Kamilla Thingholm Bünning

Tae-Hyun Kim

Vanessa Prevot

Claude Forano

Ulla Gro Nielsen

The effect of Zr(IV) addition on the phosphate removal properties of MgAl-LDH

Keywords: Layered double hydroxides, phosphate, sorption, environmental remediation, solid state NMR spectroscopy

Recovery of phosphate from wastewater represents a sustainable alternative to phosphate rock, a limited resource. Magnesium aluminum layered double hydroxides (MgAl-LDH), are promising sorbents due to their high phosphate removal capacity by ion-exchange (26-124 mgP/g).

Furthermore, an increased sorption capacity has been reported by the addition of zirconium (Zr(IV)) into the LDH lattice. However, ambiguity exists especially in relation to the incorporation of Zr and the stability of the MgAl-LDH. To understand how Zr(IV)-addition affects the phosphate sorption properties (both capacity and mechanism) on the atomic level, solid state multi-nuclear NMR spectroscopy was combined with complimentary characterization techniques to study a series of MgAl-LDH with Zr to Al ratios of 1:9 and 1:1, which had the Mg to (Al+Zr) ratio fixed at 2:1. The LDH were synthesized by coprecipitation and subsequently aged or hydrothermally treated to improve the crystallinity (particle size). No evidence of Zr(IV) incorporation in the LDH was observed, instead Zr was present as Zr(IV)-hydroxide (X-ray amorphous) and crystalline Zr(IV) oxide after aging and hydrothermal treatment, respectively. The segregation of Zr(IV)and resulting lower Al content in the MgAl-LDH decreased the phosphate sorption capacity from 45-54 to 16-20 mgP/g. Zr (hydr)oxide impurities contributed with less than 5 mgP/g. Moreover, 31 P MAS NMR spectroscopy quantified showed that the MgAl-LDH contribute to less than 60% of the total P removal capacity.

Introduction 3

The global phosphorous (P) challenge receives increasing attention since phosphorous in the form of orthophosphate (H x PO 4

(3-x)-), is a fertilizer vital for global food security [START_REF] Cordell | The story of phosphorus: Global food security and food for thought[END_REF][START_REF] Cordell | Life's Bottleneck: Sustaining the World's Phosphorus for a Food Secure Future[END_REF][START_REF] Jupp | Phosphorus recovery and recycling-closing the loop[END_REF]Reitzel et al., 2019). Traditionally, phosphate has been extracted from phosphate rock [START_REF] Cordell | The story of phosphorus: Global food security and food for thought[END_REF][START_REF] Jupp | Phosphorus recovery and recycling-closing the loop[END_REF], a non-renewable resource, and depletion is in the worst-case predicted within the next century [START_REF] Jupp | Phosphorus recovery and recycling-closing the loop[END_REF][START_REF] Walan | Phosphate rock production and depletion: Regional disaggregated modeling and global implications[END_REF]. A promising alternative is the recovery of phosphate accumulated in municipal and industrial wastewater at wastewater treatment plants [START_REF] Bunce | A Review of Phosphorus Removal Technologies and Their Applicability to Small-Scale Domestic Wastewater Treatment Systems[END_REF][START_REF] Egle | Phosphorus recovery from municipal wastewater: An integrated comparative technological, environmental and economic assessment of P recovery technologies[END_REF]. Current recovery approaches include the use of sludge and struvite (NH 4 MgPO 4 •6H 2 O) as a fertilizer, whereas vivianite (Fe 3 (PO 4 ) 2 •8H 2 O) is an emerging technology [START_REF] Hall | Phosphorus speciation and fertiliser performance characteristics: A comparison of waste recovered struvites from global sources[END_REF][START_REF] Lamastra | Sewage sludge for sustainable agriculture: contaminants' contents and potential use as fertilizer[END_REF][START_REF] Rahman | Production of slow release crystal fertilizer from wastewaters through struvite crystallization -A review[END_REF][START_REF] Talboys | Struvite: a slow-release fertiliser for sustainable phosphorus management?[END_REF][START_REF] Wilfert | The Relevance of Phosphorus and Iron Chemistry to the Recovery of Phosphorus from Wastewater: A Review[END_REF]. The use of selective and ideally reusable phosphate (ad)sorbents is a promising, but less developed technology [START_REF] Wu | Selective Phosphate Removal from Water and Wastewater using Sorption: Process Fundamentals and Removal Mechanisms[END_REF]. Layered double hydroxides (LDH) have great potential due to a high affinity for orthophosphate and ion-exchange capacity (26 -124 mgP/g), depending on, e.g., the phosphate speciation (H 2 PO 4 -, HPO 4 2-

, PO 4 3-

) and LDH composition (Table S1) [START_REF] Ashekuzzaman | Study on the sorption-desorption-regeneration performance of Ca-, Mg-and CaMg-based layered double hydroxides for removing phosphate from water[END_REF][START_REF] Luengo | High sorption of phosphate on Mg-Al layered double hydroxides: Kinetics and equilibrium[END_REF]Lundehøj et al., 2019a;Lundehøj et al., 2019b;[START_REF] Novillo | Evaluation of phosphate removal capacity of Mg/Al layered double hydroxides from aqueous solutions[END_REF]. Phosphate can subsequently be recovered and the LDH reused or the phosphate loaded LDH can directly be applied as a fertilizer [START_REF] Bernardo | Controlled Release of Phosphate from Layered Double Hydroxide Structures: Dynamics in Soil and Application as Smart Fertilizer[END_REF][START_REF] Borges | Potential Sustainable Slow-Release Fertilizers Obtained by Mechanochemical Activation of MgAl and MgFe Layered Double Hydroxides and K 2 HPO 4[END_REF][START_REF] Everaert | Phosphate-Exchanged Mg-Al Layered Double Hydroxides: A New Slow Release Phosphate Fertilizer[END_REF].

LDH have the general formula [M(II) 1-x M(III) x (OH) 2 ] x+ (A x/n n-)•yH 2 O (x typically 0.2 -0.33), whereas M(II) and M(III) are divalent and trivalent cations and A n-is a charge compensating anion located in the interlayer together with water [START_REF] Duan | Structural aspects of Layered Double Hydroxides[END_REF][START_REF] Forano | Layered double hydroxides (LDH)[END_REF][START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF]. Subsequently, the LDH will be abbreviated as M(II)M(III)-LDH. Especially, the charge density (x), intercalated anion (A n-), particle size, pH, and competing anions are important parameters related to the removal capacity and stability of the LDH.

For environmental applications, mainly the non-toxic MgFe -, CaAl -, CaFe -, and MgAl-LDH are relevant [START_REF] Jaberi | Using Ca-Fe layered double hydroxide transformation to optimise phosphate removal from waste waters[END_REF][START_REF] Ashekuzzaman | Study on the sorption-desorption-regeneration performance of Ca-, Mg-and CaMg-based layered double hydroxides for removing phosphate from water[END_REF][START_REF] Bekele | Sequestration of orthophosphate by Ca 2 Al-NO 3 layered double hydroxide -Insight into reactivity and mechanism[END_REF][START_REF] Kim | An investigation of the phosphate removal mechanism by MgFe layered double hydroxides[END_REF][START_REF] Luengo | High sorption of phosphate on Mg-Al layered double hydroxides: Kinetics and equilibrium[END_REF]Lundehøj et al., 2019b;[START_REF] Novillo | Evaluation of phosphate removal capacity of Mg/Al layered double hydroxides from aqueous solutions[END_REF]. MgAl-LDH is the most promising for phosphate removal, since the other LDH are highly unstable upon phosphate exposure [START_REF] Jaberi | Using Ca-Fe layered double hydroxide transformation to optimise phosphate removal from waste waters[END_REF][START_REF] Bekele | Sequestration of orthophosphate by Ca 2 Al-NO 3 layered double hydroxide -Insight into reactivity and mechanism[END_REF][START_REF] Kim | An investigation of the phosphate removal mechanism by MgFe layered double hydroxides[END_REF].

To enhance the function of LDH such as the phosphate sorption capacity, modification of LDH with high-valent cations, especially Zr(IV) has been proposed. The rationale is that the Zr(IV)

will increased the cation layer charge and thereby the anion exchange capacity as Zr(IV) is speculated to replace Al(III) [START_REF] Arias | Preparation of NiAlZrterephthalate LDHs with high Al and Zr content and their mixed oxides for cyclohexane dehydrogenation[END_REF][START_REF] Chitrakar | Uptake properties of phosphate on a novel Zr-modified MgFe-LDH(CO 3 )[END_REF][START_REF] He | Ni-based catalysts derived from Ni-Zr-Al ternary hydrotalcites show outstanding catalytic properties for low-temperature CO 2 methanation[END_REF][START_REF] Koilraj | Phosphate uptake behavior of ZnAlZr ternary layered double hydroxides through surface precipitation[END_REF][START_REF] Liu | The application of Zr incorporated Zn-Al dehydrated hydrotalcites as solid base in transesterification[END_REF][START_REF] Miyauchi | Phosphate Adsorption Site on Zirconium Ion Modified MgAl-layered Double Hydroxides[END_REF][START_REF] Nowicki | Transesterification of rapeseed oil to biodiesel over Zr-dopped MgAl hydrotalcites[END_REF][START_REF] Poonoosamy | Zr-containing layered double hydroxides: Synthesis, characterization, and evaluation of thermodynamic properties[END_REF][START_REF] Velu | New Hydrotalcite-like Anionic Clays Containing Zr 4+ in the Layers: Synthesis and Physicochemical Properties[END_REF][START_REF] Wang | Influence of Zr on the performance of Mg-Al catalysts via hydrotalcite-like precursors for the synthesis of glycerol carbonate from urea and glycerol[END_REF][START_REF] Wang | NiAlZrW hydrodesulfurization catalysts derived from tungstate intercalated NiAlZr layered double hydroxides[END_REF]. For example, incorporation of a Zr(IV) instead of a Al (III) in the brucite layer would require one HPO 4 2-instead of half a HPO 4 2-for charge balance. This leads to a higher anion-exchange capacity.

An increased P removal capacity has also been reported for Zr(IV) modified LDH as compared to the corresponding M(II)M(III)-LDH in phosphate solution, seawater, and wastewater (Table 1). However, most studies compare the P removal capacity of the Zr-modified LDH with the parent LDH, which has carbonate as interlayer anion, c.f., Table 1. This suppresses the phosphate removal by the anionexchange pathway, due to the high affinity of the LDH for carbonate [START_REF] Forano | Layered double hydroxides (LDH)[END_REF]Lundehøj et al., 2019b;[START_REF] Nuryadin | Phosphate adsorption and desorption on two-stage synthesized amorphous-ZrO 2 /Mg-Fe layered double hydroxide composite[END_REF]. Therefore, the increased phosphate removal capacity obtained by addition of Zr can instead be explained by the co-presence of impurities such as Zr(IV) (hydr)oxides, which also have a significant phosphate removal capacity (Table 1). Studies have also investigated the physical mixtures of MgAl-LDH and Zr-(hydr)oxide with the goal to either prepare nano-sized particle (ad)sorbents as Zr-(hydr)oxide on the surface of LDH can suppress crystal growth [START_REF] Nuryadin | Phosphate adsorption and desorption on two-stage synthesized amorphous-ZrO 2 /Mg-Fe layered double hydroxide composite[END_REF] or to modify the layer charge by the introduction of Zr(IV)-O 2-acid base pairs [START_REF] Motandi | Application of zirconium modified layered double hydroxide and calcination product for adsorptive removal of phosphate from aqueous solution[END_REF]. Here an increased P removal capacity for a mixed system was also reported (Table 1).

Several studies have concluded a successful incorporation of Zr(IV) into the LDH.

However, the successful incorporation of Zr(IV) into the LDH structure was mainly based on the analysis of low-quality powder X-ray diffractograms and/ or the qualitative variation of the M-M distance derived from the (110) reflection [START_REF] Velu | New Hydrotalcite-like Anionic Clays Containing Zr 4+ in the Layers: Synthesis and Physicochemical Properties[END_REF][START_REF] Wang | Influence of Zr on the performance of Mg-Al catalysts via hydrotalcite-like precursors for the synthesis of glycerol carbonate from urea and glycerol[END_REF]. Thus, the insertion of Zr(IV) was, in part, concluded based on an expansion of the unit cell parameter a with increasing Zr(IV) content in ternary M(II)AlZr-LDH [START_REF] Chitrakar | Synthesis and phosphate uptake behavior of Zr 4+ incorporated MgAl-layered double hydroxides[END_REF][START_REF] Koilraj | Phosphate uptake behavior of ZnAlZr ternary layered double hydroxides through surface precipitation[END_REF][START_REF] Shen | Simultaneous incorporation of palladium and zirconium ions in Mg-Al layered double hydroxides by co-precipitation[END_REF][START_REF] Velu | New Hydrotalcite-like Anionic Clays Containing Zr 4+ in the Layers: Synthesis and Physicochemical Properties[END_REF][START_REF] Wang | Influence of Zr on the performance of Mg-Al catalysts via hydrotalcite-like precursors for the synthesis of glycerol carbonate from urea and glycerol[END_REF]. A slight reduction in interlayer spacing (7.9 to 7.8 Å)

based on visual inspection of PXRD data was ascribed to an increased charge of the cation layer for NiAl-LDH with nitrate as interlayer ion, i.e. successful incorporation of Zr(IV) into the LDH [START_REF] Wang | NiAlZrW hydrodesulfurization catalysts derived from tungstate intercalated NiAlZr layered double hydroxides[END_REF]. However, for LDH with nitrate as interlayer ion, a lower Al 3+ results in a decreased interlayer spacing, as nitrate ion can be parallel to the cation layer [START_REF] Karthikeyan | Efficient exfoliation of layered double hydroxides; effect of cationic ratio, hydration state, anions and their orientations[END_REF]. However, a reduced Al content in a M(II) 1-x Al x -LDH will give a similar structural trend. Thus, it is not possible to distinguish between i) the incorporation of Zr(IV) into the LDH and ii) the formation of both MgAl-LDH and a Zr (hydr)oxide phase only by PXRD and bulk chemical analyses.

In more rigorous studies, the use of advanced structural characterization techniques and in depth data analyses have clearly shown, that Zr(IV) was not incorporated into the LDH, but instead a mixture of LDH and Zr(OH) 4 (or ZrO 2 ) was obtained [START_REF] Intissar | Reinvestigation of the Layered Double Hydroxide Containing Tetravalent Cations: Unambiguous Response Provided by XAS and Mössbauer Spectroscopies[END_REF][START_REF] Miyauchi | Phosphate Adsorption Site on Zirconium Ion Modified MgAl-layered Double Hydroxides[END_REF]. For example [START_REF] Intissar | Reinvestigation of the Layered Double Hydroxide Containing Tetravalent Cations: Unambiguous Response Provided by XAS and Mössbauer Spectroscopies[END_REF] investigated the local environment of metal cations for a series of M(II) 0.75 Al 0.25-y M(IV) y (OH) 2 (CO 3 ) (y+0.25)/2 'nH 2 O (M(II) = Mg, Co; M(IV) = Sn, Zr) LDH using X-ray absorption spectroscopy (XAS) and Mössbauer spectroscopy combined with detailed data analysis.

This clearly confirmed that the first metal ion coordination sphere for both Zr(IV) and Sn(IV) was dominated by M(IV)-M(IV) correlations, i.e., the formation of separate Sn(IV) and Zr(IV) phases.

Similarly, [START_REF] Miyauchi | Phosphate Adsorption Site on Zirconium Ion Modified MgAl-layered Double Hydroxides[END_REF] showed that ZrMgAl-LDH synthesized by coprecipitation had the same k 3 -weighted Zr-K edge as ZrO 2 in the extended X-ray absorption fine structure (EXAFS) spectra.

Additionally, zirconium oxide impurities are generally only observed after hydrothermal treatment above approximately 120°C [START_REF] Chitrakar | Synthesis and phosphate uptake behavior of Zr 4+ incorporated MgAl-layered double hydroxides[END_REF], an approach usually employed to increases the LDH particle size and thereby stability [START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF]. Such impurities were not observed, when the LDH was aged below 100C, a more common post synthesis treatment (Table 1). However, amorphous Zr phases such as Zr(OH) 4 or ZrO 2 may still be present and not observed by Powder X-ray Diffraction (PXRD), the routine characterization method.

A suite of bulk and atomic level characterization methods is necessary to link the phosphate removal capacity and pathways to the LDH properties (chemical composition, particle size, etc.). For example, inductively coupled plasma (ICP) spectroscopy and PXRD provides information about the bulk content and crystalline phases, respectively, whereas solid state NMR spectroscopy (SSNMR) gives detailed insight into the atomic level structure of the LDH and (amorphous) impurities and/ or other phases [START_REF] Nielsen | Solid state NMR studies of layered double hydroxides[END_REF]. SSNMR combined with PXRD, electron microscopy, and elemental analysis is crucial to obtain detailed insight into the LDH composition and anion-exchange properties [START_REF] Kim | An investigation of the phosphate removal mechanism by MgFe layered double hydroxides[END_REF]Lundehøj et al., 2019a;[START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF].

In contrast to previous studies, we have performed a detailed investigation on how Zr(IV) addition affects the phosphate removal capacity and pathways for MgAl-LDH with chloride (Cl -) as the interlayer anion prepared by two common post-synthesis treatments (aging and hydrothermal treatment). This provided detailed insight into the different phosphate removal pathways and their relative contributions. Thus, our goal was to assess if Zr(IV) addition will improve the performance of MgAl-LDH based sorbents with a view to reusability. This was obtained by combining conventional phosphate sorption isotherm and kinetics experiments with characterization of the LDH before and after phosphate exposure using PXRD, FT-IR, inductively coupled plasma -optical emission spectrometry (ICP-OES), zeta (ζ) potential measurements, electron microscopy (SEM, TEM), and multinuclear MAS NMR spectroscopy.

Experimental section

Materials

MgCl 2 •6H 2 O (≥98% purity), AlCl 3 •6H 2 O (≥99% purity), ZrO(NO 3 ) 2 •xH 2 O (≥99%
purity), NaOH (≥97% purity), NaCl (≥99% purity) and K 2 HPO 4 (≥ 98% purity), purchased from Sigma-Aldrich, and HCl (≥37%) purchased from Fisher Scientific, were used for the synthesis and sorption experiments without further purification.

Methods

Sample preparation

The metal-and alkaline salts were dissolved in decarbonated water prepared by boiling milli-Q water with nitrogen (N 2 ) flow for ca. one hour and then cooled to 25-35 °C under a nitrogen (N 2 ) flow before use. MgAl-and Zr doped MgAl-LDH were synthesized by coprecipitation at constant pH using earlier reported procedures [START_REF] Chitrakar | Synthesis and phosphate uptake behavior of Zr 4+ incorporated MgAl-layered double hydroxides[END_REF] with the following modifications: 500 mL of a metal ion solution containing Mg 2+ , Al 3+ , and Zr 4+ with a total metal ion concentration of 0.6 M and 500 mL of an alkaline solution containing 1.2 M NaOH and 0.6 M NaCl were prepared. For each sample, the individual metal ion concentrations and mole percent are given in Table 2. 400 mL of the metal solution was added at a rate of 1.33 mL/min using a titrator (905 Titrando®; Metrohm AG, Herisau, Switzerland) into a 1 L three-necked-round-bottom flask which already contained 100 mL of decarbonated water under magnetic stirring (600 -800 rpm). Simultaneously, the alkaline solution was added to maintain the pH at 10.0(2) at a rate like that for the metal solution. All synthesis were performed under a N 2 flow to minimize carbonate impurities.

Table 2. The absolute concentrations of the metal cation solutions (mol/L) used in the syntheses with the relative percentage of Mg, Al and Zr (mol%) in the parenthesis. Moreover, the metal ion composition (mol%) for Mg, Al, and Zr for the synthesized LDH and these LDH after phosphate exposure. n. a. = not applicable.

After addition of the metal-and alkaline solutions, the obtained solids were isolated by centrifugation (10.000 rpm, 10 min) using a Sigma Laboratory Centrifuge (2-16P) and then roughly divided into two batches: the first batch was redispersed into 350 mL of decarbonated water and aged at 80°C in a 500 mL three-necked-round-bottom flask under reflux and magnetic stirring for 16 h. The second batch was redispersed into decarbonated water (350 mL) and subsequently hydrothermally treated in multiple 70 mL Teflon® lined steel autoclaves (ca. 2/3 filled) for 16 h at 150°C in a Binder GmbH oven. Subsequently, both batches, were washed with 40 mL deionized water in 50 mL conical centrifuge tubes (Sarstedt, 114×28 mm) and shaken thoroughly followed by removal of the supernatant after centrifugation (10.000 rpm, 10 min). The washing was repeated thrice. Finally, zirconium hydroxide (Zr(OH) 4 ) and zirconium oxide (ZrO 2 ) were synthesized using 500 mL of a 0.2 M ZrO(NO 3 ) 2 •xH 2 O and 500 mL of 1.2 M NaOH solution using the same titrator, volume, metal-solution rate, and pH as above. After synthesis, the batch was also divided into two and the post-synthesis was either aging or hydrothermal treatment. Aging resulted in the formation of amorphous Zr(OH) 4 , whereas crystalline ZrO 2 was obtained after hydrothermal treatment. All solid products were dried at 50°C overnight (≈ 16-18 h) and ground to a fine powder using an agate mortar and pestle before analyses.

Table 2 summarized the nomenclature for all materials and their synthetic compositions.

Briefly, MgAl-A and MgAl-HT corresponds to conventional MgAl-LDH with either aging (A) at 80°C

or hydrothermally treatment (150°C, HT), respectively. The Zr modified LDH are named after their ideal composition based on the reactant ratioss: Zr0.1-A and Zr0.1-HT corresponds to the LDH synthesized with Zr 4+ and Al 3+ concentration of 0.02 mol/L and 0.18 mol/L, respectively. This corresponds to an Al to Zr ratio of 9 to 1; Al/Zr = 9/1), and ideally a Mg 2 Al 0.9 Zr 0.1 -LDH, whereas Zr0.5-A and Zr0.5-HT are MgAl-LDH synthesized with 0.1 mol/L Zr 4+ and 0.1 Al 3+ (Al/Zr = 1/1, ideally Mg 2 Al 0.5 Zr 0.5 -LDH). Al/Zr = 1 was chosen to match earlier studies (Table 1) whereas the ratio Al/Zr = 9 were selected to investigate if a small Zr(IV) doping was masked by the Zr(IV) impurities in the Al/Zr = 1 samples. For samples obtained after P exposure a "-P" is added as a suffix, for example MgAl-HT-P is MgAl-HT after phosphate exposure.

Phosphate isotherm batch experiments

The phosphate sorption isotherm experiments were performed in 15 mL polypropylene conical centrifuge tubes (Sarstedt, 120×17 mm) containing 10.00 mL phosphate solution and 10.0(1) mg LDH, which were shaken at 400 rpm using an orbital shaker for 24 h at room temperature (≈ 20C).

All phosphate isotherm experiments were performed as duplicates. S1). The pH of the solutions was adjusted to 7.5 (67% HPO 4 2-, 33% H 2 PO 4 -) using 0.1 M HCl. To mimic industrial applications of LDH sorbents, the pH was not actively controlled. Moreover, pH control is known to drive the dissolution of the LDH with the lowest dissolution observed at pH 8 [START_REF] Imran | Dissolution kinetics of pyroaurite-type layered double hydroxide doped with Zn: Perspectives for pH controlled micronutrient release[END_REF]. After 24 h, the supernatant was collected using a 2 mL disposable pipette and filtrated through a 0.45 μm PTFE syringe filter. All phosphate solutions were prepared in milli-Q water.

The ortho-phosphate concentration was quantified by the molybdate blue method [START_REF] Nagul | The molybdenum blue reaction for the determination of orthophosphate revisited: Opening the black box[END_REF] using a UV-Vis-spectrometer (UV-600PC; VWR, U.S.A) at 880 nm. The equilibrium phosphate sorption (q e (mgP/g)) capacity were then calculated using Eq. 1 and plotted as a function of the equilibrium concentration (C e (mgP/L)), i.e., the concentration in the solution remaining at the end of the experiment. Lastly, the sorption isotherms were modelled by the Langmuir (Eq. 2) and Freundlich (Eq. 3) models using Origin (Version 2017, OriginLab Corporation).

q e = (C o -C e )•(V / m) (Eq. 1) q e = (Q m K L C e )/(1 + K L C e )(Eq. 2)
13

q e = K F C e 1/n (Eq. 3)
Where C 0 is the initial concentration (mgP/L), V (L) is the total volume and m (g) is the mass of LDH.

Q m is the maximum monolayer coverage (mgP/g), K L is the Langmuir isotherm constant (ml 3 /mg) related to the affinity of binding sites, K F (mgP/g) is the Freundlich constant (related to the sorption capacity) and n is related to the sorption intensity [START_REF] Foo | Insights into the modeling of adsorption isotherm systems[END_REF].

Powder X-ray diffraction

PXRD were recorded on a Rigaku Miniflex 600 X-ray diffractometer using Cu Kα radiation (λ = 1.5406 Å) with K -filter and a D/Tex Ultra-high-speed silicon strip detector using a voltage of 40 kV and a current of 15 mA from 2θ = 5 to 70°, with a step size of 0.02° at 10 degrees per minute. Le Bail fitting was performed to determine the cell-parameters using the FullProf software [START_REF] Rodríguez-Carvajal | Recent advances in magnetic structure determination by neutron powder diffraction[END_REF]. The structural model used a Zn 2 Al-Cl LDH (ICSD code 155051) as starting structures (where Zn were exchanged for Mg) (ICSD code 155051) [START_REF] Lombardo | A Novel Integrated X-ray Powder Diffraction (XRPD) and Molecular Dynamics (MD) Approach for Modelling Mixed-Metal (Zn, Al) Layered Double Hydroxides (LDHs)[END_REF]. For Zr0.5-HT the CIF for ZrO 2 (ICSD code 105553 [START_REF] Katz | X-Ray Diffraction Powder Pattern of Metastable Cubic ZrO 2[END_REF] was also used in the refinement. Both CIF were found through search-and-match in the PXRD software PDXL2. Generally, the parameters refined were W, X, U, cell parameters (a and c), shape1, B ov , zero shift, and the background. The background was chosen manually by linear interpolation and described by the Pseudo-Voigt function. A Le Bail refinement was chosen instead of Rietveld refinement due the complexity of the reflection in mid angles (2θ ≈ 30 -50°), resulting from stacking faults and/ or mix of multiple polytypes. Next, the average crystallite size was estimated using the Scherrer equation (Eq. 4) for the (003) reflection and experimental broadening was corrected for using the (111) reflection at 2θ = 28.5° (FWHM = 0.10°)

for a Si standard. The full-width-half-max (FWHM) used for calculation of crystallite size were likewise calculated in WinPLOTR in FullProf suite.

14

B(2θ 003 ) = (K•λ)/( • cos (θ) ) (Eq. 4)
Were B(2θ) is the FWHM of the (003) peak, K is the crystallite shape constant (here approximated to 0.9 assuming a spherical morphology [START_REF] Forano | Layered double hydroxides (LDH)[END_REF], λ is the wavelength, θ is the diffraction angle, and is the crystallite diameter in Ångstrøm (Å).

Solid State 1 H, 27 Al, and 31 P MAS NMR spectroscopy

Single pulse 27 Al and 31 P magic angle spinning (MAS) NMR spectra and 27 Al triple quantum magic angle spinning (3QMAS) NMR spectra were recorded on a JEOL ECZ500CR 500

MHz NMR spectrometer (11.7 T) using a 3.2 mm double resonance MAS NMR probe with 15 kHz spinning speed for the single pulse and 14 -15 kHz for the 3QMAS experiments. The relaxation delay was optimized for each sample but were generally 1 s for the single pulse 27 Al MAS NMR (< 10° pulse width for quantitative excitation) and 30 -90 s for single pulse 31 P MAS NMR (45° excitation pulse).

The 27 Al 3QMAS experiment were recorded using a three-pulse z-filter sequence [START_REF] Amoureux | Triple, quintuple and higher order multiple quantum MAS NMR of quadrupolar nuclei[END_REF].

Single pulse 1 H MAS NMR spectra were recorded on an Agilent Inova 600 MHz NMR spectrometer (14.1 T) using a 1.6 mm triple resonance MAS NMR probe and 30 -32 kHz spinning speed. The 1 H, 27 Al, and 31 P and NMR spectra were referenced using H 2 O (δ iso ( 1 H) = 4.6 ppm), 1.0 M solution of AlCl 3 (δ iso ( 27 Al) = 0 ppm) and concentrated phosphoric acid (85% H 3 PO 4 ; δ iso ( 31 P) = 0 ppm), respectively. Deconvolution of the 27 Al single pulse MAS NMR spectra were performed in Quadfit [START_REF] Kemp | QuadFit-A new cross-platform computer program for simulation of NMR line shapes from solids with distributions of interaction parameters[END_REF] using two sites: one with C Q = 1.6 MHz and η = 0 (LDH) and a second site with C Q = 3.3 MHz and η = 0.2 (amorphous aluminum hydroxide, will be discussed later) [START_REF] Jensen | A solid state NMR study of layered double hydroxides intercalated with para-amino salicylate, a tuberculosis drug[END_REF][START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF][START_REF] Staal | Competitive reactions during synthesis of zinc aluminum layered double hydroxides by thermal hydrolysis of urea[END_REF]. The 27 Al 3QMAS NMR spectra were processed in ssNake (v1.1), [START_REF] Van Meerten | ssNake: A cross-platform open-source NMR data processing and fitting application[END_REF] whereas single-pulse 1 H and 31 P MAS NMR spectra were analyzed by deconvolution in MestReNova (ver. 14.2.1.).

Zeta potential -and hydrodynamic size measurements

The zeta (ζ) potential of MgAl/Zr composite, Zr(OH) 4 , and ZrO 2 were determined by electrophoretic light scattering utilizing a Zetasizer nano ZS analyzer from Malvern Panalytical fitted with a 4 mW 632.8 nm "red" laser. For the measurement, powdered samples were dispersed (ca. 0.1 mg/mL) in deionized water and stirred 24 h and then sonicated for 15 min at room temperature. The Zeta potential values were calculated by determining the electrophoretic mobility and then applying the Henry equation.

Transmissions electron microscopy

Transmission electron microscopy (TEM) images were acquired with a JEOL 2100Plus microscope (200 kV) fitted with LaB 6 electron gun. Elemental analysis of individual particles was acquired by energy dispersive X-ray spectroscopy. 20 μL of an aqueous suspension of 1 mg sample per mL water was deposited on a carbon grid covered with a forward carbon film and dried at 40°C under air.

Elemental analysis

Inductively coupled plasma optical emission spectrometry (ICP-OES) was used for the quantification of Mg, Al, Zr, and P.. Approximately 0.02 g LDH and 10.00 mL of concentrated nitric acid (HNO 3 , 65%) were mixed followed by microwave digestion at 200°C for 15 min. The resulting

Results and discussion

The effect of the Zr content on the phosphate removal capacity of MgAl-LDH

The successful synthesis of hydrotalcite LDH was confirmed for all samples based on the observation of the characteristic ( 003), (006), and (110) reflections by PXRD (Figure 1). However, the phosphate sorption capacity of the LDH samples will be presented prior to detailed analysis of the PXRD data. Sorption will in the following be used to describe the phosphate removal properties of the LDH materials since multiple processes contribute including surface sorption, ion-exchange, and the removal of P by impurity phases. The individual contribution cannot be distinguished unambiguously from the sorption data alone. The phosphate removal capacity as a function of both the time and the concentration were obtained from sorption kinetics and isotherms. The sorption isotherm does not reach a clear plateau, even at 400% TAC (Figure 2). Visual inspection of the isotherm shows that two sequential processes contribute (Figure 2). First, one process at low equilibrium concentrations, below a C e of approximately 75 mgP/L, and a second above. This is explained by the presence of multiple phosphate removal mechanisms: both ion-exchange, adsorption to the LDH surface (e.g., charge interactions, grafting), and adsorption to impurities (details in the next section) may result in phosphate removal. Moreover, dissolution of the LDH and/or Zr(IV) (hydr)oxides followed by precipitation of phosphate phases (e.g., Al-P, Mg-P) may also contribute. The contribution from two or more pathways explains the inadequately fitting by both the Freundlich (R 2 = 0.78 to 0.90) and Langmuir (R 2 = 0.56 to 0.91) (Figure S2, Table S2). Thus, the sorption capacity is estimated visually (Table 1) at the clear break-off in the isotherm due to these non-ideal fits, which is at about C e of. Noteworthy, the kinetic sorption experiments showed a quick phosphate uptake and reached an equilibrium within the first hour (Figure S1).

In contrast to previous studies (Table 1), an increased Zr(IV) content in the LDH samples is accompanied by a drastic decrease in the phosphate adsorption capacity: For Zr0.5-A and Zr0.5-HT the experimental sorption capacity is only 20(2) and 16(2) mgP/g, respectively. The MgAl-A and MgAl-HT (conventional MgAl-LDH) exhibit the highest phosphate removal capacities of 54(2) and 45(2) mgP/g, respectively (see also Table 1). To understand this trend, detailed characterization of the samples was performed. and Langmuir isotherms (Figure S2) with the parameters given in Table S2. Multiple processes contribute to the sorption capacity which was estimated visually at the break-off point (Table 1, see text). The error-bars are based on a duplicate of sorption experiments with triplicate determination of the phosphate concentration.

3.2.Characterization of the synthesized LDH with and without Zr doping

The parent LDH with and without Zr were characterized using PXRD, ICP-OES, FT-IR, TEM, SEM, 1 H, 25 Mg and 27 Al MAS NMR, and zeta potential measurements. The unit cell parameters for the pristine LDH were obtained by Le Bail fitting (Tables 3 andS3, Figure S3). For the LDH after phosphate sorption experiments, these were estimated based on the ( 003) and ( 110) reflections due to the increased complexity of the PXRD diffractograms. Agreement factors generally show a good fit with χ 2 values of 2 -8 and R-and R f -factors of < 3% (Table S3). Aged samples are less crystallinity (i.e., lower intensity, increased FWHM; Table 3) as compared to the samples after hydrothermal treatment (Figure S1), an observation in agreement with earlier studies [START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF] Table 3. Properties of the synthesized samples and these after phosphate exposure based on PXRD, SSNMR spectroscopy, ICP-OES, and zeta potential measurements. For a comparison, the zeta potential for Zr(OH) 4 and ZrO 2 were -18(5) and -8(4) mV respectively.

a The cell parameters obtained from Le Bail fitting (Figure S3), b Cell parameters based on the position of the ( 003) and ( 110) reflections. FWHM is for the (003) reflection. n. a. = Data not available. τ = is the crystallite size approximated from the Scherrer equation using the (003) reflection.

The a-cell parameter (the M-M distance) increased ca. 0.03 Å at highest Zr(IV) doping ( treatment leads to the formation of additional crystalline phases (Figures 1 andS3). For MgAl-HT only boehmite (AlO(OH)) is detected, which is formed by dehydroxylation of Al hydroxides during the hydrothermal treatment [START_REF] Pushparaj | Structural Investigation of Zn(II) Insertion in Bayerite, an Aluminum Hydroxide[END_REF]. In contrast, two new reflections are observed at 2θ ≈ 32° and 51° for the Zr modified LDH (Zr0.1-HT, Zr0.5-HT), which are assigned to ZrO 2 formed by dehydroxylation of Zr(OH) 4 . This is supported by the conversion of amorphous Zr(OH) 4 to crystalline ZrO 2 , when hydrothermally treated similar to the LDH samples. To identify the presence of Zr(OH) 4 in the aged samples FT-IR, TGA, TEM, SEM, and SSNMR were performed.

FT-IR spectra (Figure S5) of the pristine LDH, both with and without Zr(IV), exhibit the typical LDH bands including hydroxides (3500 -3600 cm -1 ), interlayer water (1600 -1650 cm -1 ), and the metal oxides bands, MO 6 , (696 -717 cm -1 ) and OMO bands (500 -400 cm -1 ); M = metal cation [START_REF] Goh | Application of layered double hydroxides for removal of oxyanions: A review[END_REF][START_REF] Staal | Competitive reactions during synthesis of zinc aluminum layered double hydroxides by thermal hydrolysis of urea[END_REF]. Minor carbonate contamination is also observed at 1350-1380 cm - 1 due the high affinity of LDH for carbonate [START_REF] Forano | Layered double hydroxides (LDH)[END_REF][START_REF] Goh | Application of layered double hydroxides for removal of oxyanions: A review[END_REF]. When the Zr(IV) content is increased from 0.1 to 0.5 the intensity of the AlO 6 stretching mode (850-950 cm -1 ) decreased strongly for both the aged and hydrothermally treated LDH samples. This indicates depletion of Al in the LDH cation layer corresponding to the reduced reactant concentration (Table 2).

From the single pulse 27 Al MAS-(Figure 3, Figures S6-7) and 3QMAS NMR two resonances are identified: Al in the LDH (δ iso ( 27 Al) ≈ 10 -12 ppm) and Al in amorphous Al hydroxide (AOH) (δ iso ( 27 Al) ≈ 5 -10 ppm) (Lundehøj et al., 2019b;[START_REF] Staal | Competitive reactions during synthesis of zinc aluminum layered double hydroxides by thermal hydrolysis of urea[END_REF]. The concentration of AOH is higher in the aged samples (30%) compared to hydrothermally treated (20%) samples, in accordance with earlier studies [START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF][START_REF] Staal | Competitive reactions during synthesis of zinc aluminum layered double hydroxides by thermal hydrolysis of urea[END_REF]. The adjusted Mg:Al ratio in the LDH phase was calculated using ICP-OES and 27 Al NMR (or 1 H MAS NMR; Table S4). They allresults in Mg:Al ratios within the known stable range for LDH (0.2 -0.33) [START_REF] Forano | Layered double hydroxides (LDH)[END_REF], as summarized in Table 3. TGA (Figure S8) and 1 H MAS NMR (Figure 4, Figures S9-11) probed the thermal degradation and water content: Zr0.5-A exhibited a mass loss of 22(2)% and Zr0.5-HT of only 11(2)% below 200C implying a higher water or hydroxide content in the aged sample compared to the hydrothermally treated. Moreover, 1 H MAS NMR showed an increased intensity around 5 ppm, which is ascribed to the overlap of the 1 H NMR resonances from Zr(OH) 4 (δ iso ( 1 H) = 4.8 ppm; Figure S9) and water in the LDH (δ iso ( 1 H) = 4.5 -5.0 ppm; Table S4 andFigures S10 -11). Deconvolution of the 1 H MAS NMR spectra showed resonances at δ iso ( 1 H) ≈ 4.8(2) ppm for Zr0.5-HT (26(5)%) and for Zr0.5-A (37(5)%) (Figure 4).

which overlap with H 2 O (see also Figure S9). Thus, Zr(OH) 4 must be present in Zr0.5-A. To further probe this, SEM provided insight into particle size, morphology, and homogeneity (Figure S12). Scanning electron microscopy (Figure S12) showed that MgAl-A and MgAl-HT consist of hexagonal, plate-like particles with a width ranging from 200 to 600 nm, as typically observed for LDH [START_REF] Pushparaj | How the Method of Synthesis Governs the Local and Global Structure of Zinc Aluminum Layered Double Hydroxides[END_REF]. In addition, LDH with Zr added (Zr0.1-A, Zr0.5-A, Zr0.1-HT, Zr0.5-HT) contain smaller ill-defined (< 100 nm) particles (see white arrows on Figure S12b, c, e, and f), which are assigned to Zr phases (ZrO 2 and Zr(OH) 4 for Zr0.5-HT and Zr0.5-A respectively). Their presence is also supported by TGA and 1 H MAS NMR.

Further characterization by transmission electron microscopy (TEM) of selected samples (Figure 5), show that the hydrothermal treatment increases the particle size and improves the shape homogeneity of the LDH-HT particles as compared to the particles aged at 80°C (Figure 5a andb).

Hydrothermal treatment prevents aggregation and favors a face-to-face orientation of individual hexagonal platelets while for Zr0.1-A the particles are in dense,disordered agglomerates. EDX analysis (Figure S13) reveal an increase in the Mg:Al ratio in the LDH from 2.0 to 2.5, for Zr0.1-A and Zr0.1-HT respectively.. ZrO 2 nanoparticles (10 nm) are clearly observed on the surface of Zr0.1-HT platelets as well as dispersed black spots (Figure 5B). The absence of these dark spots on the TEM images of Zr0.1-A confirms the formation of amorphous Zr(OH) 4 nanoclusters most likely covering the LDH surface as also observed by 1 H MAS NMR, TGA, and SEM analysis.

It is clear that the hydrothermal treatment converts these highly dispersed Zr(OH) 4 nanoclusters into ZrO 2 nanoparticles, which are very evident in the TEM images, as illustrated for Zr0.5-A (Figure S14c andd). The sorption of phosphate on both the Zr0.1-A and Zr0.1-HT samples forces the collapse of individual particles into densely packed aggregates most likely due to the change in surface charge properties and inversion of the zeta potential. These Zr-based nanoparticles are still observed after P sorption (Figures S14c andd). The presence of Al hydroxides or boehmite rods in addition to Zr0.1-A and Zr0.1-HT-P particles (Figures14) supported the results from 27 Al MAS NMR spectra, which show a significant AOH content (Table 3). Moreover, 25 Mg MAS NMR spectra supported a higher Mg:Al ratio in the LDH than the targeted 2:1 (Figure S15), in agreement with 27 Al-, 1 H MAS NMR, and ICP-OES, as the second order quadrupolar line shape is more undefined as the Mg:Al ratio increases [START_REF] Sideris | Identification of Cation Clustering in Mg-Al Layered Double Hydroxides Using Multinuclear Solid State Nuclear Magnetic Resonance Spectroscopy[END_REF].

To summarize, Zr is segregated as amorphous Zr(OH) 4 for Zr0.1-and Zr0.5-A and as crystalline ZrO 2 for Zr0.1-HT and Zr0.5-HT, i.e., Zr is not incorporated into the LDH structure. The presence of ZrO 2 and Zr(OH) 4 is expected to influence the phosphate sorption capacity. Therefore, the theoretical sorption capacity can be estimated using the Mg:Al ratio from 27 Al MAS NMR and ICP-OES (Table S1) combined with the AOH and Zr(IV) content from ICP-OES. From the detailed characterization, it can be unambiguously concluded that the samples contain three phases: 1) MgAl-LDH 2) AOH, and 3) Zr(OH) 4 (aged samples) or ZrO 2 (hydrothermally treated). Based on 27 Al MAS NMR and ICP-OES the contribution from each phase can be calculated. The adjusted Mg:Al ratio in the MgAl-LDH and their removal capacities are summarized in Table S1. The LDH phase represents 90-98 mass% of the sample. The AOH and Zr (hydr)oxide content corresponds to 1-10 mass%, with Zr(OH) 4 /ZrO 2 contribution 2-6 mol% based on ICP-OES. Thus, based on the Zr (hydr)oxide content, this phase accounts for less than 5 mgP/g (Table 1). Therefore, mainly the LDH and the AOH contribute to the sample's phosphate removal capacity. Finally, the decreased sorption capacity is a direct consequence of the decrease in Al content.

3.3.Characterization of the LDH after phosphate sorption

The phosphate loaded LDH (MgAl-A-P, MgAl-HT-P, Zr0.1-A-P, Zr0.1-HT-P, Zr0.5-A-P, and Zr0.5-HT-P) were characterized in detail to understand the stability of the LDH during P exposure and the pathway of removal.

As expected, phosphate sorption on the surface of aged and hydrothermally treated samples reversed the charge of the platelets. Thus, the zeta potential value changes from 29 -27 mV (MgAl-A and MgAl-HT) to -17 mV (MgAl-A-P and MgAl-HT-P) (Table 3). No significant different is observed between the MgAl-LDH and the lowest Zr-doping level. Increasing the Zr(IV) content to 0.5 in the aged LDH leads to a clear change of surface charge from +29 mV to -12 mV. Hydrothermal treatment leads to phase segregation and formation of ZrO 2 , which has limited effect on the mean (bulk) zeta potential (Table 3).

PXRD showed an increased stacking disorder and decrease in crystallinity of the phosphate loaded LDH (Table 3), also observed in the 1 H-and 25 Mg MAS NMR (Figures S10-11, S15), and in agreement with earlier studies of MgAl-LDH (Lundehøj et al., 2019a). The lower resolution of the 1 H MAS NMR spectra prevented unambiguous deconvolution and thereby determination of the Mg:Al ratio. However, 27 Al MAS NMR (Figure S16), FT-IR (Figure S5) and PXRD (Figure 1) supported preservation of the LDH structure, but with an increased stacking disorder.

For example, the M-M distance in the LDH (unit cell parameter a) does not change after phosphate exposure (Table 3 and Figure S4). Similarly, ICP-OES (Table 1) and 27 Al MAS NMR shown no significant change in the bulk composition of Mg, Al, and Zr or relative concentration of the LDH, respectively.

The c unit cell parameters, i.e., the interlayer spacing, increases ca 0.5 Å after P sorption, which combined with the increase in the FWHM of the (00l) reflections (Table 3) reflects the intercalation of phosphate into the LDH as previously observed [START_REF] Costantino | Intercalation and grafting of hydrogen phosphates and phosphonates into synthetic hydrotalcites and a.c.-conductivity of the compounds thereby obtained[END_REF][START_REF] Hou | Hydration, expansion, structure, and dynamics of layered double hydroxides[END_REF]Lundehøj et al., 2019a). Moreover, the ν 3 (PO 4 3-

) vibration [START_REF] Badreddine | Ion exchange of different phosphate ions into the zinc-aluminum-chloride layered double hydroxide[END_REF] observed by FT-IR (Figure S5) also proves phosphate related to the material and the negative surface charge (-9 to -19 mV; Table 3) indicates surface complexation. To summarize, characterization show no changes in the sample composition (i.e., limited dissolution and degradation) of the LDH layer after phosphate exposure but strong surface modifications. P MAS NMR spectra were recorded (Figure 6). Deconvolution of the 31 P MAS NMR spectra of LDH can be challenging due to broad resonances and/ or overlapping sites as observed previously [START_REF] Borges | Potential Sustainable Slow-Release Fertilizers Obtained by Mechanochemical Activation of MgAl and MgFe Layered Double Hydroxides and K 2 HPO 4[END_REF][START_REF] Hou | Hydration, expansion, structure, and dynamics of layered double hydroxides[END_REF]Lundehøj et al., 2019a;Lundehøj et al., 2019b). Generally, each LDH here contains four or five different P sites, i.e., five different phosphorous local environments. These sites are site 1: δ iso ( 31 P) = 6.8 to 7.8 ppm, site 2: δ iso ( 31 P) = 2.9 to 4.2 ppm, site 3: δ iso ( 31 P) = 0.6-1.7 ppm, site 4 δ iso ( 31 P) = -0.8 to -2.2 ppm and finally site 5 δ iso ( 31 P) = -4.1 to -6.4 ppm (details in Table S5)

Sites 2 (20 -31%) and 3 (21 -36%) account for most of the intensity and are therefore assigned to intercalation of ortho-phosphate in the LDH, given that it is the main removal pathway. This is agreement with phosphate related to a ZnAl-LDH were found at δ iso ( 31 P) = 4.5(5) and 0.6(3) ppm doing phosphate sorption at pH 5 (Lundehøj et al., 2019b). Furthermore, two sites at δ iso ( 31 P) = 3.2 ppm for HPO 4 2-and a site at 6.2 ppm for PO 4 3were identified for MgAl-LDH based on visual inspection [START_REF] Hou | Hydration, expansion, structure, and dynamics of layered double hydroxides[END_REF]. These reported δ iso ( 31 P) does not fully match the values for site 2 and site 3 (Table S5), but determination of the isotropic resonances in spectra with overlapping sites with overlapping resonances is less precise. Therefore, Sites 2 and 3 are assigned to intercalation of H x PO 4

(3-

x)-species.

Sites 4 (10 -26%) and site 5 (20 -28%) are assigned to phosphate removed by AOH, and the Zr phase(s), Zr(OH) 4 for aged samples and ZrO 2 for hydrothermally treated samples, respectively.

This was based on a comparison with 31 P MAS NMR spectra recorded for Zr(OH) 4 -P and ZrO 2 -P (Figure 6c andd) combined with previous studies (Lundehøj et al., 2019a;Lundehøj et al., 2019b).

Differentiation between the AOH, Zr(OH) 4 , and ZrO 2 cannot be done because of broad and overlapping resonances and is beyond the scope of this work.

Finally, Site 1 (4 -12%; δ iso ( 31 P) = 6.8 to 7.8 ppm) has the lowest intensity and is not detected for Zr0.5-A or Zr0.5-HT. Furthermore, this site is most prominent for aged samples, Therefore, this site is tentatively assigned to dissolution of the LDH, as the smaller particles are less stable. The lack of site 1 for Zr0.5-A and Zr0.5-HT is explained by the high Mg:Al ratio in the LDH.

Phosphate sorbed to impurities (AOH, Zr(OH) 4 , and ZrO 2 ) is the major pathway (41 -54%) for aged samples in accordance with 27 Al-and 1 H MAS NMR. For a comparison, the impurities contribute 34 -38% for hydrothermally treated samples. Thus, the impurity content in the LDH must be controlled, otherwise the phosphate will be strongly bound to the impurities instead of readily released from the LDH. This will decrease the recyclability of the LDH sorbents. S5). Dissolution and re-precipitation process (blue), H x PO 4 (3-x)-removed by the LDH (orange and green) and the contribution from impurities, i.e., AOH, ZrO 2 , Zr(OH) 4 (grey). mgP/g). Single pulse 31 P MAS NMR showed that 42 -62% of the ortho-phosphate was intercalated in the LDH by ion-exchange. However, 31 P MAS NMR also showed that up to 54% of phosphate was removed by impurity phases or dissolution of the LDH. Thus, phosphate has a high affinity towards the common impurities and the synthesis methods for LDH with limited impurities (coprecipitation followed by hydrothermal treatment) is important for the reusability of the LDH based sorbents. Our study clearly show that large particle size (crystallinity) is favored and that impurity phases should be minimized. Moreover, it is emphasized that phosphate removal pathways cannot be reliably assessed based on sorption studies only. Detailed characterization of the LDH sorbent before and after phosphate exposure is paramount for reliable assessment of the LDH sorbent performance and stability.

Conclusions

Figure 1 .

 1 Figure 1. PXRD diffractograms of the synthesized LDH samples and these after phosphate exposure

Figure 2 .

 2 Figure 2. Phosphate sorption isotherm, which is inadequately modelled by both the Freundlich (shown)

Figure 3 .

 3 Figure 3. A representative deconvolution of a single pulse 27 Al MAS NMR spectrum using two phases: δ iso ( 27 Al) ≈ 10 ppm (LDH, pink) and δ iso ( 27 Al) centered at ≈ 7 ppm (AOH, green). The 27 Al MAS NMR

Figure 4 .

 4 Figure 4. Deconvolution of the 1 H MAS NMR spectra of a) Zr0.5-A and b) Zr0.5-HT with the

Figure 5 .

 5 Figure 5. Two representative TEM images for each of a) Zr0.1-A, b) Zr0.1-HT), c) Zr0.1-A-P, and d)

3. 4 .Figure 6 .

 46 Figure 6. Experimental 31 P MAS NMR spectra of the LDH samples after phosphate sorption (a, b, e, f),

Figure 7 .

 7 Figure 7. The relative intensity between the different 31 P sites and their assignment based on deconvolution of the 31 P MAS NMR spectra (TableS5). Dissolution and re-precipitation process

  Detailed insight into the phosphate removal capacity and stability of a series of MgAl-LDH with different Zr(IV) modifications was achieved using global and local characterization techniques. Our results clearly demonstrate, that Zr(IV) cannot be incorporated into the LDH lattice and thereby improve the phosphate removal capacity. Instead separate phase of Zr(OH) 4 or ZrO 2 were present for the aged and hydrothermal treated samples, respectively. Especially detection of the X-ray amorphous Zr(OH) 4 in the aged samples required advanced characterization techniques ( 1 H-and 27 Al MAS NMR, SEM, TEM and TGA). The unsuccessful incorporation of Zr(IV) together with the decreased Al doping resulted in a drastic decrease in the phosphate removal capacity (45 -54 to 16 -20

Table 1 .

 1 Phosphate removal capacities (mgP/g) determined for Zr-doped LDH and the corresponding parent M(II)M(III)-LDH reported in literature and the current work. The chemical composition (M(II), M(III), anion) of the LDH, which was prepared by co-precipitation, the post synthesis treatment employed (aged or hydrothermal treated, HT), and the water medium tested (seawater, wastewater, phosphate solution) and the phosphate concentration(s) used (mgP/L). The P removal capacity for Zr(OH) 4 and ZrO 2 are given for a comparison. Finally, whether the conclusion was successful Zr doping into the LDH or not (yes/ no). RT = room temperature. HT = hydrothermal treatment. * = two phase system (M(II)M(III)-LDH and Zr-phase) purposely prepared.

	Cations	Treatment	Anion Solution	M(II)/(M(III)+Zr)	M(III):Zr	P removal	Zr in LDH	References
				(mgP/ L)			(mgP/ g)	layer?
	Mg, Al	Aged (RT)	CO 3 2	P enriched seawater	3	1	25	Yes	(Chitrakar et al., 2007)
		Aged (RT)	Cl -	(0 -0.33)	3	1	9	Yes
		Aged (RT) Aged (RT) Aged (RT)	2-CO 3 2-CO 3 Cl -	Phosphate (1 -10)	Not available 3 3	NA 1 1	1 39 22	NA Yes Yes
		Aged (RT) Aged (RT)	2-CO 3 Cl -	Phosphate (100)	3 3	1 1	50 30	Yes Yes
	Zn, Al Mg, Al	Aged (65°C) Aged (65°C) Aged (RT)	2-CO 3 2-CO 3 CO 3 2-	Phosphate (10 -250) P enriched seawater	2 -4 2 -4 4	2.5 NA 1	25 -91 9 -60 18	Yes NA No	(Koilraj and Kannan, 2010) (Miyauchi et al., 2009)
		Aged (RT) HT (120°C) HT (120°C)	2-CO 3 2-CO 3 CO 3 2-	(0.33)	4 4 4	NA 1 NA	1.3 10 0.6	NA No NA
	Mg, Fe	Aged (RT) Aged (RT)	2-CO 3 Cl -	P enriched seawater (1 -30)	2 -4 Not available	1 NA	15 -27 ≈ 3	No NA	(Chitrakar et al., 2010)
		Aged (RT) Aged (RT)	2-CO 3 Cl -	Phosphate (100)	2 -4 Not available	1 NA	20 -35 ≈ 50	No NA
	Mg, Fe	Aged (80°C)	CO 3 2-	Phosphate (1 -50)	Not available	2 -0.5	35	No *	(Nuryadin et al., 2021)
	Mg, Al	Aged (80°C) Aging (RT) Aging (RT)	2-CO 3 2-CO 3 CO 3 2-	Phosphate (5 -200)	Not available Not available Not available	NA NA Not available	18 70 99	NA NA No *	(Motandi et al., 2022)
	ZrO 2	Not available	None	Seawater (0.33)	Not available	Not available	1 -12	Not available (Miyauchi et al., 2009)
	Zr(OH) 4 Not available	None	Synthetic wastewater (10)	Not available	Not available	19 -30	Not available (Johir et al., 2016)
	Zr(OH) 4 Not available Mg, Al HT (120°C)	None Cl -	Phosphate (7 -114) Phosphate (18 -224)	Not available 2	Not available No Zr	38 45(2)	Not available (Luo et al., 2017) No Zr This work
		Aging (80°C)	Cl -		2	No Zr	54(2)	No Zr
		HT (120°C)	Cl -	Phosphate (18 -224)	2	9	43(2)	No	This work
		Aged (80°C)	Cl -		2	9	47(2)	No
		HT (120°C)	Cl -	Phosphate (18 -224)	2	1	16(2)	No	This work
		Aged (80°C)	Cl -		2	1	20(2)	No

NA, not applicable as Zr was not used during the synthesis.

Table 3

 3

		a (Å)	c (Å)	FWHM (°) τ (Å) AOH (%Al)	x ( 27 Al+ICP)	x ( 1 H)	Zeta (mV)
	MgAl-A a	3.048(2)	23.303(3)	0.641(2)	124	29.0(3)	0.27(2)	0.23(2)	29(5)
	MgAl-HT a	3.060(2)	23.617(3)	0.3249(6)	246	22.6(3)	0.28(2)	0.26(2)	27(6)
	Zr0.1-A a	3.078(2)	23.417(3)	0.647(2)	123	29(2)	0.25(2)	0.20(2)	28(5)
	Zr0.1-HT a	3.076(2)	23.576(3)	0.341(6)	234	19(1)	0.25(2)	0.27(2)	25(6)
	Zr0.5-A a	3.080(2)	24.245(3)	0.526(2)	152	20(2)	0.18(2)	0.22(2)	12(4)
	Zr0.5-HT a	3.092(2)	24.211(3)	0.2415(4)	330	< 5	0.20(2)	0.18(2)	30(5)
	MgAl-A-P b	3.043(3)	24.859(4)	1.593(7)	50	32.0(2)	0.26(2)	n. a.	-17(4)
	MgAl-HT-P b	3.054(3)	24.650(4)	0.775(2)	103	23.1(5)	0.29(2)	n. a.	-17(4)
	Zr0.1-A-P b	3.047(3)	24.947(4)	2.30(12)	35	27(1)	0.26(2)	n. a.	-19(4)
	Zr0.1-HT-P b	3.043(3)	23.935(4)	0.943(3)	85	26.0(1)	0.26(2)	n. a.	-18(4)
	Zr0.5-A-P b	3.075(3)	23.861(4)	0.526(2)	152	21.8(3)	0.18(2)	0.21(2)	-11(4)
	Zr0.5-HT-P b	3.073(3)	23.750(4)	0.2484(9)	331	9.5(3)	0.20(2)	n. a.	-9(4)

, Figures S4). Diffractograms of MgAl-A and the Zr modified samples after aging (Zr0.1-A, Zr0.5-A) only contain LDH reflections (beside Al(OH) 3 impurities). In contrast, hydrothermal

Acknowledgments

The authors acknowledge The Danish Research Foundation (DFF Grøn Omstilling; grant: 95-305-23601-01130) and The Danish Research Council -Technology and Production Science (DFF-7017-00262) for financial support. The following people are thanked for their valuable help, discussions, and assistance with experimental data: lab. technicians Carina Lohman (ICP-OES) and Christian Brandt Jørgensen (SSNMR), Dr. Christian Henriksen (PXRD and Le Bail refinement), Dr Arkadiusz Jaroslaw Goszczak (SEM), Dr. Anders Bruhn Arndal Andersen (SSNMR) Mr. Lucas Urbano José (valuable discussions).

Data availability

Data will be available upon request.

Declaration of Competing Interest

The authors declare no competing interest.

Appendix A. Supplementary data

Supporting information provide additional information about PXRD, Le Bail refinement, FT-IR, SEM, 1 H, 25 Mg, 31 P, 27 Al 1D MAS NMR together with 2D MAS NMR ( 27 Al 3QMAS) and kinetics experiments together with isotherm refinement parameters.


Manon Boulet

Guerric Gilbert

Yoan Renaud

Martina Schmidt-Dengler

Emilie Auxerre-Plantié

Romane Bertrand

Xinsheng Nan

Tomasz Jurkowski

Mark Helm

Laurence Vandel
email: laurence.vandel@uca.fr

Emilie Plantié

Lucas Waltzer
email: lucas.waltzer@uca.fr.

Adenine methylation is very scarce in the drosophila genome and not erased by the Ten Eleven Translocation dioxygenase

Keywords: Epigenetics, DNA methylation, N6-methyladenine (6mA), Ten Eleven Translocation

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Introduction

Until recently, N 6 -methyl-2'-deoxyadenosine (also called N6-methyladenine or 6mA) was considered to be essentially restricted to the genome of prokaryotes, where this modification plays a well-established role in the restriction-modification system and other processes such as DNA replication or transcription [START_REF] Sanchez-Romero | The bacterial epigenome[END_REF][START_REF] Wion | N6-methyl-adenine: an epigenetic signal for DNAprotein interactions[END_REF]. Since 2015, several reports detected the presence of 6mA in the DNA of different eukaryotic organisms [START_REF] Alderman | N(6)-Methyladenine in eukaryotes[END_REF][START_REF] Boulias | Means, mechanisms and consequences of adenine methylation in DNA[END_REF], including in metazoans [START_REF] Greer | DNA Methylation on N6-Adenine in C. elegans[END_REF][START_REF] Koziol | Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications[END_REF][START_REF] Liu | Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig[END_REF][START_REF] Wu | DNA methylation on N(6)-adenine in mammalian embryonic stem cells[END_REF][START_REF] Xiao | N(6)-Methyladenine DNA Modification in the Human Genome[END_REF][START_REF] Xie | N(6)-methyladenine DNA Modification in Glioblastoma[END_REF]Zhang et al., 2015a). Although a small fraction of all adenines seems methylated at N6 position (from 0.4% to 0.0001% or below), it was proposed that this modification participates in eukaryotic genome regulation [START_REF] Wu | The epigenetic roles of DNA N(6)-Methyladenine (6mA) modification in eukaryotes[END_REF]). Yet, the significance of 6mA in eukaryotes and the enzymes involved in its metabolism remain controversial with several studies questioning the existence and/or the level of this modification, particularly in metazoans [START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF]Kong et al., 2022;[START_REF] Liu | N(6)-methyladenine is incorporated into mammalian genome by DNA polymerase[END_REF][START_REF] Musheev | The origin of genomic N(6)methyl-deoxyadenosine in mammalian cells[END_REF][START_REF] O'brown | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA[END_REF][START_REF] Schiffers | Quantitative LC-MS Provides No Evidence for m(6) dA or m(4) dC in the Genome of Mouse Embryonic Stem Cells and Tissues[END_REF]. Part of the controversy stems from the technologies used to detect 6mA [START_REF] Boulias | Means, mechanisms and consequences of adenine methylation in DNA[END_REF][START_REF] Li | The exploration of N6deoxyadenosine methylation in mammalian genomes[END_REF].

Notably, antibody-based techniques such as dot blot or DNA immunoprecipitation followed by sequencing (DIP-seq) have been particularly called into question to study low levels of 6mA [START_REF] Bochtler | DNA adenine methylation in eukaryotes: Enzymatic mark or a form of DNA damage?[END_REF][START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF][START_REF] Lentini | A reassessment of DNAimmunoprecipitation-based genomic profiling[END_REF]. If liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) provides a sensitive method to identify 6mA and measure its absolute levels unambiguously, bacterial contaminations can affect the results [START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF]Kong et al., 2022;[START_REF] O'brown | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA[END_REF]. Finally, single-molecule real-time sequencing (SMRT-seq) can detect 6mA presence (and location) on genomic DNA but is prone to give rise to a high false discovery rate when 6mA is rare [START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF][START_REF] O'brown | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA[END_REF][START_REF] Xiao | N(6)-Methyladenine DNA Modification in the Human Genome[END_REF].

Notwithstanding, 6mA presence appears strongly supported in drosophila genome [START_REF] He | 6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda[END_REF][START_REF] Ismail | Drosophila Tet Is Expressed in Midline Glia and Is Required for Proper Axonal Development[END_REF][START_REF] Shah | Adenine Methylation in Drosophila Is Associated with the Tissue-Specific Expression of Developmental and Regulatory Genes[END_REF][START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF][START_REF] Ye | MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing[END_REF]Zhang et al., 2015a), where this modification was described to be associated with transposable element silencing and activation of gene transcription [START_REF] He | 6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda[END_REF][START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a). Unexpectedly, 6mA demethylation in drosophila was attributed to the Ten Eleven Translocation (TET) enzyme (Zhang et al., 2015a), a member of the 5-methylcytosine (5mC) dioxygenase family [START_REF] Iyer | Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids[END_REF].

Here, we re-evaluated 6mA levels in drosophila and reassessed the impact of TET on this mark. Using LC-MS/MS, we show that 6mA is present at very low levels in drosophila in axenic conditions and that the absence of TET does not lead to any consistent increase in 6mA levels in the larval central nervous system, nor in the whole larva, the embryo or the adult brain. Furthermore, our genetic and molecular analyses suggest that TET is not involved in 6mA demethylation and that its function during drosophila development is largely catalytic-independent.

Results and discussion

Previously reported levels of 6mA measured by LC/MS-MS in drosophila ranged from 0.07% to 0.0006% (6mA/A), with the highest levels in the early embryo [START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a). However, a recent study reported much lower levels (0.0002%) even in early embryos and showed that initially reported "high" levels of 6mA were likely due to bacterial contaminations (Kong et al., 2022). Indeed, the contamination of genomic DNA (gDNA) by bacterial DNA is a major confounding factor for LC-MS/MS experiments [START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF]Kong et al., 2022;[START_REF] O'brown | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA[END_REF]. Moreover, the presence of intracellular bacteria can also be a source of 6mA [START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF]. Along that line, it is worth noting that the genome of Wolbachia, a frequent endosymbiont in drosophila, codes for DNA Adenine methyltransferases [START_REF] Saridaki | Wolbachia prophage DNA adenine methyltransferase genes in different Drosophila-Wolbachia associations[END_REF]. In addition, 6mA derived from exogenous sources might be incorporated into gDNA via the salvage pathway [START_REF] Musheev | The origin of genomic N(6)methyl-deoxyadenosine in mammalian cells[END_REF], and independently of autonomously-directed adenine methylation [START_REF] O'brown | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA[END_REF][START_REF] Schiffers | Quantitative LC-MS Provides No Evidence for m(6) dA or m(4) dC in the Genome of Mouse Embryonic Stem Cells and Tissues[END_REF]. To exclude these possible sources of contamination, we generated germ-free drosophila and reared the larvae on chemically-defined ("holidic") food devoid of exogenous DNA contribution (see Methods). The absence of exogenous or endosymbiotic bacteria in the resulting flies was confirmed by PCR (Fig. 1a). In these conditions, we observed 0.00025% of 6mA in whole larvae (Fig. 1b) and 0.0005% in the larval central nervous system (CNS) (Fig. 1c). Noteworthy, this corresponds to around 200 to 400 methylated adenines per haplogenome. In addition, similar levels of 6mA were measured in the CNS when non-axenic larvae were reared on classic medium without antibiotic treatment (Fig. 1c), suggesting that contamination by exogenous sources is not a major problem in this tissue. These results confirm the presence of a very small fraction of methylated adenines in drosophila DNA.

Changes in 6mA levels following genetic manipulations of putative adenine methylases or demethylases have brought further credence to the existence and role of this modification in metazoans [START_REF] Boulias | Means, mechanisms and consequences of adenine methylation in DNA[END_REF]. In drosophila, the absence of TET (also called DMAD, for DNA Methyl Adenine Demethylase) was associated with a strong increase in 6mA levels in embryo or adult ovary and brain [START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a). In addition, in vitro experiments suggested that drosophila TET can demethylate 6mA [START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a). However, that TET mediates 6mA oxidation is at odd with the well-characterized function of this family of enzymes in 5mC oxidation in metazoans [START_REF] Lio | TET methylcytosine oxidases: new insights from a decade of research[END_REF]. Moreover, the other enzymes involved in methyladenine oxidation/demethylation belong to the AlkB family [START_REF] Boulias | Means, mechanisms and consequences of adenine methylation in DNA[END_REF][START_REF] Xu | Reversal of nucleobase methylation by dioxygenases[END_REF], which is related to, but distinct from the TET family [START_REF] Iyer | Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids[END_REF][START_REF] Jia | Large-scale examination of functional and sequence diversity of 2-oxoglutarate/Fe(II)-dependent oxygenases in Metazoa[END_REF]. Indeed, conserved residues involved in TET 5mC recognition differ from those found within the AlkB family and may not be able to accommodate a purine residue instead of a pyrimidine [START_REF] Hu | Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation[END_REF]Iyer et al., 2009;[START_REF] Parker | Insights into the Biochemistry, Evolution, and Biotechnological Applications of the Ten-Eleven Translocation (TET) Enzymes[END_REF][START_REF] Xu | Reversal of nucleobase methylation by dioxygenases[END_REF]. Nevertheless, the fact that the drosophila genome presents extremely low levels of 5mC and does not code for any 5mC DNA methyltransferase [START_REF] Iyer | Natural history of eukaryotic DNA methylation systems[END_REF][START_REF] Krauss | DNA methylation in Drosophila--a critical evaluation[END_REF] prompted the idea that TET could catalyze other forms of DNA modifications and notably 6mA oxidation/demethylation in the absence of its canonical substrate (Zhang et al., 2015a).

As tet was shown to be highly expressed in the larval CNS [START_REF] Delatte | RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine[END_REF]Wang et al., 2018), we first focused on its impact on 6mA in this tissue. Yet, we found that the levels of 6mA measured by LC-MS/MS in the absence of TET (tet null , an allele that abolishes tet transcription [START_REF] Delatte | RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine[END_REF], were similar to wild type in the larval CNS using either axenic flies raised on holidic medium or non-axenic flies raised on classic medium (i.e. conventional conditions) (Fig. 1c). Of note, TET loss did not show any impact on 6mA level either in whole larvae (Fig. 1b). As previous experiments showing an increase in 6mA in the absence of TET were performed under conventional conditions with tet DMAD1 /tet DMAD2 mutant alleles [START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a), which introduce a premature stop codon in tet open reading frame before its catalytic domain (Zhang et al., 2015a), we repeated the analyses with this allelic combination. Yet, we did not find any increase of 6mA levels in the larval CNS in this setting either (Fig. 1d). Moreover, consistent with previous results showing that TET does not control 5mC oxidation in drosophila (Zhang et al., 2015a), we observed very low levels of 5mC (around 0.001%) in the larval CNS and no increase upon TET loss (Fig. 1e). In addition, the first product of 5mC oxidation, 5hmC (5-hydroxymethylcytosine), was below the detection limit (0.00001%). These results suggest that TET is not involved in 6mA (or 5mC) demethylation in the drosophila larval CNS.

To test whether the lack of impact of TET on 6mA levels that we observe here contrary to previous studies [START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a), could be due to a tissue-specific effect or to breeding conditions, we assessed 6mA levels in embryos and adult brains using conventional or germ-free flies (see Methods). As tet null homozygote mutation is pupal lethal (Zhang et al., 2015a), only tet DMAD1/DMAD2 could be used for adult brains. The presence/absence of bacterial contaminants in conventional versus germ-free stocks was validated by PCR (Supplementary Figure S1a). Moreover, by performing gDNA sequencing, we found around 2% of bacterial DNA contaminant in gDNA in conventional flies versus less than 0.003% in germ-free flies (Supplementary Figure S1b). Hence, possible traces of bacterial contaminations in axenic samples should have negligible impact on LC-MS/MS measurements. LC-MS/MS analyses showed that 6mA levels were higher in embryos (Fig. 1f) or adult brains (Fig. 1g) using conventional flies as compared to their germ-free siblings. They were also more variable across samples in non-axenic conditions. It is thus likely that 6mA levels measured in non-axenic conditions do not solely reflect endogenous 6mA in the drosophila genome and variations between genotypes should be interpreted with caution. Still, we did not observe any significant increase in 6mA levels in the absence of TET in non-axenic conditions (Fig. 1f,g). Importantly, the same observation was made in axenic conditions (Fig. 1f,g). All together, we did not find evidence that TET loss caused an increase in 6mA levels in embryos, whole larvae, larval CNS or adult brain.

As an alternate method to study TET impact on 6mA, we used SMRT sequencing [START_REF] Boulias | Means, mechanisms and consequences of adenine methylation in DNA[END_REF]. We generated SMRT-seq data on CNS gDNA from three biological replicates of wild type and tet null larvae. As genome coverage is an important parameter to analyze SMRT-seq data, we first merged the three replicates to increase read density. In the resulting fusion datasets, around 95% of all the adenines were covered at least 25x (Supplementary Figure S2 and Table S1). The detection of 6mA by SMRT-seq is based on a modification quality value (mQV or QV), reflecting the consistency by which a specific modification is observed at a given position in a subread. Using standard parameters (coverage ≥25x and QV≥20), we found respectively, 0.158% and 0.172% of potential 6mA in the CNS of wild type and tet null larvae (Fig. 2a, Supplementary Table S2), which is much more than expected based on LC-MS/MS measurements. However, when we considered the methylation status of these adenines in the individual samples, only 2.6% were labeled as 6mA in all 3 replicates and 13.7% in at least two replicates (Fig. 2b, Supplementary Figure S3 and Table S3), consistent with the idea that SMRT-seq gives a high rate of false positives in organisms containing low 6mA levels [START_REF] Douvlataniotis | No evidence for DNA N (6)-methyladenine in mammals[END_REF][START_REF] Kong | Navigating the pitfalls of mapping DNA and RNA modifications[END_REF][START_REF] Schadt | Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases[END_REF][START_REF] Xiao | N(6)-Methyladenine DNA Modification in the Human Genome[END_REF]. Interestingly, increasing the QV strongly ameliorated the fraction of replication and drastically reduced the proportion of potential 6mA both in wild type and tet null datasets, whereas increasing the coverage had little effect (Fig. 2a, b, Supplementary Figure S3 and Table S3). We thus analyzed SMRT-seq results from wild type and tet null replicates using a coverage≥25x and increasing QV. However, we did not observe any significant differences either in the percentage of 6mA/A or in the fraction of methylation of these potential 6mA even with stringent QV values (Fig. 2c, d and Supplementary Table S4). Hence, while SMRTseq data are noisy, as cautioned in previous studies, they did not reveal an increase in 6mA levels in the absence of TET.

To directly test whether TET demethylates 6mA, we then assessed its activity in vitro. Accordingly, the recombinant catalytic domain of drosophila TET was incubated with double-stranded DNA (dsDNA) containing either a 5mC or a 6mA modification and the level of modified bases was quantified by LC-MS/MS at different time-points. Under our experimental conditions, 5mC levels were drastically reduced in 1 min with the concomitant appearance of 5mC oxidation products (5hmC and 5fC) (Fig. 3a). In sharp contrast, the level of 6mA remained constant even after 30 min of incubation (Fig. 3b). Hence, contrary to 5mC, 6mA does not seem to be a good substrate for drosophila TET in vitro. Of note, only traces of 5mC, 5hmC, 5fC or 6mA were observed when recombinant TET was incubated with non-modified dsDNA, indicating that the levels measured in the presence of modified dsDNA were not due to contaminations (Supplementary Figure S5). Besides, it is worth mentioning that a distant TET homolog in the fungus Coprinosis cinerea was recently shown to oxidize both 5mC and 6mA [START_REF] Mu | A fungal dioxygenase CcTet serves as a eukaryotic 6mA demethylase on duplex DNA[END_REF]). Yet, its peculiar capacity to bind and demethylate 6mA requires key residues within its catalytic domain which are not conserved in other TET homologs including in drosophila (Supplementary Figure S6). These observations support our results showing that drosophila TET does not demethylate 6mA.

Although previous reports suggested that TET controls fly viability, ovarian development or adult brain formation by demethylating 6mA [START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a), the functional importance of TET enzymatic activity has never been tested genetically as available tet alleles either abolish its expression or delete the whole catalytic domain. In view of our results and to address this issue, we generated a catalytic dead mutant allele of tet (tet CD ). Accordingly, the conserved HxD ironbinding motif required for the catalytic activity of TET/AlkB dioxygenase family of enzymes [START_REF] Hu | Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation[END_REF][START_REF] Tahiliani | Conversion of 5-methylcytosine to 5[END_REF][START_REF] Xu | Reversal of nucleobase methylation by dioxygenases[END_REF] was mutated by CRISPR/Cas9-mediated homologous recombination using a tet-GFP knock-in which allows to tag all protein TET isoforms with GFP (Fig. 4a,b). Of note, immunostaining in the larval CNS confirmed that TET is widely expressed in this tissue and showed that the H1947Y/D1949A mutation does not alter TET expression or its nuclear localization (Fig. 4c-h). Strikingly, while tet null homozygote individuals die at the pupal stage [START_REF] Delatte | RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine[END_REF], we found that tet CD/CD as well as tet CD/null pupae had a normal hatching rate and gave rise to viable adult flies (Fig. 4i). We did not observe lethality of tet CD/CD individuals at earlier developmental stage either (Supplementary Figure S7). Then, we assessed whether this mutation affected adult wing positioning, ovarian development or mushroom body formation as reported upon TET loss of expression (Wang et al., 2018;[START_REF] Yao | Active N(6)-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons[END_REF]Zhang et al., 2015a). Yet, tet CD/CD flies did not exhibit the "held out" wing positioning defects present in tet DMAD1/DMAD2 adult escapers (Fig. 4j, j', j''). Similarly, atrophied ovaries or mushroom body projection defects observed in the absence of TET expression were not reproduced when only its catalytic activity was impaired (Fig. 4k, k', k'' and4l, l', l''). Thus, TET function in drosophila seems essentially independent of its enzymatic activity, indicating that TETmediated regulation of 6mA level, if it truly happens, is not essential either for fly development.

Conclusions

In sum, our results confirm that 6mA is present only at very low levels in the drosophila genome. With only a few hundred methylated adenines per haplogenome, we argue that 6mA is unlikely to play a major regulatory function in normal conditions. In addition, we did not find any evidence that TET loss promotes 6mA accumulation. Rather, our results strongly suggest that this conserved enzyme is not a methyladenine demethylase and that its catalytic activity is largely dispensable for drosophila development. Further experiments will be necessary to firmly establish whether regulated adenine methylation/demethylation takes place and what are the enzymes involved, not only in drosophila but also in other metazoans.

Methods

Fly strains and breeding

The following D. melanogaster strains were used: w 1118 (control, Bloomington), tet null [START_REF] Delatte | RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine[END_REF], tet DMAD1 , tet DMAD2 (Zhang et al., 2015b). The tet-GFP knock-in line was generated by InDroso Functional Genomics (Rennes, France) using CRISPR/Cas9-mediated homologous recombination to insert the EGFP in frame with the last amino-acid of TET. Similarly, the catalytic dead tet CD flies were generated by CRISPR/Cas9-mediated homologous recombination in the tet-GFP background to mutate TET HRD motif into YRA (dm6: chromosome 3L:2,791,624 "A" to "C" and 3L:2,791,631 "C" to "T"). In both cases, the resulting flies were validated by sequencing.

Unless otherwise specified, stock maintenance and sample collection were performed using classic fly medium (75 g/l organic corn flour, 28 g/l dry yeast, 40 g/l sucrose, 8 g/l agar, 10 ml/l Moldex 20%) with a 12 h dark:light cycle. Germ-free drosophila lines were generated as described [START_REF] Sabat | A Protocol to Generate Germ Free Drosophila for Microbial Interaction Studies[END_REF]. Briefly: embryos were collected on grape juice agar plates, dechorionated with 2.7% bleach for 2-3 min, washed in sterile ddH2O and transferred to standard fly medium supplemented with antibiotics (50 µg/ml amoxicillin, tetracyclin, kanamycin and puromycin) for at least two successive generations. When "holidic" medium was used to avoid any source of contamination by exogenous DNA, embryos from germ-free adults were collected on grape juice agar plates, dechorionated, washed with ddH2O and transferred to a chemically-defined medium, using the amino acid ratio of the FLYAA recipe [START_REF] Piper | Matching Dietary Amino Acid Balance to the In Silico-Translated Exome Optimizes Growth and Reproduction without Cost to Lifespan[END_REF], together with antibiotics to maintain axenic conditions. All crosses and larvae collections were performed at 25°C.

Viability assays

Embryos were collected at 25°C from 1-week-old flies over 8h on grape juice agar plates. For each genotype, batches of 100 embryos were transferred to corn flouryeast-agar plates; the number of first instar larvae was counted after 30h, the number of pupae was counted at day 9 and the number of hatched adults was counted from days 10 to 15. Each experiment was repeated at least four times.

Immunostainings

Third instar larvae or adult fly brains were dissected in 1X Phosphate Buffer Saline (PBS) and fixed for 25 min in PBS containing 4% paraformaldehyde. Fixed samples were washed rapidly twice with PBS and 3 times for 15 min with PBS-0.3% Triton X-100 (PBT) before being pre-incubated for 1h in PBT-1% bovine serum Albumin (BSA, Sigma). Samples were incubated overnight at 4°C with primary antibody diluted in PBT-1% BSA, washed 3 times for 15 min in PBT, and incubated with respective secondary antibodies diluted in PBT-1% BSA for 3h at room temperature or overnight at 4°C. Samples were washed in PBT and mounted in Vectashield-DAPI (Vector Laboratories). Images were acquired using a Leica LSM800 confocal microscope. The following antibodies were used: goat anti-GFP (Abcam, 1/500), mouse anti-Fasciclin II (DSHB, 1/25), donkey anti-goat Alexa fluor 488 (Invitrogen, 1/1000), donkey antimouse Cy3 (Jackson Immuno, 1/1000).

Bacterial contamination assays

The presence of bacterial DNA contamination in parental and "germ-free" derived stocks was checked by PCR using universal primers targeting bacterial 16S rDNA (16S-s: 5'-AGAGTTTGATCCTGGCTCAG-3', 16S-r: 5'-GGTTACCTTGTTACGACTT-3' (Weisburg et al., 1991)) and primers amplifying the wsp gene from the endosymbiont Wolbachia (wsp-s: 5'-TGGTCCAATAAGTGATGAAGAAAC-3', wsp-r: 5'-AAAAATTAAACGCTACTCCA-3' (as from [START_REF] Jeyaprakash | Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species[END_REF]). The presence of any contaminant was also checked by DNA sequencing: for each sample, genomic DNA from 10 adult flies (5 males/5 females) was extracted using DNeasy Blood & Tissue Kit (Qiagen). DNA was resuspended and sheared in 1X TE (0.1 mM EDTA, 10 mM Tris HCl pH 8.0) by sonication for 20 min (30 sec ON/30 sec OFF) using the Bioruptor Pico (Diagenode) to obtain an average size of ~300pb. DNA libraries were prepared from 1µg DNA using the NEBNext Ultra II DNA Library Prep Kit (Illumina) following manufacturer's instructions and sequencing was performed by Novogene (Cambridge, UK) using NovaSeq 6000 (paired-end, 150pb). Between 15 to 19 million reads were obtained per sample. To assess the presence of contamination, the resulting reads were first aligned to the drosophila reference genome (dm6 Ensembl release 70) with Bowtie2. Unaligned reads were then processed for blast search to bacteria, viral and fungal genomes using the DecontaMiner tool [START_REF] Sangiovanni | From trash to treasure: detecting unexpected contamination in unmapped NGS data[END_REF].

LC-MS/MS analyses

For DNA purification, whole larvae, bleach-dechorionated embryos or dissected adult brains of the required genotypes were washed in sterile PBS, crushed in lysis buffer (100 mM Tris-HCl pH 9, 100 mM EDTA, 1% SDS), incubated at 70°C for 30 min and then in 1 M potassium acetate at 4°C for 30 min. After centrifugation at 12000 g for 20 min, the supernatant was digested with RNAse A and RNAse H for 3 h at 37°C, extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with isopropanol. DNA from dissected third instar larval brains (around 100 per sample)

was extracted using DNeasy Blood & Tissue Kit (Qiagen), digested with RNAse A and RNAse H for 3 h. In both types of extraction, DNA was precipitated with 500 mM ammonium acetate (pH 6.2). DNA pellets were dissolved in ddH2O, and their concentration, as the absence of RNA contamination, were checked on a Qubit 3.0 fluorometer (Invitrogen).

Up to 1200 ng of DNA per sample were digested to nucleosides using 0.6 U nuclease P1 from P. citrinum (Sigma-Aldrich), 0.2 U snake venom phosphodiesterase from C. D2, 15 N2-5hmC 262->146; 5fC 256->140. For absolute quantification, internal and external calibrations were applied as described previously [START_REF] Kellner | Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution[END_REF], except for 6mA and 5fC, for which only external calibration was performed.

SMRT sequencing analyses

Around 250 brains from w 1118 or tet null third instar larvae were dissected for each sample. Tissues were crushed in lysis buffer (100 mM Tris-HCl pH 9, 100 mM EDTA, 100 mM NaCl,1% SDS), digested with RNase A for 15 min at room temperature and 30 min at 65°C, before being incubated with 4 volumes of 3.2 M LiCl, 0.9 M KAc for 30 min at 4°C. After centrifugation at 12000 g for 20 min at 4°C, the supernatant was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and DNA was precipitated with isopropanol. 5 µg of DNA were used to prepare each sequencing library.

SMRT-seq was performed at the Gentyane Sequencing Platform (Clermont-Ferrand, For each sequenced sample, SMRT-seq reads were aligned using pbmm2 tool (https://github.com/PacificBiosciences/pbmm2) on Drosophila genome (dm6 Ensembl release 70). For each condition (wild type or tet null ), a fusion of alignments of the three biological replicates was done using « samtools merge » [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF]. 6mA detection was performed on individual and on merged samples with IpdSummary tool from KineticTools (http://github.com/PacificBiosciences/kineticsTools) applying the following parameters: --identify m6A --numWorkers 16 --pvalue 0.01 --identifyMinCov 5 -methylFraction. To detect 6mA with higher confidence, we applied several thresholds on coverage and modificationQV (QV) with homemade scripts in bash and R. 6mA genomic repartition was obtained using Annotatepeaks tool from Homer suite [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[END_REF]. To find associated motifs for 6mA, we used MEME web application [START_REF] Bailey | MEME SUITE: tools for motif discovery and searching[END_REF]. The different heatmaps were generated with R package "pheatmap" (V.1.0.12). Other graphics were mainly done with ggplot2 (V.3.3.2) R package or Prism9. The SMRT-seq data are deposited under GEO accession number GSE206852.

Purification of drosophila TET catalytic domain

The catalytic domain of drosophila TET (dTET) was cloned in pET28a expression vector. The His-tagged protein was overexpressed in E. coli BL21 (DE3) CodonPlus RIL cells for 17 h at 16°C. Cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 20 mM imidazole, 500 mM NaCl, 1 mM DTT, 10% glycerol and supplemented with protease inhibitor 0.2 mM PMSF) and disrupted using Bandelin Sonoplus ultrasonic homogenizer. The cell lysates were cleared by centrifugation (Lynx 600 (Thermo), Fiberlite F21-8x50y) at 38.300 g for 30 min at 4°C and the supernatant was loaded onto an affinity column packed with Ni-NTA agarose beads (Genaxxon, Germany). The column was washed with wash buffer (50 mM HEPES pH 7.5, 20 mM imidazole, 500 mM NaCl, 1 mM DTT, 10% glycerol) then the recombinant protein was eluted with elution buffer (50 mM HEPES pH 7.5, 250 mM imidazole, 500 mM NaCl, 1 mM DTT, 10% glycerol). Purified protein was dialyzed against dialysis buffer I (50 mM HEPES pH 7.5, 1 mM DTT, 300 mM NaCl and 10% glycerol) followed by dialysis buffer II (50 mM HEPES pH 7.5, 1 mM DTT, 300 mM NaCl and 50% glycerol).

TET activity assays

The double-stranded DNA substrates were prepared by annealing forward oligos and reverse complement counterpart by heating at 95°C followed by bringing temperature to RT slowly on the heat block in Annealing Buffer (10mM Tris-HCl pH7.5 100mM NaCl). Forward oligos sequences containing a 5mC or 6mA modified nucleotide were the following: 5mC 5'-GTAAGTCTGGCA5mCGTGAGCCTCAGAG-3', 6mA 5'-GTAAGTCTGGCG6mAGTGAGCCTCAGAG-3'. The reaction was performed with 0.5 μM DNA substrate and 2 μM recombinant dTET in Reaction Buffer (50 mM HEPES pH 6.8, 100 μM Ammonium ion(II) sulfate hexahydrate, 1 mM, α-ketoglutarate, 1 mM ascorbic acid and 150 mM NaCl) at 37 °C. Reaction was stopped at different time points by adding 2µl of 0.5 M EDTA to a 40µl volume reaction followed by heating at 90°C for 5 min. Samples were treated with proteinase K for 1 h at 50°C and precipitated with ethanol. The level of 6mA, 5mC, 5hmC and 5fC was analyzed by LC-MS/MS as described above. larval CNS q v 2 0 q v 3 0 q v 4 0 q v 5 0 q v 6 0 q v 7 0 q v 8 0 q v 9 0 q v 1 0 0 q v 1 1 0 q v 1 2 0 0.0

  adamanteus (Worthington), 0.2 U bovine intestine phosphatase (Sigma-Aldrich), 10 U benzonase (Sigma-Aldrich), 200 ng Pentostatin (Sigma-Aldrich) and 500 ng Tetrahydrouridine (Merck-Millipore) in 5 mM Tris (pH 8) and 1 mM MgCl2 for 2 h at 37°C. 1000 ng of digested DNA were spiked with internal standard (D3-5mC and D2, 15 N2-5hmC, 250 fmol each) and subjected to analysis by LC-MS (Agilent 1260 Infinity system in combination with an Agilent 6470 Triple Quadrupole mass spectrometer equipped with an electrospray ion source (ESI)). The solvents consisted of 5 mM ammonium acetate buffer (pH 5.3, adjusted with acetic acid; solvent A) and LC-MS grade acetonitrile (solvent B; Honeywell). A C18 reverse HPLC column (SynergiTM 4 µM particle size, 80 Å pore size, 250 × 2.0 mm; Phenomenex) was used at a temperature of 35°C and a constant flow rate of 0.5 mL/min was applied. The compounds were eluted with a linear gradient of 0-20% solvent B over 10 min. Initial conditions were regenerated with 100% solvent A for 5 min. The four main nucleosides were detected photometrically at 254 nm via a diode array detector (DAD). The following ESI parameters were used: gas temperature 300°C, gas flow 7 L/min, nebulizer pressure 60 psi, sheath gas temperature 400°C, sheath gas flow 12 L/min, capillary voltage 3000 V, nozzle voltage 0 V. The MS was operated in the positive ion mode using Agilent MassHunter software in the MRM (multiple reaction monitoring) mode. Therefore, the following mass transitions were used to detect the respective modifications: 6mA 266->150; 5mC 242->126; D3-5mC 245->129; 5hmC 258->142;

France

  ) with a PacBio Sequel Sequencer (Pacific Biosciences, Menlo Park, CA, USA).The SMRTBell libraries were prepared using a SMRTbell Express 2 Template prep kit, following the manufacturer's recommendations. High Molecular Weight Genomic DNA (5 µg) was sheared with the 40 kb program using a Diagenode Megaruptor (Diagenode) to generate DNA fragments of approximately 30 kb. Assessment of the fragment size distribution was performed with a Femto Pulse (Agilent Technologies, Santa Clara, CA, USA). Sheared genomic DNA was carried into enzymatic reactions to remove single-strand overhangs and to repair any damage that may be present on the DNA backbone. An A-tailing reaction followed by the overhang adapter ligation was conducted to generate the SMRTBell templates. After a 0.45X AMPure PB beads purification, the samples were size-selected using the BluePippin (Sage Science, Beverly, MA, USA) to recover all the material above 15 kb. The samples were then purified with 0.45X AMPure PB Beads to obtain the final libraries of around 30 kb. The SMRTBell libraries were quality inspected and quantified on a Femto Pulse and a Qubit fluorimeter with Qubit dsDNA HS reagent Assay kit (Life Technologies). A ready-tosequence SMRTBell Polymerase Complex was created using a Binding Kit 3.0 (PacBio) and the primer V4, the diffusion loading protocol was used, according to the manufacturer's instructions. The PacBio Sequel instrument was programmed to load a 6 pM library and samples were sequenced on PacBio SMRTCells v2.0 (Pacific Biosciences), acquiring one movie of 600 min per SMRTcell.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. SMRT-seq analysis of larval CNS gDNA does not reveal an increase in 6mA in the absence of TET. (a) Percentage of adenines identified as 6mA in the wild type fusion dataset depending on the QV and coverage values used for 6mA selection. The dashed grey line indicates the level of 6mA measured by LC-MS/MS (0.0005%). (b) Influence of the QV and the coverage values on the proportion of 6mA identified in the wild type fusion dataset and in the three original samples. (c) Percentage of adenines

Figure 3 .

 3 Figure 3. TET does not oxidize 6mA. (a, b) In vitro assays showing TET activity profile on 5mC (a) and 6mA (b) containing double stranded oligonucleotide substrates. The levels of 5mC and its oxidised products (5hmC and 5fC) are represented relative to 5mC level at t=0. The levels of 6mA are represented relative to 6mA level at t=0. Error bars denote standard deviations from 3 independent experiments.

Figure 4 .

 4 Figure 4. TET catalytic activity is largely dispensable in drosophila. (a, b) Schematic representation of tet locus (a) and main protein isoforms (b). (a) tet is transcribed from two alternative promoters giving rise to tet-long (tet-l) and tet-short (tet-s) isoforms. Filled boxes represent exons; non-coding exons (UTR) are depicted in green, coding exons in black or according to their domain-associated color. Introns are represented as gray lines (not to scale). The tet null, DMAD1, DMAD2 and catalytic dead (CD) alleles are depicted in red. The location of the GFP insertion generated by CRISRP/Cas9-mediated knock-in is also indicated. (b) The conserved domains of TET are colored; pink: CXXC DNA binding domain, orange: Cystein-rich domain, blue: double-stranded ß helix (DSBH) domain, red: HxD (iron binding motif). Amino acid positions are indicated according to the longest TET-l and TET-s isoforms. (c-h) Expression pattern of the wild type and catalytic-dead versions of TET in the larval CNS. tet-GFP (c-e) and tet CD -GFP (f-h) knock-in lines were used to detect TET proteins by confocal imaging after immunostaining against GFP (green). Nuclei were labelled with DAPI (blue). (c, f): stitched images showing dorsal views of the entire CNS. Scale bar: 100 µm. (d, e, g, h): high-magnification views of TET expression in the ventral nerve cord (d, g) or the central brain (e, h). DAPI only and GFP only channels are presented in the middle (') and lower ('') panels, respectively. Scale bar: 10 µm. (i) Percentage of adult flies of the indicated genotypes hatching from their pupal case. Means and standard deviations from 4 independent experiments. (j-l) Wing positioning (j), ovaries (k) and mushroom bodies (l) of wild type adult flies (ctr: tet-GFP) as compared to flies lacking TET expression (tet 1/2 : tet DMAD1/DMAD2 adult escapers) or TET

  Figure 2

  Figure S5

  Figure S6ccTET 318 IVNRATPIHRDTSGPIEGMDILVTGGN 344 JBP1 205 NERFRTASHTDNGDFDNGYGVLAVLKG 230 dTET 1939 CLDFCAHSHRDLHNMQDGCTVHVALLK 1965 hTET1 1664 CLDFCAHPHRDIHNMNNGSTVVCTLTR 1690 hTET2 1374 CLDFCAHAHRDLHNMQNGSTLVCTLTR 1400 hTET3 1069 CMDFCAHAHKDQHNLYNGCTVVCTLTK 1095 * *

Acknowledgements

We are grateful to Dr. A. Molaro for critical reading of the manuscript and to the members of the iGReD for helpful discussions. We also thank Dr V. Gautier and the Gentyane platform at the Clermont-Ferrand INRAE for PacBio sequencing and the iGReD CLIC imaging facility for help with confocal experiments. We thank the Drosophila Bloomington Stock Center, Dr. R. Steward (Piscataway), Dr. D. Chen (Beijing) for fly stocks, as well as the Developmental Study Hybridoma Bank for antibodies. This project was supported by grants from the Agence Nationale de la Recherche (ANR-17-CE12-0030-03), Fondation ARC (PJA20171206371) and I-Site Cap20-25 (EpiMob) to LW. MB was supported by fellowships from the Université Clermont-Auvergne and the Fondation pour la Recherche Médicale (FRM). GG was supported by fellowships from the Université Clermont-Auvergne and the Ligue Nationale Contre le Cancer. MH was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) TRR-319 TP C03, SPP1784, HE 3397/13-2 and HE 3397/14-2.

Competing interests

The other authors declare no competing interests.

Authors' contribution

LV and LW conceived the study. MB, GG, YR, LV and LW designed the experiments. MB, GG, EP, MSD, RB and XN performed the experiments. MB, GG, YR, MSD, TJ, MH, LV and LW analyzed the results. LW and LV wrote the manuscript with inputs from all co-authors. All authors read and approved the final manuscript. enzymatic activity (tet CD ). (l-l") Immunostaining against Fas2 on adult brains was used to label mushroom body a, b and g lobes. (k-k") Scale bar 500µm. (l-l") Scale bar 50µm. S1. Genome coverages of wild type and tet null fusin datasets in SMRT-seq. S2. Percentages of 6mA/A according to QV and coverage cutoff in wild type and tet null fusion datasets. S3. Proportions of replicated 6mA (triplicated and/or duplicated). S4. Percentages of 6mA /A and 6mA fractions of methylation according to QV in the three wild type or three tet null datasets (coverage ≥25x).

Supplementary

Supplementary Table

Supplementary Table

Supplementary Table

Supplementary Table