M Abboudi

W H Jeffrey

J.-F Ghiglione

Mireille Pujo-Pay

L Oriol

Richard Sempere

B Charrière

F Joux

R Sempéré

F Charrière

Joux

Effects of Photochemical Transformations of Dissolved Organic Matter on Bacterial Metabolism and Diversity in Three Contrasting Coastal Sites in the Northwestern Mediterranean Sea during Summer

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Introduction

Bacteria are key organisms in carbon cycling in aquatic ecosystems, acting as a sink (mineralization of dissolved organic carbon [DOC] to CO 2 ) or as a link (production of biomass that can be transferred through the microbial food web) [START_REF] Cotner | Small players, large role: microbial influence on biogeochemical processes in pelagic aquatic ecosystems[END_REF]. Microbial metabolism and diversity are mainly driven by the concentration and quality/composition of dissolved organic matter (DOM) and dissolved inorganic nutrients (e.g., [START_REF] Eiler | Heterotrophic bacterial growth efficiency and community structure at different natural organic concentrations[END_REF]). Natural solar radiation, especially ultraviolet radiation (UV-B [280-315 nm], UV-A [315-400 nm]), has been found to induce chemical transformations of DOM with the production of a variety of photoproducts, including carbon dioxide, carbon monoxide, ammonium, phosphate, and numerous LMW organic compounds (for a review, see [30]). The origin and chemical composition of DOM strongly influences its photoreactivity. The light-absorbing fraction of DOM, chromophoric dissolved organic matter (CDOM), from both terrestrial and autochthonous origins, is the primary absorber of sunlight in aquatic ecosystems and plays an important role for most photochemically mediated processes in surface waters [30]. These phototransformations are likely to affect bacterial production (BP) by changing the quantity and bacterial availability of DOM (for a review, see [START_REF] Kieber | Photochemical production of biological substrates[END_REF]). Contrasting results have been observed depending on the origin of DOM, and the observed effect of the phototransformations of DOM on bacterial growth is probably the net result of two opposite processes: photochemical increase in the lability of initially refractory substances and photochemical decrease in the lability of freshly produced algal carbon [START_REF] Obernosterer | Competition between biological and photochemical processes in the mineralization of dissolved organic carbon[END_REF][START_REF] Obernosterer | Contrasting effects of solar radiation on dissolved organic matter and its bioavailability to marine bacterioplankton[END_REF][START_REF] Tranvik | Contrasting effects of solar UV radiation on dissolved organic sources for bacterial growth[END_REF].

Most previous investigations have used bacterial biomass changes and/or bacterial protein (or DNA) production as criteria to evaluate the effects of photransformations on the availability of DOM [30]. BP, however, represents only a part of the DOC used by bacteria, the other part being consumed by respiration. Bacterial growth efficiency (BGE) is estimated by comparing gross with net production, where gross production is measured as BP plus respiration or DOM utilization [START_REF] Giorgio | Bacterial energetics and growth efficiency[END_REF]. Recent studies have demonstrated that phototransformations of DOM affected the BGE in different aquatic ecosystems by a change in anabolic and catabolic pathways [START_REF] Amado | Complementary pathways of dissolved organic carbon removal pathways in clear-water Amazonian ecosystems: photochemical degradation and bacterial uptake[END_REF][START_REF] Anesio | Effect of humic substance photodegradation on bacterial growth and respiration in lake water[END_REF][START_REF] Mccallister | Effects of sunlight on decomposition of estuarine dissolved organic C, N and P and bacterial metabolism[END_REF][START_REF] Pullin | Effects of sunlight and hydroxyl radical on dissolved organic matter: bacterial growth efficiency and production of carboxyclic acids and other substances[END_REF][START_REF] Smith | Photochemical transformations of riverine dissolved organic matter: effects on estuarine bacterial metabolism and nutrient demand[END_REF]. One question not investigated in previous studies is the extent that the changes in bacterial metabolism may be accompanied by a shift in bacterial community structure. Shifts in the source (terrestrial versus phytoplankton) and lability of DOM can result in shifts in bacterioplankton activity and ultimately in shifts in bacterioplankton diversity. Such effects have been observed during different temporal scales including seasonally [START_REF] Ghiglione | Spatial and temporal scales of variation in bacterioplankton community structure in the NW Mediterranean Sea[END_REF][START_REF] Schauer | Seasonal changes in the taxonomic composition of bacterioplankton in a coastal oligotrophic system[END_REF] and short-term events such as phytoplanktonic blooms [START_REF] Castberg | Microbial population dynamics and diversity during a bloom of marine coccolithophorid Emiliania huxleyi (Haptophyta)[END_REF][START_REF] Fandino | Variations in bacterial community structure during a dinoflagellate bloom analyzed by DGGE and 16S rDNA sequencing[END_REF]. Thus, it might be reasonable to assume that photochemical transformation of DOM taking place during exposure to solar radiation may result in a shift in the composition of the bacterioplankton community structure and/or activity.

The importance of photochemical transformation in aquatic ecosystems is dependent on a combination of CDOM concentrations and intensity of sunlight. Coastal lagoons are semi-enclosed systems with both marine and fluvial components. Depending on the importance of these two influences, salinity, nutrients and CDOM concentra-tions, optical characteristics, and productivity of the lagoons may vary considerably [START_REF] Conde | Changes in UV penetration associated with marine intrusions and freshwater discharge in a shallow coastal lagoon of the Southern Atlantic Ocean[END_REF]. The proportion of autochthonous CDOM (e.g., derived from algae) versus terrestrial CDOM also fluctuates with river discharges and exchange with the open sea [START_REF] Conde | Changes in UV penetration associated with marine intrusions and freshwater discharge in a shallow coastal lagoon of the Southern Atlantic Ocean[END_REF]. The Mediterranean coast of France is bordered by a series of coastal lagoons, and some of them are associated with important economic activities (shellfish farming, tourism) and high anthropogenic pressures (nutrient loadings, pollutants). These waters contrast with the surrounding oligotrophic coastal waters in the Mediterranean Sea. Moreover, these areas are subjected to significant surface UV irradiances during spring and summer seasons (up to 43.5 KJ m 2 per day in UV-B range; F. Joux, unpublished data).

The aim of this study was to investigate the effects of natural solar radiation in summer on DOM originating from two lagoons and coastal waters in the Northwestern Mediterranean Sea in terms of changes in (1) chemical and optical characteristics of the waters, (2) bacterial metabolism, and (3) diversity of bacteria growing on irradiated DOM.

Methods

Study Sites and Sample Collection

Water samples were collected in June 2005 from two lagoons (Canet and Leucate) and one coastal station (SOLA station) of the northwestern Mediterranean Sea (Fig. 1). Canet Lagoon covers 6 km 2 with a mean depth of 0.2 to 0.5 m. The basin of the system is drained by three rivers and extends over 260 km 2 . The lagoon is separated from the sea by a sand bar that opens artificially through a single channel. Leucate Lagoon covers 54 km 2 with a mean depth of 2 m and is heavily used for shellfish farming (oysters and mussels). There is no river input, and the lagoon connects to the sea in three places. SOLA is a shallow (maximum depth, ∼26 m) station located ∼500 m offshore of Banyulssur-Mer, France (42°29′N, 03°08′E). Surface waters were sampled by submerging an acid-washed polycarboante bottle by hand ∼30 cm below the surface and with a Niskin bottle at 5-m depth for the lagoons and the coastal station, respectively. Samples were then transferred into 20-L acidwashed polycarbonate Nalgene carboys and transported to the laboratory. Subsamples were collected for determination of salinity (Guideline 8410 portable salinometer, accuracy ±0.01), chlorophyll a and bacterial abundance.

Sample Preparation and Irradiation of DOM

Samples were prefiltered onto precombusted (450°C, 24 h) 142-mm GF/A filters (1.6-μm size particle retention, Whatman) and then filtered through a prerinsed 0.2-μm pore-size Nuclepore polycarbonate membrane (142 mm). Filtered samples were stored at 4°C in the dark for no more 15 h before irradiation. Filtered seawater was then distributed into three experimental treatments chosen to alter the spectral characteristics of solar radiation. Dark and photosynthetically active radiation (PAR) treatments were established by placing filtered seawater in four 1-L Pyrex tubes (7-cm diameter) covered with aluminum foil and Courtgard film (50% cut-off at 400 nm), respectively. Full sun treatment was established by placing filtered seawater in four 1-L quartz tubes (7-cm diameter). All glassware was precombusted (450°C, 24 h) before use. The different tubes and bottles were placed in a recirculating water bath (0.1-m depth) maintained at in situ temperature (19±1°C) and were exposed to natural solar radiation during a sunny day 8 h. Incident irradiance was measured in the PAR (400-700 nm), UV-A (315-400 nm), and UV-B (280-315 nm) using a broad band ELDONET radiometer (Real Time Computer, Inc., Ger.). Over the course of the incubation, PAR, UV-A, and UV-B doses were 11,000, 1700 and 37 KJ m -2 , respectively. Before and after sunlight exposure, subsamples were collected for DOC, dissolved organic nitrogen (DON), and dissolved organic phosphorus (DOP), nutrients (NH þ 4 , NO À 3 , NO À 2 , PO 3À 4 ) and DOM optical properties. NH þ 4 , DON, and DOP were measured only before irradiation. The remaining water was used as media for biodegradation experiments (see below).

Experimental Set-up for Biodegradation

Quartz and Pyrex tubes containing filtered water from both irradiated (full sun and PAR exposures) and dark treatments were inoculated (10% final volume) with a bacterial community from the same origin (un-irradiated 0.8-μm filtered water). A portion of the sample water mixture from each light treatment was then dispensed into six 100-mL biological oxygen demand (BOD) bottles. All BOD bottles were washed with 10% HCl and rinsed with Milli-Q water before use. The BOD bottles were allowed to overflow ∼3 times the volume of the bottle, ensuring that no bubbles remained in the bottles, and capped free of headspace with ground glass stoppers. The remaining fraction of the water (∼2-L for each light treatment) was then transferred into a 3-L glass bottle. The BOD bottles and the glass bottles were incubated in the dark at 18±1°C in a water bath (to prevent gas exchange) and in an incubation chamber, respectively. At time zero and then at 39 h, three BOD bottles from each replicate incubation were fixed with Winkler reagents for the determination of O 2 concentration. Subsamples were drawn from each glass bottle at time zero and then at different intervals up to ∼120 h for the determination of bacterial abundance, production, and community structure.

Analytical Methods

Chlorophyll a (Chl a) concentrations were obtained from water samples (50-200 mL in duplicate) filtered through 47-mm GF/F filters (Whatman) and frozen in liquid nitrogen until analysis. Pigments were extracted in 90% acetone for 12 h in the dark at 4°C, and the concentration of Chl a was measured by fluorometry using a Turner-Design 10-AU [START_REF] Lorenzen | Determination of chlorophyll and pheopigments: spectrometric equations[END_REF].

Sixty-milliliter samples for nitrate NO manual method [START_REF] Koroleff | Direct determination of ammonia in natural waters as indophenol blue[END_REF] with a spectrophotometer (Spectronic 401). Measurement accuracy was ±0.05 μM. Twenty-milliliter duplicate samples for DOC were filtered through two precombusted (450°C, 24 h) 25-mm GF/F filters, transferred into precombusted glass tubes, poisoned with 85% H 3 PO 4 (final pH=2), closed with Teflon lined screw caps, and were stored in the dark at room temperature until analysis. DOC was analyzed using the high temperature catalytic oxidation (HTCO) technique [START_REF] Cauwet | HTCO method for dissolved organic carbon analysis in seawater: influence of catalyst on blank estimation[END_REF][START_REF] Sugimura | A high-temperature catalytic oxidation method for the determination of non-volatile dissolved organic carbon in seawater by direct injection of a liquid sample[END_REF] using a Shimadzu TOC-V analyzer. Before starting analyses and between sets of samples, an international certified reference sample for DOC concentration (available for the international community) was analyzed to check the calibration of the analyzer and its stability over time. Analytical accuracy of measurements gave a coefficient of variation (CV) of less than 2%.

Samples for DON and DOP were filtered through two precombusted (450°C, 24 h) 25-mm GF/F filters (Whatman). Samples were collected directly in Teflon bottles and immediately frozen (-20°C) and stored for later analyses. DON and DOP were simultaneously determined by the wet oxidation procedure [START_REF] Pujo-Pay | Excretion of dissolved organic nitrogen by phytoplankton assessed by wet oxidation and 15 N tracer procedures[END_REF][START_REF] Pujo-Pay | Improvement of the wet oxidation procedure for simultaneous determination of particulate organic nitrogen and phosphorus collected on filters[END_REF]. DON (±0.1 μM) and DOP (±0.02 μM) concentrations were determined by sample oxidation (30 min, 120°C) corrected for NH þ 4 , NO À 3 þ NO À 2 , and PO 3À 4 concentrations, respectively. DOM fluorescence was determined with a PerkinElmer LS55 spectrofluorometer using a 1-cm quartz cuvette (excitation wavelength, 350 nm; emission wavelength, 450 nm) standardized with a quinine sulfate solution (1 QSU=1 ppb quinine sulfate in 0.05 M H 2 SO 4 ) [START_REF] Obernosterer | Contrasting effects of solar radiation on dissolved organic matter and its bioavailability to marine bacterioplankton[END_REF]. DOM absorption at 350-nm wavelength was determined with a Hitachi U-3010 spectrophotometer using a 10-cm cuvette. Absorbance was measured against Milli-Q water as blank. Absorption coefficients a 350 (m -1 ) were calculated as a 350 =2.303 D/L, where D is the absorbance at 350-nm wavelength and L is the pathlength of the absorbance cell in meters. The a 350 (m -1 ) has been used to quantify CDOM because it represents the middle of the UV-A region, where the overlap of the solar irradiance spectrum and a CDOM yields the highest absorption rates relative to the photobleaching [START_REF] Kowalczuk | Characterization of CDOM in an organic-rich river and surrounding coastal ocean in the South Atlantic Bight[END_REF].

For determination of bacterial abundance by flow cytometry, duplicate 3-mL samples in cryovials were preserved in buffered formalin (2% final concentration). The samples were gently mixed and left in the dark at room temperature for 10 min before quick-freezing in liquid nitrogen and storage at -80°C. The samples were later thawed at room temperature, stained with SYBR Green I [final concentration 0.01% (v/v) of the commercial solution; Molecular Probes Inc., OR] for at least 15 min at 20°C in the dark and analyzed on a flow cytometer (FACScan, Becton Dickinson, San Jose, CA) equipped with a 488 nm, 15 mW argon laser. Bacteria were detected on a plot of green fluorescence (515-545 nm) versus right angle light scatter (SSC), using the green fluorescence as threshold parameter. Fluorescent beads (1.0 μm; Polysciences Inc., Warrington, PA) were added to each sample analyzed to normalize SSC and green fluorescence. Analytical accuracy of measurements gave a CV of less than 5%.

BP was measured by 3 H-leucine incorporation [START_REF] Kirchman | Leucine incorporation as a measure of biomass production by heterotrophic bacteria[END_REF] as modified by Smith and Azam [START_REF] Smith | A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3 Hleucine[END_REF]. Samples (2.5 mL in triplicate) were added to a sterile polystryrene snap cap tube (5 mL), containing 8 nM [4,5-3 H]-leucine (specific activity 117 Ci mmole -1 , PerkinElmer) and 32 nM of unlabelled leucine. One killed control was prepared for each assay by addition of 250 μl 50% trichloroacetic acid (TCA), 15 min before the addition of leucine. Tubes were incubated in the dark at 18°C for 1 h and then incorporation terminated by transferring replicate 1-mL samples from each tube in microcentrifuge tubes containing 100 μl of 50% TCA. Samples were stored for at least 1 h at 4°C and then centrifuged for 15 min at 12,000×g. The precipitate was rinsed once with 5% TCA and once with 70% ethanol. The precipitates were resuspended in 1.0 mL of liquid scintillation cocktail (FilterCount, PerkinElmer) and radioactivity determined by liquid scintillation counter (LS 5000CE Beckman). Leucine incorporation rates were converted into carbon production using the conversion factor of 1.55 kg C produced by mole of leucine incorporated [START_REF] Kirchman | Leucine incorporation as a measure of biomass production by heterotrophic bacteria[END_REF]. Analytical accuracy of measurements gave a CV of less than 4%.

Bacterial respiration (BR) rates were determined by following the decrease in dissolved oxygen (DO) during incubation experiments. Concentrations of DO were measured via an automated titration procedure using a Metrohm 716 equipped with a potentiometric electrode. At 0 and 39 h, three BOD bottles were fixed with Winkler reagents for determination of DO concentrations [START_REF] Carrignan | Measurement of primary production and community respiration in oligotrophic lakes using Winkler method[END_REF]. The whole BOD bottle was titrated to increase sensitivity of the rate estimates. Analytical accuracy of measurements gave a CV of less than 0.5%.

Data Calculation for Bacterial Growth

Bacterial carbon demand (BCD) was calculated in the first 39 h during the biodegradation experiments as the sum of the bacterial biomass production and the decrease in oxygen expressed in carbon using a respiratory quotient of 0.8. Bacterial biomass production was calculated from the geometric trapezoidal integration of leucine incorporation expressed in carbon, rather than from bacterial concentration, because changes in biovolume during growth were not measured. BGE was calculated as the ratio between bacterial biomass production and BCD [START_REF] Giorgio | Bacterial energetics and growth efficiency[END_REF]. BCD and BGE were the average of all possible combinations between the independent measurements of BP and the BR.

DNA and RNA Extractions for CE-SSCP Analysis

Community structure of total and metabolically active bacterial cells was analyzed at the start of the biodegradation experiments and after 111 h of incubation for SOLA and Canet Lagoon and 87 h for Leucate Lagoon (i.e., when cells were in stationary phase). Samples (500 mL) were filtered onto 0.22-μm pore size polycarbonate filters (Nuclepore) and stored at -20°C. For analysis, frozen filters were cut with sterilized scissors into small strips and vortexed briefly in 840 μl of alkaline lysis buffer (50 mM Tris hydrochloride pH 8.3, 40 mM EDTA, and 0.75 M sucrose). Cell lysis was accomplished by an initial incubation for 45 min at 37°C after adding 50 μl of freshly prepared lysosyme solution (20 mg mL -1 ), and a second incubation at 55°C for 1 h after adding 100 μl of 10% sodium dodecyl sulfate and 10 μl of proteinase K (20 mg mL -1 ). Six hundred microliters of lysate was treated with 10 μl of a 100-mg mL -1 RNase A solution (Qiagen) before DNA extraction with the DNeasy Tissue kit (Qiagen). DNA was used as a template for PCR amplification of the variable V3 region of the 16S rDNA (Escherichia coli positions 329-533), as previously described [START_REF] Ghiglione | Spatial and temporal scales of variation in bacterioplankton community structure in the NW Mediterranean Sea[END_REF]. Four hundred microliters of lysate was treated with DNase I for RNA extraction using an SV Total RNA Isolation kit (Promega). The efficiency of the DNA removal from RNA was checked by amplifying each RNA extract by PCR (conditions as indicated below), and PCR reactions that did not give a product were used for complementary (cDNA) synthesis. RNA was reverse transcribed (RT) into firststrand cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega), according to manufacturer instructions. PCR amplification of cDNA was performed with the same conditions as for DNA. Capillary electrophoresis SSCP-based single strand conformational polymorphism (CE-SSCP) was performed as previously described [START_REF] Ghiglione | Spatial and temporal scales of variation in bacterioplankton community structure in the NW Mediterranean Sea[END_REF] using the ABI 310 Genetic Analyzer (Applied Biosystems), equipped with a capillary tube (47 cm×50 μm) filled with a polymer composed of GeneScan polymer (Applied Biosystems) 5.6%, glycerol 10% and Tris/borate/EDTA (TBE) 1×. Analysis of the electropherograms was performed by using the software SAFUM (http://www.montpellier.inra.fr/narbonne/francais/ equipes/equipe-em/safumaccueil.htm).

Results

Initial Characteristics and Light-induced Changes in Chemical and Optical Characteristics of Waters

There were marked differences in the initial characteristics of the samples from the two lagoons and from the coastal water (Table 1). Canet Lagoon is a eutrophic lagoon characterized by high DOC and Chl a concentrations (892 μM and 70 μg L -1 , respectively) and a low salinity (7.5; Table 1). Leucate Lagoon is characterized by moderate DOC and Chl a concentrations (234 μM and 2.2 μg L -1 , respectively) and a high salinity (34.5; Table 1). SOLA is an oligotrophic marine coastal station with low DOC and Chl a concentrations (80 μM and 0.3 μg L -1 , respectively; Table 1). The elemental ratio of DOC/DON ranged from ∼12 (SOLA and Leucate Lagoon) to ∼18 (Canet Lagoon; Table 1). DOP concentrations were undetectable for all the water types (Table 1). Inorganic nitrogen and phosphorus concentrations were very low for Leucate Lagoon and SOLA station, whereas high concentration of inorganic phosphorus was measured for the Canet Lagoon (Table 1). The DOC concentrations in the lagoons sustain higher bacterial biomass (4.39×10 9 and 23.6×10 9 bacteria per liter for Leucate and Canet Lagoons, respectively) compared to the coastal station (0.62×10 9 bacteria per liter). CDOM fluorescence and absorbance at 350-nm wavelength (a 350 ) varied widely between the three systems with higher values measured at Canet, then Leucate, and then SOLA (Table 1). Fluorescence and absorbance of CDOM were linearly correlated to DOC (r 2 =1.00 for both parameters) for the three locations (Fig. 2). When CDOM fluorescence and absorbance were normalized to DOC concentrations, values were comparable between the two lagoons (40.8-50.9 and 8.67-9.14 for normalized fluorescence and absorbance, respectively) and closer to those measured at SOLA (14.5 and 5.75 for normalized fluorescence and absorbance, respectively) than for absolute values (Table 1). This indicates that the concentration of CDOM is highly variable between the three systems but that contribution of CDOM to the total DOM is more consistent.

Using the absorbance at 305, 360, and 550 nm measured in 0.2-μm filtered waters before exposure (data not shown), we estimated the transmission of UV-B, UV-A, and PAR, respectively, across the quartz tube (full sun condition; Table 2). UV transmission differed noticeably between the three locations. Whereas ∼95% of UV-B penetrated the sample from the oligotrophic SOLA coastal station, only ∼71 and ∼29% was transmitted for the Leucate and Canet Lagoons, respectively.

A loss of 27 μM in DOC (3% of the initial value) was observed for Canet Lagoon after full sun treatment (Table 3). Effects of photobleaching on CDOM fluorescence were greater than changes in absorbance (Table 3). Whereas a significant loss of fluorescence was measurable for Leucate and Canet Lagoons (22 and 33%, respectively), we only observed a significant decrease in a 350 by 10% for Canet Lagoon (Table 3). Loss of CDOM fluorescence and absorbance was attributable to UV radiation in the lagoon waters (i.e., no effects were measurable in the PAR treatment). No significant changes were observed in nutrient concentrations (NO À 3 , NO À 2 , PO 3À 4 ) after irradiation (Table 3).

Effect of Photochemical Transformations on Bacterial Growth and Diversity During Biodegradation Experiments

After the inoculation of the original bacterial community to each of three water types after the light treatments, an exponential bacterial growth was observed until ∼63 h for Leucate Lagoon and ∼87 h for SOLA and Canet Lagoon (Fig. 3). The different light treatments on 0.2-μm filtered samples modified neither the bacterial growth rate nor the bacterial concentration in stationary phase for SOLA and Canet samples. For Leucate samples, a decrease in growth rate and maximum bacterial concentration was observed in the full sun treatment compared to dark and PAR treatments. Flow cytometry analyses also gave information on the cell size (using the SSC as proxy) and the cellular DNA content (using the FL1 fluorescence as proxy) of the bacteria during the experimental cultures. The data clearly show that bacteria growing in the full sun treatment were larger and contained more DNA than bacteria growing in the dark and PAR treatments for all the water types studied (Fig. 3).

CE-SSCP fingerprinting revealed different patterns of bacterial community structure for total (16S rDNA) and active (16S rRNA) depending on light treatment and sample location. The initial community structure of total and metabolically active bacterial cells of each environment differed markedly from those obtained at the end of the growth for the different light treatments and the dark control (Fig. 4). The unweighted pair group method with arithmetic mean (UPGMA) dendrogram of the CE-SSCP profiles showed that dark and PAR treatments clustered together but to a lower extent with FS for SOLA and Canet (Fig. 4A andC), whereas dark, PAR, and FS clustered together for Leucate (Fig. 4B). Detailed analysis of the CE-SSCP profiles for SOLA shows that not all the population were able to respond during the regrowth experiment to DOM bioavailability changes, as revealed by the decrease of the observed number of 16S rDNA ribotypes [START_REF] Farjalla | Photochemical reactivity of aquatic macrophyte leachates: abiotic transformations and bacterial response[END_REF] for the full sun compared to the dark and the PAR treatments (19 and 18 ribotypes, respectively; Figs. 4A and5). In contrast, the modifications to the total community structure (16S rDNA) observed with the light treatments did not change the number of ribotypes compared to the dark treatment for Canet and Leucate Lagoons (Fig. 4B andC). The same trends were observed when 16S rRNA was used as a template, with a more pronounced difference between dark and PAR clusters compared to full sun at SOLA and Canet Lagoon, and between the three treatments for Leucate Lagoon, as determined by the higher Euclidean distance between samples (Fig. 4). In this case, full sun treatment resulted in a lower number of 16S rRNA ribotypes compared to the dark and PAR treatments for both SOLA and Canet Lagoon (Fig. 4A andC).

Effect of Photochemical Transformations on Bacterial Metabolism During Biodegradation Experiments

During the biodegradation experiments, we observed a net stimulating effect of the full sun treatment on the BP for SOLA and Canet Lagoon (Fig. 6a,c). This stimulatory effect was primarily attributable to phototransformations induced by UV, as PAR and dark BP were more similar. In contrast, we observed an inhibitory effect of full sun and PAR treatments on BP for Leucate Lagoon (Fig. 6b). Highest values of BP were measured after 15-h incubation for all treatments except Canet Lagoon with full sun where the BP increased until 39 h of incubation. Integrated values of BP for the first 39 h of incubation transformed into carbon are given in Table 4. Light treatments resulted in no change in BR for SOLA and Leucate Lagoon, whereas a significant increase of BR was observed with the full sun treatment for Canet Lagoon (Table 4). Changes in BCD were noticeable only in the case of the full sun treatment for Canet Lagoon (+74% relative to dark treatment; Table 4).

Values of BGE ranged from 10 to 37% in dark treatments for the different water types with higher values for lagoon waters (Table 4). Light treatments had contrasting effects on BGE derived from the BP and BR measurements. Full sun treatment had a stimulating effect on BGE for SOLA, resulting in BGE value that was 120% higher than the corresponding value for the dark treatment (Table 4). By contrast, PAR and full sun treatments had a similar and negative effect on BGE for both Leucate and Canet Lagoons, resulting in BGE values that were 20 to 40% lower than the corresponding values in the dark treatments (Table 4).

Discussion

Photochemical Transformations DOC, CDOM fluorescence, and absorption were affected differently by exposure to sunlight. Indeed, we found that DOC concentrations decreased more slowly than absorption or fluorescence values and that CDOM fluorescence losses outpaced CDOM absorption losses during photobleaching. These patterns agree with those previously noted by others for light-induced degradation of natural organic matter (e.g., [START_REF] Buffam | Effect of ultraviolet light on dissolved nitrogen transformations in coastal lagoon water[END_REF][START_REF] Moran | Carbon loss and optical property changes during long-term photochemical and biological degradation of estuarine dissolved organic matter[END_REF]) and may be caused by the presence of a reservoir of DOC resistant to photodegradation in contrast to the reservoir of CDOM. We did not detect changes in NO À 3 and PO À 4 concentrations after light exposure as has been previously reported (e.g., [START_REF] Smith | Photochemical transformations of riverine dissolved organic matter: effects on estuarine bacterial metabolism and nutrient demand[END_REF]). However, we cannot conclude that there were no changes in nutrients, as we did not measure ammonium photoproduction, which has been frequently reported in photochemical studies (e.g., [START_REF] Buffam | Effect of ultraviolet light on dissolved nitrogen transformations in coastal lagoon water[END_REF][START_REF] Smith | Photochemical transformations of riverine dissolved organic matter: effects on estuarine bacterial metabolism and nutrient demand[END_REF]).

Bacterial Response to Photochemical Transformations

Effects of DOM phototransformations on net production may be interpreted differently depending on whether we consider bacterial cell density or BP measurements. We hypothesized that the discrepancies observed between these two measures could be partly explained by a change in bacterial biovolume, which may vary according to the different light treatments of DOM based on the observation that bacteria growing in full-sun-exposed DOM had a higher biovolume relative to other light treatments and that they incorporated more leucine for the same cells produced. Although, we did not measure the biovolume of bacteria during the experimental cultures using epifluorescence microscopy [START_REF] Fry | Determination of biomass[END_REF], flow cytometry analyses provided infor- Bacterial Metabolism and Diversity of Photoaltered DOM mation on cell size and DNA content by the mean of side scatter and SYBR-green fluorescence, respectively. Both of these parameters have been correlated to the biovolume of aquatic bacteria [START_REF] Gasol | Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities[END_REF]. We observed that bacteria growing with DOM irradiated by full sun were characterized by a higher side scatter and SYBR-green fluorescence. This indicates that cell numbers alone were insufficient to determine net production and demonstrate that a single theoretical factor may not be appropriate to convert cell numbers to biomass (e.g., [START_REF] Amado | Complementary pathways of dissolved organic carbon removal pathways in clear-water Amazonian ecosystems: photochemical degradation and bacterial uptake[END_REF]). In contrast to our observation, however, Farjalla et al. [START_REF] Farjalla | Photochemical reactivity of aquatic macrophyte leachates: abiotic transformations and bacterial response[END_REF] did not observe differ-ences in biovolume for bacteria growing on irradiated and non-irradiated leachates from two aquatic macrophytes. The experimental design used in this study allows us to determine the contribution of UV radiation and PAR to these changes. Changes in BGE seem to be due primarily to UV radiation for the coastal water and PAR for the coastal lagoons. The changes in BP and BR induced by DOM phototransformations resulted in an increase of BGE for the oligotrophic coastal station (120%) and a decrease for the coastal lagoons (20 to 40%), whereas BCD only increased for the more eutrophic lagoon (74%). However, the BGE We have compared our results for changes of bacterial metabolism caused by DOM phototransformations to other studies (Table 5). Most have observed an increase of the BCD accompanied by a BGE decrease after DOM photo-transformations. Positive effect on BGE has been observed only for lake water with high humic concentrations [START_REF] Anesio | Effect of humic substance photodegradation on bacterial growth and respiration in lake water[END_REF][START_REF] Reche | Interaction of photobleaching and inorganic nutrients in determining bacterial growth on colored dissolved organic carbon[END_REF] or for leachates from submersed macrophytes [START_REF] Anesio | Bacterial growth on photochemically transformed leachates from aquatic and terrestrial primary producers[END_REF]. According to Anesio et al. [START_REF] Anesio | Bacterial growth on photochemically transformed leachates from aquatic and terrestrial primary producers[END_REF], the initial bacterial availability of DOM can partly explain the positive or negative response effect as well as the magnitude of bacterial response to the photochemical modifications of DOM. The higher the PAR ( 18) FS ( 14)

Dark (19) A Init (25)
Dark ( 22) PAR ( 23 bioavailability of DOM before irradiation, the more negative the bacterial response to photochemical modifications of BGE. Smith and Benner [START_REF] Smith | Photochemical transformations of riverine dissolved organic matter: effects on estuarine bacterial metabolism and nutrient demand[END_REF] observed that for different highly colored 'blackwater' systems (a 350 =41 to 117 m -1 ), exposure of DOM to sunlight increased the BCD but decreased the BGE. The decrease in BGE appeared predictable from the initial DOM optical characteristics: The importance of the decrease in the BGE was positively correlated to the initial a 350 value [START_REF] Smith | Photochemical transformations of riverine dissolved organic matter: effects on estuarine bacterial metabolism and nutrient demand[END_REF]. Our results agree with the main conclusions of these two relationships: (1) a negative change in BGE was observed after irradiation to full sun for the lagoons waters characterized by a higher BGE measured in the dark treatment (an index of the bacterial availability of DOM) and a higher CDOM content, and (2) a positive change in BGE was observed after irradiation to full sun for the oligotrophic coastal water characterized by a lower BGE measured in the dark treatment and a lower CDOM content. Decreasing BGE values upon exposure can be partially explained by the highly oxidized nature of several of the quantitatively important LMW photoproducts identified in different studies [START_REF] Bertilsson | Photochemical transformation of dissolved organic matter in lakes[END_REF][START_REF] Pullin | Effects of sunlight and hydroxyl radical on dissolved organic matter: bacterial growth efficiency and production of carboxyclic acids and other substances[END_REF]. In general, although these oxidized molecules (e.g., organic acids) are readily available for bacterial utilization, they are expected to be utilized at a lower BGE compared with less oxygenated, more reduced substrates for energetic reasons [START_REF] Pullin | Effects of sunlight and hydroxyl radical on dissolved organic matter: bacterial growth efficiency and production of carboxyclic acids and other substances[END_REF]. Increasing BGE upon exposure may also be explained by the photoproduction of free dissolved sugars and amino acids [START_REF] Buffam | Effect of ultraviolet light on dissolved nitrogen transformations in coastal lagoon water[END_REF][START_REF] Kovac | Characterization of macroaggregates and photodegradation of their water soluble fraction[END_REF], which are expected to be used with a higher BGE [START_REF] Giorgio | Bacterial energetics and growth efficiency[END_REF].

Limiting nutrients during bioassays may interfere with the response of bacteria to phototransformed DOM [START_REF] Reche | Interaction of photobleaching and inorganic nutrients in determining bacterial growth on colored dissolved organic carbon[END_REF]. During a set of experiments where DOM and bacteria were exposed simultaneously to natural sunlight (i.e., conditions different from those usually used herein where exposure of DOM was conducted before the addition of bacteria), Reche et al. [START_REF] Reche | Interaction of photobleaching and inorganic nutrients in determining bacterial growth on colored dissolved organic carbon[END_REF] observed a positive interaction of CDOM photobleaching and N and P enrichments on bacterial abundance, production, and BGE. These interactions indicate that conversion of carbon from substrate into bacterial biomass was higher than expected from the addition of individual components. Increased nutrient loading in aquatic ecosystem could facilitate the utilization of the photoproducts and transfer of carbon to the food web via bacteria. Most of the experiments conducted to measure changes in bacterial metabolism after addition of phototransformed DOM have used nutrient additions (N and P) at the start of the biodegradation to avoid any nutrient limitations during growth (Table 5). We did not add nutrients in this study to measure the response of bacteria to "natural" conditions; consequently, we are aware that a part of the photoproducts may have not been used because of limiting nutrients, in particular for SOLA and Leucate Lagoon for which PO 4 was undetectable.

Links Between Changes in Bacterial Metabolism and Diversity

Microbial communities may respond to a varying supply of substrates either by physiological adaptation and/or by changes in the community composition. Even small additions of organic substrates may trigger a shift in the composition of the microbial community and an accompa- nying change in the BGE [START_REF] Eiler | Heterotrophic bacterial growth efficiency and community structure at different natural organic concentrations[END_REF]. To our knowledge, this is the first study comparing the changes in bacterial community structures and BGE induced by phototransformation of DOM. In this study, we used 16S rDNA and rRNA-based CE-SSCP fingerprinting to resolve the community structure of total and metabolically active bacteria, respectively, as active bacteria contain more rRNA than resting or starved cells [START_REF] Kemp | Estimating the growth rate of slowly growing marine bacteria from RNA content[END_REF][START_REF] Poulsen | Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms[END_REF]. The experimental design used for the biodegradation experiments (inoculation of bacteria at 10% in water free of protozoa) resulted in an increase of bacterial abundance until a stationary phase for the three water types (Fig. 3). In parallel, both DNA-and RNA-based community structures at the beginning of the experiment differed markedly from those obtained at the stationary phase for all light and dark treatments and for all water type studied (Fig. 4). We could not exclude the possibility that growth of a few opportunistic bacteria overwhelmed the original assemblage during bottle incubations (4 to 5 d) [START_REF] Gattuso | Changes in freshwater bacterial community composition during measurements of microbial respiration[END_REF][START_REF] Massana | Changes in marine bacterioplankton phylogenetic composition during incubations designed to measure biogeochemically significant parameters[END_REF]. However, dominance of few bacterial species was not detected, as revealed by the number of ribotypes remaining almost identical (even if their positions were different) between the start and the end of experimental cultures (with the exception of the full sun treatment for SOLA station). This indicates that bacterial structure was altered during the regrowth but without dominance of a few bacterial species. Moreover, the interpretation of our data is mainly based on the comparison of the diversity at the end of the experimental cultures between the different treatments, and all include the same potential "bottle effect" on the bacterial diversity composition. We did not perform replicates of each experimental culture because of operational limitations. However, high similarities sometimes obtained in the community structures of total and metabolically active bacterial cells at the end of the experimental cultures for two different light treatments (i.e., full sun and PAR treatments for Leucate Lagoon, PAR, and dark treatments for Canet Lagoon) indicate that results may be considered as robust.

The influence of phototransformations of DOM to the bacterial diversity differs with the sampling location. The effect was most dramatic in the oligotrophic SOLA coastal station (Fig. 4,A). Bioavailability changes of DOM because of full sun treatment resulted in a decrease of the number of dominant ribotypes that was not recordable for the PAR treatment (Fig. 5). Along with the increase of both BP and BGE for the full sun compared to dark and PAR treatments at SOLA, the composition of the metabolically active bacteria (16S rRNA based CE-SSCP) was also more affected in the full sun treatment, with 78% of ribotypes present at the DNA level that were active at the RNA level. At the Leucate Lagoon, inhibition of BP and BGE in both full sun and PAR treatments compared to dark was related to the dissimilarity between dark, PAR, and full sun in community structures of both total and metabolically active bacteria (Fig. 4,B). At the eutrophic Canet Lagoon, community structures of total and metabolically active bacteria were clearly influenced by DOM bioavailability changes because of UV radiation (Fig. 4,C). These changes can be related to the increase of BP measured in the full sun treatment compared to the PAR and dark treatments but not to the decrease of BGE observed for both PAR and full sun treatments compared to dark. These results corroborated several studies suggesting that the quality and supply of organic substrates affect the function as well as the composition of bacterial diversity [START_REF] Eiler | Heterotrophic bacterial growth efficiency and community structure at different natural organic concentrations[END_REF][START_REF] Pinhassi | Coupling between bacterioplankton species composition, population dynamics, and organic matter degradation[END_REF].

Interestingly, our results show that community changes of metabolically active bacteria enhance the differences observed at the DNA level, resulting in a closer link between rRNA-based analysis and BP changes compared to rDNA-based analysis. Recent work based on the comparison between 16S rDNA and rRNA libraries indicates that the characterization of the bacterial community based on rRNA has the potential to characterize distinct ribotypes from the marine environment, which remain undetected on the DNA level [START_REF] Moeseneder | A comparison of DNA-and RNA-based clone libraries from the same marine bacterioplankton community[END_REF]. The number of ribotypes per sample can be lower, equal, or higher on the RNA level than on the DNA level depending on the detection limit of the fingerprinting technique and their relative contribution to the community in terms of cell number or metabolic activity [START_REF] Troussellier | Bacterial activity and genetic richness along an estuarine gradient (Rhone River plume, France)[END_REF]. In our study, the number of ribotypes per sample was always higher or equal on the RNA level than on the DNA level (Fig. 4). This suggests that their contribution to the community in terms of cell numbers may have been too low for detection, but because of their high metabolic activity and the accompanying 16S rRNA synthesis, these ribotypes were readily detectable at the RNA level [START_REF] Winter | Impact of UV radiation on bacterioplankton community composition[END_REF]. Metabolically active bacteria that were below the detection limit at the DNA level represent from 12 to 26% of the total number of ribotypes. These bacteria may correspond to organisms present in low cellular abundance and therefore probably not detectable on the DNA level but with higher ribosome content and thus detectable at the RNA level. In addition, most of the ribotypes present at the DNA level in each of the locations studied were active (from 74 to 79%), and a few were not detectable at the RNA level (from 0 to 12%). These results suggest that the phototransformation of DOM affects both the bacterial community structure and the composition of active bacteria and that RNA-based analysis was more sensitive to point out small changes of organic substrate availability.

In conclusion, we found that BP, BR, BCD, BGE, and bacterial community structure and related metabolically active bacteria may be altered by phototransformations of DOM for different oligotrophic to eutrophic aquatic ecosystems. Changes in total and active bacterial structures induced by phototransformations of DOM can be correlated to changes in BP. These changes may occur even after a short exposure of DOM to solar radiation and without major CDOM photobleaching and photomineralization of DOM. We encourage a conservative interpretation of our results in this initial study. Further studies will be necessary to elucidate a more detailed appreciation of potential temporal and spatial variations of the effects measured. Different results may be obtained seasonally because of changes in DOM composition, bacterial community structure, and UV intensity and penetration in the water column. Future works may also elucidate the bacterial species and functional groups most responsive to photochemically modified DOC compounds [START_REF] Langenheder | Changes in bacterial community composition along a solar radiation gradient in humic waters[END_REF].

Figure 2

 2 Figure 2 CDOM fluorescence and absorbance at 350 nm (a 350 ) of the natural water samples (lagoons and coastal water) as a function of DOC concentration. Coastal water has the lowest CDOM fluorescence and absorbance, Leucate Lagoon intermediate, and Canet Lagoon highest

Figure 3

 3 Figure 3 Changes in bacterial abundance (BA), cell size scatter (SSC), and DNA fluorescence (FL1) of bacteria during the biodegradation experiments for irradiated [full sun (open circle) and PAR

Figure 5

 5 Figure 5 Example of CE-SSCP electrophoregram corresponding to a 16S rDNA or b 16S rDNA for dark, PAR, and full sun treatments after 111 h of the experimental culture using water from SOLA. The

Figure 4

 4 Figure 4 Cluster analysis dendrogram based on comparison of 16S rDNA and 16 rRNA CE-SSCP patterns obtained from the bacterial communities before (Init) and after growth on DOM pre-irradiated by full sun (FS), PAR, or maintained in the dark (Dark). The numbers of ribotypes identified in each bacterial community are indicated in parentheses. A SOLA station, B Leucate Lagoon, C Canet Lagoon

Figure 6

 6 Changes in bacterial production (BP) during the biodegradation experiment for irradiated [full sun (open circle) and PAR (filled triangle)] and dark (filled circle) treatments. a SOLA station, b Leucate Lagoon, c Canet Lagoon

Table 1

 1 Surface water characteristics of the different stations studied

		SOLA	Leucate	Canet
	Salinity	37.7	34.5	7.5
	DOC (μM)	80	234	892
	DON (μM)	6.5	21.3	49.9
	DOP (μM)	<dl	<dl	<dl
	C/N (mol/mol)	12.3	11.0	17.9
	F (QSU)	1.16	9.55	45.4
	a 350 (m -1 ) F /DOC (QSU mM -1 )	0.46 14.5	2.14 40.8	7.73 50.9
	a 350 /DOC (m -1 mM -1 ) NO À 3 (μM) NO À 2 (μM) NH þ 4 (μM) PO 3À 4 (μM)	5.75 <dl <dl 0.08 <dl	9.14 0.14 0.07 1.52 <dl	8.67 0.17 0.02 2.15 22.4
	Chl a (μg L -1 )	0.32	2.17	69.9
	Bacteria (10 9 L -1 )	0.62	4.39	23.6

DOC Dissolved organic carbon; F fluorescence of chromophoric dissolved organic matter (excitation 350 nm, emission at 450 nm, given in quinine sulfate units); a 350 absorption coefficient at 350 nm. F/DOC and a 350 /DOC: dissolved organic carbon-normalized fluorescence and absorption, respectively; <dl below detection limit Bacterial Metabolism and Diversity of Photoaltered DOM

Table 3

 3 Changes on carbon, nitrogen, and phosphorus concentrations and optical properties in 0.2-μm filtered water samples in the different light treatments; 8 h darkness (dark treatment) or 8 h to PAR (PAR treatment) or 8 h to natural sunlight radiation (full sun) Treatment means are significantly different (t test, p<0.05) from dark treatment, for within-site comparisons only.

	Station	Light treatment	DOC (μM)	F (QSU)	a 350 (m -1 )	NO À 3 (μM)	NO À 2 (μM)	PO À 4 (μM)
	SOLA	Dark	80.5 (0.2)	1.25 (0.44)	0.41 (0.12)	<dl	<dl	<dl
		PAR	80.0 (0.7)	0.75 (0.22)	0.44 (0.22)	<dl	<dl	<dl
		Full sun	82.8 a	0.77 (0.05)	0.47 (0.03)	<dl	<dl	<dl
	Leucate	Dark	233 (0)	9.59 (0.03)	2.10 (0.03)	0.11 (0.01)	0.08 (0.00)	<dl
		PAR	229 (6)	9.56 (0.63)	2.02 (0.08)	0.12 (0.00)	0.08 (0.00)	<dl
		Full sun	228 (2)	8.17 (0.17) b	1.96 (0.09)	0.16 (0.05)	0.07 (0.00)	<dl
	Canet	Dark	877 a	44.7 (2.0)	7.73 (0.33)	0.14 (0.00)	0.03 (0.02)	22.3 (0.2)
		PAR	870 a	44.2 (0.5)	7.82 (0.34)	0.14 (0.03)	0.02 (0.00)	22.5 (0.1)
		Full sun	850 a	35.5 (0.5) b	7.34 (0.10) b	0.13 (0.02)	0.02 (0.01)	21.5 (0.6)
	All data are means from duplicate samples (range).
	<dl Below detection limit
	a No duplicate available.

b

Table 4

 4 Bacterial production (BP), bacterial respiration (BR), and bacterial carbon demand (BCD=BP+BR) integrated for the first 39 h of experimental cultures Bacterial growth efficiency (BGE) was derived from BP and BR measurements (BGE=BP/[BP+BR]). All data are means (standard deviation). a Treatment means are significantly different from dark treatment, for within-site comparisons only (t test for BP and BR and U test for BCD and BGE, p<0.05)

	Station	Light treatment	BP (μg C L -1 d -1 )	BR (μg C L -1 d -1 )	BCD (μg C L -1 d -1 )	BGE (%)
	SOLA	Dark	2.7 (0.1)	24.4 (10.4)	27.0 (10.4)	9.9 (4.8)
		PAR	3.6 (0.1) a	24.7 (7.1)	28.2 (7.1)	12.6 (5.0) a
		Full sun	5.7 (0.1) a	19.5 (1.7)	25.2 (1.7) a	22.5 (4.4) a
	Leucate	Dark	16.2 (1.3)	36.6 (16.6)	52.8 (16.6)	30.7 (13.0)
		PAR	6.2 (0.2) a	26.1 (8.0)	32.3 (8.0) a	19.2 (6.1) a
		Full sun	8.9 (1.6) a	40.3 (9.7)	49.2 (9.8) a	18.1 (6.0) a
	Canet	Dark	17.9 (0.3)	30.9 (10.3)	48.8 (10.3)	36.6 (9.9)
		PAR	18.6 (0.3) a	45.0 (8.7)	63.6 (8.7) a	29.3 (5.1) a
		Full sun	22.8 (0.2) a	62.2 (9.5) a	85.0 (9.5) a	26.8 (3.8) a

Table 5

 5 Synthesis of results on changes in bacterial abundance (BA), production (BP), respiration (BR), carbon demand (BCD), and growth efficiency (BGE) after irradiation of DOM from various aquatic systems to natural or artificial sunlight

	DOM origin	Irradiation conditions	Nutrients a BA BP BR	BCD BGE	Reference
	Leachates from aquatic macrophytes							[14]
	Phragmites australis	Artificial UVA+UVB	Yes	-	nd	= or -=	-
	Hydrocaris morsus-ranae			-	nd	= or -=	= or -
	Leachates from different primary							[3]
	producers

Acknowledgments We wish to thank P. Catala for the flow cytometry analysis and I. Obernosterer for her help in CDOM analysis and for constructive suggestions that improved an earlier version of this manuscript. This study represents partial fulfillment of the PhD requirements of M.A. It was part of the UVECO project ("Induction of microbial community responses and dissolved organic matter transformation by UltraViolet radiation in marine ECOsystems", http:// www.com.univ-mrs.fr/LMGEM/uveco/uveco_fr/index.htm) supported by the French CNRS-PROOF program. This study was also funded through a fellowship from the Syrian Atomic Energy Commission to M.A. and by National Science Foundation Office of International Science and Engineering US-France Cooperative Science Program grant 0340764 to W.H.J.


Fall and fragmentation of liquid metal in a viscous fluid

Jean-Baptiste Wacheul & Michael Le Bars CNRS, Aix Marseille Univ, Centrale Marseille, IRPHE, Marseille, France wacheul@irphe.univ-mrs.fr

Simulations of the solar system formation as well as the diversity of meteorites chemical composition both point toward a scenario where the late stages of Earth-like planets accretion involved massive impacts of Mars size planets [START_REF] Kokubo | On runaway growth of planetesimals[END_REF]. Due to the combination of the energy release during impact, to the intense radioactive decay of short-lived elements, and to the heat remaining from the conversion of potential energy required to form a planet, these planetary embryos were most likely molten for a large part [START_REF] Lebrun | Thermal evolution of an early magma ocean in interaction with the atmosphere[END_REF]. This also means that they were differentiated in a liquid iron core covered by a molten silicate mantle, since these two main phases of terrestrial bodies are immiscible and of significantly different densities. Following each impact, a large-scale two-phase flow occurred as the molten iron of the impactor core flowed across the silicate magma ocean to merge with the planetary embryo core. Because of the immense inertia of the process, this flow was characterized by very high Reynolds and Weber numbers, hence prone to rapid fragmentation. The quantification of the exchanges of heat and elements in this dispersed flow is crucial to the knowledge of the initial state of telluric planets.

While the dynamics of rising bubbles have been largely investigated [START_REF] Clift | Bubbles, drops, and particles[END_REF], studies of the planetary regime of two-phase flow and fragmentation are sparse. Models of Earth's formation mostly rely on asymptotic models where the initial volume of iron is either immediately spread into an ensemble of drops of capillary sizes that behave independently [START_REF] Rubie | Mechanisms of metal-silicate equilibration in the terrestrial magma ocean[END_REF], or remains as a whole until reaching the bottom of the magma ocean, developing Rayleigh-Taylor and Kelvin-Helmholtz type instabilities on its sides [START_REF] Dahl | Turbulent mixing of metal and silicate during planet accretion and interpretation of the Hf-W chronometer[END_REF]. Recently [START_REF] Landeau | Experiments on the fragmentation of a buoyant liquid volume in another liquid[END_REF][START_REF] Deguen | Turbulent metal-silicate mixing, fragmentation, and equilibration in magma oceans[END_REF] performed laboratory experiments showing that in the relevant large Weber number limit, the released buoyant fluid behaves initially like a turbulent thermal whose surface of contact with the ambient fluid has a fractal aspect and therefore a large surface area, well before fragmentation and formation of a cloud of droplets.

The viscosity of the magma ocean is a key variable in the geophysical application. We have thus constructed an experimental set-up using Galinstan, a metal liquid at room temperature, to mimic the impactor core, and a mixture of water and industrial lubricant (Ucon Oil) with a tunable viscosity, to mimic the magma ocean. A given volume of Galinstan is initially placed at the top of a 1m deep tank inside a balloon, which is suddenly popped. The following dynamics is recorded using a highspeed camera. As shown in fig. 1, changing the viscosity of the ambient fluid by a factor of 1000 does not change the mean falling speed significantly, which is in reasonable agreement with the inviscid scaling for the terminal velocity of the initial spherical mass of metal. Nevertheless, the viscosity ratio has a great influence on the fragmentation regime. At low viscosity ratio (fig. 1 center and right), the liquid metal is rolled in a turbulent vortex ring and stretched until the thin liquid metal sheet breaksup in a single event, in qualitative agreement with the fragmentation mode found by [START_REF] Landeau | Experiments on the fragmentation of a buoyant liquid volume in another liquid[END_REF]. At the higher viscosity ratio (fig. 1 left), vorticity tends to be localized in the liquid metal; it is not rolled-up but stretched in long scales so that breakup rarely happen in a single event. Instead the liquid metal is peeled away from the initial mass or separates from the rest via Rayleigh-Taylor instabilities.

When increasing the inertia of the flow, one could expect a dynamic similar to the immiscible case, corresponding to a turbulent thermal [START_REF] Bush | Particle clouds in homogeneous and stratifed environments[END_REF]. While the linear increase with depth of the mean radius of the metal blob agrees with the classical theory for turbulent thermals, this model systematically overestimates our measured velocities. We conjecture that this is due to the invalidity of the turbulent entrainment hypothesis, where the buoyant mass entrains fluid all along its surface with no significant initial momentum [START_REF] Morton | Turbulent gravitational convection from maintained and instantaneous sources[END_REF]. As shown in fig. 2, we observe that the ambient fluid is engulfed at the rear of the liquid metal only, where strong stable vortices produce important velocity gradients in the ambient fluid. This flux produces an expansion of the liquid metal sheet that works against surface tension, producing an over-pressure in the entrained liquid that could accelerate the falling velocity. The validation of this explanation now requires precise velocimetry in the ambient fluid that will be the focus of our next studies.

Figure 1 :

 1 Figure 1: Superimposition of three snapshots extracted at the same time (time interval of xxx s) from 3 videos of the fall of a mass of liquid metal with an initial 31 mm spherical radius in an ambient fluid with different viscosities. From left to right the viscosity of the ambient fluid decreases by 3 orders of magnitude. The corresponding viscosity ratio between the twofluids are 700, 80 and 0.4, and the corresponding Reynolds numbers are 20, 195 and 34000. The Weber number is 51 in all cases. See also the video at https://doi.org/10.1103/APS.DFD.2016.GFM.V0038

The authors gratefully acknowledge financial support from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 681835-FLUDYCO-ERC-2015-CoG).