Akhila Melarkode Vattekatte

Tarun Jairaj Narwani

Aline Floch

Soubika Bisoo

Nicolas K Shinada

Agata Kranjc

Jean-Christophe Gelly

Narayanaswamy Srinivasan

Dr Alexandre G De Brevern
email: alexandre.debrevern@univ-paris-diderot.fr

A structural entropy index to analyse local conformations in Intrinsically Disordered Proteins

Keywords: protein structures / structural alphabet / entropy / rigidity / flexibility IDP: Intrinsically Disorder Protein, NMR: Nuclear Magnetic Resonance, PBs: Protein Blocks, PDB: Protein Data Bank, PED: Protein Ensemble Database, SAXS: Small-angle X-ray scattering

Sequencestructurefunction paradigm has been revolutionized by the discovery of disordered regions and disordered proteins more than two decades ago. While the definition of rigidity is simple with X-ray structures, the notion of flexibility is linked to high experimental B-factors. The definition of disordered regions is more complex as in these same X-ray structures; it is associated to the position of missing residues. Thus a continuum so seems to exist between rigidity, flexibility and disorder. However, it had not been precisely described. In this study, we used an ensemble of disordered proteins (or regions) and, we applied a structural alphabet to analyse their local conformation. This structural alphabet, namely Protein Blocks, had been efficiently used to highlight rigid local domains within flexible regions and so discriminates deformability and mobility concepts. Using an entropy index derived from this structural alphabet, we underlined its interest to measure these local dynamics, and to quantify, for the first time, continuum states from rigidity to flexibility and finally disorder. We also highlight non-disordered regions in the ensemble of disordered proteins in our study.

Introduction

Proteins are essential biological macromolecules involved in most of the cellular functions. The amino acid residue sequence dictates the functional three-dimensional structure of a protein. The access to the 3D structure allows apprehending at an atomistic level the biological functions and thus defines the dogma of sequence to structure extended to function [START_REF] Shenoy | Proteins: sequence to structure and function--current status[END_REF]. Analyses of protein 3D structures have been done extensively using regular secondary structures, namely the α-helix (or 3.6 13 h x) h βsheet [START_REF] Richardson | The anatomy and taxonomy of protein structure[END_REF], initially proposed by Pauling and Corey [START_REF] Eisenberg | The discovery of the alpha-helix and beta-sheet, the principal structural features of proteins[END_REF]Pauling and Corey, 1951;Pauling et al., 1951). Nonetheless, secondary structure definition cannot be seen without ambiguity (see [START_REF] Colloc'h | Comparison of three algorithms for the assignment of secondary structure in proteins: the advantages of a consensus assignment[END_REF][START_REF] Fourrier | Use of a structural alphabet for analysis of short loops connecting repetitive structures[END_REF][START_REF] Martin | Protein secondary structure assignment revisited: a detailed analysis of different assignment methods[END_REF]Tyagi et al., 2009a;Tyagi et al., 2009b)). Therefore, proposition of local protein structure libraries independent of classical secondary structures are of much interest. Amongst, structural alphabets [START_REF] De Brevern | New assessment of a structural alphabet[END_REF][START_REF] Karchin | Hidden Markov models that use predicted local structure for fold recognition: alphabets of backbone geometry[END_REF][START_REF] Offmann | Local Protein Structures[END_REF], Protein Blocks (PBs) have shown significant applications in structural bioinformatics [START_REF] De Brevern | Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks[END_REF][START_REF] Joseph | A short survey on Protein Blocks[END_REF], e.g. threading approaches [START_REF] Ghouzam | Improving protein fold recognition with hybrid profiles combining sequence and structure evolution[END_REF][START_REF] Ghouzam | ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles[END_REF] or protein structure superimposition and comparison [START_REF] Leonard | mulPBA: an efficient multiple protein structure alignment method based on a structural alphabet[END_REF][START_REF] Tyagi | A substitution matrix for structural alphabet based on structural alignment of homologous proteins and its applications[END_REF].

The sequencestructure -function dogma had to be redefine when it was accepted that a non-negligible part of the protein structures are not ordered and are likely to be unfolded in solution, under native, functional conditions [START_REF] Mitic | Structural disorder of plasmid-encoded proteins in Bacteria and Archaea[END_REF][START_REF] Pavlovic-Lazetic | Bioinformatics analysis of disordered proteins in prokaryotes[END_REF]Uversky, 2002a;Uversky, 2002b;[START_REF] Uversky | Why are "natively unfolded" proteins unstructured under physiologic conditions?[END_REF][START_REF] Wright | Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm[END_REF]. These

Intrinsically Disordered Proteins (IDPs) [START_REF] Habchi | Introducing protein intrinsic disorder[END_REF], do not have a well-defined 3-D structure but rather adopt an ensemble of conformations that are functional in solution. IDPs exist as dynamic ensembles, within which atom positions and backbone angles exhibit random temporal fluctuations [START_REF] Dunker | Intrinsically disordered protein[END_REF][START_REF] Tompa | Intrinsically unstructured proteins[END_REF]. To describe these ensembles, different experimental and computational approaches are available. In this field,

Nuclear Magnetic Resonance provides quantitative residue-level information on structure and dynamics of IDPs as structural ensembles [START_REF] Kragelj | Ensemble Calculation for Intrinsically Disordered Proteins Using NMR Parameters[END_REF]. Small-angle X-ray Scattering (SAXS) and electron microscopy (EM) gives an in-situ ensemble model describing the conformational behaviour of the disordered region, e.g. intrinsically disordered C-terminal domain of nucleoprotein is essential for transcription and replication of the measles virus [START_REF] Jensen | Intrinsic disorder in measles virus nucleocapsids[END_REF]. Finally, Molecular Dynamics (MDs) is used to refine and propose such ensembles [START_REF] Robustelli | Application of the maximum entropy principle to determine ensembles of intrinsically disordered proteins from residual dipolar couplings[END_REF]. Hence, the sequencestructurefunction dogma is challenged by the fact that structure is always in a dynamic state where (a) it may acquire or lose rigid, flexible and deformable states [START_REF] Carugo | Atomic displacement parameters in structural biology[END_REF][START_REF] Thorpe | Protein flexibility and dynamics using constraint theory[END_REF] and (b) is composed of ordered and disordered regions [START_REF] Uversky | Intrinsic disorder here, there, and everywhere, and nowhere to escape from it[END_REF].

The structural alphabet PBs have been used to analyse structural flexibility in Integrins [START_REF] Goguet | In silico analysis of Glanzmann variants of Calf-1 domain of alphaIIbbeta3 integrin revealed dynamic allosteric effect[END_REF][START_REF] Jallu | Modeling and molecular dynamics of HPA-1a and -1b polymorphisms: effects on the structure of the beta3 subunit of the alphaIIbbeta3 integrin[END_REF][START_REF] Jallu | Modeling and molecular dynamics simulations of the V33 variant of the integrin subunit beta3: Structural comparison with the L33 (HPA-1a) and P33 (HPA-1b) variants[END_REF], in Duffy Antigen Chemokine

Receptor protein (named now ACKR1) (de Brevern et al., 2005), impact of Post-Translational Modifications [START_REF] Craveur | Investigation of the impact of PTMs on the protein backbone conformation[END_REF], KISS1R [START_REF] Chevrier | PRR repeats in the intracellular domain of KISS1R are important for its export to cell membrane[END_REF], and in NMDA Receptor

Channel Gate [START_REF] Ladislav | The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate[END_REF]. Besides, it has also been used with experimental data (Schneider et al., 2014a;Schneider et al., 2014b). PBs have shown to be crucial in identifying rigid positions encompassed in flexible regions, i.e. local protein conformations that are rigid but are enclosed in deformable zones [START_REF] Craveur | Protein flexibility in the light of structural alphabets[END_REF].

With the understanding of flexibility in globular proteins, we extend here such analyses to the cases of IDPs from PED 3 : Protein Ensemble Database [START_REF] Varadi | pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins[END_REF]. This dataset was assembled by specialists from various groups in the aim to have an open access database for structural information on IDPs and denatured protein ensembles based on NMR, SAXS data and MD simulations. Thus, it perfectly fits to see if some regions of these IDPs are more rigid than expected, as seen in the case of the multisite phosphorylation of some kinases [START_REF] Xiang | Phosphorylation drives a dynamic switch in serine/arginine-rich proteins[END_REF].

Materials and Methods

Data sets. All the IDP structures were taken from PED 3 : Protein Ensemble Database, the database of conformational ensembles describing flexible proteins (http://pedb.vib.be/index.php, accessed 15 th May 2018) [START_REF] Varadi | pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins[END_REF]. They consist of 24 entries describing different IDPs with different technique, i.e. SAXS and NMR, NMR alone, SAXS alone and Molecular Dynamics [START_REF] Allison | A relationship between the transient structure in the monomeric state and the aggregation propensities of alpha-synuclein and beta-synuclein[END_REF][START_REF] Bacot-Davis | Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase[END_REF][START_REF] De Biasio | p15PAF is an intrinsically disordered protein with nonrandom structural preferences at sites of interaction with other proteins[END_REF][START_REF] Marsh | Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints[END_REF][START_REF] Mertens | Conformational analysis of a genetically encoded FRET biosensor by SAXS[END_REF][START_REF] Mittag | Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase[END_REF][START_REF] Ozenne | Mapping the potential energy landscape of intrinsically disordered proteins at amino acid resolution[END_REF]Sanchez-Martinez and Crehuet, 2014;[START_REF] Sivakolundu | Disordered p27Kip1 exhibits intrinsic structure resembling the Cdk2/cyclin A-bound conformation[END_REF][START_REF] Sterckx | Small-angle X-ray scattering-and nuclear magnetic resonance-derived conformational ensemble of the highly flexible antitoxin PaaA2[END_REF][START_REF] Weeks | Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6[END_REF].

A dataset of 169 X-ray globular ordered protein structures was used to compare to these proteins. This dataset was curated from Protein Data Bank (PDB), [START_REF] Berman | The Protein Data Bank[END_REF] and was extracted using ASTRAL 2.03 at 40% sequence identity [START_REF] Fox | SCOPe: Structural Classification of Proteins--extended, integrating SCOP and ASTRAL data and classification of new structures[END_REF]. Their structure resolutions were better than 1.5 Å, without presence of heteroatoms, missing residues, alternate or modified residues in the chain. Only globular proteins, with chain length ranging between 50 and 250 residues, were selected. Three molecular dynamic (MD) simulations were performed for each protein structure with GROMACS 4.5.7 software [START_REF] Pronk | GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit[END_REF], using AMBER99sb force field [START_REF] Van Gunsteren | Biomolecular Simulation: The GROMOS96 Manual and User Guide[END_REF]. Each protein structure was put in a periodic dodecahedron box, using TIP3P water molecules [START_REF] Jorgensen | Quantum and statistical mechanical studies of liquids. 25. Solvation and conformation of methanol in water[END_REF], and neutralised with Na + or Cl -counter ions. The system was then energetically minimised with a steepest-descent algorithm for 2000 steps. The MD simulations were performed in isothermal-isobaric thermodynamics ensemble (NPT), with temperature fixed at 300 K and pressure at 1 bar. A short run of 1ns was performed to equilibrate the system, using Berendsen algorithm for temperature and pressure control [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF]. The coupling time constants were equal to 0.1 ps for each physical parameter. Then, a production step of 50 ns was done using Parrinello-Rahman algorithm [START_REF] Parrinello | Polymorphic transitions in single crystals: A new molecular dynamics method[END_REF] for temperature and pressure control, with coupling constants of T=0.1 ps and P=4 ps. All bond lengths were constrained with LINCS algorithm [START_REF] Hess | LINCS: a linear constraint solver for molecular simulations[END_REF], which allowed an integration step of 2 fs. The PME algorithm [START_REF] Darden | New tricks for modelers from the crystallography toolkit: the particle mesh Ewald algorithm and its use in nucleic acid simulations[END_REF] was used for long-range electrostatic interactions using a cut-off of 1 nm for non-bonded interactions.

This protocol was applied on each of the 169 protein chains. Conformations were saved every 10 ps. For each MD simulation, the secondary structures were analysed and the structural deviation of each snapshot from the initial structure was measured. Trajectory analyses were done with the GROMACS software, in-house Python and R scripts. Root mean

squ s (R SD) m squ f uc u s (R Sf) w c mpu Cα
atoms. Normalized RMSfs and normalized B-factors were computed as in Bornot et al. study [START_REF] Bornot | Predicting protein flexibility through the prediction of local structures[END_REF].

Protein Blocks. Protein Blocks is the most widely used structural alphabet composed of 16 local prototypes [START_REF] De Brevern | Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks[END_REF], it is employed to analyse local conformations of protein structures from the Protein Data Bank (PDB) [START_REF] Berman | The Protein Data Bank[END_REF]. Each PB is ch c z by h φ ψ h g s f f c s cu s u s. PBs g reasonable approximation of all local protein 3D structures [START_REF] Joseph | A short survey on Protein Blocks[END_REF] and are very efficient in tasks such as protein superimpositions [START_REF] Gelly | iPBA: a tool for protein structure comparison using sequence alignment strategies[END_REF][START_REF] Leonard | mulPBA: an efficient multiple protein structure alignment method based on a structural alphabet[END_REF] and Molecular Dynamics (MDs) analyses [START_REF] Goguet | In silico analysis of Glanzmann variants of Calf-1 domain of alphaIIbbeta3 integrin revealed dynamic allosteric effect[END_REF][START_REF] Jallu | Modeling and molecular dynamics simulations of the V33 variant of the integrin subunit beta3: Structural comparison with the L33 (HPA-1a) and P33 (HPA-1b) variants[END_REF][START_REF] Ladislav | The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate[END_REF]. PB assignment was carried out for every residue from every structure / structural model extracted from PED 3 using our PBxplore tool [START_REF] Barnoud | PBxplore: a tool to analyze local protein structure and deformability with Protein Blocks[END_REF], available at GitHub (https://github.com/pierrepo/PBxplore). From this description, we have used a recognized measure that helps in quantifying the flexibility of each amino acid called N eq (N eq means for equivalent Number of PBs) [START_REF] De Brevern | Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks[END_REF]. N eq is a statistical measurement similar to entropy, and represents the average number of PBs a residue may adopt at a given position. N eq is calculated as follows [START_REF] De Brevern | Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks[END_REF]:

Neq = exp(-f x x=1 16 å ln f x ) (1)
Where, f x is the frequency of PB x at the position of interest. N eq value can vary between 1 and 16. A N eq value of 1 indicates that only one type of PB is observed, while a value of 16 indicates an equal probability for each of the 16 PBs, i.e. random distribution. It must be noticed that N eq was originally proposed for prediction purpose, for which random frequencies of 1/16 for each PB can be seen. As the observed distribution of PBs (see [START_REF] De Brevern | New assessment of a structural alphabet[END_REF]) ranges between 0.8% to more than 30%, the expected maximum N eq is around 10 and not 16 in ordered proteins. IDPs do not follow the same expected frequency distribution and so N eq higher than 10 can be expected. PBxplore tool allows the assignment of PBs, the calculation of N eq and the creation of sequence logos of PBs (WebLogo) [START_REF] Crooks | WebLogo: a sequence logo generator[END_REF]. RMSf reflects a large average distance to this barycentre conformation, and so not always a high flexibility. With PBs, the N eq values are at these positions low, underlying the fact that the region is rigid (i.e. in this case even can be noted as mobile). This approach allows a simple view of these cases. This approach allows straightforward analyses and interpretations of these cases. Moreover, N eq values are comparable between proteins when it is slightly more complex for RMSf and B-factors.

Interest of Protein

Analyses. The analyses were done using Python programming language v.2.7.10 (Foundation), and R software v.3.3.3 [START_REF] Team | R: A language and environment for statistical computing[END_REF] while 3D visualisation was done using MacPyMOL software v.1.7. 2.2 (DeLano, 2002;[START_REF] Schrodinger | The PyMOL Molecular Graphics System[END_REF].

Results

Analyses of local conformations in the IDP dataset vs Ordered dataset. As mentioned on the website, PED 3 contains 25,473 protein structures of 60 ensembles in 24 entries. Out of these, 6 entries have data from both SAXS and NMR, 7 from only SAXS, 10 from only NMR and one from Molecular Dynamics.

The entries were analysed separately as single chains and Protein Blocks were assigned to each protein structure [START_REF] De Brevern | Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks[END_REF] using PBxplore tool [START_REF] Barnoud | PBxplore: a tool to analyze local protein structure and deformability with Protein Blocks[END_REF].

As mentioned in the Methods section, it is possible to compute an entropy index, namely N eq , from the PB distribution; N eq ranges between 1 (only one PB seen at a defined position) and 16 (all 16 PBs are seen with equal frequency, depicting random distribution). A similar analysis was performed on 169 globular proteins for which 3 independent molecular dynamics simulations of 150 ns were performed [START_REF] Narwani | Dy m cs f m b y f α-, 310-π-helices[END_REF]. Figure 1 shows the two magnified N eq distributions of N eq > 1.0 with an inset distribution plot showing whole statistics (see also Supplementary Figure 1). Indeed, the observations are mainly directed by rigid local conformations, i.e. N eq value of 1.0, that correspond to 60% of the residues from ordered globular protein dataset [START_REF] Narwani | Dy m cs f m b y f α-, 310-π-helices[END_REF] and 58% from IDP dataset (see Sup Table 2). The tendencies change drastically for N eq > 2.0 as only 8% of the residues of globular protein dataset (see Figure 1A) and 36% of the residues of IDP dataset fall into this group (see Figure 1A); almost none of the residues in globular protein dataset (i.e. 0.82%)

have N eq > 4.0, and none have N eq > 8.0, while 31% of IDP residues have N eq > 4.0 and 15%

have N eq > 8.0. From this it is evident that the entropy index N eq is appropriate related to the question of p s' order and disorder as hypothesized previously [START_REF] Craveur | Protein flexibility in the light of structural alphabets[END_REF].

We would like to stress that N eq is a particular measure, a local one. Indeed, as we have shown in [START_REF] Narwani | Discrete analyses of protein dynamics[END_REF], on the ordered dataset the correlation between normalized Bfactor and normalized RMSf is of 0.43, a similar value is also as seen in our previous study and many others [START_REF] Bornot | A new prediction strategy for long local protein structures using an original description[END_REF]. Correlation with N eq , is only of 0.24 and 0.14. These low values are also expected since N eq does a local conformation analyses with more than 60% of residues having a N eq of 1.0, meaning that single local protein conformation remains unchanged during MD simulations time. A very low N eq of 1.0 can be found associated with high B-factor and RMSf as seen in [START_REF] Craveur | Protein flexibility in the light of structural alphabets[END_REF][START_REF] Goguet | In silico analysis of Glanzmann variants of Calf-1 domain of alphaIIbbeta3 integrin revealed dynamic allosteric effect[END_REF]. It defines then a mobile element, i.e. a rigid region encompassed between two deformable regions, which act as hinges.

Influence of data on IDP dataset. As expected, there is a huge variation among the N eq values of IDPs from PED 3 . Average N eq for entry is equal to 4.6, where 11 entries have N eq higher than 6.2 and 11 entries have N eq lower than 2.2 (see Figure 2A). The protein chains in the disordered dataset have an average length of 110 residues and does not influence the entropy values (see Figure 2B), as the longer protein sequences have an average N eq of 4.1 while the shortest having 4.9. The number of models for each PED 3 entries has a slight impact on the N eq values as seen in Figure 2C. It is thus implied that the number of occurrences influences N eq values. Nonetheless, the relationship is not so straightforward, as with 130 models, the unbound p27 KID domain (PED2AA) has an average N eq of 1.7 [START_REF] Sivakolundu | Disordered p27Kip1 exhibits intrinsic structure resembling the Cdk2/cyclin A-bound conformation[END_REF], while with 32 occurrences, the structural ensemble of phosphorylated Sic1

(PED1AAA) has an average N eq of 7.6 [START_REF] Mittag | Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase[END_REF]). An enlarged inset plot of Fig 2 .C is shown in the Supplementary Figure 2, as the number of models can be substantial (e.g.

13718 for Sendai virus phosphoprotein ensemble (PED4AAB) (Sanchez-Martinez and Crehuet, 2014)) and in this case, N eq values are high. The 8 entries with more than 500 models have an average N eq value of 7.8 while the others have an average N eq of 3.1.

Does the IDP dataset contain only IDPs?

The above analyses might seem to categorize the proteins from PED 3 database. However, it is in fact more complex: as seen in the Figure 3 (see also Supplementary Figure 3), some entries are completely disordered, while others are rigid and third are intermediate cases.

While it is simple to say that a N eq of 1 is associated to a rigid residue and a N eq of 12 is in a disordered region, it is more difficult to classify the precise delineation of flexibility and disorder as these terms are too definitive for the characterization of a continuous value. We have so decided through a visual inspection of every entry (see Sup Data 1), that a N eq higher than 8 must to be considered as completely disordered, while by looking at our multiple previous molecular dynamic simulations [START_REF] Craveur | Protein flexibility in the light of structural alphabets[END_REF][START_REF] Goguet | In silico analysis of Glanzmann variants of Calf-1 domain of alphaIIbbeta3 integrin revealed dynamic allosteric effect[END_REF][START_REF] Jallu | Modeling and molecular dynamics of HPA-1a and -1b polymorphisms: effects on the structure of the beta3 subunit of the alphaIIbbeta3 integrin[END_REF][START_REF] Jallu | Modeling and molecular dynamics simulations of the V33 variant of the integrin subunit beta3: Structural comparison with the L33 (HPA-1a) and P33 (HPA-1b) variants[END_REF][START_REF] Narwani | Dy m cs f m b y f α-, 310-π-helices[END_REF][START_REF] Narwani | Discrete analyses of protein dynamics[END_REF], we found that N eq close to 4 can be considered as flexible. By extending this simple approach, a N eq of 6 can be an intermediate state between highly flexible and disorder. The difficulty to precisely define without ambiguity a defined threshold was always critical in analysis of flexibility. For instance, Schlessinger and Rost decided to use a strict threshold between rigid and flexible region, but also have proposed a non-strict threshold with a different value [START_REF] Schlessinger | PROFbval: predict flexible and rigid residues in proteins[END_REF]. Our threshold values are so provided as them, but can be discussed in regards to the analysed proteins. Coarsely, we can classify the 24 entries into 4 groups (see Table 1): (i) completely disordered (8 entries), (ii) partially disordered (7 entries), (iii) mainly/largely flexible (5 entries), and (iv) composed of some rigid regions (4 entries).

Some completely disordered proteins have an impressive N eq values of > 7.5, such as structural ensemble of phosphorylated Sic1 (N eq 7.6) [START_REF] Mittag | Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase[END_REF] (see Figure 3A) and Sendai virus phosphoprotein ensemble (N eq 8.6) (Sanchez-Martinez and Crehuet, 2014) (see Figure 3B). Sometimes, some positions are observed to be more rigid than expected, see position 102 of the measles nucleoprotein [START_REF] Ozenne | Mapping the potential energy landscape of intrinsically disordered proteins at amino acid resolution[END_REF] with N eq of 1.2, i.e. rigid, while average N eq is characteristic of disorder about 8.4 (see Figure 3C). The latter example corresponds to a disorder protein with a rigid region. This mobile region is helical (see Sup Data 1). A similar observation was found for a solution-state ensemble f β-synuclein [START_REF] Allison | A relationship between the transient structure in the monomeric state and the aggregation propensities of alpha-synuclein and beta-synuclein[END_REF]. The average N eq is of 6.3, leading to its characterization as a completely disordered protein. However, some regions in the C-terminus are below 4, especially near position 120 with a N eq of 1, a highly rigid position for a helical structure (see Figure 3D), which is quite interesting for a terminal region. It is more striking, in case of a punctual variant fragment of heat shock protein β6 [START_REF] Weeks | Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6[END_REF], which is characterized by a rigid central region formed by residue position 20 to 95 enclosed by disordered termini (see Figure 3E). This example reflects most of the members of the second group of partial disordered proteins (see Table 1). It also corroborates with other studies in the field. Indeed, half of the protein structures lack the electron density data at their extremities due to high thermal displacement [START_REF] Faure | Protein contacts, inter-residue interactions and side-chain modelling[END_REF][START_REF] Faure | Analysis of protein contacts into Protein Units[END_REF][START_REF] Jacob | A tale of two tails: why are terminal residues of proteins exposed?[END_REF].

The third group can be considered as flexible and not disordered as they have a lower average N eq representing some rigid regions. The phosphorylated Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase (entry PED5AAD [START_REF] Bacot-Davis | Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase[END_REF])) is a perfect example with an average N eq of only 1.8, a maximum N eq of only 4 and with five regions entirely rigid (see Figure 3F). The last group encompasses two rigid protein entries both very interesting cases: (i) singly phosphorylated mengovirus leader protein (PED4AAD, see Figure 3G low N eq but also flexible regions and disordered regions with higher N eq .

Discussion

IDPs and IDRs are dynamic ensembles [START_REF] Dunker | Intrinsically disordered protein[END_REF][START_REF] Habchi | Introducing protein intrinsic disorder[END_REF][START_REF] Tompa | Intrinsically unstructured proteins[END_REF][START_REF] Uversky | Why are "natively unfolded" proteins unstructured under physiologic conditions?[END_REF]. They have been widely analysed for the notions of rigidityflexibility and orderdisorder. The transition can be seen as a continuum from rigidity to flexibility to disorder. However, this continuum may mask interspersed rigid regions entrapped in highly flexible or disordered region, namely mobile regions as shown by [START_REF] Craveur | Protein flexibility in the light of structural alphabets[END_REF][START_REF] Goguet | In silico analysis of Glanzmann variants of Calf-1 domain of alphaIIbbeta3 integrin revealed dynamic allosteric effect[END_REF] in/for the case of Calf-1 domain of integrin α IIb β 3 .

In this study, we have shown that the use of our structural alphabet [START_REF] De Brevern | Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks[END_REF][START_REF] Offmann | Local Protein Structures[END_REF] and an entropy index allows a precise analysis of flexibility and can be extended to identify disorder. PB analysis of ensembles from PED 3 database (see Sup

Table 3) enables us to propose gradient between disorder (a very large number of local backbone conformations being sampled, i.e. N eq of 8) and flexibility (fairly limited number of local protein conformations, i.e. N eq of 4, see Table 1 and Sup Data 1). Moreover, it is possible to efficiently localize a mobile region in IDPs, as for example PED7AAC (see Figure

3C [START_REF] Ozenne | Mapping the potential energy landscape of intrinsically disordered proteins at amino acid resolution[END_REF]) or PED6AAC, an ensemble of conformations of tau protein that has some extended structure property and one position with N eq of 2 when the average N eq of the protein is 8.1.

Even with the limited number of examples; N eq entropy is able to clearly distinguish backbone conformations, allowing the identification of mobile regions inside disordered regions. Thus, providing a metric not only to differentiate between flexible and disordered region but also to detect rigid regions encompassed by flexible regions. This work could be extended further owing to the physiological significance of the mobile regions in the IDPs and the biological implications of the different groups. In perspectives, we would like to go further on larger datasets of IDPS and IDRs, using NMR data, and combine it with molecular simulations. A very interesting approach would be to add information from NMR data using IDR assignment as proposed in [START_REF] Ota | An assignment of intrinsically disordered regions of proteins based on NMR structures[END_REF]. Another question will be to select proper IDRs in X-ray s uc u s. I , G z 's g up [START_REF] Zhang | Between order and disorder in protein structures: analysis of "dual personality" fragments in proteins[END_REF] underline the existence of Dual Personality Fragments (DPFs), i.e. fragments that are found both in disordered IDRs and ordered regions. The selection of IDRs must be done with certainty and should not be biased by DPFs.

Legends

Figure 1. Global probability distribution. N eq values for A) IDP proteins (from PED 3 database) and B) globular ordered proteins (from the ordered dataset). For clarity the main plots represent frequencies less than 3% for IDP and 0.2% for ordered dataset. The inset plot in both represents the global distribution for all N eq values. Completely disordered PED1AAA, PED4AAA, PED6AAA, PED6AAC 1 , PED7AAC

  Blocks to distinguish mobility from flexibility. Analysis of flexibility can be done using experimental B-factors taken from X-ray structures or Root Mean Square fluctuation (RMSf) extracted from Molecular Dynamics simulations. These measures provide a continuum from rigid (low RMSf or B-factors) to flexible (high RMSf or B-factors). However, it has a limitation when a mobile element (i.e. rigid) is encompassed in a flexible region. The B-factors and RMSf values are high but the protein region is not flexible at all. RMSf is a Euclidean distance of all different conformations of the proteins observed during the dynamics. It is based on (i) the selection of a representative median conformation, and then (ii) the superimposition of all snapshots on this representative conformation. A high

(

  Bacot-Davis et al., 2014)), and (ii) the unphosphorylated mengovirus leader protein (PED3AAD, see Figure 3H (Bacot-Davis et al., 2014)). They have some rigid regions with

Figure 2 .

 2 Figure 2. Conformational entropy analysis. A) average N eq per entry, B) average N eq versus chain length and C) average N eq versus number of models (for clarity, the main plot represents number less than 200, Sup Fig 2 show all the distribution).

Figure 3 .

 3 Figure 3. 3D visualisation and N eq distribution of some characteristic entries. A) PED1AAA -structural ensemble of phosphorylated Sic1 (Mittag et al., 2010), B) PED4AAB -Sendai virus phosphoprotein ensemble (Sanchez-Martinez and Crehuet, 2014), C) PED7AAC -Ensemble of the free form measles nucleoprotein (Ozenne et al., 2012), D) PED1AAD --synuclein: solution-state ensemble (Allison et al., 2014), E) PED1AAB -punctual variant fragment of Heat shock protein -6 (Weeks et al., 2014), F) PED5AAD -phosphorylated Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase (Bacot-Davis et al., 2014), G) PED4AAD -singly phosphorylated mengovirus leader protein (Bacot-Davis et al., 2014), and H) PED3AAD -unphosphorylated mengovirus leader protein (Bacot-Davis et al., 2014).

Table 1 .

 1 Classification of PED 3 entries. Classified into 4 groups based on conformational entropy index N eq .

	1 , PED8AAC, PED9AAA,

Acknowledgments

We would like to thank Charlotte Périn and Professor Catherine Etchebest as the g z s p c p s f B b '2016 B b '2018 for fruitful discussions. This work was supported by grants from the Ministry of Research (France), University de Paris, University Paris Diderot, Sorbonne, Paris Cité (France), National Institute for Blood Transfusion (INTS, France), National Institute for Health and Medical Research (INSERM, France), IdEx ANR-18-IDEX-0001 and labex GR-Ex. The labex GR-Ex, reference ANR-11-LABX-0051 s fu by h p g m "I s ss m s ' " f h F ch National Research Agency, reference ANR-11-IDEX-0005-02. TJN, NS and AdB acknowledge to Indo-French Centre for the Promotion of Advanced Research / CEFIPRA for collaborative grant (number 5302-2). NSh acknowledges support from ANRT. AMV is supported by Allocation de Recherche Réunion granted by the Conseil Régional de la Réunion and the European Social Fund EU (ESF). MM and NM acknowledge to project grants No. 174021 and 44006 from Ministry of Education, Science and Technological Development, Republic of Serbia

The authors were granted access to high performance computing (HPC) resources at the French National Computing Centre CINES under grant no. c2013037147, no. A0010707621 and A0040710426 funded by the GENCI (Grand Equipement National de Calcul Intensif). Calculations were also performed on an SGI cluster granted by Conseil Régional Ile de France and INTS (SESAME Grant).

Conflict of interest

The authors have no conflict of interest to declare. JCG and ADB are associated with IBL, Paris, France.


A Ricard
email: ricard@laplace.univ-tlse.fr

J P Sarrette

Transmission of N-atoms Produced by N 2 Flowing Microwave Afterglows through Hollow Tubes: Advanced Study

Keywords: Spectroscopy, N-atoms transmission, hollow tubes, γ-destruction probability. 31

Transmission rates (T N ) of N-atoms through small diameters tubes (1.5 and 3 mm int dia and 9, 50 and 80 cm length for silicone tubes, 1.5 mm int dia and 6.5 cm length for stainless steel tubes) are measured in late N 2 and Ar-N 2 flowing after glows of microwave plasmas in continuous and pulsed gas injection modes at a flow rate of 1 and 3 slpm, a gas pressure from 2 to 4 Torr for N 2 and 20 Torr for Ar-1%N 2 and a plasma power varying from 150 to 300 Watt. From the experimental T N values, it is deduced the γ-destruction probability inside the tube walls as being γ = (1.0-1.6)10 -3 for the silicon tubes and γ = (1.6-2.0) 10 -2 for the stainless steel tubes.

INTRODUCTION

Production of N-atoms has been previously studied in flowing N 2 microwave post-discharges at medium gas pressures (1-10 Torr) [START_REF] Villeger | Synergy between N and O atom action and substrate temperature in a sterilization process using a flowing N 2 -O 2 microwave post-discharge[END_REF]. Bacteria inactivation by N-atoms in such post-discharge reactors has been largely studied [START_REF] Villeger | Synergy between N and O atom action and substrate temperature in a sterilization process using a flowing N 2 -O 2 microwave post-discharge[END_REF][START_REF] Villeger | Serilization of E-coli bacterium in a flowing N 2 -O 2 postdischarge reactor[END_REF][START_REF] Villeger | Treatment of flat and hollow substrates by a pure nitrogen flowing post discharge. Application to bacterial decontamination in low diameter tubes[END_REF].

In the cited previous works, the N atoms are produced by microwave plasmas at power of 100-300 Watt. The plasma is located inside a quartz tube of internal diameter (i.d) 5 mm and extends along a few centimeters outside the cavity (surfatron) gap for a gas pressure between 1 and 30 Torr and a flow rate between 0.5 and 2.0 slpm. In these conditions, the plasma gas temperature reaches up to 1000 K near the surfatron gap [START_REF] Merel | Influence of the field frequency on the nitrogen atom yield in the remote plasma of an high frequency discharge[END_REF], needing an air cooling of the quartz tube. After a few centimetres of post-discharge, the flowing gas temperature is returned to the room temperature.

After the plasma zone, early and late afterglows appear and the late afterglow extends in the postdischarge reactor where the sterilization of bacteria is studied.

It is presently reported the results obtained for the transmission of N-atoms produced in the N 2 late afterglow through small diameters hollow tubes.

These studies are of interest to appreciate the possibility of N-atoms sterilization inside hollow silicon and stainless steel tubes.

THE EXPERIMENTAL SETUP

The flowing microwave post-discharge reactor described in previous studies [START_REF] Villeger | Synergy between N and O atom action and substrate temperature in a sterilization process using a flowing N 2 -O 2 microwave post-discharge[END_REF][START_REF] Villeger | Serilization of E-coli bacterium in a flowing N 2 -O 2 postdischarge reactor[END_REF][START_REF] Villeger | Treatment of flat and hollow substrates by a pure nitrogen flowing post discharge. Application to bacterial decontamination in low diameter tubes[END_REF] is reproduced in Fig. 1. A two-stage oil rotary pump with a pumping speed of 48 sm 3 /h (800 slm) is setup after a 5 litre post-discharge reactor. The N 2 flow rate is controlled from 1 to 3 slm using flow meters. The pressure inside the reactor is adjusted by means of a throttle valve above the pump (see Fig. 1). Optical Emission Spectroscopy (OES) along the afterglow tubes was performed using an optical fibre connected to an Acton Spectra Pro 2500i spectrometer (grating 600 gr/mm, slit aperture 0.15 mm, counting time 1 s), equipped with a Pixis 256E CCD detector (front illuminated 1024x 256 pixels). The absolute N-atom density was obtained using the NO titration method [START_REF] Villeger | Synergy between N and O atom action and substrate temperature in a sterilization process using a flowing N 2 -O 2 microwave post-discharge[END_REF][START_REF] Villeger | Serilization of E-coli bacterium in a flowing N 2 -O 2 postdischarge reactor[END_REF][START_REF] Villeger | Treatment of flat and hollow substrates by a pure nitrogen flowing post discharge. Application to bacterial decontamination in low diameter tubes[END_REF][START_REF] Merel | Influence of the field frequency on the nitrogen atom yield in the remote plasma of an high frequency discharge[END_REF], by injecting an Ar-2%NO gas mixture in the post-discharge tube (see Fig. 1).

The transmission of N-atoms through hollow tubes was studied by using a modified Teflon junction (Fig. 1), machined to receive between 3 and 9 hollow tubes of dia. 1-3 mm.

The hollow tubes insertion in the Teflon junction produces pressure variations between the plasma tube and the 5 litre reactor. Such pressure variations were analysed from the afterglow emission at z = 20 cm (the z = 0 point being taken in the quartz tube, at the 5 to 18 mm enlargement position), by conserving a constant pressure in the 5 litre reactor.

The spectrum recorded at z = 3 cm without the hollow tubes and reproduced in Fig. 2 shows the emission of the N 2 1 st positive, v = -4 sequence. Domination of the 11-7 band at 580 nm is characteristic of full late afterglow conditions, where the N-atom three body recombination process N + N + M → N 2 (B,v=11) + N 2 is at the origin of the intense N 2 (B,v=11) → N 2 (A,v=7) + h 580nm emission.

In consequence, as demonstrated in refs [START_REF] Merel | Influence of the field frequency on the nitrogen atom yield in the remote plasma of an high frequency discharge[END_REF][START_REF] Zerrouki | Determination of N and O -atom and N 2 (A) metastable molecule densities in the afterglows of N 2 and N 2 -O 2 microwave discharges Contrib[END_REF][START_REF] Zerrouki | Determination of N and O-atoms and N 2 (A) metastable molecule densities in the afterglows of N 2 -H 2 , Ar-N 2 -H 2 and Ar-N 2 -O 2 microwave discharges[END_REF][START_REF] Zerrouki | Determination of N and O-atoms, of N 2 (A) and N 2 (X, v>13) metastable molecules and N 2 + ion densities in the afterglows of N 2 -H 2 , Ar-N 2 -H 2 and Ar-N 2 -O 2 microwave discharges[END_REF] for pressures higher than 2 Torr, the I 580nm intensity is directly proportional to the square of the N-atom concentration [N]:

I 580 = k [N] 2 (1)
where k is a constant including reaction rates and characteristics of the used optical acquisition system, that can be determined by NO titration. To obtain the N-atom absolute density, the constant k in eq.1 was determined by NO titration [START_REF] Villeger | Treatment of flat and hollow substrates by a pure nitrogen flowing post discharge. Application to bacterial decontamination in low diameter tubes[END_REF]. Briefly, an Ar-2%NO gas mixture is introduced at z = 20 cm, at low flow rate:

Step 1: For a NO flow rate Q(NO) less than that the N-atom flow rate of the afterglow Q(N), a violet emission is produced by the following reactions:

N + NO → N 2 + O (a) N + O + N 2 → NO(B) + N 2 (b)
inducing the decrease of the I 580nm emission and the increase of the NO β violet bands.

Step 2: The injected NO flow rate is gradually increased, consuming stoechiometrically the Natoms. When Q(NO) becomes higher than Q(N), the N 2 1 st positive and NO β violet bands emission disappear and the NO 2 * green continuum emission is observed.

All of the N-atoms are consumed by reaction (b) and the produced O-atoms now react with the NO molecules in excess following the process:

O + NO + N 2 → NO 2 * + N 2 . (c)
At the extinction point where no emission is observed, the injected NO flow rate Q(NO ext ) equals the N-atom flow rate of the afterglow Q(N), relation (2) then allows to obtain the absolute N-atom concentration [N]:

[N]/[N 2 ] = Q(N)/Q(N 2 ) = Q(NO ext )/Q(N 2 ) (2)
With the used spectrometer and optical fiber (see set-up above), the k constant in (1) was found to be k = 0.6 (±0.3) 10

-26 counts cm 6 [START_REF] Ricard | Active species densities in R/X%N 2 and R/X%(N 2 -5%H2)( R=Ar,He)microwave early afterglows[END_REF]. According to (1), the k constant can then be used to determine the N-atom absolute concentration by simply recording the (I 580 ) 1/2 value.

Fig. 2. Spectrum of the N 2 1 st positive bands recorded between 560 and 620 nm at 3 cm from the inlet of the i.d. 18 mm afterglow quartz tube

The evolution of the I 580 intensity observed at z = 20 cm with the gas pressure measured in the 5 litre reactor (see Fig. 1) is reproduced in Fig. 3 for several N 2 flow rates without the silicon tubes in the Teflon junction.

It is deduced that the N-atom density at z= 20 cm, given by (I 580 ) 1/2 /k (see ( 1)) always shows a maximum value depending on the gas pressure and can be optimized for every used flow rate. With increasing N 2 flow rates, the optimized N-atom density is obtained for higher gas pressures and the maximum [N] density is found at 10 Torr for Q N2 = 0.25 slpm.

In consequence, at 1 slpm and 100 Watt, between 2 and 20 Torr, the I 580 measurement gives both the absolute N-atom density but also the gas pressure. At lower flow rates, as the I 580 maximum is shifted to lower pressures, the gas pressure determination is limited to 10 Torr for 0.25 slpm, 7 Torr for 0.125 slpm and 4 Torr for 0.064 slpm.

In presence of the silicon tubes, the I 580 intensity at z = 20cm reported in Fig. 3 will allow to determine both the upstream absolute N-atom density and the gas pressure for a given flow rate. At high N 2 pressure (p > 5 Torr), it was not always possible to switch on the N 2 discharge at a power less than 300 Watt. Nevertheless, as described in [START_REF] Ricard | Time varying afterglow emission and gas pressure in a pulsed N 2 gas microwave flowing discharge at reduced pressure[END_REF], it has been found that the plasma can be switched on at higher pressure by pulsing the gas flow. For that purpose, a Parker electromagnetic valve was setup between the mass flow controller and the microwave cavity in Fig. 1.

When the valve is closed, the continuous pumping decreases the pressure in the tubes and the discharge can be switched on as the gas pressure and it was maintained at high pressure when the valve was opened (at a constant time of 2 10 -2 sec).

An Ar-N 2 gas mixture was also studied since the discharge is easier to sustain in this mixture than in pure N 2 at high pressure [START_REF] Ricard | Optical spectroscopy to control a plasma reactor for surface treatments[END_REF].

PLASMAS AT HIGH GAS PRESSURES

Plasmas in Pulsed N 2 Gas

The microwave cavity was first tuned with a continuous gas inlet of 1 slpm and an injected power between 100 and 300 Watt. Then, by pulsing the gas with a δt pulse varying between 0.1 to 2.0 s and a Δt period of 1 and 2 s, it was successively produced two plasmas at high (2-3 Torr) and low (0.4-0.5 Torr) pressure [START_REF] Ricard | Time varying afterglow emission and gas pressure in a pulsed N 2 gas microwave flowing discharge at reduced pressure[END_REF].

By checking the band intensity ratio N 2 + 391 nm / N 2 380 nm near the surfatron gap, it was found that the microwave cavity correct tuning remained with the gas on, as for the continuous case with a negligible reflected power. This N 2 + /N 2 band intensity ratio can be effectively considered as a signature of plasma ionization rate, a constant value indicating that identical plasma conditions were reached.

At low pressure, when the gas was off, the reflected power was found important, varying from 30 to 90 Watt for an incident power from 100 to 150 Watt [START_REF] Ricard | Time varying afterglow emission and gas pressure in a pulsed N 2 gas microwave flowing discharge at reduced pressure[END_REF].

In these conditions, a strong late afterglow of N 2 1 st pos. bands (showing the typical emission at 580 nm illustrated in Fig. 2 and resulting of 3-body N-atoms recombination) was observed at high pressure but not at low pressure. Time fluctuations of the integrated afterglow intensity with the gas pressure were studied using a photodiode (600-900 nm) and are reproduced in Fig. 4.

It was established that for long pulses (0.4-1.0 sec), the N-atom density increased up to the end of gas pulse as do the plasma length.

A maximum of the N-atom density was obtained for time period Δt twice the pulse time δt. The maximum pressure variations were found for long pulses. For the following experimental conditions: incident power 200 Watt, N 2 flow rate 1 slpm, pumping speed 800 slpm, Δt ~ 2 δt = 2 s, it was determined a mean N-atom density of 9 10

Plasmas in Ar-N 2 Gas Mixture

Homogeneous N 2 microwave post-discharges can be obtained in the continuous mode at reduced gas pressures (2-10 Torr). When the pressure is increased, the plasma became unstable and a 300 W power is not sufficient to sustain the discharge. Adding Ar to N 2 allowed obtaining a stable discharge from low pressure up to atmospheric gas pressure with N 2 percentage below 23% with a microwave power limited to 300 Watt [START_REF] Ricard | Optical spectroscopy to control a plasma reactor for surface treatments[END_REF].

The gas temperature was estimated from the rotational structure of the N 2 775 nm band giving the rotational temperature as described in [START_REF] Ricard | Time varying afterglow emission and gas pressure in a pulsed N 2 gas microwave flowing discharge at reduced pressure[END_REF]. Before the Teflon junction, it was determined a temperature of 360 K in the Ar Torr, 5 slpm and 150 Watt. This temperature is similar to the one obtained Torr, 1 slm, 100 Watt.

In pure N 2 plasma, the electron energy distribution function is depleted above 3 eV because of the low mean vibrational excitation of N 2 molecules [START_REF] Capitelli | Non-equilibrium vibrational kinetics[END_REF]. In contrast, in Ar gas, the first excitation threshold being 11 eV, the extension of the electron energy is highe plasma, the ionization in Ar-N 2 gas mixture is enhanced, particularly at low N conditions of an Ar discharge dominate.

TRANSMISSION OF N-ATOMS IN HOLLOW TUBES

Silicone Tubes

It was first experimented 9 tubes of silicones of i.d. 1.5 mm and o.d. 2 mm located inside the Teflon junction of Fig. 1 where 9 holes of dia.2 mm were drilled. The tubes were placed and if necessary coiled inside the 5 litres reactor.

For 150 long tubes, it was not possible to produce the N it was necessary to pulse the gas with Δt ~ 2 δt = 2 s at 200 Watt to switch on the N 3.1.).

In these pulsed conditions, the afterglow main emission (N 50 cm inside the 9 tubes. Teflon junction, it was determined a temperature of 360 K in the Ar-1%N 2 Torr, 5 slpm and 150 Watt. This temperature is similar to the one obtained in pure N 2 (330 plasma, the electron energy distribution function is depleted above 3 eV because of the low molecules [START_REF] Capitelli | Non-equilibrium vibrational kinetics[END_REF]. In contrast, in Ar gas, the first excitation threshold being 11 eV, the extension of the electron energy is higher. Consequently, compared with pure N gas mixture is enhanced, particularly at low N 2 percentage where the conditions of an Ar discharge dominate. The length of the tubes was then reduced to 50 cm and the afterglow was detected at the tubes outlet, in the 5 litre reactor.

ATOMS IN HOLLOW TUBES

In conditions of Δt ~ 2 δt = 2 s, 200 Watt, 1 slpm, the gas pressure was found varying from 0.8 to 2.0 Torr in the reactor and the N-atom density was obtained from the I 580nm intensity (see eq. ( 1)) after calibration by NO titration.

It was found [N] = 3.4 10 15 cm -3 at 1 cm before the Teflon junction. In the reactor, the N-atom density was evaluated from the I 580nm intensity corrected from the change of emitting length between the Pyrex tube (dia. 1.8 cm) and the reactor chamber (dia. 15 cm).

It was deduced

[N] = 1.2 10 14 cm -3
after 50 cm of silicon tubes length. The N-atom transmission T N was thus evaluated to be 3.5%.

A second experiment was undertaken with a 3 holes (dia. 4 mm) Teflon junction, allowing connecting 3 silicon tubes of i.d. 3 mm (o.d. 4 mm) and length from 80 to 250 cm.

For these larger diameter tubes, the N 2 plasma was switched on in the continuous mode.

At a flow rate of 1 slpm, a pressure of 4 Torr in the 5 litre reactor, a power of 150 Watt and silicon tube lengths of 80 cm, it was determined a N-atom density of 2.7 10 15 cm -3 before the Teflon connector, corresponding to a transmission rate T N = 8.5%.

At a low flow rate of 0.125 slm, a power of 100 Watt and silicon tube lengths of 60 cm, it was obtained a small increase of T N from 3.9% at 1 Torr to 4.9% at 3 Torr.

To increase the plasma ionization at high flow rates (i.e. high gas pressures), it was experimented as discussed in 3.2., an Ar-1%N 2 gas mixture.

With this mixture, a discharge was switched on in continuous mode at 150 W, 3 slpm, 20 Torr and with 80 cm length silicon tubes. The method described in part 2 was extended to the Ar-N 2 gas mixture, allowing obtaining the N-atom density before the Teflon connector. It was found [N] = 8.2 10 15 cm -3 and T N = 8.5% at the outlet of the 80 cm silicone tubes, indicating an increase by a factor 3 of the N-atom density and the same T N value, when compared to the pure N 2 experiments at 150 W, 1 slpm and 4 Torr.

Stainless Steel Tubes

9 stainless tubes of i.d. 1.5 mm and o.d. 2 mm, of 6.5 cm in length 6.5 cm were inserted in the Teflon junction as the silicon tubes in the previous experiment.

A continuous N 2 afterglow was obtained at 300 W, 1 slpm and 1.7 Torr in the 5 litre reactor. In these conditions, the N-atom density was measured to be 8 10 15 cm -3 before the Teflon junction and 10 14 cm -3 in the reactor, at the exit of the 9 stainless tubes, corresponding to a N-atom transmission of 1.3%.

In the pulsed mode, with Δt ~ 2 δt = 2 s, 200 Watt, 1 slpm, for a gas pressure varying between 0.9 and 2.3 Torr, the N-atom transmission was increased to 3.5-4.0%.

Transmission rates T N through silicone tubes of i.d. 1.5 and 3 mm and lengths of 9, 10 and 80 cm in pulsed and continuous modes obtained in pure N 2 and with the Ar-1%N 2 mixture are reported in Table 1a.

Table 1b reports the T N rates measured through stainless tubes of i.d. 1.5 mm and 6.5 cm in length in pulsed and continuous N 2 conditions.

Destruction Probability γ of the N-atoms on the Tube Walls

Estimating that below 15 Torr, the main loss process of the N-atoms along the tubes is due to recombining collisions on the tube walls, it can be written:

[N] z = [N] 0 exp -[ γ (c'/2R) (z/v) ] ( 3
)
where z is the axial distance in the silicon tube, [N] 0 is the N-atom density at z 0 =20 cm, c' is the thermal gas velocity at 300K (c' = 5 10 4 cms -1 ), R is the radius of 1 tube and v the axial gas velocity .

The axial gas velocity is deduced from the N 2 flow rate Q by:

Q(p) = S v (4)
where Q(p) is the N 2 flow rate at pressure p(Torr): Q(p) = 760 Q 0 / p (Q 0 is the standard flow rate at 760 Torr and 300K) and S is the tube internal surface.

When the flow rate was decreased to 0.125 slpm, a quasi constant pressure was measured in the 5 litre reactor (1-3 Torr) and before the Teflon junction (2-3 Torr, obtained from I 580nm intensity measurements-see Fig. 3).

With eq. ( 4), it is calculated at 2 Torr an axial gas velocity of 3. s up to 60 cm. It is then determined from eq. ( 3) and (4), a γ value varying from 1.2 to 0.8 10 -3 as the pressure in the reactor increased from 1 to 3 Torr. Such values are 3 times lower than published values for Nylon [START_REF] Sarrette | Determination of nitrogen atoms loss probabilities on copper, aluminium, alumina, brass and nylon surfaces[END_REF]: γ = 3-2 10 In N 2 , for 1 slpm and 150 Watt, the pressure before the Teflon junction is found from Fig. 3 to be 10 Torr. For a pressure of 4 Torr in the 5 litre reactor, the mean pressure in the tubes is 7 Torr, the axial velocity is 8 10 , close to the published value for Nylon at 4 Torr : γ = 2 10 -3 [START_REF] Sarrette | Determination of nitrogen atoms loss probabilities on copper, aluminium, alumina, brass and nylon surfaces[END_REF].

Fig. 1 . 2 s

 12 Fig. 1. Post discharge set up with the emplacement of the Teflon junction connecting the quartz and the Pyrex parts of the 18 mm i.d. tubes. The picture shows details of a modified Teflon junction used in the transmission experiments, allowing the gas to flow through three silicone tubes

Fig. 3 .

 3 Fig. 3. Variation of I 580 intensity in z =20 cm with N 2 gas pressure without the silicon tubes inside the Teflon junction, for several flow rates at 100 Watt

  conditions) and a pressure variation Δp = 2.3-0.5 Torr.

Fig. 4 .

 4 Fig. 4. Time variations of the gas pressure in electric field of the EM valve (3), obtained by the Labview program. The photodiode and electric field signals are in arbitr

Recent

  Advances in Science and Technology Research Vol.3 atoms Produced by N2 Flowing Microwave Afterglows through Hollow Tubes: Advanced Study 26 Time variations of the gas pressure in Torr (1), of the photodiode signal (2) and of the electric field of the EM valve (3), obtained by the Labview program. The photodiode and electric field signals are in arbitrary units. Discharge parameters: 150 Watt, 1 slpm, Δt = 2 s, δt = 1s

  It was first experimented 9 tubes of silicones of i.d. 1.5 mm and o.d. 2 mm located inside the Teflon 1 where 9 holes of dia.2 mm were drilled. The tubes were placed and if necessary it was not possible to produce the N 2 plasma at 300 Watt in continuous mode and it was necessary to pulse the gas with Δt ~ 2 δt = 2 s at 200 Watt to switch on the N 2 In these pulsed conditions, the afterglow main emission (N 2 1 st pos. around 580 nm) extended up to

  Recent Advances inScience and Technology Research Vol.3 Flowing Microwave Afterglows through Hollow Tubes: Advanced Study T (s) Torr (1), of the photodiode signal (2) and of the electric field of the EM valve (3), obtained by the Labview program. The photodiode and : 150 Watt, 1 slpm, mixture for 20 330-350 K) at 5 plasma, the electron energy distribution function is depleted above 3 eV because of the low molecules [11]. In contrast, in Ar gas, the first excitation threshold Consequently, compared with pure N 2 percentage where the It was first experimented 9 tubes of silicones of i.d. 1.5 mm and o.d. 2 mm located inside the Teflon 1 where 9 holes of dia.2 mm were drilled. The tubes were placed and if necessary plasma at 300 Watt in continuous mode and discharge (see pos. around 580 nm) extended up to

Table 1 .

 1 Transmission rates T N of N-atoms obtained through: (a) silicone tubes of i.d. 1.5 and 3 mm and lengths of 9, 10 and 80 cm in N 2 (pulsed and continuous modes) and in Ar-1%N 2 continuous modes. (b) stainless steel tubes of i.d. 1.5 mm and 6.5 cm in length, N

  time in the silicon tubes of 1.6 10

1 .

 1 With T N = 8.5% at 80 cm, it is deduced γ = 1.6 10

He received a Ph.D. degree in

29

With such γ values, the N-atom density at the exit of the 3 silicon tubes of i.d. 3 mm and 250 cm in length can be calculated. It is found concentrations of 1.3 10 12 cm -3

for N 2 at 1 slm, 150 W, 4Torr in the 5 litre reactor.

Eqs. ( 3) and (4) were applied to the results obtained in the stainless steel tubes in part 4.2.

From the I 580nm measurement, it is estimated a pressure of 10-16 Torr before the Teflon junction for 4 Torr in the 5 litre reactor .The mean pressure in the tubes is taken between 7-10 Torr. Such a gas pressure is sufficiently low to neglect the N+N+M volume losses when compared to N-atom destruction on the tube walls.

With a N-atom transmission of 1.3% in continuous mode after 6.5 cm of tube length, it is calculated

This value is 3-4 times higher than published values in pulsed RF discharge for stainless steel [START_REF] Adams | Surface and volume loss of atomic nitrogen in a parallel plate of discharge reactor[END_REF]: γ = 5 10 -3 at 5 Torr and γ = 6 10 -3 at 3 Torr.

CONCLUSIONS

Transmission rates of N-atoms through hollow tubes in the flowing late afterglow of a N 2 microwave discharge were measured for silicon tubes of i.d. 3 mm and a length up to 250 cm and for stainless steel tubes of i.d 1.5 mm and 6.5 cm in length. Hollow tubes were inserted through holes drilled a Teflon junction of i.d. 18 mm placed in the N 2 afterglow flow before a 5 litre reactor. Depending on the gas flow, a gas pressure variation is produced before and after the Teflon junction. To switch on the plasma at high gas pressure, it was experimented a pulsed N 2 gas mode and Ar-N 2 gas mixtures. The pulsed gas mode was of interest to produce the plasma at lower gas pressure when the gas was off and to maintain it at high pressure when the gas was in.

Compared with the continuous mode, the N-atom transmission through the 1.5 mm i.d stainless steel tubes was found to increase with the pulsed gas mode.

An Ar-1%N 2 gas mixture allows to produce plasmas at high flow rates and gas pressures with higher N-atom densities than in pure N 2 .

From the measured variations of the N-atom density before and after the tubes, the γ destruction probabilities of the N-atoms on the tube walls has been determined: γ = (1.0-1.6) 10 -3 for the silicon tubes and γ = (1.6-2.0) 10 -2 for the stainless steel tubes. These probabilities are in good agreement with published values for the silicon tubes but not for the stainless steel tubes which appears to be 3-4 higher.

Such results are of interest for the sterilization of hollow tubes with N 2 and Ar-N 2 late flowing afterglows.

COMPETING INTERESTS

Authors have declared that no competing interests exist.


Hongbo Liu

Stefan Norra
email: stefan.norra@kit.edu

Qiyan Ying

Congyu Li

Eric Lichtfouse

Enhanced removal of antimony in dyeing wastewater by mixing Fe 3 O 4 with manganese sand filter material

Keywords: antimony removal, dyeing wastewater, dynamic filter, heavy metal, iron and manganese mixture

Wastewaters from the printing and dyeing industries contain many contaminants in particular antimony (Sb) that end up in the environment. Both manganese sand filter and Fe 3 O 4 have good removal effect on Sb, and are cheap and easy to obtain. We made a filter material by mechanically mixing the manganese sand filter material and ferro-ferric oxide (Fe 3 O 4 ). The Fe-Mn oxide filter material was analyzed by X-ray diffraction. We studied the filtration of real wastewater from a dyeing wastewater resource recovery facility in Suzhou, China, containing Sb at high concentration of 410 μg/L, using dynamic tests in adsorption columns during 7 days. We tested the effects of filter material volume filling ratio, the empty bed contact time (EBCT), pH, and back washing on the removal of Sb. Results show that the addition of Fe 3 O 4 enhanced the removal of Sb, reaching 85% of initial Sb. When the initial influent pH of the raw water is 3.0, the volume filling ratio of filter material is 60%, the EBCT is 20 min, and the developed dynamic Fe-Mn oxide filter has the best removal effect on Sb. Daily back washing of the filter keeps a Sb removal rate of about 80%.

Introduction

Large amounts of printing and dyeing wastewaters have been produced by the printing and dyeing industries in recent years [START_REF] Xi | Adsorption of antimony on kaolinite as a function of time, pH, HA and competitive anions[END_REF]. On August 4, 2018, the discharge of industrial waste has forced the closure of wastewater resource recovery facilities due to excessive Sb levels found in a river of Suzhou, China. Sb pollution has thus become a major problem. In the dyeing industry, it is necessary to use Sb compounds such as antimony acetate and ethylene glycol antimony as catalysts to produce the polyester fiber by poly-condensation reactions [START_REF] Jiang | Metal pollution and metal sustainability in China[END_REF][START_REF] Wang | Microwell confined iron oxide nanoparticles in honeycomblike carbon spheres for the adsorption of Sb (III) and sequential utilization as a catalyst[END_REF]Wang, Ye, Yu, & Jing, 2018). Previous studies have shown that the toxicity of Sb varies with Sb valence, for example, the toxicity of Sb(III) is ten times higher than that of Sb(V) [START_REF] Ungureanu | Arsenic and antimony in water and wastewater: Overview of removal techniques with special reference to latest advances in adsorption[END_REF].

Strict regulations on Sb contents in drinking water have been implemented worldwide [START_REF] Herath | Antimony as a global dilemma: Geochemistry, mobility, fate and transport[END_REF]. For example, the U.S. Environmental Protection Agency sets a maximum limit of 6 μg/L for Sb in drinking water [START_REF] Herath | Antimony as a global dilemma: Geochemistry, mobility, fate and transport[END_REF], whereas it is 10 μg/L in the European Union [START_REF] Davies | Pollution control in United States: Evaluating the system[END_REF]. Because of the largest antimony reserves and rapid development, China is suffering from environmental pollution caused by the exploitation of antimony ores [START_REF] Wen | Sources, migration and transformation of antimony contamination in the water environment of Xikuangshan, China: Evidence from geochemical and stable isotope (S, Sr) signatures[END_REF]. Chinese authorities have decreased the limit of Sb emission in water from 100 μg/L in 2012 to 50 μg/L in 2015 and 5 μg/L in 2019 [START_REF] Gao | Simultaneous evaluations of occurrence and probabilistic human health risk associated with trace elements in typical drinking water sources from major river basins in China[END_REF].

Sb can be removed from the waters by precipitation, coagulation/flocculation, adsorption and electrochemical methods. Experiments show that combining different methods to remove antimony at the same time can achieve better results, such as dual-functional electroactive filter system [START_REF] Liu | Simultaneous oxidation and sorption of highly toxic Sb(III) using a dual-functional electroactive filter[END_REF]Liu, Wu, et al., 2019). Coagulation and sedimentation are simple and easy to operate, but require a large amount of precipitant; these processes produce a large amount of Sb containing sludge and thus cause secondary pollution. Electrochemical methods have the advantages of a short reaction time and less secondary pollution, but it is more suitable for treating high-strength wastewater [START_REF] Li | Antimony contamination, consequences and removal techniques: A review[END_REF][START_REF] Ungureanu | Arsenic and antimony in water and wastewater: Overview of removal techniques with special reference to latest advances in adsorption[END_REF].

Adsorption is also widely used to remove Sb due to high adsorption efficiency and easy to operate [START_REF] Mubarak | Antimony (Sb)-pollution and removal techniques-critical assessment of technologies[END_REF][START_REF] Saleh | Effective adsorption of antimony (III) from aqueous solutions by polyamide-graphene composite as a novel adsorbent[END_REF]. For instance, the manganese sand filter is cheap and stable for long-term operations. The real wastewater used in this study was based on the adsorption of manganese sand filter material as the main method to remove Sb. The main component of the manganese sand filter material is manganese dioxide, a common adsorbent in water treatments [START_REF] Liu | Manganese-catalyzed C-H activation[END_REF][START_REF] Xu | Removal of Pb(II) from aqueous solution by hydrous manganese dioxide: Adsorption behavior and mechanism[END_REF][START_REF] Zhang | Adsorption of clofibric acid from aqueous solution by graphene oxide and the effect of environmental factors[END_REF].

Manganese dioxide has rich and varied crystal morphology, a strong oxidation ability, and specific adsorption for heavy metals [START_REF] Chon | Fabrication of Fe/Mn oxide composite adsorbents for adsorptive removal of zinc and phosphate[END_REF][START_REF] Islam | Manganese oxides and their application to metal ion and contaminant removal from wastewater[END_REF]. Iron oxide composite adsorption materials are also widely used to treat wastewater with heavy metals and organic pollutants for their advantages of low-cost, high adsorption capacity and uniform particle dispersion [START_REF] Nizamuddin | Iron oxide nanomaterials for the removal of heavy metals and dyes from wastewater, nanoscale materials in water purification[END_REF][START_REF] Yang | Preparation and application of iron-based composite materials for the removal of antimony from aqueous solution[END_REF]. Fe 3 O 4 magnetic particles are widely used because of their simple preparation, low-cost, nontoxic, and high specific surface area [START_REF] Kumari | Heavy metals [chromium (VI) and lead (II)] removal from water using mesoporous magnetite (Fe 3 O 4 ) nanospheres[END_REF].

When manganese and iron oxides are combined as ferromanganese oxides, Sb can be removed by both oxidation and adsorption [START_REF] Bai | Antimony oxidation and adsorption by in-situ formed biogenic Mn oxide and Fe-Mn oxides[END_REF]. The removal of Sb ions from water using Fe and Mn bimetallic oxides as adsorbents has been shown [START_REF] Shi | Modeling coupled kinetics of antimony adsorption/desorption and oxidation on manganese oxides[END_REF]; however, few investigations have tested these oxides during dynamic processes [START_REF] Ochirkhuyag | Development of a high-performance cake-less continuous filtration system[END_REF]. Moreover, previous studies have often produced adsorbents by costly and complex chemical synthesis, which is often costly. The use characteristics of materials can only be determined through dynamic experiments, and the actual operating conditions can be simulated through dynamic experimental data to determine the amount of materials added and the composition ratio. Therefore, this study combines the advantages of both materials to explore the feasibility of this low-cost and easy-to-use synthesis method. Our objective was therefore to design a filter which filled with a mixture of Fe 3 O 4 and manganese sand filter material to remove Sb in real wastewater and to explore the effect of filter material volume filling ratio, EBCT, pH, and back washing on its removal efficiency.

Materials and Methods

Dyeing wastewater

Real wastewater was taken from the secondary effluent of a dyeing wastewater resource recovery facility that uses a contact oxidation process in Suzhou, China. It is detected that pH in the wastewater was 9.0-9.5, and COD was 1,090.0 mg/L; the main valence of Sb is +5, and the concentration was 410 μg/L. From the effluent water quality index, it was found that the contact oxidation method has a good removal effect on chemical oxygen demand (COD) and NH 3 -N, but does not remove Sb efficiently. Wastewater plant originally used manganese sand filter material as the main method to remove Sb. On this basis, we modified the filter material to improve the removal rate of antimony.

Characterization of the manganese sand filter material

The study subject in this paper is the mixture of Fe 3 O 4 and manganese sand filter material. The main component of the manganese sand filter material is MnO 2 . Because manganese sand filter and iron oxide are often used as water treatment materials, neither material will introduce secondary pollution (Liu, Liu, et al., 2015). The particle size of the study manganese sand filter is 0.5 mm, containing MnO 2 as the main composition and a small amount of SiO 2 . It has a strong adsorption capacity to heavy metals. The picture and XRD of the manganese sand filter material can be referred to Supporting Information Figure S1.

Synthesis of the Fe-Mn oxide filter material

We used a physical, gentle blending, stratified dosing method to prepare the materials, which does not require complicated chemical synthesis. Firstly, the manganese sand filter material and Fe 3 O 4 were mixed at a mass ratio of 3:1, and ultrasonically dispersed with pure water for 2 hr; then, the composite filter was taken out and dried; subsequently, the dried composite filter was put into adsorption columns for dynamic investigations.

Preparation of filters

Treatment of real Sb-containing wastewater was studied in a filter column packed with the Fe-Mn oxide filter material (Figure 1). The diameter of the filter column is 3.16 cm, and the total volume is 39.19 cm 3 . The volume filling ratio refers to the percentage of the filter volume to the total volume of the filter column. A continuous flow filtration process was used to study dynamic performance of the Fe 3 O 4 enhanced manganese sand filter.

Optimization of operational parameters

The volume filling ratio of filters, EBCT, pH, and effects of back washing was investigated at room temperature with dynamic tests. The test results are based on the change in antimony content in the influent and effluent water, and converted to the percentage of antimony removal. Firstly, the effect of the filter volume filling ratio on the removal of Sb was studied by testing the different volume filling ratios of the synthesized Fe 3 O 4 -MnO 2 sand material in the filter column. The running filling ratios are 30%, 40%, 50%, 60%, and 70%, respectively. After the optimal filling ratio of 60% was found, this filling ratio was used to carry on the follow-up tests. Then, three EBCTs of 10, 20, and 30 min were tested and optimized. EBCT refers to the amount of water treated per unit volume of particulate filler in unit time and is generally expressed in min. The effect of influent pH on the removal rate was studied by adjusting the initial pH of real wastewater to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with 0.1 mol/L HCl or NaOH solution, respectively. Finally, the effect of back washing on the removal efficiency was investigated. Two identical filter columns were operated in parallel; one of the filter columns was operated without back washing, and the other was back washed every day.

Samples were taken every day, passed through a 0.45 μm filter, and then, the Sb concentration was measured by an inductively coupled plasma optical emission spectrometer (ICP-OES, PE Optima 8000, PerkinElmer, Waltham, Massachusetts,) with forward power of 1.15 kW and auxiliary gas of 0.5 L/min.

Results and Discussion

We studied the effect of adding Fe 3 O 4 in a manganese sand filter on Sb adsorption in real Sb-contaminated wastewaters using dynamic adsorption experiments during 1-7 days. We tested the effects of filter column filling ratio, empty bed contact time, influent pH, and back washing.

Kinetics of the study filter

The kinetics of single manganese sand filter and Fe 3 O 4mixed manganese sand filter were studied by static test with preconfigured Sb pure solution. The results are shown in Figure 2. The adsorption equilibrium capacity of the Fe 3 O 4mixed manganese sand filter for Sb is 0.66 mg/g, which is slightly higher than the adsorption equilibrium capacity of Sb by single manganese sand filter (0.63 mg/g). At the same time, the R 2 values of both materials reached 0.99. Therefore, the pseudo-second-order adsorption kinetic equation can be used to describe the adsorption kinetics of Sb.

Performance of the manganese sand filter material mixed with or without Fe 3 O 4

Dynamic Sb removal of the study filters is shown in Figure 3. The manganese sand filter material has the lowest Sb removal rate. When the manganese sand filter material and Fe 3 O 4 are mixed, the removal rate has been improved obviously. It can be indicated from the figure that compared with the simple manganese sand filter, mechanical mixing of the manganese sand filter and Fe 3 O 4 has a significant increase in Sb removal rate. This is because Fe 3 O 4 has a higher specific surface area for better removal of Sb, but it is easy to agglomerate and affect the removal effect, so mixing it with manganese sand filter material can exert the better removal effect of Fe 3 O 4 on Sb. However, single Fe 3 O 4 is prone to corrosion and agglomeration, resulting in a significant decrease in its adsorption capacity (Yang et al., 2017). Therefore, this study considers manganese sand filter material as the main adsorption carrier. By grafting a small amount of Fe 3 O 4 to the manganese sand filter, the adsorption site of the material can be increased, improving Sb adsorption on the surface of the manganese sand filter [START_REF] Islam | Manganese oxides and their application to metal ion and contaminant removal from wastewater[END_REF]. Compared with other synthetic filters, the composite manganese-Fe 3 O 4 sand filter can be prepared without complicated synthesis process.

Dynamic Sb removal by filters is shown in Figure 3. Results show that the Fe 3 O 4 manganese sand filter induces a higher Sb removal of 75% versus 83% for the manganese sand filter at day 1. This finding is explained by the magnetic properties and high specific surface area of Fe 3 O 4 . Our results are supported by a previous report showing that grafting a small amount of Fe 3 O 4 to a manganese sand filter improves Sb adsorption [START_REF] Islam | Manganese oxides and their application to metal ion and contaminant removal from wastewater[END_REF]. Grafting Fe 3 O 4 to a manganese sand filter is better than that using Fe 3 O 4 alone because Fe 3 O 4 is prone to corrosion and agglomeration, resulting in a significant decrease in its adsorption capacity [START_REF] Yang | Preparation and application of iron-based composite materials for the removal of antimony from aqueous solution[END_REF]. Moreover, compared with other synthetic filters, the composite manganese-Fe 3 O 4 sand filter can be prepared without complicated synthetic steps.

Effect of volume filling ratios

The filter material volume filling ratio is an important parameter affecting performance of the filter. Excessive filling is likely to cause clogging of the filter material. If the volume filling ratio is too small, the adsorption site is insufficient and the effluent is not satisfactory. Therefore, this investigation conducted dynamic tests under five different filling ratios. When the volume filling ratio is 60%, saturated adsorption can be achieved with a Sb removal rate above 80% (Figure 4). Found through subsequent tests the removal rate of the composite adsorbent has a significant upward trend when rising the volume filling ratio from 30% to 60%, implying that the adsorption position of the study filter was still at an insufficient state. When the filling ratio is increased from 60% to 70%, the removal rate did not increase significantly, indicating that the filter medium has reached a saturated state. Although the removal rate did not increase significantly when the filling ratio reached 70%, the filter column is more likely to occur plugging and blockage problems during operation. This is because that the increase of filling ratio is also controlled by the size of filter medium. If the size is too small, the larger filling ratio will reduce the water permeability of the filter column and lead to poor performance (Liu, Liu, et al., 2015). Considering the economic benefits, the optimal filling ratio is determined as 60% for the study dynamic composite filter. At this time, the optimal removal rate of the filter column is 90%. When the filter column is continuously operated for 7 days without back washing, the removal rate of the filter column can still be maintained at about 77%.

Effect of empty bed contact time

Subsequently at room temperature, real Sb wastewater was put into the adsorption column with the optimized filter filling ratio of 60%. Performances of the Fe 3 O 4 -MnO 2 sand material dynamic filter under the empty bed contact time of 10, 20, and 30 min were investigated, respectively. The results are shown in Figure 5.

Results show that reducing EBCT increases the filtration rate, resulting in poor Sb removal when the contact time is less than 20 min. Performance of the Fe 3 O 4 -MnO 2 sand material dynamic filter was only slightly enhanced when the EBCT was increased from 20 to 30 min, indicating that the maximum utilization rate of adsorbed particles was almost achieved when the EBCT reached 20 min. A previous study has also shown that the best adsorption capacity is achieved for EBCT of 20 min [START_REF] Shakya | Simultaneous removal of arsenic, iron and nitrate in an attached growth bioreactor to meet drinking water standards: Importance of sulphate and empty bed contact time[END_REF].

The adsorption process is controlled by the time of the surface contact, the diffusion of the adsorbate in the filter space, and adsorption by electrostatic attraction or chemical bonds [START_REF] Gheju | Progress in understanding the mechanism of CrVI removal in Fe0based filtration systems[END_REF]. If EBCT is larger than 20 min, and the contact time between the solid phase and the liquid phase in the adsorption zone is sufficient, inducing high adsorption efficiency. Therefore, for our composite adsorbent of Fe 3 O 4 mixed with manganese sand filter material, we found that 20 min is the optimal EBCT. Under this condition, the optimal Sb removal is 91%.

Significantly, the use of Fe 3 O 4 -reinforced manganese sand filter material can significantly reduce the material synthesis cost while obtaining a good adsorption effect, which is very suitable for practical engineering.

Effects of the initial pH value

We tested the effect of pH by changing the real wastewater pH with NaOH or HCl. Initial influent pH is a major factor affecting the adsorption of pollutants. Figure 6 shows an increase of Sb removal by the Fe 3 O 4 -manganese sand filter from 88.5% at pH 3 to 70.0% at pH 10. This finding is explained by the fact that Sb mainly is in the form of the [Sb(OH) 6 ] -anion in water. Indeed, at acidic pH, surfaces are more protonated, thus inducing electrostatic attraction of anions. Lower pH might weaken the protonation of the adsorbent surface, thereby reducing the amount of adsorbed contaminants [START_REF] Liu | Adsorption of antimony (V) onto Mn (II)-enriched surfaces of manganese-oxide and Fe Mn binary oxide[END_REF][START_REF] Yang | Removal of Sb (V) from aqueous solutions using Fe-Mn binary oxides: The influence of iron oxides forms and the role of manganese oxides[END_REF].Therefore, for the composite Fe 3 O 4 -manganese sand filter, the pH of the influent should be acidic. When the pH is 3.0, the optimal removal rate of the filter column can reach 89%. After 7 days of continuous operation, the removal rate of the filter column can still be maintained above 80% (Figure 6).

Effects of back washing

As an important operational parameter for filter columns, back washing has a great influence on Sb removal. Back washing is the process of washing the filter material with reverse water flow to restore the normal operation of the filter column. Water flows through the bottom of the filter column in the reverse direction to flush out the blocking material in the filter. In this study, two filter columns with identical operating conditions were used for 7 days of operation. One of the filter columns was back washed every 24 hr while the other column was operated without back washing. Back washing at the initial stage of the reaction does not have a significant effect on performance of the filter column, but the positive influence is gradually amplified when the filter column is operated to the third day (Figure 7). When the filter is operated for a period of time, the surface and gap of the filter material are gradually saturated by the contaminants, resulting in a decrease in the removal rate of the Sb and an increase in the head loss. The removal rate of Sb in the wastewater will be significantly reduced when the head loss decreased rapidly, resulting in a continuous decline in quality of the filtered wastewater. However, when back washing is implemented every day, the removal rates of the Fe-Mn oxide filters remain constant and there is no obvious agglomeration observed in the filter media. This is because after back washing, the solid material covered on surface of the filter can be removed, and the performance of the filter can be restored [START_REF] Gheju | Progress in understanding the mechanism of CrVI removal in Fe0based filtration systems[END_REF].

Wastewater generated by back washing is not suitable for reuse due to heavy load of suspended solids. The use of polyaluminium chloride (PAC) as a coagulant treating discharged wastewater from back washing is recommended. Figure 7 indicated that the Fe 3 O 4 -mixed manganese sand filter was a promising technology removing Sb from wastewater, with an optimal removal rate of 92%. The filter can be stably operated for at least 7 days without back washing. The filter column should be back washed every 24 hr to remove pollutants on surface and gap of the filter material so that it can be regenerated, thereby maintaining a stable and excellent filtration performance.

Conclusions

This study has developed and explored the Fe-Mn oxide filter material which is low-cost, easy-to-synthesize, and easy-tooperate. Manganese sand filter material and Fe 3 O 4 both have a good removal effect on Sb, but Fe 3 O 4 is not suitable for practical wastewater treatment due to its shortcomings such as agglomeration. Fe 3 O 4 is attached to the surface of the manganese sand filter by mechanical mixing, which enhances the adsorption effect of the manganese sand filter and avoids the agglomeration of Fe 3 O 4 . The investigation shows that manganese sand filter mixed with Fe 3 O 4 is suitable for improving the antimony removal efficiency of the manganese sand filter. Optimized parameters for the developed dynamic Fe-Mn oxide filter are as follows: The initial influent pH of the raw water is 3.0, the volume filling ratio of filter material is 60%, and the EBCT is 20 min. The adsorption column should be back washed every day. Optimal removal rate of the filter column against Sb is 92%. This paper provides a method for upgrading the antimony removal of manganese sand filter media for printing and dyeing wastewater resource recovery facilities.

Figure 1 .

 1 Figure 1. Diagram illustration of the dynamic filtration tests.

Figure 2 .

 2 Figure 2. Pseudo-second-order kinetic curves of the study filters.

Figure 3 .

 3 Figure 3. Comparison of adsorption performance of Sb in real wastewater by different filters.

Figure 4 .

 4 Figure 4. Comparison of adsorption performance of Sb in real wastewater under different filter material filling ratios.

Figure 5 .

 5 Figure 5. Comparison of adsorption performance of Sb in real wastewater under different empty bed contact times.

Figure 6 .

 6 Figure 6. Comparison of adsorption performance of Sb on real wastewater under different pH conditions.

Figure 7 .

 7 Figure 7. Effect of back washing on Sb adsorption performance of the filter column.

Acknowledgments

This work was financially supported by the Shanghai Science International Cooperation Project (STCSM, 18230712300).

Project (STCSM), Grant/ Award

Conflict of interest

The authors have declared no conflict of interest.

Supporting Material