been evaluated as the ratio of lysed cells (VLC) to prokaryotic production (Danovaro et al., 2008a). The hourly carbon released by viral shunt from prokaryotes (VICR) was calculated assuming 79 fg C per cell (Saint-Béat et al., 2013) and weighted by the total Prokaryotic Carbon Demand (PCD) considering a bacterial growth efficiency of 31% in order to investigate the impact of viruses on C cycle (wVICR, Pinto et al., 2013).

Ex situ experiment: viral lysis

To address and quantify the viral impact on benthic prokaryotes and microphytobenthos, only the free pore-water viruses were tested in order to avoid the time-consuming chemophysical treatment during benthic extraction and to limit the input of mineral or organic matter detached from the biofilm. Pore-water viruses ('benthic viruses', Vb) were harvested by centrifugation at 3500 g for 10 min (Jouan CR412) of fresh sediment (1 cm top-surface sediment), then filtered through a 0.2 µm membrane to eliminate all other microbes. Additionally, viruses in the water column ('planktonic viruses', Vp) were isolated from other organisms by filtration of the overlying seawater through a 0.2µm filter. In May 2010, heatinactivated Vb (boiled and cooled 3 times) were tested.

Sub-samples of fresh sediment (6 x 2 ml) were incubated in 6-well microplates (Falcon), humidified top-down either with 250 µl of virus-free pore-water ('Control') or 250 µl of benthic or planktonic viruses ('Vb or Vp treatment'). Virus-free pore-water was obtained by ultrafiltration of the virus-rich filtrate using a centrifugation filter device (Centricon Plus-70 Ultracel PL-30, Millipore). The 6-well-microplates were exposed to natural light at ambient temperature in order to maintain the in situ migratory behaviour of MPB cells. Time-series sampling was performed daily in triplicate at the corresponding time of mid low tide in the field (using a 1 ml syringe corer after homogenization of the well, with a coefficient of variation of 13.41%). The impact of pore-water viruses on the prokaryotes was estimated over three consecutive days in May 2009 and 2010. Daily viral production and virus-mediated mortality of prokaryotes were calculated for the concomitant period of prokaryotic decrease and viral increase [START_REF] Luef | Online program "Vipcal" for calculating lytic viral production and lysogenic cells based on a viral reduction approach[END_REF]. Total prokaryotic loss was estimated as the net decrease in abundance (i.e net prokaryotic production; [START_REF] Middelboe | Spatial distribution and activity of viruses in the deep-sea sediments of Sagami Bay, Japan[END_REF].

In May 2009, viral lysis activity of Vb was also evaluated on MPB over seven days and compared to Vp lysis activity. To counteract the possible evaporation of water from the sediment, 250 µl of virus-free pore water was added to each well at day 3.

Photosynthetic activity of the MPB ex situ: Maxi-Imaging-PAM chlorophyll fluorescence measurements

Chlorophyll fluorescence measurements were performed with the Maxi-version of an Imaging-PAM chlorophyll fluorometer (I-PAM, Walz, Effeltrich, Germany) on a 6-well microplate, which occupies the total surface of the fluorescence image (10×13 cm; Fig. S3).

Three wells (one horizontal row) used for one kind of treatment only (control, Vb-or Vp-) enabled instantaneous triplicate measurements (Fig. S3). The photosynthetic activity of the MPB was assessed by rapid light curve (RLC) measurements [START_REF] Perkins | The application of variable chlorophyll fluorescence to microphytobenthic biofilms[END_REF]. RLCs were obtained by the application of a series of 11 sequential short light exposures (20 s) with increasing irradiance from 0 to 1250 µmol photons. m -2 s -1 . At each irradiance, F m ' and F t were recorded. F m ', the maximum fluorescence yield, was measured by applying a saturation pulse (800 ms, 2800 µmol photons. m -2 s -1 ); F t , the steady-state fluorescence, was continuously monitored throughout each 20 s light step. F 0 ', the minimum fluorescence yield, was measured at irradiance 0 µmol photons. m -2 s -1 by measuring non-actinic light solely.

Two main parameters were computed from the RLCs: (i) ΦPSII, the effective quantum yield of photosystem II (PSII), was calculated for the 0 µmol photons m -2 s -1 irradiance as ΦPSII = (F m '-F t )/F m '. As no adaptation to the dark was performed before the measurement in order to avoid vertical migration of the motile microalgae, Ft (or F 0 ') and Fm' were close to their respective dark-adapted values F0 and Fm, so that ΦPSII at this irradiance is close to the standard fluorescence index F v /F m , i.e. the maximum photosynthetic efficiency of PSII [START_REF] Ralph | Fluorescence as an assay to understand aspects of the physiology of light regulation. Fluorescence as an assay to understand aspects of the physiology of light regulation[END_REF], and (ii) NPQ, the non-photochemical quenching of chlorophyll fluorescence, was calculated as NPQ = F m -F m ' / F m ' [START_REF] Ralph | Fluorescence as an assay to understand aspects of the physiology of light regulation. Fluorescence as an assay to understand aspects of the physiology of light regulation[END_REF]. The NPQ kinetics were further measured during a short (5 min) light exposure of 280 µmol photons m -2 s -1 , which was close to the intensity necessary to saturate photosynthesis (249 ± 50 µmol photons m -2 s -1 ) for the control MPB biofilm, as measured using the RLCs.

Statistical analysis

All statistics were performed with Excel and Prism 4 softwares or Minitab for nested ANOVA. Regression analysis was performed for prokaryote abundance and prokaryote loss against viral abundance and VPR, respectively, using log-transformed data.

III RESULTS

Efficient protocol for viral and bacterial extraction and counting from sediment

To extract viruses, sonication has been rejected because it emulsified the mud-samples and the sediment was disrupted into smaller particles that decreased the accuracy of the microscopic observation of viruses. Therefore in the test of July 2011 (Fig. 1), the microscopic abundance significantly lowered (ANOVA, p=0.0002) as well as cytometric counts (ANOVA, p<0.0001). The method of extraction (with or without sonication, nested ANOVA) accounted for 73.8% of total variance by microscopy and for 78.9% by cytometry. The accuracy of the protocol used for viruses (July 2011 test, n=3) was illustrated by a coefficient of variation (CV) of 16.4 ± 8.4 % instead of 19.3 ± 5.6 % with the sonication step, and this difference in CV was confirmed by flow cytometry analysis (2.6 ± 1.9% vs 13.3 ± 4.5%).

The addition of EDTA [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF]Carreira et al., 2015) was not conclusive for the same reason while vortexing has made the grids opaque for observation. After having tested our protocol on several extraction-steps, its efficiency seemed satisfactory. The first two steps (S1 and S2) involving pyrophosphate-shaking detected up to 99.6% of extractable viruses; a third step removed only 0.4% of the total cumulative number. The first and second steps extracted 63.9± 6.9 % and 36 ± 1 % (n=34, in situ data of 2008) of the extractable viruses, respectively. Overall to date, all acquired data posteriorly validated that the first S1-extraction corresponded to 64.1 ± 8.1 % of extractable viruses (n= 56, Fig. 2). For samples of 2009-2010 after a preliminary confirmation of the percentage of extractability, only one step was performed for in vitro counts and the initial extractable virus numbers was then corrected for the miscounting based on the determined 64/36% ratio of S1/S2 extraction efficiency.

All samples were stored at -20°C for a week and no factor was ever applied for correcting the viral loss due to fixation with formaldehyde and conservation. Indeed a time-point comparison of the decay of viruses included into muddy samples and stored for 15 days at 4°C, -20°C and -80°C (Fig. 3), revealed (i) no significant variation in virus counts after 8 days of storage at each or other temperature (p>0.05), even if curiously the number of extracted viruses from samples stored at 4°C was higher after 1 day, (ii) irrespective of temperature, a significant loss of viruses between 8 d and 15 d of storage (p<0.0001) and thus significant lowered T 15 values compared to initial field value (p<0.001) and (iii) at each time point, a higher preservation in refrigerated samples (4°C) than in frozen samples (-20°C and -80°C, p<0.001) without any difference between the two freezing temperatures (p>0.05). However, the percentage of S1 recovery declined over time in line with increased negative temperature of preservation (Fig. 3 B,C,D).

A significant fraction of the prokaryotes was not eluted with the extraction procedure used for viruses, despite three sequentially repeated extractions. Prokaryotes were thus counted independently from viruses with a protocol previously used in our laboratory for benthic prokaryotes [START_REF] Pascal | Seasonal variation in consumption of benthic bacteria by meio-and macrofauna in an intertidal mudflat[END_REF]. Our protocol consists in ultrasound treatment of highly diluted sediments followed by a DAPI staining (Fig. S2). The counting procedure was not disturbed by the accumulation of sediment particles inappropriately masking the microscopic slides.

Monthly survey of viral and prokaryotic abundance

On a monthly average, the abundance of viriobenthos was 60-fold higher than that of virioplankton (60.3 ± 20.3, n=7). The abundance of benthic prokaryotes varied between 244 ± 13 (May 2009-July 2008) and 1945 times higher (February 2008) than those in the water column.

In 2003-2004, at all stations along the transect, viral abundance peaked to 2.43 ± 0.46 x 10 9 viruses ml -1 in September then dropped to c. 0.9-1 x 10 9 ml -1 in winter (Fig. 4A). The sampling date accounted for 77.2% of the total variance in viral abundance while spatial location accounted only for 5.7%.

A large morphological diversity in virus-like particles (VLPs) was observed using transmission electron microscopy (Fig. S4). The majority of VLP showed icosahedral shape, only few pleomorphic particles or elongated capsids were observed. Some filamentous forms of 0.6-1.2 µm were observed, notably in February and September (3-8%). Excluding filamentous VLPs, the capsid size was on average 77.4 ± 34.9 nm. Tailed viruses accounted for 4-21% of the particles and theirs capsids were on average 96.4 ± 25.7 nm in size. Because of the low frequency of tailed VLPs, their morphotypes were not discriminated between myovirus, siphovirus or podovirus shape; however the largest particles could be address to Myoviridae. The distribution of capsid diameters provided accurate seasonal morphological comparisons (Fig. 4B). Throughout the year, VLPs less than 65 nm in size constituted up to 36-49% of the total particles, depending on the frequency of the < 45nm-sized particles (17-19% in winter versus 5-9% in summer and autumn). The largest size-class (> 125 nm) represented 4-21% of the total VLPs varying from 0.36 (March) to 3.85 x 10 8 (June). In September, the 65-85 nm-sized particles was 5-fold higher than in the others months and predominated the viral community (42%). Tailed viruses accounted for 9-33% of their sizeclass. It was noticeable that, in February 2004, 22% of the VLPs were longer than 105 nm, and 38% of these were tailed viruses.

In situ hourly survey of viral and prokaryotic abundance during an emersion period in winter and summer

In the top-surface sediment of the mudflat, viral abundance at the beginning of the emersion was c. 1.91 ± 0.22 x 10 9 ml -1 in February 2008 and c. 6.30 ± 0.47 x 10 9 ml -1 in July 2008 (Fig. 5A). Prokaryotes numbers were 3.19 ± 0.45 x 10 9 cells ml -1 and 8.48 ± 1.37 x 10 8 cells ml -1 , respectively (Fig. 5B). Consequently, the Virus to Prokaryotes Ratio (VPR) was on average 0.85 ± 0.49 (n=6) in February and 9.61 ± 3.31 (n=8) in July (Table 1).

Viral abundance increased during the 3 h of diurnal emersion on the 19 th and 20 th February 2008 (+47% and 9%, respectively; Fig. 5A) with no clear tendency for prokaryotes, resulting in an insignificant relationship between viruses and prokaryotes (p>0.05). On average, the hourly viral production (VP) was 2.23 x 10 8 viruses ml -1 h -1 , and was responsible for 0.22 ± 0.04 % of prokaryotic loss (in terms of prokaryotic standing stock, PSS). In summer, significant viral replication occurred on the 17, 18 and 19 July 2008 (t test, p = 0.0037; +22.41 ± 4.74 %) while prokaryotes concomitantly decreased (-5.44 ± 1.97 % h -1 ; Fig. 5B). Viruses accounted for 84% of the variation in prokaryotic abundance (log Prokaryotes=-1.63 log Viruses + 24.90, r 2 =0.84, n=6, p=0.01). In July 2008, VP was 4.39 ± 1.42 10 8 viruses ml -1 h -1 , representing a loss of 1.52 ± 0.56 % of PSS. Virus-mediated prokaryotic lysis accounted for 28.99 ± 5.61% of observed prokaryotic cell loss (i.e. the net prokaryotic growth). Moreover, considering that the gross prokaryotic production was 3.21 % and 3.84% of the standing stock per hour (Production/Biomass; P/B), in winter and summer 2006 respectively [START_REF] Pascal | Seasonal variation in consumption of benthic bacteria by meio-and macrofauna in an intertidal mudflat[END_REF], virus-induced mortality (VIM) could account for 6.78 ± 1.44% of the prokaryotic production in February 2008 and for 39.7 ± 14.7% of prokaryotic production in July 2008. However whatever the season, the viral turnover averaged 0.099 ± 0.082 h -1 (range 0.059 -0.236), slightly higher in winter (0.14) than in summer (0.07). During the diurnal emersion, between 3.12 and 15.83 mg C m 2 would be released by viral shunt per hour (i.e. VICR) and the released C may represent around 2.1% (winter) and 12.3 % (summer) of the Prokaryotic Carbon Demand (i.e. weighted VICR). Interestingly, by using all the in situ data (February 2008, July 2008, May 2009 and May 2010), a significant negative relationship was observed between the virus to prokaryotic ratio (VPR) and the water content of the sediment (p<0.0001; Fig. 6A). A higher prokaryotic abundance was observed when the water content exceeded 58.6%, expressed in fine as a lower VPR (<2, February 2008 and May 2010 versus 4.3< in July 2008 and May 2009; Table 1). And during the emersion period and whatever the season, the viral production (VP) tended to be negatively linked to water content and the magnitude of change in VPR was positively and significantly related to viral production (r = +0.88; p=0.01). When the net positive prokaryotic growth represented more than 19.5 % of the PSS, VPR tended to decline during emersion (Fig. 6B).

Daily monitoring of top-surface spring sediments maintained ex situ: viral inoculation and virus-mediated prokaryotic lysis

In May 2009, in the top-surface mud layer, there were c. 2.81 ± 0.32 x 10 9 ml -1 viruses and c. 6.58 ± 0.08 10 8 ml -1 prokaryotes, corresponding to c. 5.30 ± 0.61 x 10 9 and c. 1.29 ± 0.01 x 10 9 g -1 dry sediment, respectively. The VPR was on average 4.27 ± 0.08. In May 2010, viral abundance was only 1.89 ± 0.34 times the prokaryotic abundance (c. 4.31 ± 0.19 x 10 9 versus c. 2.22 ± 0.07 x 10 9 ml -1 or c. 8.13 ± 0.35 x 10 9 versus c. 4.18 ± 0.14 x 10 9 g -1 dry sediment).

In response to the addition of pore-water viruses (Vb) (+7.87% in 2009 and +3.84% in 2010), a daily increase in PSS loss was observed: +7.11% in 2009 and +11.69% in 2010. In 2009 (Fig. 7E), viral abundance decreased during the first day, notably in control wells. A net viral production occurred during the second day while prokaryotic abundance was still decreasing. By day 3, viral and prokaryotic dynamics diverged between the control wells and virus-treated wells. In both cases, viruses rose back to their initial titers, while prokaryotes nearly doubled in controls compared to samples treated with viruses (Fig. 7D,E). During the 3 days of incubation, the amplitude of variation in viral abundance was more pronounced in the controls than in virus-treated wells and thus was characterized by a higher VP: 4.73 x 10 8 versus 3.93 x 10 8 viruses ml -1 d -1 . However, during the first two days, viral abundance was significantly higher in virus-treated wells, leading to an increase of 1.07% in virus-mediated loss of PSS. Otherwise, no significant relationship was observed between viral and prokaryotic abundance (r=0.32; p=0.45).

In contrast, in May 2010 (Fig. 8), prokaryotic abundance decreased significantly with increasing viral abundance (log Prokaryotes= -1.46 log Virus + 23.43, r 2 =0.89, n= 8, p<0.001) whatever the treatment. One day post inoculation, prokaryotic abundance was significantly reduced with the Vb treatment compared to controls (t test, p<0.05) while inactivated Vb had no effect (t test, p=0.5). The daily viral production, calculated over the first two days in control wells, was 6.48 x 10 8 ml -1 d -1 of fresh sediment. In virus-treated wells, an additional viral production occurred, c. 8.09 x 10 8 ml -1 d -1 , which was responsible for the supplementary lysis of 1.01% of PSS. By combining the data from May 2009 and May 2010 with the in situ data from July 2008, a significant positive relationship between loss of prokaryotic abundance and VP was estimated as: prokaryotic loss = -0.759 x VP -1.088 (r 2 = 0.79, n=5, p=0.04).

In May 2009, the influence of Vp was also tested (Fig. 7D,E). Only 6% of PSS was lost per day compared to 7.5% in the controls and 14.6% with Vb. However, daily VP reached 3 x 10 9 particles ml -1 .

Daily monitoring of top-surface spring sediments maintained in vitro: MPB dynamics and photosynthesis as a function of viral inoculation

The analysis of the microphytobenthos (MPB) was only conducted in May 2009 (Fig. 7). MPB biomass evolved similarly in the control and virus Vb-treated wells (two way ANOVA, p= 0.363; Fig. 7A). Of the 6.15 ± 0.11 x 10 5 cells ml -1 in the top-surface sediment, pennate diatoms largely dominated over centric species (stable at around 12-13%, whatever the treatment), of which 59.6 % belonged to the Pleurosigma-Gyrosigma taxon (P-G taxon) and 40.4 % to Navicula sp. In controls, a slight decline (3.1% per day) was noticeable until day 5, followed by a sharp decrease since only 57% of the original assemblage persisted at day 7 (Fig. 7A). Within the first 3 days, the P-G taxon (Fig. 7B) decreased slightly more (-9.8%) in the Vb-treated wells than in the control wells while the growth of Navicula sp. (Fig. 7C) was significantly enhanced (+42.9 %, ANOVA, p=0.045). From days 3 to 5, the P-G taxon biomass regained its level at T=0 in control and, although a net increase was observed whatever the treatment, its biomass was significantly higher in the control wells than in the Vb-treated wells (+12.9%; ANOVA, p=0.017). The abundance of Navicula sp. decreased by 30%, but the number of cells was still significantly higher in Vb-treated wells than in the controls wells (+ 18.7%, ANOVA, p=0.04). At day 7, no difference in abundance was observed whatever the treatment (ANOVA, p=0.81 and 0.16 for the P-G taxon and Navicula sp., respectively). At that time, the loss in MPB biomass from T0 on was largely due to Navicula sp. (63%) while only 28% was due to the P-G taxon.

The addition of Vp significantly changed the MPB dynamics (two way ANOVA, p<0.001) by doubling the MPB decay rate to 6.9% d -1 versus 3.1 and 3.6% for controls and Vbtreatment, respectively (Fig. 7A). P-G taxon (Fig. 7B) was significantly less represented than in the control wells over the 5 days (p<0.008). In contrast, the number of Navicula sp. cells (Fig. 7C) was only significantly lower at day 3 (ANOVA, p=0.04).

After 5 days of viral infection, the photosynthetic activity of the MPB biofilm was measured with a Maxi-Imaging-PAM fluorometer. The basic chlorophyll fluorescence emission of MPB (F t ), which illustrates the photosynthetic biomass, was similar in the control and virus-treated wells (Fig. S3). This confirmed the results of MPB-cell counting. Nevertheless, the maximum photosynthetic efficiency (F v /F m ) was clearly reduced in all virus treated-wells (Vb and Vp) compared to the control sediment (Table 2). It was noticeable that NPQ, the nonphotochemical quenching of fluorescence, which is an index of light stress photoprotection, decreased in Vb-treated sediment but not in Vp-treated sediment, compared to the control (Table 2). Measurement of the NPQ development kinetics showed that not only the extent of NPQ was reduced but that the kinetic was slowed down by Vb infection (Fig. S5). After 10 days of viral infection, the photosynthetic efficiency strongly decreased in all samples, illustrating the senescence of the MPB biofilm; there was no longer any difference between controls and virus-treated sediments (data not shown).

IV DISCUSSION

Methodological considerations

Comparative storage of sediment samples allows to conclude to a relatively good persistence of viruses at short-term (a week) at 4°C as well as freezing temperatures. While the loss of viruses may be up to 35% in 24 h (according to the exponential model of decay, [START_REF] Wen | Accurate estimation of viral abundance by epifluorescence microscopy[END_REF], a gain of viruses (+19%) was detected in 24h at 4°C like in the case of lacustrine sediment (up to 31% in 5 days, [START_REF] Duhamel | Flow cytometric analysis of bacteria and virus-like particles in lake sediments[END_REF]. Changes in the structure of the sediment matrix (organic matter and silt-clayed substratum) may modify the extractability of the viruses and their sorption capacity to the plastic tube-wall according to the extended-DLVO theory [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF]; Guttierez and N-Guyen, 2012; Wong et al, 2013). The apparent higher viral counts at 24h may be consecutive to a stark release in pore water of colloidal EPS from microalgae and bacterial cells. EPS may help to desorb viruses by reducing the minimum energy depth and increasing steric hindrance (Walshe et al, 2010; Zhao et al, 2014) and then favour their persistence by inhibiting the extracellular nuclease or protease [START_REF] Hewson | Temporal dynamics and decay of putatively allochthonous and autochthonous viral genotypes in contrasting freshwater lakes[END_REF]. They may finally limit the bias of sorption on the plastic tube-wall of tube by complexing viruses in solution [START_REF] Wong | Effect of organic carbon on sorption of human adenovirus to soil particles and laboratory containers[END_REF]. Forehand, the recurrent loss of viruses into frozen samples compared to refrigerated samples may result from the disruption of cells due to ice crystal disruption [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF] and the subsequent release of virucidal substances. Inversely no differences were reported for sediments of the Chesapeake Bay that contains greater proportion of sand and smaller percentage of organic matter [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF].

Roughly, 64% of the extractable viruses were dislodged during the initial extraction step. This is similar to values reported for estuarine sediments from other sites [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF][START_REF] Danovaro | Separation of free particles from sediments in aquatic systems[END_REF]. Given the physical properties of the sediment, an ultrasound treatment was recommended by [START_REF] Danovaro | Separation of free particles from sediments in aquatic systems[END_REF] and Carreira et al (2015). Nevertheless, in our case, the presence of very small silt-clayed particles (up to 98%; [START_REF] Blanchard | Kinetics of tidal resuspension of microbiota: testing the effetcs of sediment cohesiveness and bioturbation using flume experiments[END_REF] precluded the use of a physical treatment (ultrasound and vortex). Indeed, compared to sandy sediment, the higher total porosity and lower permeability of muddy sediment favours the enhancement of the electrostatic forces between clayed particles and virions [START_REF] Gerba | Applied and Theoretical Aspects of Virus Adsorption to Surfaces[END_REF][START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF]) and a slow desorption of viruses [START_REF] Pinto | Viriobenthos in aquatic sediments: varia bility in abundance and production and impact on the C-cycle[END_REF]. Although the adaptation of the extraction protocol we propose here for highly clayed sediments can be considered as conservative for viral extraction, it could not be validated for the extraction of prokaryotes.

Given the intertidal nature of the Marennes-Oléron bay (MOB), we made improvements to the methodology for the determination of viral production (VP). Estimates of viral increase over time is standard for marine sediments either by incubating (1) undiluted and homogenized deep-sea sediments in a Würgler-bag in anoxic conditions, (2) diluted slurry similarly to a pelagic analysis [START_REF] Glud | Virus and bacteria dynamics of a costal sediment: Implication for benthic carbon cycling[END_REF]Danovaro et al., 2008a;[START_REF] Corinaldesi | Viral decay and viral production rates in continental-shelf and deep-sea sediments of the Mediterranean Sea[END_REF] or by maintaining (3) intact Haps-cores of coastal sediments in water (Siem-Jørgensen et al, 2008). However, both the dilution-based and Würgler-bag approaches (1 and 2) suffer from methodological biases altering either the heterotrophy activity and the mineralization rate, the host-virus contact and progeny of infections or the loss of viruses by exoprotease [START_REF] Hansen | Anoxic incubation of sediment in gas-tight plastic bags: a method for biogeochimical process studies[END_REF]Danovaro et al., 2008b;[START_REF] Dell'anno | Determination of viral production in aquatic sediments using the dilution-based approach[END_REF]. Although the dilution-based technique is recommended by Dell-Anno et al. ( 2009) as the most suitable methodology to estimate VP in marine systems, we chose to deduce VP directly in the field, during the emersion period, from net temporal variations in viral abundance as reflecting the true in situ production of surface sediment. By mimicking the mudflat in a low-tide situation, the microplate approach, is quite similar to the Würgler-bag method because it includes a homogenisation step to uniformly distribute the undiluted mud into the wells. It reposes upon the same assumptions in terms of heterotrophic prokaryotes activity, biocide activity and competition with predators. Additionally 'Microplate incubation' is compatible with the use of Imaging-fluorometers to simultaneously study photosynthesis. To our knowledge, this is the first time the Imaging-PAM (I-PAM) has been applied for the assessment of the effect of viral infection on the photosynthetic activity of mudflat MPB natural assemblages. As a nondestructive technique and a rapid assay, I-PAM greatly facilitates measurements on complex samples collected in situ and maintained ex situ and allows the accurate implementation of photosynthesis regulation kinetics.

Are benthic viruses mainly prokaryotic phages or eukaryotic viruses?

Viral abundance in the mudflat of the MOB is within the range reported for marine sediments (from 10 7 to 10 11 ml -1 ; [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF] and is even closer to the results reported for freshwater and shallow marine ecosystems (9x10 9 viruses g -1 ; Danovaro et al., 2008b). The viral abundance of mudflat sediments was 60-fold higher than in the overlying water column. Such small ratios have been reported for other eutrophic bays: x22 for Moreton Bay (Hewson et al., 2001a), x14 for Niva Bay [START_REF] Middelboe | Distribution of viruses and bacteria in relation to diagenetic activity in an estuarine sediment[END_REF] and x10 for Chesapeake Bay [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF]) while higher values (from 100 to 1000) were observed in oligotrophic sites (Hewson et al., 2001a;Danovaro et al., 2008b). Because of a much higher abundance of prokaryotes in mudflat sediment, the virus to prokaryote ratio ranged 0.8-9.6 like for Dutch intertidal sediment (0.6-1.4; Careira et al, 2015); benthic VPR was lower than previous observations in the MOB water column (11.6 ± 3.7 in 2002-2003;[START_REF] Auguet | Dynamic of virioplankton abundance and its environmental control in the Charente Estuary (France)[END_REF]. This general trend (except across the mouth of Chesapeake Bay; Drake et al., 1998) suggests a low viral production from prokaryotes in the sediments, even though the high density of prokaryotes and viruses probably fosters host-virus encounters [START_REF] Filippini | Infection Paradox: High Abundance but low Impact of Freshwater Benthic Viruses[END_REF] which in turn may enhance prokaryotic resistance [START_REF] Weinbauer | Viral ecology of organic and inorganic particles in aquatic systems: avenues for further research[END_REF]. This situation is even more striking in that the higher availability of nutrients and organic matter in the sediment favours a higher activity of benthic heterotrophic prokaryotes [START_REF] Danovaro | Viral density and Virus-to-Bacterium Ratio in Deep-Sea Sediments of the Eastern Mediterranean[END_REF]. This discrepancy could be explained by several factors that may be inferred from the virusprokaryotes interaction: (i) different viral decay due to nuclease and/or protease concentrations [START_REF] Middelboe | Distribution of viruses and bacteria in relation to diagenetic activity in an estuarine sediment[END_REF][START_REF] Filippini | Infection Paradox: High Abundance but low Impact of Freshwater Benthic Viruses[END_REF] The autochthonous or allochthonous origin of benthic viruses is still a matter of debate. Some evidence supports an endogenous origin without excluding an input of pelagic viruses [START_REF] Siem-Jørgensen | Viral deynamics in a coastal sediments: seasonal pattern, controlling factors and relations to the pelagic-benthic coupling[END_REF]. However in the study case of the microphytobenthic (MPB) biofilm, the proportion of phytoviruses may be significant or even it may oversize the proportion of prokaryotic phages among the viriobenthos. The high abundance of viruses in the surface sediment without any sign of intensive viral infection of prokaryotes (low VPR) may thus originate (1) in the sorption of large particles, algal viruses, from the water column and/or (2) in the replication through benthic microalgae, all the more so since the burst-size of algal viruses (range 10 2 -10 4 ; Short, 2012) exceeds those of prokaryotes (range 3-69 in marine sediments; Danovaro et al., 2008b). Our results are congruent with both hypotheses. Indeed in the MOB intertidal mudflat, only 50% of viruses had a capsid size of less than 65 nm compared to 71% in the overlying water [START_REF] Auguet | Structure of Virioplankton in the Charente Estuary (France): transmission electron microscopy versus pulsed field gel electrophoresis[END_REF]. The sorption of viruses and the binding links on clay-and silt-particles enhances proportionally to the capsid-size [START_REF] Dowd | Delineating the Specific Influence of Virus Isolelectric Point and Size on Virus adsorption and transport through Sandy Soils[END_REF][START_REF] Chattopadhyay | Adsorption of bacteriophages on clay minerals[END_REF]. Moreover, large-sized virus particles may strongly counteract against the forces of desorption when the organic matter increases during emersion and the ionic strength decreases at rising tide [START_REF] Gerba | Effect of particulates on virus survival in seawater[END_REF]. Overall, our results support the scenario of the replication of planktonic viruses through MPB diatoms since the addition of planktonic viruses (Vp) only slightly changed the daily loss of benthic prokaryotes but significantly declined diatom microalgae biomass. This enhanced viral production at the expense of MPB diatoms, as a result of input of pelagic viruses, may suggest fluxes of viruses at the water-sediment interface via the MPB biofilm. Nevertheless, we clearly demonstrated the negative impact of benthic viruses on prokaryotes since changes in VP explained 79% of the changes in net prokaryotic growth, even though this was delayed compared to VP (power slope = 0.75). This delay sustains the idea of a related viral replication through MPB hosts, in line with the relative high frequency of large capsid-sized virions while among the algal viruses isolated to date, virions size ranged from 22 to >200nm (Short, 2012).

Viral production and prokaryotic mortality

The value of 10 7 -10 8 viruses produced ml -1 h -1 is in the range of 10 6 -10 9 viruses g -1 h -1 reported for marine sediments (Danovaro et al, 2008a;[START_REF] Corinaldesi | Viral decay and viral production rates in continental-shelf and deep-sea sediments of the Mediterranean Sea[END_REF]. Like in the deep sea sediments of Sagami Bay [START_REF] Middelboe | Spatial distribution and activity of viruses in the deep-sea sediments of Sagami Bay, Japan[END_REF], VP may be responsible in mudflat for an average of 29% of the net bacterial losses. Cell loss of 0.2-1.5% of the PSS and virus-induced mortality of 7-40% of prokaryotic production per hour, confirm the ascending gradient in viral-induced prokaryotic mortality in terms of production from coastal sediments (around 16%, e.g. 12-57% in Adriatic Sea [START_REF] Mei | Virus production and life strategies in aquatic sediments[END_REF]) and 4-41% in Central Øresund, Denmark (Siem-JØrgensen et al., 2008)) to deep-sea sediments (89%, Danovaro et al., 2008a) and positioned MOB mudflat in terms of viral impact on prokaryotic standing stock together with the sites with the lowest cell losses (0.3% : Adriatic sea [START_REF] Mei | Virus production and life strategies in aquatic sediments[END_REF]); 0.08-6.7% : Central Øresund, Denmark [START_REF] Glud | Virus and bacteria dynamics of a costal sediment: Implication for benthic carbon cycling[END_REF][START_REF] Siem-Jørgensen | Viral deynamics in a coastal sediments: seasonal pattern, controlling factors and relations to the pelagic-benthic coupling[END_REF]; <1% : deep-sea sediments [START_REF] Middelboe | Spatial distribution and activity of viruses in the deep-sea sediments of Sagami Bay, Japan[END_REF]. The highest cell losses were reported in Southern California (4-14%; [START_REF] Hewson | Viriobenthos Production and Virioplankton Sorptive Scavenging by Suspended Sediment Particles in Coastal and Pelagic Waters[END_REF]. Although there was little apparent involvement of viruses in prokaryotic mortality, the net prokaryotic loss (the so-called prokaryotic net production) was positively related to the viral production rate in MOB during the emersion period. Differences in virus origin have been related to amplitude in prokaryotic responses; prokaryotes appeared more permissive to interstitial viruses (extracted from fresh sediment) than to surficial pelagic viruses. Benthic prokaryotes would mainly be of benthic origin and their viruses too. Our bioassays confirmed that after erosion into the overlying water, benthic bacteria and their viruses would be mainly located at the water interface and not dispersed efficiently upwards in the entire water column (Guizien et al., 2014).

Impact of viruses on the MPB diatoms

In MOB, MPB diatoms may be easily eroded from mudflat and 30 to 80% of diatoms of the water column can be from benthic origin depending of hydrodynamics (Guizien et al., 2014). Also there is a strong relationship between planktonic viruses and phytoplankton [START_REF] Ory | Pelagic food web patterns: Do they modulate virus and nanoflagellate effetcs on picoplankton during the phytoplankton spring bloom?[END_REF]. Microalgae and especially diatoms have already been described as potential hosts for viral multiplication in the estuarine benthos and phytoplankton (Hewson et al., 2001b), and in monospecific cultures (planktonic diatoms, [START_REF] Nagasaki | Dinoflagellates, Diatoms, and their Viruses[END_REF][START_REF] Tomaru | First evidence for the existence of pennate diatom viruses[END_REF]; benthic diatom Navicula sp. [START_REF] Suttle | Infection of Phytoplancton by viruses and reduction of primary productivity[END_REF]. Thus in MPB biofilm associated to surficial sediment, both prokaryotic and algal viruses were logically present. Indeed for Chesapeake Bay benthic virome, 11% of the viral hits to dsDNA belonged to the Phycodnaviridae family [START_REF] Helton | Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Ramdomly Amplified Polymorphic DNA PCR[END_REF]. As a consequence, the impact of viruses on MPB is more complex than on prokaryotes, due to higher dispersion of diatoms in the entire water column. Planktonic viruses like benthic viruses may regulate diversity and photosynthetic activity of the MPB. In our experiments, during the first days of infection, Vb modified the abundance of MPB diatoms by promoting the growth of Navicula sp. while inhibiting the growth of the P-G taxon, the two dominant taxa. In contrast, Vp decreased the abundance of both taxa, with a greater impact on the P-G taxon, which thus appeared to be more sensitive to viral infection (by both Vb and Vp) than Navicula sp.. This observation is congruent with the diatom species specificity of viruses [START_REF] Nagasaki | Previously unknown virus infects marine diatoms[END_REF] and may infer a selective growth inhibition of a subset of the microalgal community [START_REF] Castberg | Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta)[END_REF]Hewson et al., 2001b;[START_REF] Larsen | Spring phytoplnkton bloom dynamics in Norvegian coastal waters: Microbial community succession and diversity[END_REF]. Some microbial/diatom species may then benefit from the subsequent lower competition for light and nutrients.

Before the lysis of cells, the selective decline of a community or a population is probably linked to the negative effect of viral infection on photosynthetic activity, as reported before for phytoplankton [START_REF] Suttle | Inhibition of photosynthesis in phytoplankton by the submicron size fraction concentrated from seawater[END_REF]Hewson et al., 2001b;[START_REF] Juneau | Effetcs of viral infection on photosynthetic processes in the bloom-forming alga Heterosigma akashiwo[END_REF]. Indeed, both Vb and Vp infection of MPB decreased its photosynthetic efficiency (F v /F m ). Additionally, Vb infection specifically decreased and slowed down the development of the photoprotective NPQ; this is in contrast to the effect of Vp infection in the bloom-forming raphidophyte Heterosigma akashiwo [START_REF] Juneau | Effetcs of viral infection on photosynthetic processes in the bloom-forming alga Heterosigma akashiwo[END_REF]. Interestingly, light intensity and UVB radiation are important factors controlling algal host-virus interactions [START_REF] Jacquet | Ultraviolet radiation on marine virus-phytoplankton interactions[END_REF][START_REF] Baudoux | Influence of irradiance on virus-algal host interactions[END_REF]. This is even more significant since it is well documented that the diatom and MPB communities have a powerful NPQ and that in reaction to a decrease in NPQ, MPB photosynthesis and behaviour are impaired [START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. The decrease in NPQ could render the cells more sensitive to environmental stresses, i.e. high light, temperature and salinity stresses [START_REF] Juneau | Combined effect of high light and high salinity on the regulation of photosynthesis in three diatom species belonging to the main growth forms of intertidal flat inhabiting microphytobenthos[END_REF][START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. Forehand for virus-infected plant models, NPQ may be a relevant 'disease signature' to diagnose the different stages of infection, increasing locally at the early stage of viral infection and decreasing at the final stage in senescent tissue [START_REF] Pérez-Bueno | Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus[END_REF][START_REF] Pérez-Clemente | Photosynthetic and antioxydant responses of Mexican lime (Citrus aurantifolia) plants to Citrus tristeza virus infection[END_REF]. Further studies will be useful to extrapolate the virus biotic effect on NPQ to photosynthetic protists.

In this context, Vp generated the highest Viral Production (VP) together with the highest impact on MPB biomass and photosynthesis but had no effect on NPQ. Despite the reduction in photosynthetic potential, the maintenance of photoprotection may support the permissiveness of cells (or sub-sets of cells) by offering a sufficient energy level for viral replication [START_REF] Juneau | Effetcs of viral infection on photosynthetic processes in the bloom-forming alga Heterosigma akashiwo[END_REF][START_REF] Baudoux | Influence of irradiance on virus-algal host interactions[END_REF]. In contrast, the viral yield during Vb infection may be limited by the decrease in the photoprotection capacity of MPB. These observations support the hypothesis of [START_REF] Baudoux | Influence of irradiance on virus-algal host interactions[END_REF] that diatom species-specific photo-acclimation/-protection capacity (defined according to their habitat of origin, [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF] may determine the differential effect of irradiance on viral propagation by influencing the burst size and/or the latent period. Overall, efficient Vp infection of MPB questions the real susceptibility of MPB diatoms to viruses in the sediment and in the water column due to their upward sediment-water transport at high-tide (i.e. resuspension in the water column) and their downward water-sediment transports when settling.

Ecological implications

The MPB biofilm of intertidal mudflats is a product of complex interactions between microalgal primary producers, bacteria and viruses. The specific algae-prokaryotes coupling, as well as the structure of the prokaryotic community and its remineralisation activity [START_REF] Glud | Virus and bacteria dynamics of a costal sediment: Implication for benthic carbon cycling[END_REF][START_REF] Haynes | Diatom-Derives Carbohydrates as factors Affecting Bacterial Community Composition in Estuarine Sediments[END_REF]Danovaro et al., 2008b) have been related to (i) the availability of labile organic matter derived from detritus [START_REF] Galois | Spatial distribution of sediment particulate organic matter on two estuarine intertidal mudflats: a comparison between Marenns-Oléron Bay (France) and the Humber Estuary (UK)[END_REF], (ii) the cellderived EPS production [START_REF] Haynes | Diatom-Derives Carbohydrates as factors Affecting Bacterial Community Composition in Estuarine Sediments[END_REF][START_REF] Bruckner | Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria[END_REF], and (iii) the virusmediated production of DOM as cellular debris and decomposed virions [START_REF] Wilhelm | Viruses and Nutrient Cycles in the Sea[END_REF][START_REF] Sutherland | The interactions of phage and biofilms[END_REF][START_REF] Dell'anno | Virus decomposition provides an important contribution to benthic deep-sea ecosystem functioning[END_REF].

Pore-water content would be one of the main factors determining the encounter rate between viruses and hosts [START_REF] Weinbauer | Viral ecology of organic and inorganic particles in aquatic systems: avenues for further research[END_REF]. For the MOB mudflat, as in soil [START_REF] Srinivasiah | Phages across the biosphère: contrasts of viruses in soil and aquatic environments[END_REF], water content (WC) was inversely correlated with the Virus to Prokaryote Ratio while Pinto et al. (2013) reported a positive relationship from global analysis of worldwide in situ data and WC was positively related to prokaryotes abundance. Nonetheless at the emersion-scale, the VPR always varied inversely to net bacterial growth, from negative to positive values, since a net viral production occurred during emersion concomitantly to the decrease in water content and in line with the negative links between porosity and VP [START_REF] Pinto | Viriobenthos in aquatic sediments: varia bility in abundance and production and impact on the C-cycle[END_REF] and viral abundance [START_REF] Helton | Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments[END_REF]. No change of the VPR over emersion occurred when the net prokaryotic increase was around 20% of PSS. Interestingly, we observed such similar features in the dynamics of the viruses and prokaryotes, at spring tide on July 21 of 2008 (outer data on Fig. 6). This was a singular day characterized by a minimum value of Chl a biomass and a high erodibility, which may be partially explained by the destabilizing effect of a more pronounced hygroscopic feature of EPS (see for details, Orvain et al., 2014b). Therefore, the occurrence of area of water retention and the breaks of cell-matrix bonds may corollary favour in fine the bacterial growth. However taking into account the viral dynamics and the VPR allows us to also postulate that phytophages may be responsible of the observed decline of MPB on July 21 supporting indirectly the bacterial growth. To round off this item, VPR may be a good integrative proxy for the description of the functioning of the microbial food-web within a complex biofilm. It reflects both the interactions between the different microbial components (virus, prokaryotes, MPB), and their respective and interlinked relationships with water content and the bioavailability of organic matter but also its hydrophobicity (notably the protein/polysaccharide ratio of EPS).

Like in water column where viral abundance is influenced by the quality, size and age of the aggregates [START_REF] Weinbauer | Viral ecology of organic and inorganic particles in aquatic systems: avenues for further research[END_REF], it may be related, in the case of intertidal mudflat, to the maturation and the structure of MPB biofilm, which is seasonally distinguishable by differences in the bioavailability of the organic matter (as detailed in Orvain et al., 2014a). Briefly, DOM was higher in the developing biofilm of July 2008 and its composition may traduce a synergetic collaboration between highly active diatoms and prokaryotic cells in the resistance to strong irradiance and salinities whereas the algal biomass and prokaryotic abundance standing stock were less abundant than in the more stabilized biofilm. In winter 2008, diatoms excreted bound EPS carbohydrate enriched in rhamnose that can promote the biostabilization of the sediment and act as bacterial development sensor [START_REF] Pierre | Biochemical composition and changes of extracellular polysaccharides (ECPS) produced during microphytobenthic biofilm development (Marennes-Oléron, France)[END_REF]Orvain et al., 2014a). Moreover the dense population of the snail Peringia ulvae in summer may also infer seasonal differences in microbes abundance due to grazing activity (Orvain et al, 2014b) and/or vertical bioturbation of sediment (as proposed for subpolar ecosystem, [START_REF] Wróbel | Low virus to prokaryote ratios in the cold: benthic viruses and prokaryotes in a subpolar marine ecosystem (Hornsund, Svalbard)[END_REF]. Nevertheless, we can postulate that, during the ingrowing of biofilm (July), the viral production was enhanced since both microalgae and prokaryotes were metabolically active (prokaryotic P/B=3.84) without allowing, nevertheless, an efficient viral turn over. On contrary, when the MPB biofilm was better structured (February) but less active (prokaryotic P/B=3.21), the prokaryotes and the microalgae grew under steady-state conditions in phase with a lower but more efficient viral production to maintain the viral stock.

Overall, this study credited the previously report of seasonal variation of the benthic viral shunt and the estimated supplies for Prokaryotic Carbon Demand (PCD), i.e. 2 and 12% of PCD, in winter and summer respectively, considering all viruses as prokaryotic phages (to be compared to 0.1-10% of PCD; [START_REF] Pinto | Viriobenthos in aquatic sediments: varia bility in abundance and production and impact on the C-cycle[END_REF]. Therefore, the impact of viruses may appear negligible for nutrition of heterotrophic prokaryotes in surface sediment worldwide compared to deeper anoxic sediment (30%, Danovaro et al., 2008a).

Concluding remarks

Mudflat viriobenthos is a highly active component of the MPB biofilm during emersion. Viral infections play an important role in the functioning of the surficial sediment of intertidal mudflat with a seasonal variability in the viral mediated mortality of prokaryotes. However, a sizeable part of benthic viruses (and probably of pelagic viruses) originates from MPB and may regulate biomass and diversity of the benthic diatoms/microalgae. Therefore, viruses must be included to current models of the functioning of the benthos-pelagos coupled foodweb of intertidal mudflats not only as bacteriophages (Saint-Béat et al., 2014) but also as phytophages albeit the partitioning between the phages of MPB and prokaryotes remains to circumscribe, as well as the exact impact of benthic and planktonic viruses on MPB and phytoplankton biomasses.

  1, 2 and 4, Fig. S1) on 5 March 2003, 18 June 2003, 30 September 2003 and 1 February 2004. Secondly, five hourly surveys were performed during the diurnal emersion period at station 2 in 2008. Three cores were taken from each 4-m 2 quadra, randomly chosen in triplicate at each time-point within a 320 m 2 study zone on the 19 and 20 February and 360 m 2 on the 17, 18 and 19 July, few days before the spring tide on the 22 February and 21 July (for details, see Orvain et al, 2014a). Samples were also harvested for ex situ experiments (15 May 2009 and 3 May 2010).

  ; Dell'Anno et al., 2015); (ii) a possible sorption on mineral matter or embedding in the EPS matrix, limiting the movement of bacteria and viruses and/or masking the viral receptors of bacterial cells (Danovaro and Serresi, 2000; Filippini et al., 2006), although polysaccharide depolymerases on viral capsids are known to degrade the EPS matrix (Sutherland et al., 2004); (iii) a hypothetical prevalence of lysogeny or chronic multiplication (Middelboe et al., 2003; Danovaro et al., 2008b); (iv) a reduction in the probability of virus-sensitive hosts encountering due to both high viral (Hewson and Fuhrman, 2007; Helton and Wommack, 2009) and bacterial diversities (Torsvik et al., 2002); and (v) a direct influx of viruses from the water column which settle, or indirectly as a result of the settlement of lysogenic prokaryotes and/or cells visibly infected by lytic viruses (Hewson and Fuhrman, 2003; Taylor et al., 2003; Danovaro et al., 2008b; Pradeep Ram et al., 2009).

Figure 1 :

 1 Figure 1 : Comparative test for viral extraction from muddy-sediment by shaking or sonication. Reports of three assays performed in triplicate and analysed by microscopy and cytometry (n=3, mean ± SE). ns= non significant, *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 2 :

 2 Figure 2 : Efficiency of the first extraction step of viruses (out of two) performed using rocking shaker and based on microscopic counts (n=3, mean ± SE). Note that in July 2011, samples have been enumerated by microscopy and cytometry.

Figure 3 :

 3 Figure 3 : Time-point persistence of viruses over 15 days during the preservation of sediment samples at 4°C, -20°C and -80°C. A: comparison of the persistent viruses at T1d, T8d and T15d to the initial count (T0) based on counting by cytometry, *: p<0.05, **: p<0.01, ***: p<0.001. B, C, D: details of the efficiency of the two extraction-steps as numbers of viruses into the first and the second supernatants (S1, S2). (n=3, mean ± SE).

Figure 4 :

 4 Figure 4 : Seasonal survey of viriobenthos at the diurnal emersion period of the Marennes-Oléron mudflat in 2003-2004: A: abundance along a 4 km cross-shore transect (stations 1 to 4; see Fig. S1); mean ± SE. B: Virus distribution by capsid class sizes; mean of the three stations surveyed. Only significant spatial heterogeneity between the three stations (two ways ANOVA) were marked as * : p< 0.05, ***: p < 0.001.

Figure 5 :

 5 Figure 5 : Hourly survey of viriobenthos (A) and prokaryotes (B) during the diurnal emersion period in February and July 2008. Abundance at the beginning of the emersion and 3 hours later at station 2 of the transect (see Fig. S1); mean of triplicate samples from three randomly chosen quadras ± SE.

Figure 6 :

 6 Figure 6 : In situ Virus to Prokaryote ratio (VPR) in the top-surface sediment: (A) Log/log relationship between VPR and the water content of the sediment, established with data averaged from triplicate samples taken in February and July 2008, May 2009 and May 2010 (B) Relationship between the variation of VPR during the 3 hours of emersion and the net prokaryotic production (% of prokaryote stock standing) in February and July 2008. With ♦: outer data of 21 July 2008, the relationship would be: Y= -1.38X+ 28.32 (r 2 =0.66; p=0.05

Acknowledgments

This work was partly supported by the French National Research Program PEVS (2000)(2001)(2002)(2003)(2004)(2005)(2006) and is part of the VASIREMI project (2007-2011) funded by the French National Research Agency (ANR-06-BLAN-0393-01). HM designed the different bioassays, analysed and conceptualised results. JL performed and analysed the photosynthetic measurements on microalgae, he was supported by the CNRS and the French national consortium CPER 'Littoral' (2007-2013), including European FEDER funds. MdC, supported by a Ph.D. fellowship from the French National Institute for Scientific Research (CNRS) and the 'Poitou-Charentes' Region, was in charge of sediment sampling and counts of prokaryotes in February and July 2008. We thank Christine Dupuy for her coordination of the VASIREMI program, M. Bréret for chlorophyll a and DOM analysis, and C. Fontaine and six undergraduate students (A. Tchechenko, G. Chereau, E. Goutanier, J. Meilland, P. Pelard and J. Ezzedine) for their help in microbial counting. This paper is a contribution to the 'biofilm' work package of the LIENSs research unit.

Table 1: Environmental data and Virus to Prokaryotes ratio within the 1cm-top surface sediment. Mean ± SD. * per g of dry sediment.


Vincent Repellin
email: repellin@emse.fr

René Guyonnet
email: guyonnet@emse.fr

Evaluation of heat treated beech by non destructive testing

Keywords:

Improvement of dimensional stability and durability is wished for the use of wood as a building material. For the last decade, retification® has been industrially developed. It consists in a stabilization and preservation of wood by heat treatment.

The aim of this study is to find simple and fast methods to characterize heat treated beech. Non destructive testing is expected to be relevant to evaluate the level of treatment and the properties for the use of heat treated wood.

Six treatments were carried out in a pilot reactor. The parameters of the retification® stage (temperature and time) were studied.

For each treatment, the non destructive tests (free oscillations in the fundamental mode, colour and dry weight loss)were performed, and the properties for use (mechanical resistance and volumetric shrinkage) measured.

Lightness and dry weight loss seem to be suitable properties to characterize beech retification® when the time parameter is fixed. However, they are not suitable for other wood species, and for retification stages with a variable duration. Moreover, the correlation with the properties for use were plotted, but presented too large dispersion to be relevant.

After correction of moisture content, the longitudinal Young's modulus of the material is slightly increased by each of the six treatments, but do not present any variation with changing parameters values. On the contrary, the mechanical resistance decreased with increasing temperature and time. Thus the dynamic Young's modulus is not reliable to evaluate the treatment and to predict the loss of mechanical resistance.

The logarithmic decrement was not increased by any of the treatments, which is in opposition with the hypothesis that retification® generates cracks and microcracks in the material.

Effects of long time at low temperature have been investigated. From these experiments, properties of treated wood may be improved significantly by choosing appropriate values of the parameters.

INTRODUCTION

Improvement of dimensional stability and durability is wished for the use of wood as a building material. For the last decade, stabilization and preservation of wood by heat treatment have been industrially developed. Moreover, these heat treatments are more environmentally friendly than the chemical ones. Retification® is one of these heat treatments that stabilizes wood and improves its durability. However, their main drawback is the loss of mechanical resistance of the material that happens at high temperature.

Some tests involving rather complicated physical or chemical techniques have been developed for the evaluation of the treatment. The first aim of this study is to find simple and fast methods that allow to characterize the heat treatment of wood. Non destructive testing could easily reach these industrial requirements. But the considered properties have to vary significantly with the parameters of the retification stage (temperature and time), in spite of wood heterogeneity.

Six treatments have been carried out in a pilot reactor. The conduct was similar for all the treatments. The changing parameters were temperature (5 minutes at 200°C, 220°C, 240°C, and 260°C)) and time (600 minutes at 200°C and 60 minutes at 220°C).

Properties related to the use of wood, such as the modulus of rupture(MOR) and the volumetric shrinkage have been measured.

Three types of non destructive tests have been done. The measurement of CIE L* a* b* colour and dry weight loss were performed. Nevertheless, the efforts were focused on the free-free flexural vibrations test. Dynamic longitudinal Young's modulus (MOE) and logarithmic decrement were measured. These non destructive tests have been carried out before and after Retification®.

MATERIALS PREPARATION

The purpose of this work was to investigate the influence of the two main parameters of retification® on the material : temperature and time of exposure. Sis treatments were carried out in a pilot kiln, at the "Ecole Des Mines De Saint Etienne". This pilot kiln regulates by PID on the temperature of its atmosphere. The specimens were 850×85×25 millimetres beech beams, carefully cut in the longitudinal direction. Before heat treatment the specimens had been hold in a climatic chamber at 65% relative humidity 20°C. The moisture content (MC) of the specimens was 12%. Each batch was composed of sixteen specimens. The kiln is equipped with eight thermocouples that allow to follow the temperature reached by the wood specimens.

Table 1 summarises the treatments parameters. The atmosphere was composed of nitrogen gas. The increase in temperature matched the standard one, used for the retification® process [START_REF] Weiland | Etude physico-chimique du traitement thermique du bois. Optimisation des paramètres du procédé de rétification®[END_REF].

Four treatments were performed with a fixed time of exposure of 5 minutes. As the wood transformation is known to begin around 200°C, we chose the following temperatures : 200°C, 220°C, 240°C and 260°C. In order to investigate the effect of long time exposure at low temperature of retification®, two treatments were carried out, at 200°C during 600 min and at 220°C during 60 min. For each treatment, non destructive tests (size, weight, colour and free vibrations) were performed before and after the treatment. After treatment, the specimens had been held until equilibrium in a climatic chamber at 65% relative humidity 20°C before being tested.

The physical properties (volumetric shrinkage, MOR, and MC) of untreated and treated wood were measured by destructive methods. For each measurement, 48 samples have been cut and tested.

EXPERIMENTAL PROCEDURE

Evaluation of properties for use

There are three main properties for the use of wood : mechanical resistance, volumetric shrinkage, and fungus resistance. In this work, we focused on the physical properties : mechanical resistance (evaluated by static bending) and volumetric shrinkage.

Mechanical resistance

The bending strength of treated wood was measured using a four points bending device. 48 beams have been tested for each batch. The measurements were done following the French normative NF B 51-008. Normalised beams of dimension 20*20*360 mm underwent a force applied on the (LR) plane.

Volumetric shrinkage

This test is carried out on cubic samples of around 2cm edge size. The volume of a cube is measured in the water saturated state and in the anhydrous state. The volumetric shrinkage is then calculated by the formula :

S 0 S V V V 100 S - × = ( 1
)
where S is the volumetric shrinkage, V S is the volume of the water saturated sample, V 0 is the volume of the dried sample.

Evaluation by non destructive testing

Non destructive evaluations can be done before and after the treatment. Consequently, they allow to get partly rid of the natural dispersion of wood, and to measure only the modifications due to the heat treatment.

Colour

The colour measurements were carried out using a MINOLTA Spectrophotometer CM-508i. The principle of colour measurement is inspired by the human vision. One colour is repaired in a three coordinate space. Thus, the device records three coordinates : L* (also called lightness), a*, and b*. They match the three following pairs of colour : from white (L*=100) to black (L*=0), from red (a=+60) to green (a*=-60) and from yellow (b*=+60) to blue (b*=-60). Thirty points have been recorded on each specimen, before and after the heat treatment.

Free oscillations

The vibrational properties were evaluated in the fundamental mode of free-free flexural vibrations. The impulse was given on the flat face of the specimens. The support were placed at the nodal location of the first vibration mode. A piezoelectric captor recorded the vibration of the sample at the end of the beam (Fig. 1). Since the length of the specimens was ten times superior to their thickness, we could do the computation of the dynamic Young's modulus following the elementary Euler-Bernouilli's theory. Two quantities could be measured : dynamic MOE and logarithmic decrement. The dynamic Young's modulus can be calculated from the following Eq. 2. It is linked to the stiffness of the material measured by static bending [START_REF] Pellerin | A vibrational approach to non destructive testing of structural lumber[END_REF]).

2 i 3 3 4 2 f ba ML ) kL ( 48 E π = (2)
Where E is the dynamic modulus of elasticity in the longitudinal direction, M is the beam weight, L its length, a its thickness, and b its width. f i is the natural frequency of vibration in the i th mode, and kL is the solution of Eq. 3 matching the i th mode of vibration. This Eq. 3 is worth in the case of free-free vibrations. 1 -cos(kL)ch(kL) = 0

(3)

Since we are working in the first mode we have :

2 1 3 3 f ba ML 9464 , 0 E = (4) or : 2 1 2 4 f a L 9464 , 0 ρ E = (5)
where E/ρ ρ ρ ρ is the specific dynamic MOE and ρ ρ ρ ρ is the specific gravity of the specimen. As the specimens were cut in the longitudinal direction, we measured the Young's modulus in the longitudinal direction.

The logarithmic decrement gives an evaluation of the internal friction of the material. It is linked to the volumetric viscous damping force, which depends on the presence of defects, of microcracks and on the MC. From the experimental point of view, we measure the amplitude of the N th oscillation, and the amplitude of the (N+n) th oscillation and calculate the logarithmic decrement following Eq. 6 :

        = δ +n N max N max A A ln n 1 (6)
Since the MOE and the logarithmic decrement should depend on the MC of the specimens, we fixed the correction relationships. In this purpose, four specimens of untreated beam were conditioned in a climatic chamber at different relative humidity. Their moisture content varied from 0 to 20% and we plot the logarithmic decrement and the specific dynamic MOE versus their MC. For the specific dynamic MOE, linear regression gave an average slope of -0.255 GPa per percent of water. For the logarithmic decrement the linear regression gave an average slope of 9 10 -4 per percent of water, but the data scattering was very large.

RESULTS AND DISCUSSION

Properties for use

Table 2 and Fig. 3 present the results of the tests.

The volumetric shrinkage decreases monotonically with increasing temperature and time of treatment. Indeed, the main effect of retification® is the destruction of the hemicelluloses [START_REF] Weiland | Etude physico-chimique du traitement thermique du bois. Optimisation des paramètres du procédé de rétification®[END_REF], which are the most hydrophilic constituents of wood. Consequently, the wood is less sensitive to moisture. The mechanical resistance decreases from 240°C. This decay matches a degradation of the material. Since the modulus of elasticity is also a mechanical property, we expect that it should vary similarly than the MOR.

Increasing time of treatment result also in decrease of the volumetric shrinkage and the MOR.

Effect of temperature of treatment

Effect of the temperature on different properties

The aim of this work is to find out properties easy to measure and that vary significantly with the level of treatment. For this purpose, a review of different properties of treated wood is presented in table 3. The colour (through the lightness L*) and the dry weight loss are the most influenced properties. They are already known to be relevant for the control of heat treatment of wood.

Concerning the four treatment of five minutes, they both vary accurately with the temperature actually reached by the specimens (Fig. 2).

Variation of ligthness and dryweight loss vs temperature (°C) y = 0,0006e 0,0377x R 2 = 0,9665 y = -0,3321x + 123,56 R 2 = 0,9679 Consequently, once the time of exposure is fixed, dry weight loss and lightness can be used to evaluate the retification® temperature. However, they can not be used to evaluate both temperature and time of retification®. Indeed, treatments 5 and 6 result approximately in the same lightness and in the same dry weight loss as treatment 3, but they involve longer time.

Nevertheless, the dry weight loss should be used carefully. In this work it varies well with temperature, because beech are broad lived tree and do not contain resin. The weight loss of softwood is known to be unreliable as it depends on their resin content. The use of colour also may not be relevant with all species of tree. Indeed, beech wood has a very homogeneous colour, which is not the case for all wood species. Moreover, correlation between the properties for use and lightness and dry weight loss were plotted, but presented too large dispersion to qualify the retification®. That is why, we proposed to evaluate the relevance of the free oscillations method. There are two observations concerning the specific dynamic Young's modulus (Table 4):

Vibrational properties

-it increases from natural to treated state, -there is no obvious variation following the parameters.

To conclude, it is not possible to evaluate the treatment with this property.

The logarithmic decrement decreases with increasing temperature of treatment from 200°C to 240°C. But the scattering of the data is so large that it is again impossible to qualify the heat treatment with this property. By destruction of the hemicellulose, heat treatment makes the wood loose its affinity for water. As a consequence, viscous damping is reduced and the logarithmic decrement decreases. Moreover, the stress strain curves of the high temperature batch (treatment 3 and 4) have the shape of brittle material. The heat treated wood behaves more like brittle material progressively with increasing values of the parameters.

During retification®, two phenomenon may influence the vibrational properties : loss of MC (that increase the MOE and decrease the logarithmic decrement), and lignocellulosic material modification. In order to investigate the influence of the material modification on the vibrational properties alone, it was necessary to calculate and to eliminate the influence of the MC.

Correction of the vibrational properties with MC

We know that the equilibrium MC of the treated wood decreases with increasing temperature and time of treatment (Table 3). Consequently, we had to do the correction of the vibrational properties values according to the variation in moisture content. Linear regression of the specific dynamic MOE of untreated wood vs. MC between 0% and 20% has been plot. For each batch, one could then compute the specific dynamic MOE of untreated wood at the same moisture content as the equilibrium moisture content of the treated wood ((Ed/ρ)untreatedcor).

The ratio ((Ed/ρ)treated)/((Ed/ρ)untreatedcor) so calculated (Table 5) allow to compare the natural and treated wood at the same moisture content.

Once the correction is carried out, one observe that MOE of wood is slightly enhanced by each of the treatments. We can conclude from this, that the modification of material result in an increase of the Young's modulus. Phenomenon involved in this modification are discussed. As well as before correction, the scattering of the increase is large, which forbids to make a difference between treatments of different parameters.

The same correction in MC was done for the logarithmic decrement. Considering the values obtained and the scattering of the data, one couldn't notice obvious difference between the decrement of anhydrous wood and the decrement of treated wood. We can conclude from this, that the measurement of the vibrational properties in the fundamental mode is not relevant to make an evaluation of the retification® of beech beams.

Mechanical and vibrational properties

Another point concerns the mechanical resistance of the treated wood. The bending strength decreases significantly from 240°C (Table 4). During the heat treatment, physical and chemical transformations of the material lead to a loss of mechanical resistance. In the spite of this, the specific Young's modulus is not modified from 200°C until 260°C. Thus, the material degradation causes reduction of mechanical resistance, but no variation of the modulus of elasticity.

The longitudinal MOE of a beam depend strongly to the microfibril angle of the S2 layer, and depend less strongly on the crystallinity of cellulose and stiffness of the amorphous matrix [START_REF] Ono | Study on Young's Modulus and internal friction of wood in relation to the evaluation of wood for musical instruments[END_REF]. These three elements of the wood structure could be slightly influenced by the heat treatment and result in this slight increase of the young's modulus.

The mechanical strength of wood is more linked to the presence of cracks, and their opportunity of initiation and propagation. Thus, the loss of mechanical resistance may be attributed to a higher number of cracks and microcracks in the treated material, or/and to a decrease of the energy necessary for a crack to initiate and propagate in the material.

With microscopic investigation of the microstructure, no obvious damage is visible on heat treated wood [START_REF] Avat | Contribution à l'étude des traitements thermiques du bois jusqu'à 300°C : transformations chimiques et caractérisation physico-chimique[END_REF]. Moreover, since the logarithmic decrement is decreased by heat treatment (or at least not increased once the moisture correction is done), the hypothesis of a higher number of microcracks in the treated beech should be rejected. Indeed, the presence of more microcracks should lead to a higher internal friction, and thus to an increase of the logarithmic decrement.

As a conclusion, more investigation is necessary to find out the influence of the retification® on the structure and microstructure of the wood, and to do the link with the macroscopic properties as mechanical resistance and vibrational properties.

Effect of long time treatment

The last point of the study was the effect of long time treatment. Treatments number 5 and 6 were performed (Fig. 3) and (Table 2). One of the benefit of long time treatment at low temperature is that the conduct of the process is easier. Indeed, no exothermic effect is expected and the temperature homogeneity is better in the pilot kiln. Moreover, a lot of reaction is involved during retification® (Bohnke 1993[START_REF] Weiland | Etude physico-chimique du traitement thermique du bois. Optimisation des paramètres du procédé de rétification®[END_REF]. By choosing low temperature and long time treatment, one intend to improve reactions that decrease volumetric shrinkage, and to slow reactions that decrease the MOR.

Treatment 5 and 6 result both in a decrease of the volumetric shrinkage and in a loss of the mechanical resistance. It seems to be difficult to get reduction of volumetric shrinkage without reduction of the bending strength. In the spite of this general observation, there is no evidence that these two effects are caused by the same reactions ; and we may find an optimum of the parameters values, where the mechanical loss would be reduced.

Concerning the volumetric shrinkage, a one hour retification stage at 220°C (batch number 5) allowed to reach the same level of stabilisation as a five minutes treatment at 240°C (batch number 3). The mechanical resistance seem to be slightly less reduced, but the dispersion of MOR is large. Nevertheless, the difference on average MOR between number 3 and 5 is statistically significant at risk 2%.

Since this difference is statistically significant, we expect that an optimum of the parameters exists to get the best compromise for the use properties. Moreover, comparing treatment 6 and 3, the volumetric shrinkage is lower for treatment 6 with approximately the same value of MOR.

According to these observations, reactions that improve the reduction of volumetric shrinkage should prevail at low temperature (until 220°C) with regard to reactions that cause mechanical degradation. Though, more investigation is necessary to find out what reaction occurs at a given temperature.

CONCLUSION

Six treatments have been carried out in a pilot reactor. The parameters of the retification® stage ( temperature and time) have been studied.

For each treatment, the non destructive tests (free oscillations in the fundamental mode, colour and dry weight loss) were performed, and the properties for use (mechanical resistance and volumetric shrinkage) measured.

Lightness and dry weight loss seem to be suitable properties to characterize beech retification® when the time parameter is fixed. However, they are not suitable for other wood species, and for retification stages with a variable duration. Moreover, the correlation with the properties for use were plotted, but presented too large dispersion to be relevant.

After correction of moisture content, the longitudinal Young's modulus of the material is slightly increased by each of the six treatments, but do not present any variation with changing parameters values. On the contrary, the mechanical resistance decreased with increasing temperature and time. Thus the dynamic Young's modulus is not reliable to evaluate the treatment and to predict the loss of mechanical resistance.

The logarithmic decrement presented a large dispersion, which does not allow to qualify retification® with this property. After moisture correction, it was not increased by any of the treatments. This observation is in opposition with the hypothesis that retification® generates cracks and microcracks in the material.

Effects of long time at low temperature have been investigated. From these experiments, it results that significant improvement of the wood properties may be expected by choosing appropriate values of the parameters.

More investigation is required to find out what reaction prevails according to the temperature, and to understand better the mechanisms that cause the loss of mechanical resistance.

  figure 1: scheme of the device used to measure free oscillations.

Figure 2 :

 2 Figure 2: dry weight loss and lightness plot against the temperature reached by each beam of treatment 1, 2,3 and4.

Figure 3 :

 3 Figure 3 : properties for use of retified® beech.

Table 1 : Experimental parameters of the treatments

 1

	Treatment number	Temperature [°C]	Time [min]
	1	200	5
	2	220	5
	3	240	5
	4	260	5
	5	220	60
	6	200	600

Table 2 : Properties for use of treated and untreated beech Treatment number

 2

		Volumetric	MOR
		shrinkage S	[Mpa]
		[%]
	Untreated	15.9 (1.4)	94.8 (15.8)
	1	15.0 (1.4)	100.4 (24.9)
	2	14.0 (1.0)	97.9 (21.1)
	3	11.7 (1.8)	74.3 (19.3)
	4	8.3 (1.2)	59.6 (21.0)
	5	11.6 (1.2)	84.6 (18.7)
	6	10.6 (1.5)	74.0 (25.6)

Table 3 : Results of non destructive tests Treatment number

 3

		Dry weight	Equilibrium	Colour :	Colour :	Colour :
		loss ∆ ∆ ∆ ∆ms	MC	lightness L*	a*	b*
		[%]	[%]
	untreated	-	12.0 (0.3)	70.7 (3.5)	7.7 (0.7)	18.6 (1.2)
	1	1.3 (0.3)	7.0 (0.8)	56.5 (2.2)	7.4 (0.6)	17.3 (0.6)
	2	2.7 (0.7)	5.3 (0.5)	49.6 (2.9)	8.1 (0.4)	17.2 (0.9)
	3	6.5 (1.7)	4.1 (0.7)	41.9 (2.9)	7.8 (0.5)	14.5 (1.4)
	4	12.8 (1.9)	3.7 (0.3)	36.2 (2.1)	6.3 (0.7)	11.3 (1.6)
	5	6.9 (1.6)	4.2 (0.5)	42.6 (2.3)	7.5 (0.3)	14.6 (1.2)
	6	7.8 (0.8)	4.9 (0.5)	41.7 (1.9)	8.5 (0.5)	16.0 (1.2)

Table 4 : Vibrational properties of heat treated beech

 4

	Treatment number	Ratio : ((Ed/ρ) ρ) ρ) ρ)treated )/ ((Ed/ρ) ρ) ρ) ρ)untreated )	Ratio : δtreated )/ (δ δ δ (δ δ δ δuntreated )
	Natural	1 (0)	1 (0)
	1	1.14 (0.06)	0.98 (0.10)
	2	1.13 (0.07)	0.86 (0.07)
	3	1.16 (0.02)	0.76 (0.09)
	4	1.14 (0.02)	0.80 (0.07)
	5	1.14 (0.02)	0.64 (0.09)
	6	1.13 (0.02)	0.85 (0.08)

Table 5 : corrected vibrational properties of heat treated beech

 5

	Treatment number	Ratio : ((Ed/ρ) ρ) ρ) ρ)treated )/ ((Ed/ρ) ρ) ρ) ρ)untreated cor)	Ratio : (δ δ δ δtreated )/ (δ δ δ δuntreated cor)
	1	1.07 (0.06)	1.12 (0.13)
	2	1.04 (0.02)	1.00 (0.10)
	3	1.04 (0.02)	0.90 (0.12)
	4	1.02 (0.01)	1.00 (0.10)
	5	1.04 (0.02)	0.77 (0.12)
	6	1.04 (0.01)	1.03 (0.11)


Guillaume Mandil

Thao Van

Henri Le

Mathieu Paris

Suard

Building new entities from existing titanium part by electron beam melting: microstructures and mechanical properties

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