Axel Canredon

Pierre Anschutz

Damien Buquet

Celine Charbonnier

David Amouroux

Emmanuel Tessier

Dominique Poirier

Stéphane Bujan

Ludovic Devaux

Benoit Gouillieux

Céline Charbonnier

Benoît Gouillieux

Sophie Gentes

Alexia Legeay

Agnès Feurtet- Mazel

Serge Galaup

Régine Maury-Brachet

Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin

Keywords: Mercury methylation, fish contamination, aquatic sediment, sulphate, freshwater lake

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

Introduction

Technological, industrial, and agricultural progress triggered both the exploitation of natural resources and the dispersion of anthropogenic contaminants. Since the 19 th century, mercury (Hg) fluxes in the environment have increased at a global scale. Typical concentrations of Hg range from 50 to 150 µg kg -1 in the continental crust [START_REF] Wedepohl | The composition of the continental crust[END_REF], from 0.1 to 2 ng L -1 in continental and ocean waters [START_REF] Mason | The biogeochemical cycling of elemental mercury: Anthropogenic influences[END_REF], and from 1 to 3 ng m -3 in the atmosphere [START_REF] Schroeder | Atmospheric Transport, Chemistry and Deposition of MercuryAtmospheric mercury-An overview[END_REF].

Mercury dynamics is characterizes by properties, such as volatility at ambient temperature, stability of its bonds with carbon and sulphur, and very high bioconcentration and toxicity [START_REF] Cossa | La dynamique du mercure[END_REF]. Methyl mercury (MeHg) is the most toxic chemical form of Hg. It is a neurotoxin for vertebrates, which leads to damage in central nervous system [START_REF] Allen | The consequences of methylmercury exposure on interactive functions between astrocytes and neurons[END_REF]. Methyl mercury passes easily through biological barriers and can bioaccumulate and biomagnify along the food chain.

Although it is present at very low concentrations in water, it can concentrate up to 10 million times in aquatic organisms such as predators [START_REF] Cossa | La dynamique du mercure[END_REF]. Human exposure to MeHg compounds occurs often through the consumption of fish and fish products [START_REF] Driscoll | Mercury as a global pollutant: Sources, pathways, and effects[END_REF]. High trophic level species of fish can present very high MeHg concentrations. The maximum tolerable level for the sale of carnivorous fish is 1 mg kg -1 wet weight according to the European Commission directive 93/351/EEC.

Mercury is naturally emitted in the environment through degassing of the Earth's crust, volcanoes, and erosion of soils and rocks. Human-related Hg emissions dominate the Hg cycle such that most of the Hg in the atmosphere is anthropogenic [START_REF] Mason | The biogeochemical cycling of elemental mercury: Anthropogenic influences[END_REF]. Natural Hg emission and re-emission mainly occurs as elemental mercury vapours (Hg 0 ). The main mercury source in aquatic environments comes from wet and dry atmospheric deposition, and via runoff from the drainage basin [START_REF] Mason | Sources, Sinks and Biogeochemical Cycling of Mercury in the Ocean[END_REF]. Globally, the main anthropogenic Hg emissions into the atmosphere originate from artisanal gold production, the combustion of coal, and solid wastes [START_REF] Pirrone | Global mercury emissions to the atmosphere from anthropogenic and natural sources[END_REF]UNEP, 2013;[START_REF] Streets | Total Mercury Released to the Environment by Human Activities[END_REF]. These emissions, coupled with long-distance transport of elemental Hg, result in a worldwide distribution of Hg deposition with a global average of 5.6 µg m -2 yr -1 [START_REF] Lamborg | Modern and historic atmospheric mercury fluxes in both hemispheres: Global and regional mercury cycling implications[END_REF].

In 2015, a large-scale sampling of piscivorous fish was conducted in order to study fish contamination by mercury in the four main lakes in Aquitaine, specifically from the north to the south, lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse (Fig. 1). The results showed an increasing Hg contamination gradient from Lake Parentis-Biscarrosse to Lake Carcans-Hourtin (south-north gradient), with values in the top predator zander (Sander lucioperca) from 2000 µg kg -1 dw in the southernmost lake to 7400 µg kg -1 dw in the Lake Carcans-Hourtin (Gentès et al., submitted). This latter study showed that trophic transfer efficiency does not explain this Hg latitudinal gradient in biota. The mercury concentration in fish suggested that the lakes underwent variable contamination pressure. However, the four lakes are located in the same geological context, they are subject to identical climate, and they do not have known local Hg sources, suggesting that different fish contamination in each lake results from distinct methylation efficiency of a background Hg flux.

Therefore, these lakes represent a natural laboratory, in which the study of biogeochemical parameters allows us to determine the impact of the main factors that control methylation of Hg. Methylation occurs through the action of sulphate-reducing bacteria [START_REF] Gilmour | Sulfate Stimulation of mercury methylation in freshwater sediments[END_REF] and, to a lesser extent, iron-reducing and methanogenic microorganisms [START_REF] Alpers | Mercury cycling in agricultural and managed wetlands, Yolo Bypass, California: spatial and seasonal variations in water quality[END_REF][START_REF] Hamelin | Methanogens: principal methylators of mercury in lake periphyton[END_REF][START_REF] Kerin | Mercury methylation by dissimilatory iron-reducing bacteria[END_REF][START_REF] Parks | The genetic basis for bacterial mercury methylation[END_REF], which are common in organic-rich sediments. Consequently, methylation is localized in the anoxic zone of aquatic environments such as biofilms, sediment, or water column.

As Aquitaine lakes are shallow, between 7 and 23 m maximum depth, the water column is generally well mixed and oxygenated. Therefore, it was expected that anoxic conditions favourable to Hg methylation occurred in anoxic sediments instead of the water column. Indeed, it has been observed recently that the bottom of the deepest parts of Aquitaine lakes are blanketed with anoxic organic muds [START_REF] Buquet | Nutrient sequestration in Aquitaine lakes (S France) limits nutrient flux to the coastal zone[END_REF].

The present study focuses on the relationship between the biogeochemistry of lakes and the distribution of mercury and MeHg in sediments. The aim was to (i) perform a mapping of lake sediments in order to link Hg concentrations and the nature of the substrate, (ii) measure concentrations of total mercury and methyl mercury in several representative sediment samples, (iii) analyse the redox compounds influencing Hg speciation in sediments, (iv) attempt to explain the differences in sediment mercury speciation between the four lakes studied, and finally (v) determine the relationship of Hg in sediments and Hg in fish.

Materials and Methods

2.1.Study site

Large lakes in Aquitaine are located close to the French Atlantic coast in the south-western part of France. They are separated from the ocean by a 5-km-wide forested coastal dune system (Fig. 1), which represents a physical barrier to streams draining the lowland of Landes de Gascogne. Most of the drainage basin of the lakes is located on the eastern side. The surface areas of the catchments are similar, between 200 and 310 km 2 (Table 1), which is relatively small. The relief of the drainage basins is very flat with an westward slope lower than 0.2%. The geology is homogeneous and consists of podzols established on Quaternary siliceous aeolian deposits [START_REF] Jolivet | Forest soils in the Gascony Landes Region: formation, history, properties and spatial varaibility[END_REF]. Soil occupation has been determined by remote sensing using maps of the French national institute of geography (IGN), and aerial photographs analysed with the Geographic Information System (GIS) ArcGIS [START_REF] Canton | Nutrient export to an eastern Atlantic coastal zone: First modeling and nitrogen mass balance[END_REF]. Pine (Pinus pinaster) forests dominate the land use and occupy more than 80% of the total surface (Fig. 1). Intensive farming covers between 5% and 20% of lake drainage basins.

Housing is confined to the coastal zone.

Lakes are fed by direct rainfall on the lake surface and by low-discharge rivers. River systems are artificial ditches dug to dry up marshes. Water level of lakes is controlled by locks in channels that connect lakes. Locks also determine the water residence time (Table 1). Lake Lacanau is located downstream from Lake Carcans-Hourtin. The lakes are connected by the 7.5-km-long Etang channel.

Waters of Lake Lacanau flow to the Arcachon Lagoon through the 18.5-km-long Porge channel. Lake Parentis-Biscarrosse is downstream from Lake Cazaux-Sanguinet. All of these lakes are shallow (less than 23 m) (Table 1). The deepest part is located on the western side of the lakes, at the base of the dunes. Only the southern lakes are deep enough to have a marked summer thermocline at about 12 m depth. The hypolimion does not become totally anoxic in the oligotrophic Lake Cazaux-Sanguinet.

Cyanobacteria blooms occur frequently in Lake Parentis-Biscarrosse due to eutrophication caused by the legacy of large amounts of phosphorus stored in lake sediment. This lake experiences short periods of bottom-water anoxia for a few weeks during summer water stratification. Less than 10% of the water volume becomes anoxic during these episodes. Dense stands of invasive aquatic plants (Egeria densa and Lagarosiphon major) are found in Lake Parentis-Biscarrosse and Lake Lacanau. They are developed in shallow-sheltered or deep-exposed areas and represent about 10% of lake surface area [START_REF] Bertrin | Effects of wind-induced sediment resuspension on distribution and morphological traits of aquatic weeds in shallow lakes[END_REF]. Only restricted vegetated areas occurs in the other two lakes. The highest biomass is present in Lake Parentis-Biscarrosse, with an estimated amount of 2800 tons [START_REF] Bertrin | Effects of wind-induced sediment resuspension on distribution and morphological traits of aquatic weeds in shallow lakes[END_REF].

2.2.Sampling

Fish were collected from the four lakes by fishing associations and federations and by a professional fisherman during autumn 2015. Three well-represented piscivorous species in the four lakes were selected: zander (Sander lucioperca), pike (Esox Lucius) and perch (Perca fluviatilis). They were placed in zip-locked bags and immediately cooled at 4°C. Sampling of lake sediment was carried out using cores and grabs. The sediment mapping of lakes was obtained from sediment grabs that were deployed along high spatial resolution N-S and E-W transects (Fig. 2). This strategy allowed us to collect samples from the different bathymetric levels. We collected the top 10 cm of sediment. We obtained 70, 55, 59, and 60 homogenized sediment samples from Lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse, respectively. For Lake Cazaux-Sanguinet the sampling location was constrained by the security perimeter of the Cazaux military base. Maps of the distribution of sediment properties, such as organic carbon or Hg content, were obtained from interpolation between grab and core samples, and bathymetry was used for extrapolation to map whole lakes. Additional grabs have been collected to validate the sediment mapping based on bathymetry.

In addition, sediment cores allowed us to define the vertical distribution of reactive compounds at the water-sediment interface. Sediment cores were collected at three to nine stations for each lake. Stations were selected according to the sedimentary map established with sediment grabs.

Cores were collected in organic deposits and sandy sediments from each lake, and also in the vegetated sediment of Lake Parentis-Biscarrosse. Some stations were sampled twice in two different seasons; in April 2014 and in January 2015 for the two northern lakes, and in spring and summer 2016 for the two southern lakes. Bottom water in contact with sediment cores was always oxic. Core sampling was carried out by scuba divers using 10-cm-diameter polycarbonate tubes. Sealed cores were immediately brought to the shore and cut horizontally into several subsamples for pore water extraction. Vertical subsampling and pore water extraction from lake sediment required specific approaches. We distinguished broadly two kinds of sediment: sandy permeable sediments and very fluid organic-rich muds. This latter sediment type was subsampled with a vertical resolution of 2 cm using Tygon tubing connected to a 100-mL syringe. Sandy permeable sediment was collected using a PVC tube pre-cut in the vertical dimension. The core was laid horizontally on its side and immediately halved into two similar parts. The sediment was cut rapidly into 1-to 2-cm-thick slices. This technique prevented the percolation of pore waters in the vertical dimension during subsampling. For each level of sandy and muddy sediment, a subsample was immediately sealed in a pre-weighed clean vial and frozen under an N2-atmosphere for further analysis of the solid fraction and determination of water content. Another subsample was put into a centrifuge vial.

Pore waters were extracted by centrifugation at 2100 g for 20 min under an inert N2atmosphere. For muddy sediments, the supernatant was immediately filtered (0.2-µm cellulose acetate syringe filter). For sandy sediments, we used 0.2-µm VivaSpin20 centrifuge vials [START_REF] Anschutz | Spectrophotometric determination of phosphate in matrices from sequential leaching of sediments[END_REF]. The possibility that traces of oxygen had affected the pore water concentrations of reduced elements during the slicing and filtration could not be excluded. Our strategy to prevent oxidation of reduced compounds was to handle the samples in less than 30 min from slicing to conditioning. One part of filtered interstitial water was frozen at -18°C, and another part was acidified with HCl at a pH close to 2 to measure dissolved manganese and iron. Soils of the Landes de Gascogne forest were sampled to estimate the geochemical mercury background levels of the area. Soils were collected every 5 cm from the surface to a depth of 1 m at three stations in young to mature forests of the Lake Cazaux-Sanguinet watershed. Lake water and stream waters of the drainage area were collected to measure dissolved sulphate, Fe, and Mn [START_REF] Buquet | Nutrient sequestration in Aquitaine lakes (S France) limits nutrient flux to the coastal zone[END_REF]. A total of 13 streams were sampled every month in 2014 and 2015. Sampling locations were along roads parallel to the lakes at about 1 km from the east shore.

Samples were also collected in headwaters close to agricultural areas. Sediment from the beds of rivers feeding the northernmost lakes was collected manually using 50-mL centrifuge tubes in order to measure total mercury concentrations. Surface lake waters were sampled every month at several stations using a 2-L Niskin bottle. Bottom waters were also sampled at deep stations, where cores were collected on the western side of the lakes. More than 50 samples were collected for each lake. For determination of dissolved sulphate, Fe, and Mn concentration, water was filtered in situ with 0.20-µm cellulose acetate syringe filters. Filtered samples were stored in polypropylene tubes in a fridge after acidification with HCl. Solutes were analysed within 3 weeks.

Analyses

Fish

Fish dissection was performed within 48 h in laboratory conditions. Dorsal muscle tissue free of skin was dissected and frozen then freeze-dried and crushed. Based on the fact that size is correlated with the age of the individuals, only zander between 50 and 70 cm (standard length), pike between 50 and 70 cm and perch between 18 and 23 cm were considered to compare Hg concentrations between lakes.

Solid fraction

The grain-size distribution was measured using a Malvern laser diffraction particle size analyser. For organic sediments, granulometry was carried out after organic matter removal with H2O2 to recover only the mineral particulate fraction. The bulk mineralogy was determined for this fraction by X-ray diffraction using a Siemens D500 X-ray diffractometer with CuKα radiation (1.5405-Å wavelength). Scans were done from 5° to 80° 2θ at 0.02° s -1 , using a 40-kV accelerating voltage and 30-mA current. Peak identification and relative abundance estimates of minerals were determined using EVA interpretation software and the DIFFRAC software package.

Particulate carbon and total sulphur were measured on freeze-dried samples by infrared spectroscopy (LECO 200 C-S analyser). Organic carbon was measured after removal of carbonates with 2 M HCl from 50 mg of powdered sample [START_REF] Etcheber | Distribution and quality of sedimentary organic matter on the Aquitanian margin (Bay of Biscay)[END_REF]. The detection limit was 0.2‰ for carbon and 0.1‰ for sulphur. The precision of measurement was better than 5%. Ascorbate solution was used to obtain the most reactive particulate Fe(III) and Mn(III,IV) fractions (Fe-asc, Mnasc) [START_REF] Kostka | Partitioning and speciation of solid phase iron in saltmarsh sediments[END_REF]. Iron and manganese content was analysed colorimetrically according to standardized techniques (Anschutz and Deborde 2016) with ± 5% precision.

Total mercury concentration was determined in 100 mg dry and crushed sediment and in fish muscle by cold vapour atomic absorption spectrometry after incineration and amalgamation, using an Advanced Mercury Analyser (Altec AMA 254). The Teflon grinding bowls for fish and the mortar for sediments were washed with HCl 3% and rinsed with milli-Q water between each sample. The analytical results were quality-checked by analysing international certified reference materials (IAEA 407 for fish and PACS-2 and MESS-3 for sediment) after each set of 10 samples. For this method, precision was ± 5% and the detection limit was 0.1 ng Hg.

Methyl mercury content in solid sediment was determined in samples of eight chosen cores collected in winter or early spring, one organic sediment and one sandy sediment for each lake.

Measurements were performed by gas chromatography coupled to inductively coupled plasma mass spectrometry (Trace Ultra GC-XII Series ICPMS, Thermofisher). Sample preparation and analytical protocol were adapted from [START_REF] Bouchet | MeHg production and export from intertidal sediments to the water column of a tidal lagoon (Arcachon Bay, France)[END_REF] and [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF]. Mercury species were extracted from 0.25 g of dry and crushed sediment in 5 mL of 6N ultrapure grade HNO3 solution using a microwave system (Discover SP-D, CEM). Digestion was achieved in CEM Pyrex vessels by 1 min of warming up to 75 °C and 3 min at 75 °C with magnetic agitation to homogenize the samples. Prior to GC injection, sample extracts were ethylated at pH 4 (NaBEt4, 5% v:v, Merseburger Spezialchemikalien), in order to produce volatile ethylated forms of Hg, then extracted in isooctane by mechanical shaking (elliptic table, 20 min) and readily separated by gas chromatography.

Quantification was performed by species-specific isotope dilution by spiking known amounts and concentrations of isotopically enriched standard solutions (MM 201 Hg and 199 Hg(II), ISC) (Rodriguez Martin-Doimeadios et al., 2004). The detection limit of the method was 0.03 ng Hg g -1 . The analytical results were continuously quality-checked by analysing reference materials certified for Hg speciation (IAEA-405 Estuarine Sediment). Relative standard deviation of MeHg concentration has been evaluated to 1.7%, from triplicate preparations and analyses of the reference material.

Dissolved compounds

The sediment water content was determined from the weight difference between wet and dry sediments. Dissolved iron (Fe 2+ ) was analysed using the colorimetric method by adding a ferrozine solution in an aliquot [START_REF] Stookey | Ferrozine-a new spectrophotometric reagent for iron[END_REF]. Dissolved manganese (Mn 2+ and Mn 3+ ) was determined colorimetrically using a Cd-TCPP complex according to [START_REF] Madison | Simultaneous determination of soluble manganese(III), manganese(II) and total manganese in natural (pore)waters[END_REF]. Dissolved Fe and Mn were determined with ± 5% precision. Dissolved sulphate was analysed by a nephelometric method based on BaSO4 precipitation using BaCl2 [START_REF] Rodier | L'analyse de l'eau, eaux naturelles, eaux résiduaires[END_REF]. The detection limit was 10 µM, and the precision of replicate samples was better than 8%.

Results

Mercury in fish

We observed the highest average total Hg concentrations in carnivorous fish from Lake Carcans-Hourtin (Table 2), with a maximum value of 6.238 mg kg -1 dw. Total Hg concentrations were two to five times lower in fish collected from Lake Parentis-Biscarrosse. We measured intermediate values in fish from Lake Lacanau and Lake Cazaux-Sanguinet. Therefore, the data revealed a decreasing gradient from north to south for total Hg in piscivorous species.

Sedimentary cover of lakes

Sediment mapping

Sediment type was strongly influenced by bathymetry. Therefore, we used bathymetry to draw the sediment map from the grab and core observations (Fig. 2). Deep areas in the western parts of the lakes presented muddy deposits. These deposits had a homogeneous appearance and a very soft consistency. The water content of this substrate was greater than 95%, making the sediment texture very fluffy. These sediments were highly organic deposits. Organic deposits were found below 5 m depth in lakes Carcans-Hourtin and Lacanau, below 7 m in Lake Cazaux-Sanguinet, and below 10 m in Lake Parentis-Biscarrosse. For these two last lakes, deep areas also presented a mixture of these organic deposits with sand, called sandy organic deposits or organic sands, depending on the proportion of sand and organic carbon (Fig. 2). Organic sands were present in sub-lacustrine channels.

These channels are the paleo-valley of the rivers that flowed to the ocean before the formation of lakes through the development of coastal dunes in the last thousand years [START_REF] Buquet | Nutrient sequestration in Aquitaine lakes (S France) limits nutrient flux to the coastal zone[END_REF]. Shallow zones presented sands with shades of green, yellow, black, or grey depending on the content of iron oxides or organic matter. The water content of sands was 20% in weight.

Vegetated areas of lakes Lacanau and Parentis-Biscarrosse consisted of sandy sediments. In sheltered areas, 2 to 10 cm of muddy sediment covered the sand.

Grain size

Sand grain size after organic matter removal with H2O2 was very homogeneous, with a median size of 362 µm, a standard deviation (SD) of 49 µm, and a standard error of the mean (s.e.) of 7 µm.

The sand was the same as the soils of the lake catchment (Fig. 3). The grain size of the mineral fraction of organic deposits was centred between 10 and 20 µm (Fig. 3). X-ray diffraction indicated that the mineral fraction consisted of quartz, clay minerals (illite and kaolinite), and feldspars. Carbonate minerals were not detected.

Total carbon and total sulphur

Particulate organic carbon (POC) and total carbon (TC) measurements on grab and core sediments gave the same concentration, indicating that TC consisted exclusively of POC and that inorganic carbonate was absent or very scarce. TC concentration ranged from 10% to 30% in organic deposits. Averaged TC concentrations (± SD) in organic sediments were 15.8% (± 3.1), 17% (± 1.4), 15.5% (± 1.7), and 15.2% (± 0.9) for lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse, respectively (Fig. 4). Concentrations ranged from 3% to 10% in sandy organic deposits, and from 1% to 3% in organic sands. TC was below 1% in sands.

Total sulphur (TS) content was also dependent on the type of sediment. TS ranged from 1% to 4% in organic deposits (Fig. 4). The mean concentrations were close to 2.5% (± 0.8% SD) except for organic sediments of Lake Parentis-Biscarrosse, in which the value was 1.2%. TS ranged from 0.1% to 1% in sandy organic deposits and organic sands. TS was below 0.1% in sands.

Particulate total mercury (HgTp)

HgTp concentrations were closely related to the nature of sediment and the organic carbon concentrations. Classes of mercury concentration fitted classes of TC content (Fig. 2). HgTp concentrations ranged from 100 to 420 µg kg -1 in organic deposits (means of 230 (± 82 SD), 203 (± 60 SD) 206 (± 68 SD), and 142 µg kg -1 (± 53 SD) for lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse, respectively). HgTp concentrations in Lake Parentis-Biscarrosse were significantly lower (ANOVA test) than in other lakes (Fig. 4). HgTp concentrations ranged from 20 to 100 µg kg -1 in sandy organic deposits. HgTp concentrations ranged from 4 to 20 µg kg -1 in organic sands. Finally, HgTp concentrations ranged from below the detection limit to 10 µg kg -1 in sands (Fig. 2).

Geochemical background levels

Sand samples from forest soil east of Lake Cazaux-Sanguinet had the same particle size distribution as the sandy sediments from lakes (Fig. 3). HgTp concentrations ranged from 0.5 to 21 µg kg -1 . Highest values were associated with soil samples enriched in organic carbon. However, HgTp values were not significantly different (ANOVA test) in lake sands and in drainage basin soils (Fig. 2).

Fine sediments collected from the beds of streams that feed Lake Carcans-Hourtin and Lake Lacanau had a mean HgTp concentration of 2.3 ± 1.8 µg kg -1 , which was much lower than values measured in lake organic sediments.

Lake sedimentary column

Cores of organic deposits had a TC concentration between 10% and 30% (Fig. 1S, Supplementary Material), like sediment grabs. TC decreased slightly with depth, except for Lake Carcans-Hourtin, which showed a sharp decrease in TC below 10 cm depth from 30% to 15%. TS concentrations were similar to values measured in grabs. Concentrations increased with depth, except in organic muds of Lake Carcans-Hourtin, where sediments below 25 cm depth were less enriched in sulphur than those above. Iron and manganese oxide concentrations extracted by leaching with ascorbate decreased with depth. Ascorbate-extracted iron was two-to three-fold higher in muddy sediments of Lake Parentis-Biscarrosse than in other lakes. HgTp concentrations were between 100 and 300 µg kg -1 in organic sediments. Shallow vegetated sediment from Lake Parentis-Biscarrosse contained up to 80 µg kg -1 HgTp in the muddy layer of the superficial sediment. This muddy layer of vegetated sediment contained about 10% TC.

Methylmercury concentrations of organic sediments were maximal in the first centimetres below the water-sediment interface (Fig. 1S, Supplementary Material). There was an increasing gradient in MeHg from the southern lakes to the northern lakes: average vertical concentrations in the top 10 cm below the sediment-water interface were 4.61 (± 2.6 SD) µg kg -1 for Lake Carcans-Hourtin, 5.14 (± 2.8 SD) µg kg -1 for Lake Lacanau, 1.56 (± 0.14 SD) µg kg -1 for Lake Cazaux-Sanguinet, and 1.1 (± 0.32 SD) µg kg -1 for Lake Parentis-Biscarrosse, which represented 2.53% (± 1.0), 1.69% (± 0.7), 0.55% (± 0.1), and 0.53% (± 0.1) of HgTp, respectively. Concentrations of TC, mercury, Fe and Mn oxyhydroxides, and sulphides were lower by a factor of 10 to 100 in sandy sediment cores compared to muddy sediments. Contrary to observations for organic sediments, mercury decreased with depth. MeHg concentrations were often below the detection limit of the GC-ICPMS method (i.e. 0.03 µg Hg kg -1 ).

Lake water and pore waters

Sandy sediment depleted in organic carbon (e.g, Fig. 1S-A and 1S-G, Supplementary Material) did not show a pronounced gradient of dissolved redox compounds below the sediment surface, except a small sulphate decrease with depth. Pore water dissolved Fe and Mn remained at concentrations similar to those measured in lake waters. Sandy sediments with an organic carbon content above 0.5% showed more pronounced redox gradients, with appearance of reduced dissolved species and rapid loss of sulphate in the first centimetres below the interface (Fig. 1S-H and 1S-J, Supplementary Material). Dissolved iron and manganese concentrations were present from the first centimetre of the sedimentary pore water column in cores that were collected in muddy sediments.

Dissolved sulphate concentrations decreased below the sediment surface in organic sediment pore water to reach the detection limit a few centimetres below the interface. Dissolved sulphate concentrations of overlying water differed depending on the lake. Mean water sulphate concentrations were 328 (± 28 SD), 221 (± 26 SD), 100 (± 19 SD), and 82 (± 17 SD) µM for lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse, respectively (N > 50 for each lake). Therefore, there was a decreasing north-south gradient of sulphate concentrations. Mean dissolved sulphate concentration of the stream waters that feed the lakes was lower than 150 µM, except for the Lake Carcans-Hourtin catchment, where streams contained up to 600 µM sulphate (Fig. 5). For this lake, sulphate concentrations were the highest in head waters close to agricultural areas. Values above 1000 µM were measured in upstream waters of the Caillava river, which drain large maize fields (Fig. 5).

Head waters that drain maize fields of the Cazaux-Sanguinet catchment were also enriched in dissolved sulphate, with mean concentrations of 300 µM.

Discussion

Mercury distribution, diagenetic processes, and mercury mobility in sediments

Mercury distribution and background concentrations

The concentration of HgTp was close to 200 µg kg -1 in organic muds and below 20 µg kg -1 in sandy sediments. Concentrations were below 2 µg kg -1 in sands poor in organic carbon. Sandy sediments had concentrations identical to those measured in forest sandy soil from the lake drainage basin. Sands were deposited before the formation of Aquitaine lakes, whereas the fine particles present in organic deposits were autochthonous sediments. Higher HgTp values in organic muds may result from a grain-size effect: deep parts of lakes accumulate fine-grained particles that consist of fine quartz and clay minerals, which are probably enriched in Hg relative to sandy sediments [START_REF] Bloom | Distribution of silver, mercury, lead, copper and cadmium in central puget sound sediments[END_REF]. However, grain size alone cannot explain the difference, because mercury has a strong affinity with organic carbon, sulphur, and Fe/Mn oxides [START_REF] Feyte | In situ adsorption of mercury, methylmercury and other elements by iron oxyhydroxides and organic matter in lake sediments[END_REF], which are highly enriched in muddy sediment. The difference in HgTp content between sands, sands enriched in organic matter, and muds is most likely explained by the organic matter content (Fig. 2), suggesting that organic matter is the initial carrier phase of Hg that accumulates in the sediment.

Diagenetic processes

Mercury has affinity with organic matter, iron/manganese oxyhydroxides, and sulphides. Therefore, it is strongly influenced by diagenetic processes [START_REF] Matty | Early Diagenesis of Mercury in the Laurentian Great Lakes[END_REF]. These processes are directly linked to benthic organic matter mineralization. Mineralization of organic matter is the result of its oxidation, carried out in presence of microorganisms, and is accompanied by reduction of electron acceptors according to a sequence of reactions where O2 is used preferentially, then nitrate when the sediment becomes depleted in dissolved oxygen. In anoxic sediments, mineralization consumes oxides and oxyhydroxides of manganese (MnO2, MnO(OH)) and iron (Fe(OH)3) present in the solid fraction of sediments, and dissolved sulphate (SO4 2-) and CO2, which generally represent both the main electron acceptors in anoxic environments [START_REF] Stumm | Aquatic chemistry[END_REF]. Anoxic sediment is generally enriched in particulate sulphur because of the precipitation of sulphide minerals as a result of sulphate reduction.

Redox species profiles in the cores (Fig. 1S, Supplementary Material) agreed with a typical diagenetic sequence. Iron and manganese oxides extracted with an ascorbate solution have the highest concentrations at the sediment surface and decrease with depth. Dissolved reduced iron and manganese appear in the top core samples. This indicates that sediments are anoxic from the first centimetre of the sediment column. Sulphate concentration decreases rapidly and reaches a value below the detection limit a few centimetres below the sediment surface, which means that the sulphate reduction process occurs effectively. Sulphide precipitate is produced as FeS, as suggested by the high particulate S content in organic deposits and the decreasing pore water iron concentration with depth.

It must be noted that the concentration gradients in organic sediments are stronger than gradients in sandy sediments, suggesting that diagenetic processes are more intense in muddy sediments. However, the decrease and disappearance of sulphate in sandy cores suggest that the small amount of organic matter present in sands is labile.

Mercury diagenesis

Mercury transformation in sediments depends on many biogeochemical processes, which may affect the ability of mercury to be methylated, or to migrate from sediment to the water column [START_REF] Schäfer | Mercury methylation in the sediments of a macrotidal estuary (Gironde Estuary, south-west France)[END_REF]. Mercury mobility depends on its speciation between solid and dissolved phases.

This distribution is intimately coupled to organic matter mineralization as Hg distribution is influenced by the presence of Mn/Fe oxyhydroxides and sulphide minerals. In oxic sediment layers, Hg mobility is generally low because it is essentially associated with the solid phase by adsorption or precipitation with Fe/Mn oxyhydroxides [START_REF] Feyte | In situ adsorption of mercury, methylmercury and other elements by iron oxyhydroxides and organic matter in lake sediments[END_REF]. During the organic matter mineralization process, Fe/Mn oxyhydroxides may be reduced, and associated Hg is released [START_REF] Matty | Early Diagenesis of Mercury in the Laurentian Great Lakes[END_REF]. In the sediment cores studied, the decrease in Fe/Mn oxyhydroxide concentrations with depth in lakes Carcans-Hourtin, Lacanau, and Cazaux-Sanguinet can be interpreted as metal oxide reduction. In Lake Parentis-Biscarrosse, the oxide concentration remains high in all organic cores, suggesting that Hg mobility is limited in this lake. In anoxic sediments, sulphate reduction leads to sulphide production, for which Hg has a strong affinity. Mercury precipitates as HgS or it is absorbed on FeS and included in pyrite [START_REF] Fitzgerald | Geochemistry of mercury in the environment[END_REF]. Grab samples show that the Hg concentration increases involved in MSR. Communities of Fe-and sulphate-reducing bacteria are also involved in MeHg demethylation processes [START_REF] Oremland | Methylmercury decomposition in sediments and bacterial cultures: involvement of methanogens and sulfate reducers in oxidative demethylation (English)[END_REF][START_REF] Bouchet | MeHg production and export from intertidal sediments to the water column of a tidal lagoon (Arcachon Bay, France)[END_REF]. It is the balance between methylation and demethylation in sediment that dictates the standing pool of MeHg (Rodríguez [START_REF] Martín-Doimeadios | Mercury methylation/demethylation and volatilization pathways in estuarine sediment slurries using species specific enriched stable isotopes[END_REF]. The progressive decrease in MeHg with depth of the sedimentary column could be related to the dominance of demethylation processes in the deep sediment, when sulphate becomes depleted [START_REF] Pak | Mercury methylation and demethylation in anoxic lake sediments and by strictly anaerobic bacteria[END_REF].

Spatial distribution

Organic sediments of the four lakes have different MeHg concentrations. The fraction of total Hg present as MeHg (%MeHg) describes a positive south-north gradient from 0.5% in Lake Parentis-Biscarrosse to 2.5% in Lake Carcans-Hourtin (Fig. 7). The proportions between 0.5% and 0.6% obtained in the southernmost lakes are consistent with those found in the literature for lacustrine or coastal systems (Table 3). Generally, MeHg concentration in sediment is less than 1% of HgTp [START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF]. MeHg proportions in northern lake sediments are high (especially Lake Carcans-Hourtin), which means that the methylation potential is high in these lake sediments.

Main factors that are known to influence the methylation rate are Hg(II) availability and bacteria activity within the sedimentary column. Sulphate inputs stimulate MSR activity. As sulphate concentrations differ between lakes, the high proportion of MeHg in northern lakes, more particularly in Lake Carcans-Hourtin, can be related to high methylation potential as sulphate concentrations are the highest (Fig. 7). Previous studies have found that sulphate addition stimulates net methylation rates in fresh water sediments (e.g, [START_REF] Gilmour | Sulfate Stimulation of mercury methylation in freshwater sediments[END_REF][START_REF] Åkerblom | Significant interaction effects from sulfate deposition and climate on sulfur concentrations constitute major controls on methylmercury production in peatlands[END_REF]. Sulphide from sulphate reduction is known to have two effects on the availability of Hg(II). At low concentrations, sulphide promotes methylation. By contrast, for higher concentrations, as may be the case in marine sediments, sulphide limits the bioavailability of Hg(II) to be methylated [START_REF] Benoit | Behavior of mercury in the Patuxent River estuary[END_REF][START_REF] Hsu-Kim | Mechanisms regulating mercury bioavailability for methylating microorganisms in the aquatic environment: A critical review[END_REF][START_REF] Johnson | Methylmercury production in a chronically sulfate-impacted sub-boreal wetland[END_REF]. This suggests that there is a range of sulphate concentration which is optimal for Hg methylation, above which methylation is inhibited. A broad range in the sulphate concentration associated with maximum efficiency of Hg methylation has been observed. For example, [START_REF] Orem | Modeling sulfate transport and distribution and methylmercury production associated with Aquifer Storage and Recovery implementation in the Everglades Protection Area[END_REF] observed maximum MeHg concentrations at sulphate concentrations of 2 and 10-15 mg L -1 in the Everglades Protection Area. Peak MeHg was observed in wetland mesocosms with a sulphate concentration of 59 and 93 mg L -1 [START_REF] Myrbo | Increase in nutrients, mercury, and methylmercury as a consequence of elevated sulfate reduction to sulfide in experimental wetland mesocosms[END_REF]. In the case of Aquitaine lake muddy sediments, MeHg concentrations increase with sulphate, suggesting that MSR is not limited, and that produced sulphide does not inhibit Hg availability. The availability of dissolved sulphide produced by MSR will depend on iron oxide availability. The reactive fraction of iron oxides determined by the ascorbate reagent is above 100 µmol g -1 in the surface sediments of the four lakes, which represents at least, when considering sediment water content, 4000 µmol L -1 , suggesting that the sulphide produced is efficiently trapped as Fe sulphide phases with this abundant available reactive Fe.

Temperature is an important control factor for methylation process. Low temperatures will slow down methylation [START_REF] Hintelmann | Levels of total mercury and methylmercury compounds in sediments of the polluted Elbe River: influence of seasonally and spatially varying environmental factors[END_REF][START_REF] Ramlal | Sites of Methyl Mercury Production in Remote Canadian Shield[END_REF]. The cores studied were sampled in different seasons, but MeHg was measured only in cores collected in winter and early spring. Higher concentrations of MeHg in summer may be expected, although the effect of temperature on biotic demethylation is not well known. Gradients of dissolved sulphate between the water column and the sediment were sharper when the water temperature was the highest at a given station sampled in two seasons, suggesting that the sulphate reduction rate, and the related methylation, was more efficient in summer and autumn.

The gradient of mercury contamination in carnivorous fish muscles between the four lakes (Table 2) fits well with sediment MeHg concentrations, since the highest levels were measured in fish from northern lakes. Therefore, the muddy sediment compartment may be one of the main sources of MeHg which accumulates in the trophic chain (e.g. [START_REF] Hammerschmidt | Biogeochemistry of methylmercury in sediments of long island sound[END_REF]. Periphyton microbial communities of aquatic macrophytes of Lake Cazaux-Sanguinet have also been recognized to potentially methylate Hg (Gentès et al., 2013). This study was based on experimental batches placed under high environmental Hg concentrations and anoxic conditions. Hence, through dietary bioaccumulation, the MeHg in higher trophic levels within Aquitaine lakes may be attributed to MeHg produced in anoxic muds, as observed in the present study and in the periphyton (Gentès et al., 2013), both being limited by sulphate.

Sulphate from lake waters may originate from several sources: atmospheric inputs from rain and ocean spray, rocks from the drainage area, and anthropogenic inputs. The distance from lake to ocean is the same for the four lakes studied, suggesting that the atmospheric sources by spray are similar. The lake catchment geology is also the same for the four lakes. The major difference between lake catchments is the land use. The drainage area of Lake Carcans-Hourtin has the highest proportion of agriculture areas (Fig. 1). Tributaries of this lake are enriched in dissolved sulphate, with a concentration as high as 1200 µM in ditches located upstream, in the cultivated areas (Fig. 6). Head waters collected in ditches close to agriculture surfaces of the Lake Cazaux-Sanguinet catchment are enriched in dissolved sulphate compared to waters collected downstream, but the concentrations remain below 300 µM. Thus, agricultural activity is the main source of sulphate. Discussions with farmers informed us of the fact that sulphate was supplied indirectly to croplands through liming, which allows control of soil pH. Lime comes from limestone or dolomite quarries, and in some cases in the recent past from neighbouring paper mill plants that produce lime sludge as a by-product.

Depending on lime origin, the sulphate content can be high, and lime can become a source of sulphate for lakes.

MeHg diffusion and bioavailability

Our study points out that MeHg concentration is related to sulphate concentration. Methyl mercury produced in the sediment can be transferred to the water column through molecular diffusion and resuspension of surface sediments. Hence, the amount of MeHg diffusing into the water column and thus the amount bioavailable to pelagic organisms depends, among other things, on the proportion of lake floor carpeted with organic sediments. Organic deposits account for 21%, 20%, 19%, and 18% of the surface area of lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse, respectively. These proportions are close and suggest that the proportion of organic sediment is not the variable that explains the gradient of fish contamination. Bioturbation and bioirrigation may affect the transport of Hg from sediment to water [START_REF] Benoit | Infaunal burrow densities and sediment monomethylmercury distributions in Boston Harbor, Massachusetts[END_REF]. We observed the presence of chironomid larvae in some grabs from the four lakes. The density of these organisms was low, with zero to three individuals per grab. Even if the density of benthic organisms was low, burrowing organisms may assimilate MeHg directly from underlying anoxic sediments and become vectors of MeHg to pelagic predators [START_REF] Gagnon | Diagenetic behavior of methylmercury inorganicrich coastal sediments[END_REF]. Finally, despite the occurrence of episodic bottom-water anoxia during the summer in Lake Parentis-Biscarrosse, this lake remains the one in which the fish are less contaminated with Hg. The methylation of Hg in a polymictic lake can be shifted from the sediment to the anoxic water column, because sulphate reduction is shifted in the same way to the water column [START_REF] Ramlal | Sites of Methyl Mercury Production in Remote Canadian Shield[END_REF]Wartras et al., 1995). This suggests that sulphate availability remains a key environmental parameter for Hg methylation, wherever sulphate reduction occurs, in the water column or at the sediment surface.

Sources of Hg

Land use is not impacted by activities that may represent point sources of Hg. HgTp concentrations are similar in lake sandy sediments and in watershed sandy soils, with values below 20 µg kg -1 , suggesting that the geological background level is low. Moreover, we measured HgTp concentrations of between 1 and 7 µg kg -1 in fine sediments of river beds, which is low compared to the 200 µg kg -1 measured in lakes muds, suggesting that the contribution of Hg from lake catchment is negligible. Hg distribution in lakes depends exclusively on sediment type, not on geography: there is no zone that presents a peak of HgTp concentration. This suggests the absence of a point source in lakes. Several lines of evidence suggest that Hg that accumulates in muddy sediment is mainly of atmospheric origin. Considering that the superposition principle applies to organic sediment deposits, the decrease in Hg concentrations when approaching the water-sediment interface (Fig. 1S, Supplementary Material) may be explained by the decrease in atmospheric emission in European countries in recent decades (UNEP, 2013).

South-western France is not a hot spot for atmospheric Hg emissions [START_REF] Colette | Air pollution trends in the EMEP region between 1990 and 2012[END_REF]. In the light of the concentrations measured in lake sediments, Hg fluxes are not alarming. Average atmospheric Hg flux on the Aquitaine coast is estimated by modelling as 10 µg m -2 yr -1 for the year 2001 [START_REF] Roustan | Modélisation du mercure, du plomb et du cadmium à l'échelle européenne[END_REF]. Such a flux represents 620, 200, 580, and 350 g yr -1 for lakes Carcans-Hourtin, Lacanau, Cazaux-Sanguinet, and Parentis-Biscarrosse, respectively. Considering that all Hg that reaches the lake surface is trapped in organic matter that accumulates in organic sediments, considering also the mean HgTp of each lake and the particle content of organic sediment (40 g L -1 ), we found that 1 cm of deposit contains between 1 and 1.8 years of Hg deposition. This suggests that the mean sedimentation rate is between 0.6 and 1.0 cm yr -1 . A very similar value was found for lakes Lacanau and Carcans-Hourtin using a mass balance based on dissolved inorganic nitrogen retention [START_REF] Buquet | Nutrient sequestration in Aquitaine lakes (S France) limits nutrient flux to the coastal zone[END_REF]. The higher sedimentation rate found here (1 cm yr -1 ) is for Lake Parentis-Biscarrosse. This lake has a higher phosphorus content than the other lakes [START_REF] Cellamare | Ecological assessment of French Atlantic lakes based on phytoplankton, phytobenthos and macrophytes[END_REF], which results in higher primary production. Eutrophication of the lake may explain a higher sedimentation rate of organic matter. As a consequence, the concentration of HgTp is lower in the muds of Lake Parentis-Biscarrosse than that of other lakes, even if the atmospheric flux is the same, due to a dilution effect.

Conclusion

Our study presents new results from Aquitaine lake sediments. We have established the first map of sediment distribution and we have determined several physicochemical properties of these sediments. More particularly, we showed that the bottom of the four lakes consists of sands. Sandy sediments are covered with organic muds, which accumulate only in the deepest parts of lakes and in shallow densely vegetated areas. The highest Hg concentrations are encountered in these organic deposits. However, they do not exceed reference thresholds for sediments (1100 µg kg -1 for the probable effect concentration; [START_REF] Macdonald | Development and evaluation of consensus-based sediment quality guidelines for freshwater ecosystems[END_REF]. Organic sediment traps Hg in the four lakes, which is most likely of background diffusive atmospheric origin. These sediments are hot spots for Hg methylation, due to sulphate reduction processes.

The mercury concentration gradient in the muscles of carnivorous fish is the same as the methyl mercury concentration gradient of lake sediment. This suggests that the sedimentary compartment could be a MeHg source for the water column, resulting in its accumulation along the trophic chain. Methylation rate depends on sulphate reduction activity. This activity is limited by sulphate availability. Lake Carcans-Hourtin is the most concentrated in sulphate. Lake Lacanau, whose waters come partly from Lake Carcans-Hourtin, is enriched in sulphate by this very fact. Agricultural activity is the main source of sulphate in the Lake Carcans-Hourtin drainage basin. This excess sulphate that is leached from soils instead of being absorbed and exported by plants originates likely from liming. Therefore, the use of sulphate-rich lime in the headwater drainage area has an indirect consequence on carnivorous fish Hg content in lakes located at the outlet of the catchment, because sulphate favours methylation of the background flux of atmospheric Hg in anoxic environments of lakes, such as autochthonous organic sediments, as seen in this study, or in other compartments, such as aquatic plant periphyton (e.g. Gentès et al., 2013). One question that remains unresolved focuses on the pathway that transmits MeHg produced from the sediment to fish. For that the distribution of Hg in fish and in the food web is presently conducted in Aquitaine lakes.

Figure captions

Fig 1

 1 Fig 1 Location map of Aquitaine coastal lakes and drainage basin land use. Small rings: Location of river sampling for sulphate measurements; black star: location of soil sampling.

Fig 2 Fig 3

 23 Fig 2 Sediment mapping of Aquitaine lakes. Black dots and stars show the location of grab and core samples in which Hg has been measured, respectively. Red dots are additional grabs that have been collected to validate the sediment mapping based on bathymetry. The range of total carbon (TC) concentrations and particulate total mercury (HgTp) levels for each sediment class is indicated in the legend. Fig 3 Average grain size distribution of lake sandy sediment and catchment soils (right panel), and of lake organic deposits after organic matter removal with H2O2 (left panel).

Fig 4

 4 Fig 4 Mean concentration of total carbon (TC), total sulphur (TS) and total Hg (HgTp) in organic deposits (white bars) and in sandy sediments and catchment soils (grey bars) from the four Aquitaine lakes. Error bars are standard deviations. Number of samples is indicated.

Fig 5

 5 Fig 5 Mean sulphate concentrations in streams that feed the lakes. Error bars are standard deviations. Number of samples indicated in brackets. See Figure 1 for sampling location.

Fig 6

 6 Fig 6 Total Hg (HgTp) concentrations vs total sulphur (TS) concentrations in sediment grabs collected in Aquitaine lakes.

Fig 7

 7 Fig 7 Relationship between the proportion (%) of MeHg relative to HgTp in organic sediments and average sulphate concentrations in lake water column. Error bars are standard deviations (N=5 for %MeHg, and N=15 for sulphate)

Acknowledgements

We thank Cécile Bossy, Lionel Dutruch, Rémy Sinays, and Thierry Corrège for their assistance during field and laboratory work. We acknowledge the assistance given by Frank Quenault. This research was founded by the project CLAQH (Agence de l'eau Adour-Garonne, Région Nouvelle Aquitaine), the Syndicat Intercommunal d'Aménagement des Eaux du Bassin Versant des Etangs du Littoral Girondin (SIAEBVELG), the LITTOLAC project supported by the French national program INSU-EC2CO-BIOHEFECT, and the project PSDR AQUAVIT (Région Nouvelle Aquitaine and INRA). This study has been carried out in the frame of the Investments for the future Program, within the Cluster of Excellence COTE (ANR-10-LABEX-45).

when total S concentration increases as well (Fig. 6). This trend also occurs in organic sediment cores.

Sulphur is associated with organic matter either as an initial compound or as a result of the sulphurization by sulphide of organic matter during diagenesis [START_REF] Urban | Addition of sulfur to organic matter during early diagenesis of lake sediments[END_REF]. Therefore, high organic matter concentrations correspond to high sulphur concentrations. [START_REF] Urban | Addition of sulfur to organic matter during early diagenesis of lake sediments[END_REF] found an averaged C : S ratio of organic matter between 167 and 198 based on sediment cores from 50 lakes. For lake sediments with the highest concentrations of organic carbon they found a maximum organic sulphur concentration of 260 µmol g -1 (0.83%), which is below the concentration of sulphur measured in muddy sediments (Fig. 6). This indicates that most of the sedimentary sulphur occurs as particulate sulphide, and that particulate sulphides are major carrier phases of Hg in organic sediments. Organic matter itself presents many adsorption sites for Hg, which plays a role in Hg mobility [START_REF] Benoit | Behavior of mercury in the Patuxent River estuary[END_REF]. This probably explains why organic sediments are enriched in Hg relative to organic-poor sandy sediments (Fig. 2). Organic matter is mineralized in sediments through early diagenesis processes. In anoxic lake sediments, Hg initially trapped in the fraction of organic matter that is consumed is most likely released into the interstitial waters and then quickly trapped by sulphides when sulphate reduction occurs [START_REF] Benoit | Sulfide controls on mercury speciation and bioavailability to methylating bacteria in sediment pore waters[END_REF].

Methylation

Vertical distribution

Organic sediments of Lake Carcans-Hourtin and Lake Lacanau show the highest concentrations of MeHg at the depth where the decreasing gradient of dissolved sulphate occurs. Inorganic Hg methylation takes place during Hg(II) absorption by some anaerobic microorganisms which methylate Hg in their cytoplasm [START_REF] Benoit | Sulfide controls on mercury speciation and bioavailability to methylating bacteria in sediment pore waters[END_REF][START_REF] Gilmour | Methylmercury concentrations and production rates across a trophic gradient in the northern Everglades[END_REF]. Microbial sulphate reduction (MSR) is recognized to be the most common pathway leading to mercury methylation in sediments [START_REF] Barkay | Microbial transformations of mercury: potentials, challenges, and achievements in controlling mercury toxicity in the environment[END_REF][START_REF] Compeau | Sulfate-reducing bacteria: principal methylators of mercury in anoxic estuarine sediment[END_REF]. Other anaerobic microorganisms such as iron-reducing bacteria are able to methylate Hg [START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF].

Diagenetic profiles of organic cores show that anaerobic processes occur at the depth of the maximum MeHg concentrations (Fig. 1S, Supplementary Material). This suggests that the predominant process of mercury methylation is carried out by a consortium of anaerobic microorganisms, including those


W J Nawrocki
email: w.j.nawrocki@vu.nl

B Bailleul

D Picot

P Cardol

F Rappaport

F.-A Wollman

P Joliot

The Mechanism of Cyclic Electron Flow

Keywords: Photosynthesis, biophysics, plastoquinone, cytochrome b6f, photosystem I, cyclic electron flow Highlights

Apart from the canonical light-driven linear electron flow (LEF) from water to CO2, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.

Linear and cyclic electron flow

Photosynthesis consists of a photoinduced linear electron flow (LEF), where electrons from water are transferred to NADP + . The electron-coupled proton translocation generates a proton motive force across the thylakoid membrane that is used by the CF1-F0 ATP synthase to form ATP. Photoproduced ATP and NAD(P)H then allow CO2 fixation by the Calvin-Benson-Bassham cycle. However, alternative electron transfer pathways have been documented in photosynthesis. Most notable among them are water-to-water cycles and cyclic electron flow (CEF) around Photosystem I (PSI). All these alternative pathways translocate extra protons into the lumen without producing reducing compounds, thus increasing the ATP/NAD(P)H ratio. The dynamic changes between the rates of LEF and alternative pathways are thus critical in conditions where i) NAD(P)H accumulates when the electron transfer chain is kinetically limited by the NAD(P)H oxidation pathways (carbon fixation and others), and ii) lumen acidification needs to be enhanced to protect the two photosystems via non-photochemical quenching (NPQ) and photosynthetic control at the level of cytochrome b6f (cyt. b6f).

Despite the well-recognized importance of CEF in the regulation of photosynthesis, information about its precise mechanism is still lacking. Not only is our understanding of the regulation of CEF and LEF pathways scarce, but even the cofactors involved in electron transfer from Ferredoxin (Fd) to plastoquinone (PQ) remain elusive. Here, we reassess current knowledge about CEF pathways, in particular from a kinetic and methodological standpoint in the light of our report that PGRL1 is not directly transferring electrons from Fd to PQ during CEF [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF]. We further propose a mechanism that can explain how and why the rate of CEF is not tightly controlled by the redox state of the PQ pool.

Current view on cyclic electron flow

CEF was first identified by the Arnon group ( [START_REF] Tagawa | Role of chloroplast ferredoxin in the energy conversion process of photosynthesis[END_REF] and references therein). These experiments pointed to the existence of cyclic photophosphorylation as a pathway of light-dependent ATP phosphorylation, where the net production or consumption of reducing power was null. Therefore, CEF was defined as a rerouting of reducing equivalents from the acceptor side of PSI back to its donor side. The implication of cyt b6f in CEF, also proposed by Arnon and co-workers, was further confirmed by the sensitivity of CEF to Qo site inhibitors [START_REF] Cleland | Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity[END_REF]. Later attempts to identify specific CEF transporters -in particular through genetic approaches aimed at the isolation of mutants devoid of CEF -fell short but allowed to disclose molecular bases for CEF regulation. These studies led to propose two CEF routes, which are considered by most authors to be responsible for the reduction of PQ by PSI acceptors: the NDH-1/NDH-2-, and PGR5/PGRL1-dependent pathways (e.g. ref. [START_REF] Yamori | Physiological Functions of Cyclic Electron Transport Around Photosystem I in Sustaining Photosynthesis and Plant Growth[END_REF]) (Fig. 1). For the first pathway, the components allowing the transfer of electrons from PSI acceptors to the PQ pool are homologous to the respiratory NAD(P)H:PQ oxidoreductases. In land plant chloroplasts, NDH-1 shares at least 11 subunits with the mitochondrial and bacterial NADH:UQ oxidoreductase (complex I) [START_REF] Peltier | NDH-1 and NDH-2 Plastoquinone Reductases in Oxygenic Photosynthesis[END_REF]. Chloroplast NDH-1 complex however lacks an NADH binding module and has been proposed to use Fd as a substrate [START_REF] Yamamoto | An Src homology 3 domain-like fold protein forms a ferredoxin binding site for the chloroplast NADH dehydrogenase-like complex in Arabidopsis[END_REF]. It probably pumps additional protons per electron transferred [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF], similarly to mitochondrial and bacterial complexes I. In some microalgae, NDH-2 is present instead of NDH-1. It resembles mitochondrial Ndi1 [START_REF] Iwata | The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water-and lipid-soluble substrates[END_REF], and is a monotopic membrane protein [START_REF] Blobel | Intracellular protein topogenesis[END_REF] that inserts into the stromal leaflet of the thylakoid membrane, and therefore is unable to pump protons to the lumen upon oxidation of its substrate, NAD(P)H [START_REF] Jans | A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas[END_REF]. Yet, the enzymatic pathway allowing the electron transfer from PSI acceptors back to the electron transfer chain upstream cytochrome b6f is still a matter of debate. Any legitimate candidate (enzyme or combination of enzymes) for the closing of the cycle must display a turnover rate in agreement with the highest rates of CEF measured in vivo, i.e. ~100 and ~60 electrons per second per PSI in plants [START_REF] Joliot | Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves[END_REF][START_REF] Joliot | Regulation of cyclic and linear electron flow in higher plants[END_REF] and in green algae (from the companion paper, [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF]).

The role of NDH-1 in CEF is doubtful from many perspectives. It is highly sub-stoichiometric with regards to PSI (1:100 ratio [START_REF] Burrows | Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes[END_REF]), therefore its rate would necessarily need to exceed 10 4 e -.s -1 per complex to sustain transitory rates of CEF in plants in the order of a hundred of electrons per second per PSI [START_REF] Joliot | Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves[END_REF][START_REF] Joliot | Regulation of cyclic and linear electron flow in higher plants[END_REF], a value up to 4 orders of magnitude higher than experimentally measured in vivo for this enzyme [START_REF] Trouillard | Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant[END_REF]. Experimental data shows that the absence of NDH-1 does not alter the qE (pH-dependent NPQ) -and therefore its proton-pumping contribution is negligible -in plants [START_REF] Shikanai | Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I[END_REF], consistent with its measured rate being in the order of only one tenth (0.1) e -.s -1 .PSI -1 in vivo [START_REF] Trouillard | Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant[END_REF]. Similarly, NDH-2 -the algal counterpart of NDH-1 -runs at 2.5 e -.s - 1 .PSI -1 [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF][START_REF] Nawrocki | Chlororespiration controls growth under intermittent light[END_REF], a rate which is far too sluggish to sustain the rate of 60 e -.s -1 .PSI -1 for CEF that we report in the companion paper [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF]. Finally, an NDH-2 mutant only showed limited changes in electron transfer with a slower PQ re-reduction in darkness [START_REF] Jans | A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas[END_REF] or in the light, exclusively when the competing PQ-reducing PSII activity was severely affected [START_REF] Mignolet | Function of the chloroplastic NAD(P)H dehydrogenase Nda2 for H(2) photoproduction in sulphur-deprived Chlamydomonas reinhardtii[END_REF]. It is thus likely that the activity of NDH enzymes is limited to chlororespiration [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF](Fig. 1).

The other most studied putative CEF route is described as "PGR5/PGRL1-dependent". The two molecular actors of this pathway were discovered in mutagenized plants exhibiting distinctly low steady-state qE, and thus ΔpH across the thylakoid membrane, leading to their name (proton gradient regulation 5 and pgr5-like 1, respectively) [START_REF] Munekage | Cyclic electron flow around photosystem I is essential for photosynthesis[END_REF][START_REF] Dalcorso | A Complex Containing PGRL1 and PGR5 Is Involved in the Switch between Linear and Cyclic Electron Flow in Arabidopsis[END_REF]. However, despite a plethora of proof of their involvement in the regulation of CEF [START_REF] Yamori | Physiological Functions of Cyclic Electron Transport Around Photosystem I in Sustaining Photosynthesis and Plant Growth[END_REF][START_REF] Munekage | Cyclic electron flow around photosystem I is essential for photosynthesis[END_REF][START_REF] Nandha | The role of PGR5 in the redox poising of photosynthetic electron transport[END_REF][START_REF] Godaux | Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii<[END_REF][START_REF] Alric | Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) involvement of the PGR5-PGRL1 pathway under anaerobic conditions[END_REF][START_REF] Johnson | Proton Gradient Regulation 5-Mediated Cyclic Electron Flow under ATP-or Redox-Limited Conditions: A Study of Delta ATPase pgr5 and Delta rbcL pgr5 Mutants in the Green Alga Chlamydomonas reinhardtii[END_REF][START_REF] Munekage | PGR5 Is Involved in Cyclic Electron Flow around Photosystem I and Is Essential for Photoprotection in Arabidopsis[END_REF], there is still no clear evidence of their direct role in the transfer of electrons from the PSI acceptors to the PQ pool. On the contrary, several observations make this very unlikely. PGR5 is a small, stroma-soluble protein, binding no cofactors [START_REF] Toshiharu | Cyclic Electron Transport Around Photosystem I: Genetic Approaches[END_REF], and it is found tethered to the thylakoids by PGRL1. The maximal rate of CEF does not change in the absence of the former at the beginning of the induction of photosynthesis, showing that while PGR5 is involved in fine-tuning of CEF, its presence is not required for a fully efficient Fd:PQ electron transfer [START_REF] Nandha | The role of PGR5 in the redox poising of photosynthetic electron transport[END_REF]. PGRL1 has been proposed to fulfil the role of the elusive FQR [START_REF] Hertle | PGRL1 is the elusive ferredoxin-plastoquinone reductase in photosynthetic cyclic electron flow[END_REF], directly reducing quinones, and could almost stoichiometrically bind to the fraction of cyt. b6f found in the stroma lamellae [START_REF] Albertsson | A quantitative model of the domain structure of the photosynthetic membrane[END_REF]. However, the FQR role is difficult to reconcile with its sub-stoichiometric ratio relative to PSI, with about 0.3 proteins per PSI, or 0.15 PSI -1 if it indeed dimerises in vivo [START_REF] Hertle | PGRL1 is the elusive ferredoxin-plastoquinone reductase in photosynthetic cyclic electron flow[END_REF]. Crucially, we show in the accompanying article [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF] that a Chlamydomonas pgrl1 mutant exhibits no differences in the maximal transitory rate of CEF, which would be expected if PGRL1 was a bona fide CEF Fd:PQ oxidoreductase. Instead, we observed that the PSI acceptor side was altered in its contribution to charge recombination within PSI and to the rerouting of reductants towards CEF, both of which being lower than in the WT. These traits rather argue for an indirect role of PGRL1 in CEF, similarly to that of PGR5 [START_REF] Dalcorso | A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis[END_REF], by regulating either the fate of PSI acceptors or the redox state of the stroma. Such a role would go in line with the observations that PGRL1 is required for recruitment of PGR5 into PGRL1/PGR5 complex [START_REF] Dalcorso | A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis[END_REF]. Fig. 1. A scheme of CEF and chlororespiration, and the molecular actors involved in these processes. PGRL1 (dashed) could almost stoichiometrically bind to cyt. b6f to regulate CEF. Note that chlororespiration rates are so low that the only contribution of this process is observed in darkness [START_REF] Nawrocki | Chlororespiration controls growth under intermittent light[END_REF].

Cyt. b6f is a well-known plastoquinone reductase

The lack of evidence for sustained CEF through the two pathways above strongly suggests another pathway for rerouting reductants produced by PSI to its donor side: namely a direct PQ reduction by cyt. b6f (Figs 1,2A). Originally proposed by Mitchell and reiterated afterwards [START_REF] Joliot | Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves[END_REF][START_REF] Mitchell | The protonmotive Q cycle: a general formulation[END_REF], the classical Q-cycle involves a double reduction of PQ at the Qi site. Initially, such a mechanism included one electron provided by the bH haem (following a PQH2 oxidation at the Qo site) and the second electron provided by a stromal donor, such as Fd, possibly through the later discovered redox-active ci haem which is absent in mitochondrial and bacterial cyt. bc1 [START_REF] Joliot | Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves[END_REF][START_REF] Lavergne | Membrane potential-dependent reduction of cytochrome b-6 in an algal mutant lacking Photosystem I centers[END_REF][START_REF] Stroebel | An atypical haem in the cytochrome b(6)f complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF]. This early mechanism was mostly abandoned at the expense of the modified Q-cycle, in which the electrons used for PQ reduction at the Qi site are stored on the two b haems [START_REF] Cramer | The Q cycle of cytochrome bc complexes: A structure perspective[END_REF][START_REF] Crofts | The Q-cycle -A Personal Perspective[END_REF][START_REF] Baniulis | Structure-Function of the Cytochrome b6f Complex †[END_REF].

Nonetheless, for the purpose of rapid CEF (see above), it is possible to envisage the abovementioned scenario of Fd --[stromal haems] electron transfer similar to the classical Q-cycle. This is because once Fd is placed in a putative binding site on the stromal side of cyt. b6f (Fig. 2A), its iron-sulfur cluster has an edge-to-edge distance to the PQ or the haems in the Qi site that is short enough to allow efficient electron transfer between them, as defined by the Moser-Dutton ruler [START_REF] Page | Natural engineering principles of electron tunnelling in biological oxidation-reduction[END_REF]. Accordingly, Fd:NAD(P)H oxidoreductase (FNR) was shown to bind to the b6f and at least to regulate CEF [START_REF] Joliot | Regulation of cyclic and linear electron flow in higher plants[END_REF][START_REF] Zhang | Ferredoxin : NADP(+) oxidoreductase is a subunit of the chloroplast cytochrome b(6)f complex[END_REF][START_REF] Mosebach | Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii[END_REF]. It could therefore act as a tether for Fd in the vicinity of stromal haems of the b6f [START_REF] Mosebach | Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii[END_REF]. One could further argue that FNR is involved directly in CEF as a cofactor and mediates the electron transfer between Fd and stromal haems: indeed Fd in an Fd:FNR complex has a redox potential of -400 to -500 mV, when FNR alone stands at -350 mV [START_REF] Correll | Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin[END_REF], and the bH and ci haems are at -30 mV and +100 mV, respectively (note that in dark-adapted algae the redox difference between the latter two is much smaller with the ci haem being more reduced than the bH haem; see ref. [START_REF] Alric | Spectral and redox characterization of the heme ci of the cytochrome b6f complex[END_REF] for a discussion). Functional measurements of CEF in vivo are in agreement with a strong involvement of FNR in the process [START_REF] Joliot | Regulation of cyclic and linear electron flow in higher plants[END_REF][START_REF] Hald | Feedback regulation of photosynthetic electron transport by NADP(H) redox poise[END_REF].

If indeed the cyt. b6f is the FQR involved in CEF, there might not be a single cofactor specific to CEF -the ci and bH haems seem to form an ensemble in any type of Q-cycle. Crucially, this is in line with (i) the absence of mutants fully devoid of CEF but not of LEF; (ii) site-directed mutagenesis in the vicinity of the ci haem heavily affecting overall cyt. b6f function (personal communication, C. de Vitry, F.-A. Wollman); and (iii) specific binding of CO to the ci haem strongly impeding LEF [START_REF] Joliot | The low-potential electron-transfer chain in the cytochrome bf complex[END_REF].

In vascular plants the maximal rate of CEF is comparable with that of LEF, making it difficult to determine which exact step of those two interdependent pathways is limiting in vivo [START_REF] Joliot | Regulation of cyclic and linear electron flow in higher plants[END_REF]. It remains a possibility that the PQH2 oxidation in the Qo site constitutes the slowest step of the electron transfer in both CEF and LEF. On the other hand, we have shown that in Chlamydomonas the maximal CEF rate is lower, around 60 instead of ~100 e -.s -1 .PSI -1 [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF]. We speculate that in this case, the Fd --[stromal haems] transfer could be limiting for the overall rate.

Given the differences in architecture of the cyt. b6f in Chlamydomonas due to the presence of regulatory/associated proteins [START_REF] Takahashi | PETO Interacts with Other Effectors of Cyclic Electron Flow in Chlamydomonas[END_REF][START_REF] Buchert | The labile interactions of cyclic electron flow effector proteins[END_REF][START_REF] Terashima | Calcium-dependent regulation of cyclic photosynthetic electron transfer by a CAS, ANR1, and PGRL1 complex[END_REF] and the borderline distance for electron tunnelling between Fd and the stromal haems, some minor changes of the stromal side of the cytochrome could affect the rate of the Fd --[stromal haems] electron transfer.

Dimeric structure of cyt. b6f complex and its functional implications Most functional studies are satisfactorily interpreted in the framework of a monomeric complex, but tridimensional structures showed that the two cyt. b6f monomers are strongly intertwined in the dimer [START_REF] Stroebel | An atypical haem in the cytochrome b(6)f complex[END_REF][START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF][START_REF] Cramer | Transmembrane Traffic in the Cytochrome b 6 f Complex[END_REF] (Fig. 2B). On one hand, the soluble domain of the Rieske protein of one monomer interacts with the other one. On the other hand, the interface between monomers spans the transmembrane space and forms a V-shaped cavity filed with lipids. The tip of this Vshaped cavity is on the lumenal side, next to the entrance of the Qo site of one monomer whereas the Qi site of the other monomer is positioned toward the stroma where the crevice becomes wider.

Crucially, these structural features may deeply impact cyt. b6f activity. The PQ/ PQH2 bound to either site could easily slide between the Qi and the Qo sites of opposite monomers. The helix D of cyt.b6 also forms the narrowest part of the crevice next to the two-fold symmetry axis and creates a potential hindrance to the transfer of quinones between the Qo and Qi sites of the same monomer. Interestingly, the two bL haems have an edge-to-edge distance of 14-15 Å, sufficiently close to allow interdimer electron transfer [START_REF] Swierczek | An electronic bus bar lies in the core of cytochrome bc1[END_REF][START_REF] Lanciano | Intermonomer electron transfer between the low-potential b hemes of cytochrome bc(1)[END_REF]. All these features are shared not only by all cyt. b6f complexes, but also by all cyt. bc1 complexes and are probably a general feature of most other Rieske/cytochrome b complexes. However, some features, potentially critical for the CEF function discussed here, differ between the cyt. b6f and proteobacterial-related cyt. bc1 complexes. The Qo site entrance in the cyt. b6f complex harbors the phytyl chain of a chlorophyll molecule that modifies the accessibility of the active site [START_REF] Hasan | Traffic within the Cytochrome b6f Lipoprotein Complex: Gating of the Quinone Portal[END_REF]. Most importantly, the Qi site of the cyt. b6f complex has the additional haem ci that pushes the Qi pocket toward the stroma by about 6 Å, the position of haem bH staying identical. The amphipathic helices of cyt. b6, delimiting the cavity from the stromal side, and most likely the membrane boundaries, are shifted by an equivalent distance. Despite these changes, the hydrophobic residues of the amphipathic helix that faces the crevice are well conserved, underlying the critical importance of this intermonomer space.

Regulation of CEF/LEF partitioning -involvement of the dimeric cyt. b6f

The model for CEF, proposed by Mitchell and others [START_REF] Joliot | Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves[END_REF][START_REF] Mitchell | The protonmotive Q cycle: a general formulation[END_REF] and further discussed here in the light of structural information, has strong implications regarding the competition between LEF and CEF. Due to its transmembrane interfacial cavity, the cytochrome b6f complex offers a niche for quinones, which should be in slow equilibrium with the rest of the PQ pool. Indeed, it has been shown earlier by Joliot and Joliot [START_REF] Joliot | Mechanism of proton-pumping in the cytochrome b/f complex[END_REF] that, in reducing conditions, a quinone readily produced (oxidized) at the Qo site has an absolute priority to shuttle to the Qi pocket of the very same protein complex rather than to escape and diffuse to other Qi sites in its vicinity. In anoxic Chlorella cells, subsaturating laser flashes lead to the oxidation of a small proportion of cyt. f and to the rapid generation of an electric field caused by a quinone reduction by the low potential chain [START_REF] Joliot | Mechanism of proton-pumping in the cytochrome b/f complex[END_REF]. This indicates that the quinone generated at the Qo site successfully shuttles to the Qi site of the same cyt. b6f complex to act as an electron acceptor from the reduced bH and bL haems. However, transfer of the nascent quinone between the Qo and Qi sites from a same monomer may be challenging due to the obstacle formed by D helices of cyt. b6 (see above and in Fig. 2). We thus further propose that a direct electron transfer between the bL haems, experimentally detected in cyt. bc1 complexes [START_REF] Swierczek | An electronic bus bar lies in the core of cytochrome bc1[END_REF], may alleviate this difficulty by "coupling" the two monomers within the dimeric complex. The nascent quinone at Qo, which has reduced the bL haem in monomer A ("bL(A)") could be next transferred to the Qi site of monomer B, Qi(B), which is adjacent to the Qo site (A), through the internal cavity of the dimer (see Fig. 2b). Electron tunnelling between haems bL(A) and bL(B) could then occur (its rate, according to the Moser-Dutton ruler [START_REF] Page | Natural engineering principles of electron tunnelling in biological oxidation-reduction[END_REF][START_REF] Moser | Nature of biological electron transfer[END_REF] is within the measured rates of CEF in reducing [START_REF] Alric | Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) involvement of the PGR5-PGRL1 pathway under anaerobic conditions[END_REF] and oxidising conditions [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF] in the order of 60 e -.s -1 .PSI -1 , especially because of the lack of an electron acceptor in the Qi(A) pocket. Would this happen, the two haems of the low potential chain of the B monomer would be reduced, thus allowing a reduction of the Qi(B)-bound quinone and the observation of the electrogenic bL(B) -> bH(B) electron transfer despite an initial oxidation of the high potential chain in the A monomer. Such intermonomer electron transfer would not necessarily need to continuously compete with the more efficient monomeric turnover [START_REF] Hong | Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc1 complex[END_REF] if one considers it a mean to 'prime' the system during a transition from reducing to oxidising conditions. Unfortunately, a molecular dynamics study, which could reveal preferential intracomplex quinone shuttling, similar to diffusional exploration of quinone channels in PSII supercomplex [START_REF] Van Eerden | Exchange pathways of plastoquinone and plastoquinol in the photosystem II complex[END_REF], has not been conducted yet.

Consequently, we further propose that in vivo, nascent PQH2, reduced at the Qi site, have a high probability to be reoxidized at the adjacent Qo site of the same cyt. b6f dimer, i.e. without entering the "PQ pool". Such a mechanism provides heterogeneity in the pool of PQ/PQH2, and uncouples LEF and CEF at this step. Indeed, the LEF and CEF routes to the Qo site involve different pools of PQ/PQH2: the LEF route from PSII involves the pool of PQ/PQH2 outside of the cytochrome b6f, whereas the CEF route would only require the plastoquinol in the cavity of the cyt. b6f complex. The reduction of a quinone at the Qi site would be independent of the overall redox state of the PQ pool and thus would not compete for PQ reduction by PSII. In such a CEF model, previous proposals [START_REF] Allen | Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain[END_REF][START_REF] Alric | The plastoquinone pool, poised for cyclic electron flow?[END_REF] that the highest efficiency of CEF is achieved when 50% of the PQ pool is oxidized, and 50% in reduced state would not be required. It would allow CEF to be efficient also under high light, when the PQ/PQH2 pool is strongly reduced, as supported by experimental data [START_REF] Joliot | Cyclic electron transfer in plant leaf[END_REF]. Fig. 2. Electron and plastoquinone transfer within cyt. b6f. (a), A scheme of possible cyclic electron transfer within cyt. b6f. A side view of the cyt. b6f monomer from the dimerization surface perspective. The stromal and luminal extremities of the thylakoid membrane are depicted in grey (OMP). Separate subunits of the monomer are coloured individually as indicated in the figure. The electron transfer pathways are shown as yellow arrows. Ferredoxin in its putative binding mode is shown in a red cartoon form. (b) PQ/PQH2 transfer within cyt. b6f dimer. Side view of the complex. One cyt. b6f monomer is in orange, while the other one is blue. Possible route for the quinone between the Qo and Qi side are represented in green for transfer in the same monomer, which implies crossing the narrow portion of the crevice, or in green/orange for transfer in the opposite monomer, which uses a broader and shorter path.

Therefore, we consider that a control of the routing of electrons in the stroma is sufficient for a modification of CEF/LEF partitioning, without a need for considering the redox state of the plastoquinone pool. Competition between CEF and LEF for reduced Fd is, in our view, the hub where those two pathways require some regulatory processes. As outlined above, Fd -is readily oxidized by the CBB cycle and also by Flv proteins in microalgae and cyanobacteria [START_REF] Ilik | Alternative electron transport mediated by flavodiiron proteins is operational in organisms from cyanobacteria up to gymnosperms[END_REF]. We showed that such an oxidation is decreased at low oxygen concentrations when CEF increases.

Furthermore an increase in the duration of CEF also was observed in an Flv mutant of moss [START_REF] Gerotto | Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens[END_REF], yet it was interpreted as a compensatory increase in CEF rather than a decrease in electron leak.

The regulation between CEF and LEF could be achieved by increasing the probability that Fd stays in proximity of the stromal side of the cyt. b6f, through its anchoring by the cyt. b6f -bound FNR, as had been proposed earlier [START_REF] Joliot | Regulation of cyclic and linear electron flow in higher plants[END_REF]. Recently, FNR was shown to be a target of the redoxsensitive STN kinase, a transmembrane enzyme which interacts with cyt. b6f [START_REF] Dumas | A stromal region of cytochrome b6f subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas<[END_REF][START_REF] Schonberg | Identification of STN7/STN8 kinase targets reveals connections between electron transport, metabolism and gene expression[END_REF]. STN being activated upon reduction of the PQ pool, one can imagine a tentative model for an FNR phosphorylation-dependent CEF regulation -but there is a need to produce functional and biochemical data regarding the regulation of Fd-FNR-b6f binding before such a model can be critically assessed. Fd is at the very crossroads of photosynthetic electron transfer, donating electrons not only to CEF and LEF, but also to other metabolic pathways such as nitrite and sulphide reduction [START_REF] Hanke | Plant type ferredoxins and ferredoxin-dependent metabolism[END_REF]. Furthermore, there are multiple Fd isoforms, with varying redox potentials and concentrations, which interact with multiple isoforms of FNR, some of which are soluble and some membrane-bound [START_REF] Hanke | Plant type ferredoxins and ferredoxin-dependent metabolism[END_REF][START_REF] Mulo | Chloroplast-targeted ferredoxin-NADP+ oxidoreductase (FNR): Structure, function and location[END_REF]. It is obvious that a strict regulation of this hub is necessary both for photosynthesis and for poising the redox state of the entire stroma, making it easy to imagine that CEF is also governed at this level.

Supercomplexes: can they really drive CEF?

A dynamic heterogeneity among electron transfer complexes that would favour CEF also has been sought based on the formation of PSI-cyt. b6f supercomplexes [START_REF] Iwai | Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis[END_REF]. Originally proposed to account for the elevated rates of CEF observed in anoxic conditions in Chlamydomonas, it is poorly consistent with our recent study where we compared maximal rates of CEF in conditions that would favour -or not -such supercomplex formation [START_REF] Nawrocki | Cyclic electron flow in Chlamydomonas reinhardtii<[END_REF]: we found no evidence for changes in CEF maximal rates although we did observe that a lower oxygen availability increased the duration of CEF due to a slowdown of the electron leakage from the electron transfer chain.

Nevertheless, the biochemical identification of supercomplexes associating cyt. b6f, PSI and a number of CEF-related proteins has stimulated much speculations as to the way CEF may function [START_REF] Takahashi | PETO Interacts with Other Effectors of Cyclic Electron Flow in Chlamydomonas[END_REF][START_REF] Terashima | Calcium-dependent regulation of cyclic photosynthetic electron transfer by a CAS, ANR1, and PGRL1 complex[END_REF][START_REF] Iwai | Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis[END_REF][START_REF] Yadav | Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH[END_REF][START_REF] Takahashi | Cyclic electron flow is redoxcontrolled but independent of state transition[END_REF][START_REF] Johnson | Proton gradient regulation 5-mediated cyclic electron flow under ATP-or redox-limited conditions: a study of DeltaATpase pgr5 and DeltarbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii[END_REF]. The hypothesis of a supercomplex-borne CEF process raises several mechanistic and stoichiometric issues. Cyt. b6f being a dimer, a cyt. b6f dimer-PSI supercomplex uses twice as much cyt. b6f as PSI. The overall cyt. b6f:PSI stoichiometry is about 0.7 [START_REF] Anderson | Cytochrome b6f complex: Dynamic molecular organization, function and acclimation[END_REF], with less than half of cyt. b6f present in the appressed regions of the membrane [START_REF] Vallon | Lateral redistribution of cytochrome b6/f complexes along thylakoid membranes upon state transitions[END_REF]. In consequence, less than 20% of PSI are available for supercomplex formation.

From a structural standpoint, PSI from Chlamydomonas placed in state II in anoxia, where supercomplex formation would be favoured, binds multiple PSII antenna on the side where its own peripheral antenna, LHCa, are not attached [START_REF] Drop | Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii[END_REF]. This leaves a place for binding of a cyt. b6f dimer only on the edges of the PSI complex. Such putative supercomplex between the b6f and PSI was present in less than 1% of PSI particles as assessed with single-particle cryo-EM after thylakoid solubilisation ( [START_REF] Steinbeck | Structure of a PSI-LHCIcyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions[END_REF], Supporting Information). Furthermore, in cyt. b6f-PSI supercomplex particles isolated from land plants, it was found that a majority of the complexes bound a cyt. b6f in a monomeric form [START_REF] Yadav | Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH[END_REF]. Recent biochemical data finally calls into question the significance of green bands of high molecular weight in sucrose density gradient preparations from Chlamydomonas thylakoids. These should not be mistaken for cyt. b6f-PSI supercomplex formation, since various PSI complexes can migrate in these sucrose density regions. [START_REF] Buchert | The labile interactions of cyclic electron flow effector proteins[END_REF].

As for the kinetic rationale for supercomplex formation, one can take the well-documented supercomplex in mitochondria, the NADH dehydrogenase-cyt. bc1, as a case study. Through flux control analysis, this complex was suggested to provide a kinetic advantage for NADH oxidation due to a purported trapping of the lipid-soluble electron carrier, ubiquinone (UQ), allowing its oxidation before it diffuses away within the mitochondrial membrane [START_REF] Genova | Functional role of mitochondrial respiratory supercomplexes[END_REF]. However, recent structural data argues against the UQ trapping in a mitochondrial respirasome and provides an "open" structure, at least for the UQ reduction site in the NADH dehydrogenase (see [START_REF] Letts | The architecture of respiratory supercomplexes[END_REF] and the discussion within). Moreover, the distance between the cyt. c reduction site on cyt. bc1, and its oxidation site on cyt. c oxidase (COX), is too large to propose an electron transfer without a release of cyt. c from the respirasome. Finally, the lack of kinetic advantage from complex formation between bc1 and COX has been experimentally demonstrated in yeast [START_REF] Trouillard | Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain[END_REF]. The conclusions from respiratory system indicates that a presence of supercomplex does not infer a kinetic advantage for electron transfer.

We suggest that a similar situation does hold for CEF supercomplexes between PSI and cyt. b6f [START_REF] Iwai | Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis[END_REF][START_REF] Yadav | Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH[END_REF]. A trapping of water-soluble CEF carriers, Fd and PC would be required to prevent an electron leak to LEF. Such a trapping is unlikely because the distances between cyt. f and the PC binding site in PSI, and between FA/FB iron-sulfur clusters and the hypothetical Fd-binding site close to the Qi site are too large to allow intracomplex electron transfer without a release and diffusion of these soluble carriers (at least 100 Å, see [START_REF] Yadav | Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH[END_REF][START_REF] Drop | Consequences of state transitions on the structural and functional organization of photosystem I in the green alga Chlamydomonas reinhardtii[END_REF]). Moreover, the kinetic limitation in both cases (presence or absence of PSI-b6f complex) still would be set by the rate of PQH2 oxidation within the membrane, at the Qo site of the cyt. b6f. Such a system could at best contribute to the regulation of the CEF/LEF ratio, but it would not control the maximal rate of CEF. Finally, the complexes between PSI and NDH are even more unlikely to have functional relevance for CEF not only due to the extremely low rates of NDH, as discussed above, but critically due to the absence of the PQ:PC oxidoreductase within such complex [START_REF] Yadav | Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH[END_REF][START_REF] Kouril | Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex[END_REF][START_REF] Peng | Efficient Operation of NAD(P)H Dehydrogenase Requires Supercomplex Formation with Photosystem I via Minor LHCI in Arabidopsis[END_REF].

To conclude, the mechanism and actors of cyclic electron flow around Photosystem I remain far less well established than the photosynthetic community generally assumes. It is crucial to better consider the kinetics of this process in order to propose appropriate assumptions as to its mechanism and regulation. While the contribution of auxiliary CEF proteins such as PGR5 and PGRL1 is now firmly grounded, the actual mechanism of CEF is probably very different from what is presently discussed in the literature. It is of note that the functional insights provided by the 3D structure of cyt. b6f have been overlooked up to now. The quinone cavity inside the cyt. b6f dimer stands like a treasure trunk that has not yet revealed its CEF secrets. This is a thrilling time for the bioenergetics of photosynthesis.