Montserrat Miranda-Olvera

Rafael Arcos-Ramos

Mauricio Maldonado-Domínguez

Lionel Salmon

Gábor Molnár

Azzedine Bousseksou

María Del

Pilar Carreón-Castro

Design and synthesis of benzothiadiazole-based molecular systems: self-assembly, optical and electronic properties

A set of small benzothiadiazole (BTD)-based derivatives with a D-A-D architecture was synthesized and characterized as building blocks of organic semiconducting materials by applying an experimental-theoretical approach. The four derivatives tend to self-assemble into highly ordered crystalline solids which, with varying degrees of responsiveness to mechanical and thermal stimuli. The featured derivatives exhibit absorption maxima in solution and molar extinction coefficient values related to -* electronic transitions with minor solvatochromic response, displaying broad fluorescence profiles with large Stokes shifts and high quantum fluorescence yields. In the solid-state, the BTD derivatives display absorption maxima in the visible range between and intense fluorescence emission for the n-butoxy and fluorene derivatives. The stability of the one-electron reduced and oxidized forms of all compounds was assessed by means of cyclic voltammetry which, complemented by DFT calculations allowed identifying one BTD derivative (BuO-BTD) as a strong candidate for use as electron transport layer in organoelectronic devices.

Introduction

Multifunctional materials for organic electronics can be readily accessed through the engineering of small supramolecular building-blocks capable to modulate their self-assembly into highly ordered arrangements in the solid-state. In this sense, -conjugated organic systems have attracted considerable interest during the past decades due to their wide structural versatility and remarkable optical and electronic properties.

1-5

Principally, -conjugated small-organic molecules are evolving active-layer components for applications in organic photovoltaics reaching equivalent photo-conversion efficiencies to that of polymers.

6-8

A useful strategy to improve charge transport through conjugated frameworks is based on designing molecular structures with electron-donor and electron-withdrawing moieties on a discrete entity. In general, for donor-acceptor (D-A) architectures, the HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) band gap can be tuned to enhance holes and electrons transport through the -conjugated system, due to the efficient charge transfer processes from the ground excited state and light absorption increase.

9-13 Despite of numerous advances in organic semiconducting materials development; solution-processable compounds with chemical stability and high performance are still required. Among the representative electron-accepting small-organic molecules used in this field, benzothiadiazole (BTD) based conjugated molecules display remarkable electric and optical responsivity, 14-18 coupled to a fair chemical stability and high electron affinity. 14, 19 In this context, the BTD-fragment has been used as a molecular design motif in the development of molecular and polymeric materials for organic electronic applications. Due to the electron-withdrawing nature of the BTD core, it can be exploited as a building block for n-type semiconductors. The symmetrical functionalization with aryl-or heteroaryl-moieties facilitates intramolecular charge transfer (ICT) processes which allow the fine-tuning of their optical and electronic properties.

20-23

In addition, 4,7-diaryl-benzothiadiazole derivatives substituted with flexible alkoxy chains display interesting liquid crystal properties. Several BTD-based smectic and nematic liquid crystals have been used as highly dichroic fluorescent dyes for liquid crystal displays and as active layers in organic field effect transistors (OFETs).

20,24-26

Small -conjugated structures based on benzothiadiazole (BTD) derivatives can afford self-assembled supramolecular arrangements.

27- 29 In particular, rod-shaped donor-acceptordonor (D-A-D) architectures where BTD acts as a central electron-withdrawing (A) core, attached to alkyl/alkoxy (R, OR) electron-donor (D) units, have been extensively used as liquid crystals.

30-32

The (alkoxy-substituted-phenyl)-benzothiadiazole derivatives are well-known fluorescent dyes with -conjugated rodshaped structures that display liquid crystal properties. 24 These types of fluorescent dyes have been emerged as promising supramolecular building blocks of homogeneous thin-films with reduced defects and with multi-layered -stacked arrangements useful for charge transport processes.

33-34

Charge transport in semiconductor organic solids is facilitated when periodic -stacking interactions are present, and evidence suggests that it is most favored when the stacking vector is aligned with that of the charge transport;

35-36

noteworthy, this kind of supramolecular organization is often present in a wide variety of BTD-based derivatives.

In general, the structural variation of the electron-donor substituents modifies the capacity of these molecules to selfassemble in single-crystals or liquid crystals and allows thus to fine-tune their optical and electronic properties. In the present work, we selected the 4,7-diphenyl-substitutedbenzothiadiazole as basic core, due to its remarkable luminescent response in solution and we explored the effect of the different supramolecular arrangements on the optical and electronic properties of 4,7-bis(alkoxy-substituted-phenyl)and 4,7-bis(dibenzofuran(DBF)/fluorene(FL)-substituted)benzothiadiazoles.

Results and Discussion

Synthesis

We synthesized four BTD-derivatives (with methoxy, butoxy, fluorene and dibenzofurane substituents) using Pd-catalyzed Suzuki coupling reaction in moderate to good yields (Scheme 1). Each compound was purified by successive recrystallization with CHCl 3 /toluene mixture and characterized by liquid NMR (

Solid-state characterization. Thermal analysis and X-Ray diffraction experiments

Freshly recrystallized samples (from chloroform) of BTD derivatives were studied by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) to ascertain their thermal stability upon heating in the range 25-800 °C (see ESI).

In all DSC experiments, a sharp endothermic transition that corresponds with the visual observation of the melting point was found; no additional transitions due to solvent loss or phase change were found in the 20-100°C range.

In order to explore the influence of the donor groups on the solid-state organization of the BTD derivatives, we attempted to grow single crystals of the different compounds with chloroform, toluene and chloroform/toluene mixture. For BuO-BTD, high-quality single-crystals suitable for X-ray diffraction analysis have been obtained after slow evaporation of a toluene solution in a partially open glass vial. BuO-BTD crystallizes in the orthorhombic space group Pnca with Z = 12 and two independent units per asymmetric unit (see ESI for crystal data). The two molecules are antiparallelly aligned and display slightly different torsion angles between the butoxysubstituted-phenyl and the BTD core (35.17/35.82° and 36.88°). In one of the molecules, the alcoxy-substituted-phenyl are involved in intermolecular interactions (C-H•••N, 2.66 Å) with the benzothiadiazole core producing a one-dimensional zig-zag arrangement, locating the BTD rings in a parallel displaced manner (Figure 1a). In the other molecule, C-H•••(ca. 2.82 Å) interactions between neighboring phenyl rings produce a -stacked columnar arrangement locating the BTD rings in an antiparallel mode with interplanar distances (centroid-centroid) of ca. 3.8 Å (Figure 1b). These onedimensional arrangements are self-assembled through the butyl-chains (interidigation) in a highly dense supramolecular structure that contains different intermolecular contacts between planar -units (Figure 1c). Powder X-Ray diffractograms of the butoxy-derivative displayed intense and sharp peaks between 10 to 50 degrees, which only vary in intensity upon mechanical or thermal stimuli. For fluorene derivative, the diffractogram of the crystalline powder displays two important peaks at 13.2° and 26.1° with corresponding spacing's of 6.7 and 3.4 Å, respectively. The theta value can be assigned to face-to-face distances between adjacent -conjugated entities in 1Dsupramolecular arrangements. Grinding this crystalline sample significantly affects the diffraction pattern with the appearance of peaks at 17.7 and 23.9° and the disappearance of the previous peaks. On the other hand, the thermal treatment (at 100 °C) does not produce a significant change. In the case of dibenzofuran-derivative, the diffractogram of the crystalline powder display well-resolved peak including two main peaks at 13.2° and 17.2° (corresponding to spacings of 6.7 and 5.2 Å). The mechanical treatment considerably modifies the powder pattern, reducing the intensity of the main peak at 13.2°, and inducing the appearance of peaks at 31.6 and 35.9° (2.8 and 2.5 Å, respectively); while thermal treatment slightly affects the diffraction peaks up to 20°. Finally, dimethoxy-derivative diffractograms of crystalline, ground, and annealed samples present larger number of wellresolved peaks, suggesting a more complex degree of structural order or a coexistence of crystalline phases. In particular, the thermal treatment elicits an improvement change of the crystallinity as indicated by the appearance of diffraction peaks between 30° and 50°.

Optical properties

Initially, we investigated how the different electronic nature of the electron donor groups attached to the benzothiadiazole core influence the optical properties of the BTD-based derivatives by means of optical spectroscopy (UV-Vis absorption and fluorescence) (Figure 3). The relevant data are summarized in table 1. Absorption profiles of BTD-based derivatives were determined in different organic solvents at room temperature (Table S2). The four derivatives display absorption bands assigned to -* transitions of the BTD core and to charge transfer (CT) transitions. In general, absorption maxima ( max ) of compounds were located between 405-416 nm (FL-BTD), 373-387 nm (DBF-BTD), 381-388 nm (MeO-BTD), and 406-415 nm (BuO-BTD). The changes in the solvent polarity afforded slight solvatochromic shifts with no clear dependence on the environment polarity suggesting that the transition dipole moment is only weakly affected by the differential functionalization.

In addition, all derivatives exhibit intense fluorescence in solution with emission maxima ( max ) located between 518-532 nm (FL-BTD), 486-491 nm (DBF-BTD), 495-498 nm (MeO-BTD), and 527-534 nm (BuO-BTD). As expected, the emission wavelengths are strongly influenced by the electron-donor nature of the different substituents following a similar trend than the absorption. The relative fluorescence quantum yields ( f ) were determined using quinine sulfate as a reference. In general, The absorption spectra of crystallized, ground, and annealed powders display red-shifted absorption wavelengths in contrast with the absorption spectra of diluted solutions.  max is located around 469 nm (FL-BTD), 478 nm (BuO-BTD), 448 nm (DBF-BTD), and 455 nm (MeO-BTD) (figure 3). The ground powders are slightly different as compared with the crystallized and annealed powders. The red-shifting can be attributed to the -stacking interactions established between the BTD derivatives. The solid-state emission spectra of crystallized, ground, and annealed powders are illustrated in Figure 4. In general, strong intermolecular interactions, such as -stacking, in combination with densely-pack arrangements led to emission quenching. The well-known fluorescence displayed by alkoxy-substituted benzothiadiazoles 24,25 such as BuO-BTD is still present in the fluorene-substituted derivative. We expect that the inclusion of the extended aromatic moiety will favor the formation of aggregates of -stacked units in the solid state while retaining the emissive behavior in solution. For all compounds, the emission maxima ( max ), located at 540 nm (FL-BTD), 546 nm (BuO-BTD), 532 nm (DFB-BTD), and 516 nm (MeO-BTD) are red-shifted when compared to the fluorescence in solution.

Nevertheless, the Stokes shifts were less pronounced than in solution, between 61 and 86 nm, the fluorene and butoxy derivatives displaying the largest values (Table 1).

Electronic properties and quantum chemical computations

The capacity of BTD-based derivatives to self-assemble into stacked layered supramolecular arrangements can be exploited to fine-tune the optical properties of these compounds, as described In addition, the electrochemical properties of BTD-based building blocks were determined by cyclic voltammetry (Figure 6). Each compound can be easily reduced to a stable radical anion due to the strong accepting behavior of the benzothiadiazole moiety, and reduction is reversible in all scenarios. Further, the formal reduction potential of this event (defined as the average of the peak cathodic and anodic potentials) is virtually insensitive to the identity of flanking donors, highlighting the chemical stability of the BTD subunit radical anion. The electrochemical behavior of the donor groups, in contrast, differs for each molecule. In BuO-BTD, two discrete and reversible oxidation steps are discernible; these can be unambiguously ascribed to stepwise electron removal from each flanking group. The system FL-BTD affords an electroactive, yet dissimilar product as evinced by the starkly different profile observed during cathodic and anodic sweeps. The identity of the corresponding product remains admittedly unclear. Finally, both MeO-BTD and DBF-BTD display irreversible, fast two-electrons oxidation, yielding nonelectroactive products as seen in the lack of a reduction peak during reverse scanning. HOMO energy was determined using the oxidation potential that is related to the ionization potential (IP), because it implies the minimum energy necessary to remove an electron from the system. Likewise, we determined the LUMO energy using the reduction potential, which is related to electronic affinity (EA) due that it implies the energy gained by dropping an electron from the vacuum level to the lowest unoccupied molecular orbital.

38-39

The oxidation and reduction potentials were obtained by using as a reference ferrocene in dichloromethane (DCM). The value for ferrocene (Fc) with respect to the vacuum level is estimated at -4.8 eV, determined from -4.6 eV for the standard electrode potential E° of a normal hydrogen electrode (NHE) on the zero-vacuum level, and 0.2 V for Fc versus NHE. 40 The values for the HOMO and LUMO levels were obtained through equations ( 1) and ( 2) as follows:

( The EA was calculated as the finite difference between neutral and anion energies, and the IP was obtained as the finite difference between cation and neutral energies. 41 The calculated IP/EA energies were: 5.45/2.71 eV (FL-BTD), 5.34/2.75 eV (BuO-BTD), 5.79/2.88 eV (DBF-BTD) and 5.72/2.79 eV (MeO-BTD). The HOMO/LUMO energy values obtained were: -6.01/-3.55 (FL-BTD), -6.00/-3,49 eV (BuO-BTD), -6.32/-3.53 (DBF-BTD) and -6.05/-3.47 (MeO-BTD). Derived from these results, the electrochemical energy gap (EgEc) values are 2.46 (FL-BTD), 2.51 (BuO-BTD), 2.79 for (DBF-BTD) and 2.58 for (MeO-BTD).

The calculated and experimental IP and EA energies values are comparable to the typical semiconductors of interest in organic electronics. Threshold values of IP > 5.7 eV and EA > 3 eV have been proposed as markers of potentially efficient electron-transporting layers for n-type semiconductors.

42-43

Compounds DBF-BTD and MeO-BTD appear as the closest candidates to these suggested values, yet their propensity to irreversible oxidation may lead to their decomposition. In this sense, despite its relatively low IP, BuO-BTD is stable upon one-and two-electron oxidation, while showcasing an EA value close to 3 eV. Hence, this compound emerges as a promising candidate, which is also prone to facile crystallization with extensive π-stacking.

IP and EA are relevant to the hole and electron transport in a semiconductor material because in the solid-state the electrons located in the valence band (associated with the IP) can be promoted to the conduction band (associated with the EA), resulting in the formation of an electron-hole of charge carriers, which can be collected by metallic electrodes.

44

Hence, it is important that the work function of metals and IP and EA energies converge. In this context, figure 7 shows the IP (HOMO) and EA (LUMO) energies obtained from the electrochemical (EC) and theoretical (Calcd) methods, and the work function of metals used as electrodes in optoelectronic devices. 45 Taking into account that HOMO represents the donor character, if the BTD derivatives had a donor character, they can work with a gold or platinum electrode; because the electrode can accept an electron from the proximal molecule, forming a hole that will be spread throughout the material. On the contrary, if the BTD derivatives had an acceptor character, they can work with calcium or magnesium electrodes; because BTD can accept and electron from the electrode, creating a charge that will be spread throughout the material. In both cases a flow of charge carriers would be created through the material.

Finally, we determined the optical band gap between the valence band (VB) and conduction band (CB) by means of solid-state absorption using an integrating sphere and Tauc's plots (see ESI). [START_REF] Eaton | Reference materials for fluorescence measurement[END_REF] The solid samples exhibit a decrease in the energy gap for all BTD derivatives (table 3). These results are expected due to the intermolecular interactions present in the solid-state.

E g

Calcd

Experimental Materials and methods

Starting materials were purchased from commercial suppliers and used without further purification. Solvents were dried by standard methods or distilled prior to use. Solvents HPLC and spectroscopic grade were used for absorption, emission and recrystallization. Reactions were monitored by thin-layer chromatography (TLC) on pre-coated silica gel plates (ALUGRAM SIL G/UV 254 ) revealed by exposure to a UV 254 lamp.

C NMR experiments were recorded with a JEOL 400 MHz equipment using deuterated chloroform as solvent. Chemical shifts () are reported in ppm and coupling constants are reported in Hz. Melting points were measured in an Electrothermal Mel-Temp® apparatus and are uncorrected. Infrared spectra were recorded on a Perkin Elmer Spectrum 400 spectrometer as solid samples using Attenuated Total Reflectance (ATR). The signals are reported in wavenumber  (cm -1 ). High resolution mass spectrometry (HRMS) data were acquired in an Agilent Technologies ESI-TOF spectrometer.

UV-Vis absorption and emission spectroscopy

Absorption studies were performed on a UV/VIS Lambda 35 Perkin Elmer spectrophotometer. Measurements were recorded in a wavelength region of 300-800 nm and carried out in 1 cm path length quartz cells at room temperature in different solvent solutions (HPLC grade). The UV-Vis spectra profiles in solid-state were determined on a Perkin-Elmer Lambda 950 spectrophotometer with integrating sphere in a wavelength region of 400-600 nm at room temperature. Fluorescence emission spectra were acquired on a LS55 Perkin Elmer fluorescence spectrometer; the wavelength of maximum absorption was employed as the excitation radiation.

The measurements of relative fluorescence quantum yield were carried out using quinine sulfate in 0.1 M H 2 SO 4 solution (literature quantum yield is 0.54) 47 as the reference. The quantum yield was calculated according to the following equation:

QY x = QY S  (m x /m s )  (n x /n s ) 2
Where the subscript S represents standard, quinine sulfate, and X represents the sample, n refers to the refractive index for each solvent, m is the slope of the curve from the plot of the integrating fluorescence intensity against absorbance.

Quantum chemical calculations

The modelling of light absorption and emission in the studied molecules was performed using density functional theory (DFT) as implemented in Gaussian 16. 48 The hybrid functional B3LYP was used throughout the study, 49 with the augmented triple-zeta polarized basis set 6-311+g(d,p). [50][51] Solvation was modelled using the implicit conductor-like polarizable continuum (CPCM) model with the dielectric constant of CHCl 3 , PhMe and DMSO. All optimized geometries in the ground and excited states were confirmed as minima in their corresponding potential energy surface by the absence of imaginary eigenvalues upon hessian diagonalization. Excitedstate geometries were optimized with the same functional and basis set, using the time-dependent DFT formalism, targeting the first root in all cases. Electron affinity and ionization potential were estimated by computing the total electronic energies of neutral, cationic and anionic molecules of the completely optimized molecular structures.

Powder X-ray diffraction experiments

PXRD experiments were carried out using a PANalytical MPD X'PertPRO diffractometer [Cu-K α1 radiation, Ge (111) monochromator, X'Celerator detector] within the 2 range 5-90° at a scan speed of 30° per hour. The solids were subjected to thermal and mechanical treatments. The thermal treatment was carried out in a LINKAM temperature control system from 25 degrees to 100 degrees with a speed of 0.5 degrees per minute. The mechanical treatment was carried out by grinding samples during 20 min.

Thermal analysis

The thermal characterization of the BTD derivatives was carried out using a simultaneous thermal analyzer NETZSCH model STA409PC. The sample was placed in the analyzer an aluminum sample pan under nitrogen atmosphere. To obtain the thermogram, the weight loss of the sample and the temperature were recorded in four second intervals, in a temperature range of 25 to 800 ºC with a temperature ramp of 5ºC / min, in a nitrogen atmosphere and with a quantity sample of 5 to 10 mg. NETZSCH proprietary software was used to calculate the rate of weight loss versus temperature, in this way the DSC curve was obtained.

Cyclic voltammetry

Cyclic

Synthetic procedures

General procedure for the synthesis of BTD-based derivatives

In a dry, round-bottom flask, previously degassed 4,7dibromobenzo[c]-1,2,3-thiadiazole (0.29 g, 1 mmol), boronic acid (2.20 mmol) and Pd(PPh 3 ) 4 (0.10 g, 10% mmol) were added. Afterwards, toluene (25 mL) and K 2 CO 3 (4 mmol, 0.55 g in 2.5 mL of water) were added dropwise under argon atmosphere. The reaction mixture was refluxed until total starting materials consumption (16 to 24 h). Then, the reaction mixture was cooled to room temperature and evaporated to dryness. The resulting solid was dissolved in CHCl 3 (150 mL), washed with saturated NH 4 Cl (2 x 100 mL), brine solution (2 x 100 mL) and distilled water (1 x 100 mL), dried over Na 2 SO 4 anhydrous and evaporated to dryness. The crude products were purified by successive recrystallizations with CH 3 Cl/toluene mixtures.

4,7-bis(4'-butoxyphenyl)benzo[c][1,2,3]thiadiazole (BuO-BTD)

Yellow crystalline solid (82 % yield

4,7-di(9H-fluoren-2-yl)benzo[c][1,2,3]thiadiazole (FL-BTD)

Yellow crystalline solid (85 % yield

4,7-bis(3',5'-dimethoxyphenyl)benzo[c][1,2,3]thiadiazole (MeO-BTD)

Yellow crystalline solid (77 % yield). Melting point (°C): 132. 5

Conclusions

Benzothiadiazole-based D-A-D derivatives can be readily accessed through Pd-catalyzed coupling reactions in moderate to good yields. The featured compounds combine ionization potentials and electron affinities close to those recommended for n-type semiconductors, while displaying reversible reduction events in all cases due to the electronegative and stable BTD heterocycle. The solid-state ordering of the compounds is crystalline, as inferred from PXRD experiments, with minor response to thermal or mechanical stimuli. This is desirable for their incorporation into organoelectronic devices, as it suggests that the compounds retain their supramolecular structure despite external stress.

Additionally, in an integral manner, the results indicate that the BTD derivatives analyzed in this work have potential to be used as semiconductor materials, not only because of their electronic properties, but also because the compounds can be obtained through a simple one-step synthesis, the isolation of the derivatives is highly processable and they are obtained with a high degree of purity; which implies that obtaining these compounds can be easily scalable. On the other hand, it should be noted that the analyzed compounds shown high stability as revealed by the thermal analysis; a property desired in semiconductor materials.

Scheme 1 .

 1 Scheme 1. Benzothiadiazole-based D-A-D derivatives.

  FTIR and HRMS (see ESI).

Figure 1 .

 1 Figure 1. (a, b) Intermolecular interactions and (c) crystal packing of BuO-BTD.

Figure 2 .

 2 Figure 2. PXRD diffractograms of BTD derivatives of crystallized, ground and annealed (at 100°C) powders.

Figure 3 .

 3 Figure 3. UV-Vis and fluorescence profiles of BTD-based building blocks in different solvents.

Figure 4 .

 4 Figure 4. Solid-state absorption and emission spectra of BTD-based building blocks powders. Crystallized (black line), ground (red line) and annealed (blue line) powders.

  above. An initial assessment to determine the potential application of the BTD-based building blocks (FL-BTD, BuO-BTD, DBF-BTD and MeO-BTD) as organic semiconductors was based on the exploration of how the different D-A-D architectures modify the electronic properties (frontier orbital energies and redox) by means of cyclic voltammetry (CV) and quantum chemical computations. Density functional Theory (DFT) and Time-Dependent Density functional Theory (TD-DFT) calculations were performed to estimate the energy of the frontier molecular orbitals and to characterize the most relevant electronic transitions of the BTD-based building blocks. The selected computational protocol provides remarkable agreement with the observed solvent-dependent response in both UV-Vis absorption and fluorescence (see Quantum Chemical Calculations section and ESI).To visualize the electronic excitations in terms of electron-hole pairs, the natural transition orbitals of the BTD-based building blocks are depicted in Figure5.37 In all cases; the electron is promoted from the flanking donor moieties towards the central BTD unit, in a charge-transfer from the electrondonating portion to the benzothiadiazole acceptor unit of the molecules. In all cases, only small remnants of electron density in the phenylene bridge are present. According with the theoretical results, the molecular →* orbitals correspond to the HOMO and LUMO respectively. All the derivatives exhibited a maximum absorption band corresponding to HL. Relevant data for computing energy levels of the frontier orbitals in conjunction with the energy gap are summarized in tables S3-S7. The gaps of frontier orbital energies, HOMO-LUMO (EgOpt), of benzothiadiazole derivatives were extracted from the absorption spectra in solution, ranging from 2.86-2.94 eV (FL-BTD), 2.87-2.93 eV (BuO-BTD), 3.08-3.20 eV (DBF-BTD) and 3.13-3.24 eV (MeO-BTD). The calculated gaps (EgCalcd) were consistent with the experimental results; ranging from 2.95-2.98 eV (FL-BTD), 2.98-3.04 eV (BuO-BTD), 3.08-3.29 eV (DBF-BTD) and 3.23-3.313 eV (MeO-BTD). Analyzing the solvation effect on the energy gap, the lower values correspond to the experiments carried out in toluene, a non-polar aromatic solvent, while the larger values are correlated with the experiments carried out in polar solvents. We can attribute this behavior to strong solute and non-polar aromatic solvent intermolecular interactions, resulting in the stabilization of the molecule and a reduction of the HOMO-LUMO energy gap. It should be noted that the BTD derivatives with oxygen and alkyl chains also have a low energy band gap when they are dissolved in aprotic polar solvents, probably due stabilizing dipole-dipole interactions.

Figure 5 .

 5 Figure 5. Natural transition orbitals for the most relevant excitation of all featured BTDbased building blocks. Calculated at the B3LYP/6-311+G(d,p) level.

Figure 6 .

 6 Figure 6. Cyclic voltammograms of BTD-based building blocks.

Figure 7 .

 7 Figure 7. Experimental and calculated IP and EP values of BTD-based building blocks.

13 C

 13 -NMR [100 MHz, CDCl 3 ] (, ppm): 159.35 (C-4'), 154.21 (C-4a, C-7a), 132.33 (C-1'), 130.44 (C-2', C-6'), 129.77 (C-5, C-6), 127.25 (C-4, C-7), 114.64 (C-3', C-5'), 67.80 (C-8'), 31.34 (C-9'), 19.29 (C-10'), 13.89 (C-11'). HRMS (ESI-TOF): calculated for C 26 H 29 N 2 O 2 S, 433.19497. Found for 433.19499 [M + + H + ] (error: 0.03 ppm).

13 C

 13 -NMR [100 MHz, CDCl 3 ] (, ppm): 160.84 (C-3', C-5'), 153.97 (C-4a, C-7a), 139.23 (C-1'), 133.40 (C-4, C-7), 127.99 (C-5, C-6), 107.60 (C-2', C-6'), 100.40 (C-4'), 55.49 (C-8', C-10'). HRMS (ESI-TOF): calculated for C 22 H 21 N 2 O 4 S, 409.12220. Found for 409.12270 [M + + H + ] (error: 1.22 ppm).

Table 1 .

 1 Relevant

	ARTICLE
				BuO-BTD				FL-BTD			MeO-BTD			DBF-BTD
	Solvent
			Toluene	CHCl3	DMSO	Solid-state	Toluene	CHCl3	DMSO	Solid-state	Toluene	CHCl3	DMSO	Solid-state	Toluene	CHCl3	DMSO	Solid-state
	 abs (nm)	413	412	415	478	416	413	415	469	388	384	385	455	387	382	375	448
	 em (nm)	527	534	551	546	518	532	545	540	495	498	508	516	486	491	498	532
	Stokes
	shift	5237 5545 5948 2605 4733 5125 5748 2803 5571 5961 6289 2564 5264 5811 6586 3224
	 (cm	-1 )
	 f		0.57	0.43 0.36	--	0.60	0.55 0.27	--	0.77	0.71 0.05	--	0.91	0.78 0.60	--

absorption and fluorescence emission data of BTD-based building blocks in solid state and in different solvents.

Table 2 .

 2 The HOMO/LUMO energy values and electrochemical energy gap.

	1) IP = HOMO = -(E ox(onset) + 4.8) eV
	(2) EA = LUMO = -(E red(onset) + 4.8) eV
		Eox (onset)	Ered (onset)	HOMO a	LUMO a	EgEc b
		(V)	(V)	(eV)	(eV)	(eV)
	FL-BTD	1.21	-1.25	-6.01	-3.55	2.46
	BuO-BTD	1.20	-1.31	-6.00	-3.49	2.51
	DBF-BTD	1.52	-1.27	-6.32	-3.53	2.79
	MeO-BTD	1.25	-1.33	-6.05	-3.47	2.58

a Calculated from Eox (onset) and Ered (onset).

b Calculated by the difference between the HOMO and LUMO.

Table 3 .

 3 Calculated, a optical (DCM solution), electrochemical, and b optical (solid) band gap energies (Eg).

  Yellow crystalline solid (65 % yield). Melting point (°C): 306.1 -307.0. FTIR-ATR (, cm -1 ): 3039, 1587, 1577, 1452, 1262, 1187, 897, 835, 738, 515. 1 H-NMR [400 MHz, CDCl 3 ] (, ppm): 8.36 (s, 1H, H-5, H-6), 8.16 (d, J = 8.8 Hz, 1H, H-1'), 8.07 (dd, J = 11.0, 6.7 Hz, 2H, H-3', H-9'), 7.58 (t, J = 7.7 Hz, 2H, H-2', H-6'), 7.49 (t, J = 7.7 Hz, 1H, H-7'), 7.41 (t, J = 7.6 Hz, 1H, H-8'). 13 C-NMR [100 MHz, CDCl 3 ] (, ppm): 188.36 (C-4a, C-7a), 183.72 (C-4a'), 178.24 (C-4b'), 141.43 (C-4, C-7), 139.97 (C-5, C-6), 135.55 (C-4'), 130.20 (C-7'), 130.20 (C-3'), 128.67 (C-9a'), 127.35 (C-2'), 126.46 (C-9b'), 122.95 (C-9'), 120.79 (C-8'), 111.84 (C-1'), 106.40 (C-6'). HRMS (ESI-TOF): calculated for C 30 H 17 N 2 O 2 S, 469.10107. Found for 469.10086 [M

	Hz, 1H, H-5'), 7.92 (s, 1H, H-1'), 7.92-7.85 (d, J = 7.5 Hz, 1H, H-
	3'), 7.62-7.60 (d, J =7.0 Hz, 1H, H-8'), 7.47-7.39 (t, 1H, H-6'),
	7.39-7.31 (t, 1H, H-7'), 4.10 (s, 2H, H-9').	13 C-NMR [100 MHz,
	CDCl 3 ] (, ppm): 143.69 (C-4a, C-7a), 143.62 (C-2'), 142.30 (C-
	8b'), 141.14 (C-8a'), 139.97 (C-8'), 135.98 (C-4b'), 135.30 (C-
	4a'), 133.54 (C-1'), 128.10 (C-4'), 126.99 (C-4, C-7), 126.87 (C-5,
	C-6), 125.90 (C-7'), 125.10 (C-6'), 120.16 (C-3'), 120.00 (C-5'),
	37.10 (C-9'). HRMS (ESI-TOF): calculated for C 32 H 21 N 2 S, 465.14254. Found for 465.14210 [M + + H + ] (error: 0.94 ppm).
	4,7-bis(dibenzo[b,d]furan-4-yl)benzo[c][1,2,3]thiadiazole	(DBF-
	BTD)

). Melting point (°C): 290.6-291.6. FTIR-ATR (, cm -1 ): 3038, 2009, 1955, 1606, 1546, 1452, 1395, 824, 738, 517. 1 H-NMR [400 MHz, CDCl 3 ] (, ppm): 8.20 (s, 1H, H-5, H-6), 8.02 (d, J = 7.9 Hz, 1H, H-4'), 7.97 (d, J = 8.1 + + H + ] (error: 0.46 ppm).

J. Name., 2013, 00, 1-3 | 3

J. Name., 2013, 00, 1-3 | 7

J. Name., 2013, 00, 1-3 | 9

Acknowledgements

MDP Carreón-Castro acknowledges to DGAPA-UNAM (PAPIIT IN-207421) and to SEP-CONACYT-ANUIES-ECOS Francia (296682) Ingeniería de películas delgadas basadas en compuestos de transición de espín para aplicaciones en micro(opto)electromecánicos. RMMO would like to thank to CONACYT for the granted scholarship (Grant number 335895). The work has been performed within the framework of the French-Mexican International Laboratory (LIA-LCMMC). This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic through the e-INFRA CZ (ID:90140).

Notes and references

Author Contributions

Conflicts of interest

There are no conflicts to declare.


Amour Abdallah

Merzouk Belkacem

Removal of reactive textile dye from aqueous solutions by electrocoagulation in a continuous cell

Keywords: Electrocoagulation, Color, Turbidity, Red nylosan dye, Textile wastewater

In electrocoagulation (EC), coagulants are delivered in situ using the corrosion of sacrificial anodes when a DC voltage is applied. Simultaneously, electrolytic gases (H 2 ) are generated at the cathode. Aluminum and iron materials are usually used as anodes, the dissolution of which produces hydroxides and polymeric hydroxides. These coagulants are able to destabilize colloidal suspensions and emulsions, to adsorb, neutralize or precipitate dissolved polluting species, and finally to form flocs that can be removed either by settling or flotation. The purpose of this study was to investigate the effects of the operating parameters, such as initial pH, initial concentration (C 0 ), residence time (τ), current density (j) inlet flow rate (Q) on the removal of a red nylosan dye by electrocoagulation (EC) process using aluminum electrode in a continuous electrochemical reactor. FTIR analysis was conducted to interpret the functional groups involved during the electrocoagulation process.

It was found that the increasing of the current density up to 100 A m -2 had increased the dye removal efficiency and the optimum inlet flow rate for EC process was the minimum at 15 L h -1 and it has increased the electrical energy consumption.

A rate of abatement of about 90 % for turbidity and 97 % for the color was observed, when the initial concentration of the dye C 0 was lower than 300 mg L -1 , current density j = 300 A m -², conductivity κ = 2.54 mS cm -1 , inlet flow rate Q = 15 L h -1 , residence time τ = 35 min and initial pH ranged from 2.3 to 8.8. The specific electrical energy consumption was 19.5 kWh per kilogramme of removed dye.

Introduction

The textile dyeing and finishing industries generate large quantity of wastewater containing unreacted dyes, suspended solids, dissolved solids, and other auxiliary chemicals. Dye-containing effluent is toxic to the environment since dyes are stable compounds, with low biodegradability and can be carcinogenic [START_REF] Gregory | Azo dyes: Structure-carcinogenicity relationships[END_REF].

The colored wastewater released into the ecosystem is a dramatic source of esthetic pollution and perturbation in the aquatic life. Conventional methods for dealing with textile wastewater consist of various combinations of biological, physical and chemical methods [START_REF] Daneshvar | Immobilization of TiO 2 nanopowder on glass beads for the photocatalytic decolorization of an azo dye C.I. Direct Red 23[END_REF].

In recent years, investigations have been focused on the treatment of wastewaters using electrocoagulation (EC) owing to the increase in environmental restrictions on effluent wastewater. EC is a simple and efficient method for the treatment of many water and wastewaters. It has not been widely accepted because of high initial capital costs as compared to other treatment technologies. EC technique uses a direct current source between metal electrodes immersed in polluted water [START_REF] Merzouk | Using electrocoagulation-electroflotation technology to treat synthetic solution and textile wastewater, two case studies[END_REF][START_REF] El-Ashtoukhy | Removal of acid green dye 50 from wastewater by anodic oxidation and electrocoagulation -A comparative study[END_REF]. The electrical current causes the dissolution of metal plates including iron or aluminum into wastewater. The metal ions, at an appropriate pH, can form wide ranges of coagulated species and metal hydroxides that destabilize and aggregate the suspended particles or precipitate and adsorb dissolved contaminants [START_REF] Chen | Electrochemical technologies in wastewater treatment[END_REF].

The most widely used electrode materials in EC process are aluminum and iron. In the case of aluminum, main reactions are as: Anode : Al (s) → Al 3+ + 3e - [START_REF] Gregory | Azo dyes: Structure-carcinogenicity relationships[END_REF] Cathode : 3H 2 O + 3e -→ 2 3 H 2(g) + 3OH -(

Al 3+ and OH -ions generated by electrode reactions (1) and (2) react to form various monomeric species, which finally transform into Al(OH) 3(s) according to complex precipitation kinetics.

Al 3+ + 3H 2 O → Al(OH) 3 + 3H + (3)
Freshly formed amorphous Al(OH) 3(s) "sweep flocs" have large surface areas, which are beneficial for a rapid adsorption of soluble organic compounds and trapping of colloidal particles. Finally, these flocs are removed easily from aqueous medium by sedimentation or by electroflotation [START_REF] Kobya | Operating cost analysis of electrocoagulation of textile dye wastewater[END_REF][START_REF] Merzouk | Removal turbidity and separation of heavy metals using electrocoagulation-electroflotation technique. A case study[END_REF][START_REF] Mollah | Treatment of orange II azo-dye by electrocoagulation (EC) technique in a continuous flow cell using sacrificial iron electrodes[END_REF].

The electrocoagulation has been considered for a number of wastewaters in a very broad range of nature and composition: in particular oil suspensions [START_REF] Koren | State of the-art: electroflocculation[END_REF][START_REF] Sanchez-Calvo | An electrocoagulation unit for the purification of soluble oil wastes of high COD[END_REF][START_REF] Khemis | Electrocoagulation for the treatment of oil suspensions: relation between the electrode generations and the waste content[END_REF][START_REF] Kumar | Removal of arsenic from water by electrocoagulation[END_REF], wastes from textile industry [START_REF] Zongo | Electrocoagulation for the treatment of textile wastewaters with Al and Fe electrodes: compared variations of COD levels, turbidity and absorbance[END_REF][START_REF] Zodi | Treatment of the industrial wastewaters by electrocoagulation: Optimization of coupled electrochemical and sedimentation processes[END_REF][START_REF] Merzouk | Treatment characteristics of textile wastewater and removal of heavy metals using the electroflotation technique[END_REF], tannery [START_REF] Golder | Removal of Cr 3+ by electrocoagulation with multiple electrodes: bipolar and monopolar configurations[END_REF][START_REF] Heidman | Removal of Cr (VI) from model wastewaters by electrocoagulation with Fe electrode[END_REF] or food processing [START_REF] Chen | Separation of pollutants from restaurant wastewater by electrocagulation[END_REF][START_REF] Inan | Olive oil mill wastewater treatment by means of electrocoagulation[END_REF] -a list far from exhaustive.

Examination of the available literature reveals that the treatment mechanism by electrocoagulation depends on the waste nature, as observed in [START_REF] Moreno-Casillas | Electrocoagulation mechanism of COD removal[END_REF][START_REF] Khemis | Treatment of industrial liquid wastes by electrocoagulation: experimental investigations and an overall interpretation model[END_REF] and to some extents by the design of electrocoagulation device and the flow conditions. It also appears that the treatment efficiency has to be considered in terms of various criteria: TOC, COD, turbidity, color or absorbance at a given wavelength, and concentration of toxic species, e.g. arsenic [START_REF] Kumar | Removal of arsenic from water by electrocoagulation[END_REF][START_REF] Gomes | Arsenic removal by electrocoagulation using combined Al-Fe electrode systemand characterization of products[END_REF] or phosphate [START_REF] Irdemez | The effects of current density and phosphate concentration on phosphate removal from wastewater by electrocoagulation using aluminum and iron plate electrodes[END_REF]. The continuous mode of the EC process has however been less investigated, except in a few studies [START_REF] Mollah | Treatment of orange II azo-dye by electrocoagulation (EC) technique in a continuous flow cell using sacrificial iron electrodes[END_REF][START_REF] Chen | Separation of pollutants from restaurant wastewater by electrocagulation[END_REF][START_REF] Mameri | Defluoridation of septentrional of Sahara water of North Africa by electrocoagulation process using bipolar aluminum electrodes[END_REF][START_REF] Daneshvar | Decolorization of orange II by electrocoagulation method[END_REF][START_REF] Chen | Investigation on the electrolysis voltage of electrocoagulation[END_REF][START_REF] Ben Mansour | Treatment of wastewaters of paper industry by coagulationelectroflotation[END_REF], especially the conditions of floc removal. The results obtained show a good efficiency but with low operational parameters (inlet flow rate Q  20 L h -1 ).

In this regard, the objective of the paper is thus to study the operation of this new design of electrocoagulation reactor by examining the effects of the operating parameters, such us the current density (j), residence time (), initial concentration (C 0 ), initial pH and inlet flow rate (Q) on color removal efficiency of a synthetic wastewater containing red nylosan dye.

Materials and methods

Synthetic wastewater

Red nylosan dye N-2RBL (sodium 6-amino-5-

[[4-chloro-3-[[(2,4-dimethylphenyl) amino] sulphonyl]
phenyl]azo]-4-hydroxynaphthalene-2-sulphonate) was used for preparing synthetic dye wastewater. Its chemical structure is shown in Fig. 1. Principal characteristics of this compound are given in Table 1.

The synthetic wastewater was prepared by dissolving the dye into deionized water and was diluted according to the desired concentration (50 -300 mg L -1 ). The pH was adjusted to 7.5 ± 0.3 with NaOH and the conductivity was adjusted to 2500 ± 25 μS cm -1 with NaCl. The values of pH and conductivity were chosen to simulate the properties of wastewater from a textile industry. The initial concentration of 50 mg L -1 was selected because it provided a noticeable color and a Chemical Oxygen Demand (COD) value in the same order of magnitude as the effluent from the textile factory [START_REF] Merzouk | Removal turbidity and separation of heavy metals using electrocoagulation-electroflotation technique. A case study[END_REF][START_REF] Zongo | Electrocoagulation for the treatment of textile wastewaters with Al and Fe electrodes: compared variations of COD levels, turbidity and absorbance[END_REF][START_REF] Emamjomeh | Review of pollutants removed by electrocoagulation and electrocoagulation/flotation processes[END_REF]. The typical characteristics of the synthetic textile wastewater to be treated were presented in Table 2.

COD (mg O 2 L -1 ) 123
Conductivity (µS cm -1 ) 2500

Temperature (°C) 20

Suspended solids SS (mg L -1 ) 105

Color Dark red

Experimental set-up

A single reactor for both metal dissolution and solid settling has been designed and constructed as the The gaps between the two neighboring electrode plates were kept constant at 10 mm for all the experiments. The electrodes were connected in bipolar mode to a laboratory DC power supply (GPS3303 3CH ) providing current in the range of 00-10 A, that was held constant for a given experimental run.

The reactor is configured with an electrochemical chamber of nearly 3.1 L volume and a large sedimentation and flotation tank of 5.5 L volume.

The two compartments were connected by a triangular groove and the wastewater passed from the first chamber to the second one by overflow. The volume V 2 = 5.5 L of the tank and the position of outlet tube were designed to avoid the presence of solid particles in the effluent stream. The sludge was essentially recovered by flotation. The floating materials were removed continuously from the second compartment by overflow, whereas the few more dense materials could settle. The solution to be treated was continuously circulated in the flow circuit by means of a peristaltic pump ''Heidolph PD 5006, Pump drive'' providing flow in the range of 0-60 L h -1 . The influence of the various parameters on the dye removing process was achieved using synthetic wastewater in a continuous EC reactor.

Analytical methods

Samples (40 mL) were collected in the reservoir every 05 min until the steady state regime was achieved.

In practice, the reactor reaches the steady state conditions approximately at mean residence time. This is because; the reaction at the electrodes is very fast and, reactor behaves at plug flow reactor in the electrochemical cell.

Dye concentration was estimated from its absorbance characteristics in the UV-vis range (250 -800 nm), using the wavelength that provided the maximum intensity ( max = 502 nm) and a UV-vis spectrophotometer (Shimadzu-1240 S). Solution conductivity and pH were measured using a Hanna EC214 conductimeter and a Hanna pH210 pH-meter respectively. The colorimetric micro-method (HACH (Loveland, Colorado) method 8000) with an excess of hexavalent chromium and subsequent measurement of the optical density using a HACH 2400 spectrophotometer (Loveland, Colorado) was used for the soluble COD determination. For COD measurements, the calibration curves were obtained using potassium hydrogeno-phtalate (Fluka, analytical grade). The UV-visible spectra of the filtered samples

were measured with an Anthelie Light (Secomam, Domont, France) spectrophotometer; light absorbance was measured between 200 and 600 nm using 1 cm-path quartz cells. Turbidity of the waters was measured using a Hanna HI88703 spectrophotometer. Data were given in Nephelometric Turbidity Unit (NTU).

Color in terms of absorbance and turbidity removal efficiencies (Y Col , Y Tr ,) were expressed as a percentage and defined as

100 . C C C (%) Y 0 f 0   (4)
The subscripts, "0" and "f", are used to distinguish concentration in the inlet and the outlet streams of the electrocoagulation cell.

The specific electrical energy consumption per kg dye removed (SEEC) and the specific electrode consumption per kg dye (μ Al ) were calculated from the liquid absorbance as follows

This parameter depends upon the pH and the amount of other species present in solution, for example coexisting anions [START_REF] Hu | Effects of co-existing anions on fluoride removal in electrocoagulation (EC) process using aluminum electrodes[END_REF].

Results and discussion

Effect of current density on the color and turbidity removal

Since the decolorization is the main objective of the treatment of textile wastewaters, absorbance variation is the first parameter to follow up. In all electrochemical processes, current density is the most important parameter for controlling the reaction rate within the electrochemical reactor [START_REF] Zongo | Removal of hexavalent chromium from industrial wastewater by electrocoagulation : A comprehensive comparaison of aluminium and iron electrodes[END_REF]. It is well known that current density determines the production rate of coagulant, adjusts also bubble production, and hence affects the growth of flocs [START_REF] Merzouk | Using electrocoagulation-electroflotation technology to treat synthetic solution and textile wastewater, two case studies[END_REF][START_REF] Chen | Electrochemical technologies in wastewater treatment[END_REF]. To investigate the effect of current density j on the efficiency of color and turbidity removal, continuous electrocoagulation process was carried out using various current densities at fixed inlet concentration C 0 = 50 mg L -1 with influent pH 7. The absorbance decreases down from 0.38 to 0.01 and turbidity from 85 to 1.43 NTU. As clearly seen from Fig. 4 (a), the calculated specific energy consumption per kg dye removed (SEEC) increased with increasing applied current densities as expected.

Energy consumption varies with the square of the current density and generates heat by Joule effect; the SEEC values increased accordingly from 3.2 to 31.3 kWh kg -1 dye removed for 45 min operation time.

An increase in current density from 100 to 400 A m -2 yielded an increase in the efficiency of color removal from 85.5 to 99.3 %. This could be expected: when the current density increases, the amount of Al 3 + cations released by the anode and therefore of Al(OH) 3 particles also increases. However, the difference between the curves corresponding to different current densities became small when j was higher than 200 A m -2 . An optimum current density can therefore be defined. Indeed, when the increase of Y Col with j is slight, the disadvantages of working at high current densities prevail: first, the amount of sludge increases typically as j; then, the same stands for electrode material consumption, while energy consumption rises as j 2 . These trends were confirmed experimentally in Figs. 4 (a) and 4 (b) for SEEC and µ Al , respectively, and they agree qualitatively with data already reported in the literature for textile dye wastewaters [START_REF] Merzouk | Using electrocoagulation-electroflotation technology to treat synthetic solution and textile wastewater, two case studies[END_REF][START_REF] Merzouk | Removal turbidity and separation of heavy metals using electrocoagulation-electroflotation technique. A case study[END_REF][START_REF] Bayramoglu | Treatment of the textile wastewater by electrocoagulation: economical evaluation[END_REF][START_REF] Can | Treatment of the textile wastewater by combined electrocoagulation[END_REF][START_REF] Kobya | Treatment of levafix orange textile dye solution by electrocoagulation[END_REF][START_REF] Hu | Treatment of high fluoride-content wastewater by continuous electrocoagulation-flotation system with bipolar aluminum electrodes[END_REF][START_REF] Daneshvar | Decolorization of basic dye solutions by electrocoagulation: an investigation of the effect of operational parameters[END_REF][START_REF] Merzouk | Studies on the decolorization of textile dye wastewater by continuous electrocoagulation process[END_REF]. The specific electrical energy consumption (SEEC) was 19.5 kWh per kilogramme of removed dye for j = 300 A m -2 which corresponds to 96.6 % of removal turbidity.

Effect of inlet flow rate on the absorbance and color removal

In order to investigate the effect of the residence time on the absorbance and color removal efficiency, the inlet flow rate Q was progressively increased from 15 to 60 L h -1 , respectively, for j = 300 A m -2 and influent pH 7.58.

As shown in Figs. 5 (a) and 5 (b), the continuous EC process functions for all studied inlet flow rates and the color removal ratio reach 90 %.

These figures show that Y Col presents a maximum at a flow of approximately 15 L h -1 , with a value of 97.2 % after 55 min of treatment and 91.4 % with 35 min of treatment which is the residence time.

On the other hand for Q = 38.7 L h -1 , Y Col (max) is 93 % with 35 min of treatment against 92.2 % with 55 min of treatment. For Q = 60 L h -1 , Y Col (max) is 90.2 after 55 min of treatment. The optimal rate of feed could thus be taken between 15 and 38.7 L h -1 . Comparing the results obtained in this study with similar ones [START_REF] Mollah | Treatment of orange II azo-dye by electrocoagulation (EC) technique in a continuous flow cell using sacrificial iron electrodes[END_REF][START_REF] Chen | Separation of pollutants from restaurant wastewater by electrocagulation[END_REF][START_REF] Mameri | Defluoridation of septentrional of Sahara water of North Africa by electrocoagulation process using bipolar aluminum electrodes[END_REF][START_REF] Daneshvar | Decolorization of orange II by electrocoagulation method[END_REF][START_REF] Chen | Investigation on the electrolysis voltage of electrocoagulation[END_REF][START_REF] Ben Mansour | Treatment of wastewaters of paper industry by coagulationelectroflotation[END_REF][START_REF] Essadki | Electrocoagulationelectroflotation in an external-loop airlift reactor -Application to the decolorization of textile dye wastewater: A case study[END_REF], the inlet flow rates do not exceed 20 L h -1 to obtain a color removal ratio higher than 80 %.

Effect of initial concentration on the absorbance and color removal

Effect of initial pH on the absorbance and color removal

It has already been established that pH is a key operating factor influencing the performance of electrocoagulation process [START_REF] Merzouk | Removal turbidity and separation of heavy metals using electrocoagulation-electroflotation technique. A case study[END_REF][START_REF] Zongo | Electrocoagulation for the treatment of textile wastewaters with Al and Fe electrodes: compared variations of COD levels, turbidity and absorbance[END_REF][START_REF] Zodi | Treatment of the industrial wastewaters by electrocoagulation: Optimization of coupled electrochemical and sedimentation processes[END_REF][START_REF] Zongo | Removal of hexavalent chromium from industrial wastewater by electrocoagulation : A comprehensive comparaison of aluminium and iron electrodes[END_REF][START_REF] Merzouk | Studies on the decolorization of textile dye wastewater by continuous electrocoagulation process[END_REF][START_REF] Essadki | Electrocoagulationelectroflotation in an external-loop airlift reactor -Application to the decolorization of textile dye wastewater: A case study[END_REF][START_REF] Attour | Influence of operating parameters on phosphate removal from water by electrocoagulation using aluminum electrodes[END_REF][START_REF] Zodi | Direct Red 81 dye removal by a continuous flow electrocoagulation/flotation reactor[END_REF]. To examine its effect, the pH of the wastewater was adjusted to the desired value for each experiment by adding either sodium hydroxide or sulfuric acid. It is desirable that final pH values are near neutrality after EC process. Fig. 8 demonstrates pH fluctuation during EC. This was observed by several authors [START_REF] Merzouk | Removal turbidity and separation of heavy metals using electrocoagulation-electroflotation technique. A case study[END_REF][START_REF] Chen | Separation of pollutants from restaurant wastewater by electrocagulation[END_REF][START_REF] Kobya | Treatment of levafix orange textile dye solution by electrocoagulation[END_REF][START_REF] Merzouk | Studies on the decolorization of textile dye wastewater by continuous electrocoagulation process[END_REF][START_REF] Essadki | Electrocoagulationelectroflotation in an external-loop airlift reactor -Application to the decolorization of textile dye wastewater: A case study[END_REF]. Its evolution depends on initial pH: whatever the initial pH values, acidic or basic, there is an EC buffering trend toward pH around 7. This is explained by a balance between OH -production and consumption, preventing high changes in pH [START_REF] Chen | Electrochemical technologies in wastewater treatment[END_REF][START_REF] Merzouk | Studies on the decolorization of textile dye wastewater by continuous electrocoagulation process[END_REF][START_REF] Essadki | Electrocoagulationelectroflotation in an external-loop airlift reactor -Application to the decolorization of textile dye wastewater: A case study[END_REF], which is advantageous for wastewater treatments.

FTIR study

FTIR (Fourier Transformed Infra Red spectroscopy) analysis helps in understanding the reaction mechanism as it identifies the functional groups involved during the electrocoagulation process. The spectra were recorded in region of 4000 -400 cm -1 with a resolution of 2 cm -1 in an IR spectrophotometer (Perkin Elmer, RZX) (Fig. 9). The FTIR spectrum of the dye before treatment by EC (RN 1) shows peak at 3402.2 cm -1 due to presence of O-H and N-H stretching vibrations, while peak at 2923.9 cm -1 is due to C-H group of alkyl chain. The peaks at 1624 cm -1 is due to presence of carbonyl groups. The peak at 1184.2 cm -1 shows the presence of C-N (s) vibration. The bands at 952.8 cm -1 and 837 cm -1 are characteristic of C-Cl (s) vibrations. The peaks at 767.6 cm -1 and 655.8 cm -1 are due to presence of N-H vibrations due to secondary amines. Comparing the FTIR spectra of the dye before and after EC process (RN 1 and RN 2), there was a marked shift in the band from 2349.7 cm -1 to 3448.5 cm -1 . Our results suggest an opportunity for the application of continuous EC process to remove color and turbidity from wastewater. The increasing of the current density increases the kinetics rate due to the faster dissolution of aluminum. Consequently the reaction time is faster for a given removal efficiency which allows to increase the productivity of the continuous reactor by working at higher flow rate. The results showed that about 90 % for turbidity and 97 % for the color was observed, when the initial concentration of the dye C 0 was lower than 300 mg L -1 , current density j = 300 A m -², conductivity κ = 2.54 mS cm -1 , inlet flow rate Q = 15 L h -

Fig. 1 .

 1 Fig. 1. The chemical structure of Red nylosan dye

  continuous treatment EC reactor of industrial wastewaters: the long cell consisted in two chambers in series of most different volumes and flow conditions. The continuous flow pilot-scale electrocoagulation reactor is shown in Fig.2. The waste liquid is pumped into the electrochemical chamber provided with two aluminum (purity of Al was 98 %) plate anodes and cathodes (dimension 240*20*10mm) used as vertical Al electrodes. The electrodes were 100 % dipped into an aqueous solution in a Perspex reactor.

Fig. 2 .

 2 Fig.2. Experimental setup of continuous EC cell (1: wastewater tank; 2: peristaltic pump; 3: inlet of the first compartment; 4: electrodes; 5: first chamber; 6: sludge; 7: second chamber; 8: sludge exit (overflow); 9: treated effluent outlet; 10: treated water tank; 11: outfall).

  74 and an inlet flow rate Q = 25.2 L h -1 . The data monitored for 60 min operation is presented in Figs. 3 (a) and 3 (b).

Fig. 3 .

 3 Fig. 3. Evolution of absorbance (a) and turbidity (b) during the treatment for different current densities j: C 0 = 50 mg L -1 , initial pH 7.74, Q = 15 L h -1 , e = 1cm,  = 2.5 mS cm -1

Fig. 4 .

 4 Fig. 4. Effect of current density j on energy consumption (SEEC) (a) and electrode consumption ( Al ) (b): C 0 = 50 mg L -1 , initial pH 7.74, Q = 15 L h -1 , e = 1cm,  = 2.5 mS cm -1

Fig. 5 .

 5 Fig. 5. Evolution of absorbance (a) and turbidity (b) during the treatment for different inlet flow rates Q: C 0 = 50 mg L -1 , initial pH 7.58, j = 300 A m -2 , e = 1cm,  = 2.54 mS cm -1

Figures 6 (Fig. 6 .

 66 Figures 6 (a) and 6 (b) illustrate the evolution of removal efficiency of color and turbidity removal as a function of the influent dye concentration, using the optimum conditions obtained previously for current, residence time and pH. In this figure, dye solutions with different concentrations in the range 50-300 mg L -1 were treated by continuous EC process. The data shows that the continuous EC process gave satisfactory results for all the concentrations studied, as Y Col was always higher than 97.2 % and Y Tr higher than 97.4 %.

Fig. 7 (Fig. 7 .

 77 Fig. 7 (a) and (b) shows that Y Col and Y Tr exceed 93 % for all the pH studied. The maximum efficiency of color removal was observed at pH 7.74 with Y Col = 97.2 % and Y Tr = 98.3 %.

Fig. 8 .

 8 Fig. 8. Evolution of pH values during EC for different values of initial pH: C 0 = 50 mg L -1 , j = 300 A m -², Q = 15 L h -1 , e = 1cm,  = 2.5 mS cm -1

Fig. 9 .

 9 Fig. 9. FTIR spectrum of Acid Red 336 before and after the EC process

Table 1 . Main characteristics of Red nylosan dye

 1

	Color index number	Acid Red 336
	CAS registry number	71873-39-7
	Molecular formula	C 24 H 21 ClN 4 O 6 S 2 , Na
	Molecular weight	587.97 g mol -1
	UV absorption	λ max 502 nm
	Table2.

Physicochemical characteristics of the synthetic textile wastewater investigated

	Parameter	Synthetic wastewater
	pH 0	7.74
	Turbidity (NTU)	85

  1 , residence time τ = 35 min and initial pH ranged from 2.3 to 8.8. The specific electrical energy consumption (SEEC) was 19.5 kWh per kilogramme of removed dye. The EC continuous mode was determined to be an efficient and viable process for achieving a high degree of dye removal from textile wastewater.

			2349.1
	Nomenclature	2935.4
	C 0	dye concentration (kg m -3 )		952.8
	C Al	concentration of dissolved Aluminium (kg m -3 )
	COD EC	2923.9 Chemical Oxygen Demand (mgO 2 L -1 ) 2353.0 3402.2 electrocoagulation		767.6
	F FTIR	Faraday's constant (96487 C mol -1 ) Fourier Transformed Infra Red spectroscopy	1624.0	837.0	655.8
	I	3448.5 current (A)
	j	current density (A m -2 )
	M Al	molar mass of aluminum (0.0267 kg mol -1 )
	Q	inlet flow rate (L h -1 )		1184.2
	RN	red nylosan
	S	electrode surface area (m 2 )
	SEEC	specific electrical energy consumption (kW h kg -1 dye removed)
	SS	suspended solids (mg L -1 )
	t	time (h)
	U	measured potential (V)
	V	reactor volume (m 3 )
	Y Col	color removal efficiency (%)
	Y Tr	turbidity removal efficiency (%)
	Greek
	m exp	experimental weight loss of Al electrode (kg)
	m th	theoretical weight loss of Al electrode (kg)
	κ	wastewater conductivity (μS cm -1 )
	λ max	maximum wavelength of the absorption spectrum (nm)
	μ Al	specific consumption of the anode (kg Al kg -1 dye removed)
	τ	liquid residence time in the reactor (s)
	 Al	Faradic yield of Al dissolution.


Mounira Mkaddem

Jalloul Bouajila

Monia Ennajar

Ahmed Lebrihi

AND Florence Mathieu

Mehrez Romdhane

Chemical Composition and Antimicrobial and Antioxidant Activities of Mentha (longifolia L. and viridis) Essential Oils

Keywords: antioxidant activity, antimicrobial activity, essential oil, Mentha longifolia L., Mentha viridis L

The study was aimed to investigate essential oil chemical composition (gas chromatography/flame ionization detection [GC-FID] and gas chromatography/mass spectrometry [GC-MS]) and antioxidant (1,1-diphenyl-2picrylhydrazyl free radical (DPPH) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonate [ABTS] assays) and antimicrobial (Gram-positive and Gram-negative bacteria, fungi, and yeast) activities of essential oils extracted from leaves of Mentha longifolia L. and Mentha viridis. GC-MS analysis revealed that M. longifolia was constituted by pulegone (54.41%) as a major component followed by isomenthone (12.02%), 1,8-cineole (7.41%), borneol (6.85%), and piperitenone oxide (3.19%). M. viridis was rich in carvone (50.47%), 1,8-cineole (9.14%), and limonene (4.87%). The antioxidant activity by ABTS assay showed IC 50 values of 476.3 ± 11.7 and 195.1 ± 4.2 mg/L for M. longifolia and M. viridis, respectively, the DPPH assays have resulted in a moderate IC 50 (>8000 mg/L and 3476.3 ± 133 mg/L for M. longifolia and M. viridis, respectively). Antimicrobial activity showed that Listeria monocytogenes and Klebsiella pneumoniae bacteria were more inhibited by the 2 essential oils tested. Escherichia coli was least susceptible. A strong activity was also observed on fungi and yeasts. Carvone, thymol, and piperitone oxide have not been detected in Tunisian M. longifolia. Camphor is reported for the 1st time for M. viridis. Antioxidant and antibacterial activities were correlated to chemical composition.

Introduction

T he genus Mentha L. (Lamiaceae), is widely distributed in all continents (except in South America and Antarctica) [START_REF] Chambers | Mentha: genetic resources and the collection at USDA-ARSNCGR-Corvallis[END_REF]. The centers of variety of this genus are Europe, Australia, Central Asia, and North Africa [START_REF] Harley | Chromosome number in the genus Mentha L[END_REF][START_REF] Chambers | Mentha: genetic resources and the collection at USDA-ARSNCGR-Corvallis[END_REF]). It groups spontaneous and cultivated forms. It is represented by 18 species and about 100 varieties and cultivars divided into 5 sections: Audibertia, Eriodontes, Mentha, Preslia, and Pulegium. The systematic of the genus is not very elucidated because of the strong morphologic variations, levels of ploïdie (2n = 2x = 24 to 2n = 6x = 96), and hybridizations intra-and interspecific (or between spontaneous and cultivated forms).

Several studies have reported the research of phylogenic relations between different species on the basis of morphologic markers [START_REF] Malinvaud | Simple aperc ¸u des hybrides dans le genre Mentha[END_REF][START_REF]Aromatic biodiversity among the flowering plant taxa of Turkey[END_REF], chemical [START_REF] Lawrence | A study of the monoterpene interrelationships in the genus Mentha wich special reference to the origin of pulegone and menthofuran[END_REF]Chalchat and others 1997;Raeis Vasco and others 1999), caryologic [START_REF] Harley | Chromosome number in the genus Mentha L[END_REF][START_REF] Singh | Mentha-taxonomic status as interpreted through cytology, genetics and phytochemistry[END_REF], or molecular. Others researchers have aimed also the identification of specific composites of essential oils for its economic value and its use in pharmaceutical, cosmetic, food, confectionary, and beverage industries [START_REF] Hendriks | Pharmaceutical aspects of some Mentha herbs and their essential oils[END_REF]Kanatt and others 2007).

Authors Mkaddem, Ennajar, and 'Agrobiopole,BP 32 607,France. Direct inquiries to author Bouajila (E-mail: bouajila@cict.fr).

In Tunisia, the genus Mentha L. is represented by the species M. rotundifolia L., M. longifolia L. Huds., M. spicata (M. viridis) L., M. aquatica L., and M. pulegium L. [START_REF] Maire | Sur quelques plantes du Sahara occidenta L[END_REF][START_REF] Bonnier | Nouvelle flore pour la détermination facile des plantes de la région parisienne[END_REF][START_REF] Pottier-Alapetite | Flore de la Tunisie, Angiospermes dicotylédones[END_REF]. Many naturalized species (M. piperita, M. spicata, M. longifolia, and so on) are cultivated for family usages or for small commerce.

M. longifolia or wild mint is a fast-growing, perennial herb, growing wildly in the northeastern part of Tunisia and cultivated in all regions. The species possesses antimicrobial and antioxidant properties (Economou and others 1991;[START_REF] Kaur | Anti-oxidant activity and total phenolic content of some Asian vegetables[END_REF]Daferera and others 2003;Gulluce and others 2007). However, there has been no attempt to study antimicrobial and antioxidant activities of essential oils and extracts of M. longifolia from Tunisia.

M. viridis (green mint) is cultivated everywhere in Tunisia (humid to arid and semi-arid climate). It is considered as a simple variety obtained by culture of the wild mint. Green mint essential oil contains monoterpenoids like carvone, limonene, menthone, menthol, pulegone, and dihydrocarveol. Some of them were found to possess high antioxidant activity (Elmasta and others 2006). This composition can vary considerably according to the origin of the plant.

The green mint acquires a very powerful action on the nervous system [START_REF] Bruneton | Pharmacognosie-Phytochimie des Plantes Médicinales[END_REF]Adsersen and others 2006). The boiled leaves extract has an anti-infectious antiflatulence effect and antiinflammatory action (notably of the digestive system), it was counseled in the viral hepatitis and colitis, gastric acidities, aerophagia, to stimulate the digestion; furthermore, it presents some invigorating and stimulating qualities (Saleem and others 2000;Kouhila and others 2001;[START_REF] Kumar | DNA damage protecting activity and antioxidant potential of pudina extract[END_REF]Arumugam and others 2008).

Apart from being a stimulant and carminative, the mint plant is also known for its insecticidal [START_REF] Papachristos | Repellent, toxic and reproduction inhibitory effects of essential oil vapours on Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae)[END_REF], antimicrobial, antispasmodic, and antiplatelet properties [START_REF] Samarth | Mentha piperita (Linn) leaf extract provides protection against radiation induced chromosomal damage in bone marrow of mice[END_REF]Ozgen and others 2006;Tognolini and others 2006).

Actually, essential oils and their components are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers, and their exploitation for potential multi-purpose functional use (Ormancey and others 2001;Yadegarinia and others 2006). Many researchers have reported the antimicrobial, antifungal, and antioxidant properties of essential oils (Baratta and others 1998;Vardar-Unl ü and others 2003;Mimica-Dukic and others 2004;Singh and others 2005;Sacchetti and others 2005). In fact, essential oils from mint species have strong antioxidant activity and good antimicrobial activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Salmonella enteritidis (Yadegarinia and others 2006;Gulluce and others 2007;Mahboubi and Haghi 2008). However, activity against fungi Aspergillus ochraceus and yeast species as Saccharomyces cerevisiae has not been reported previously only for Menta arvensis [START_REF] Basilico | Inhibitory effects of some spice essential oils on Aspergillus ochraceus NRRL 3174 growth and ochratoxin A production[END_REF]. The Mucor ramamnianus has not been tested until now with essential oils of M. longifolia L. and M. viridis L. The search for antioxidant and antimicrobial activities of Tunisian Mentha species essential oils is also not explored.

Therefore, the objectives of this study were to analyze chemical composition of hydrodistilled essential oils of M. longifolia and M. viridis by a GC-MS system to determine the essential oils chemotypes and investigate their antimicrobial and antioxidant activities.

Materials and Methods

Chemicals used

All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-Aldrich and Fluka (Saint-Quentin, France).

Essential oil isolation

Fresh leaves of M. longifolia L. and M. viridis L. cultivated species were collected (May 2007) from the region of Gabes Tunisia.

About 100 g of fresh leaves from each sample of Mentha were placed in 400 mL distilled water and submitted to hydrodistillation for 3 h using a Clevenger-type 5 apparatus (British Pharmacopoeia 1980). The condensation of each essential oil gives 2 phases: an organic phase was formed practically by essential oils (Hpo) to which we added anhydrous sodium sulfate to eliminate water traces and an aqueous phase that contains a negligible quantity of essential oil. Essential oils were stored at 4 • C until tested and analyzed.

Gas chromatography and gas chromatography-mass spectrometry

Quantitative and qualitative analysis of essential oils was carried out by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Gas chromatography analyses were carried out on a Varian Star 3400 (Les Ulis, France) Cx chromatograph fitted with a fused silica capillary DB-5MS (5% phenylmethylpolysyloxane, 30 m × 0.25 mm, film thickness 0.25 µm). Chromatographic conditions were a 60 to 260 • C temperature rise with a gradient of 5 • C/min and 15 min isotherm at 260 • C. A 2nd gradient was applied to 340 • C at 40 • C/min. Total analysis time was 57 min. For analysis, essential oils were dissolved in petroleum ether. One microliter of sample was injected in the split mode ratio of 1 : 10. Helium (purity 99.999%) was used as carrier gas at 1 mL/min. The injector was operated at 200 • C. The mass spectrometer (Varian Saturn GC-MS/MS 4D) was adjusted for an emission current of 10 µA and electron multiplier voltage between 1400 and 1500 V. Trap temperature was 150 • C and that of the transfer line was 170 • C. Mass scanning was from 40 to 650 amu.

Compounds were identified by comparison of their KI (Kovats indices) relative to C5-C24 n-alkanes obtained on a nonpolar DB-5MS column, with those provided in the literature, by comparison of their mass spectra with those recorded in NIST 08 (Natl. Inst. of Standards and Technology) and reported in published articles and by co-injection of available reference compounds. The samples were analyzed in duplicate. The percentage composition of essential oils was computed by the normalization method from the GC peak areas, assuming identical mass response factor for all compounds. Results were calculated as mean values of 2 injections from essential oil, without using correction factors.

Free radical scavenging activity: DPPH test

Antioxidant scavenging activity was studied using 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) as described by [START_REF] Blois | Antioxidant determinations by the use of stable free radical[END_REF] with some modifications. Briefly, 1.5 mL of various dilutions of the test materials (2 mg/mL vitamin C were diluted 1/200, 1/400, 1/800, 1/1000, 1/1200; 16 mg/mL of essential oil M. longifolia were diluted 1/2, 1/3, 1/4, 1/5, 1/10; 16 mg/mL of essential oil M. viridis were diluted 1/2, 1/3, 1/4, 1/5, 1/10) were mixed with 1.5 mL of 0.2 mM methanolic DPPH solution. After an incubation period of 30 min at 25 • C, the absorbance at 520 nm was measured. The wavelength of maximum absorbance of DPPH was recorded as A (sample) , using a spectrophotometer (Helios, Unicam, Cambridge, U.K.). A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as A (blank) . The free radical scavenging activity of each solution was then calculated as percent inhibition according to the following equation:

% inhibition = ([A (blank) -A (sample) ]/A (blank) ) × 100
Antioxidant activity of standard or essential oils was expressed as IC 50 , defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate.

ABTS radical scavenging assay

The radical scavenging capacity of the 2,2-AzinoBis-3-ethylbenzoThiazoline-6-Sulphonate radical cation (ABTS) was determined as described by Re and others (1999). ABTS was generated by mixing a 7 mM of ABTS at pH 7.4 (5 mM NaH 2 PO 4 , 5 mM Na 2 HPO 4 , and 154 mM NaCl) with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using a spectrophotometer. For each sample, diluted methanol solution (2 mg/mL of vitamin C were diluted 1/200, 1/400, 1/800, 1/1000, 1/1200; 6 mg/mL of essential oil M. longifolia were diluted 1/4, 1/5, 1/10, 1/20, 1/50; 2 mg/mL of essential oil M. viridis were diluted 1/4, 1/5, 1/10, 1/20, 1/50) of the essential oil (100 µL) was allowed to react with fresh ABTS solution (900 µL), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard. The capacity of free radical scavenging was expressed by IC 50 (mg/L) values, which represents the concentration required to scavenge 50% of ABTS radicals. The capacity of free radical scavenging IC 50 was determined using the same equation previously used for the DPPH method. All measurements were performed in triplicate.

Microbial strains

All strains were obtained from the Laboratory of Chemical Engineering, Bioprocess Systems Microbiens Dept. of High Natl. School of Agronomy of Toulouse. The different essential oils were

Well-diffusion assay

Agar well-diffusion method was employed for the determination of antimicrobial activity of essential oils (Feyza and others 2009). Briefly, 15 mL of nutrient agar and CYA, respectively, for bacterial and fungal strains were inoculated with 100 µL of microbial suspension containing 10 8 CFU/mL of the indicator strain and then poured onto a petri dish and allowed to solidify. The dish was left to cool down and to solidify at room temperature for 30 min. Wells of 10 mm dia were aseptically bored into the culture medium. A sterile cox borer was used to punch wells and 15 µL of each essential oil were added to the wells. Each plate contained only 1 essential oil. Ampicillin and nalidixic acid (30 µg/well) were used as positive reference standards to determine the sensitivity of Gram-positive and Gram-negative bacterial species, respectively. Nystatin (30 µg/well) was used as positive reference standard to determine the sensitivity of fungi and yeast species. These plates, after staying at 4 • C for 2 h to allow dispersal, were incubated for 24 h at 37 • C for bacteria and 48 h at 30 • C for yeasts and fungi. The antimicrobial activity was visually appraised as inhibition zones surrounding the wells. The diameters of the inhibition zones were measured in millimeters. Tests were carried out in triplicate.

Statistical analysis

All data were expressed as means ± standard deviations (SD) of triplicate measurements. The confidence limits were set at P < 0.05. Standard deviations did not exceed 5% for the majority of the values obtained.

Results and Discussion

T he extraction yield of hydrodistilled M. longifolia essential oil was 1.3% whereas the yield of M. viridis oil was 0.8%. In the literature (Kokkini and others 1995;Elmasta and others 2006), yield values were between 1.2% and 3.2% for the 2 species essential oils. The yield variability in the total essential oil content within the 2 species of mint could have resulted from the variation of climatic factors [START_REF] Lawrence | Labiatae oils-mother nature's chemical factory. Paper Xlth, 11th Int Congress of Essential Oils, Fragrence and Flavour[END_REF]Kokkini and others 1995).

Chemical composition

The components identified in the 2 species of mint are listed in Table 1. Thirty-five components representing 99.72% of the total volatiles were identified for M. longifolia essential oil. The main components were oxygenated monoterpenes (89.18%). Pulegone (54.41% of the total essential oil) was the major compound followed by isomenthone (12.02%), 1,8-cineole (7.41%), borneol (6.85%), and piperitenone oxide (3.19%). M. longifolia including a high percentage of pulegone is reported also by Asekun and others (2007). Several compounds such as thymol (Gullus and others 2007), carvone (Kokkini and others 1995), or piperitone oxide (Ghoulami and others 2001) previously found in M. longifolia essential oil, have not been found in our samples.

Forty-nine components representing 99.18% of the total M. viridis essential oil were identified and characterized mainly by car-vone (50.47%), followed by 1,8-cineole (9.14%), limonene (4.87%), camphor (3.68%), and β-caryophyllene (3%). Oxygenated monoterpenes corresponded to the main fraction (77.22%) of the total essential oil. M. viridis was rich in carvone as reported by other studies (Akhila and others 1980;Kokkini and others 1995). Camphor content for M. viridis is reported for the 1st time.

The 2 species showed an important variation in essential oil composition due to geographical and bioclimatic factors of the region. Different essential oil chemotypes have been described for M. viridis (Kokkini and others 1995) and M. longifolia (Gulluce and others 2007).

Antioxidant activity

Free radical scavenging activity of essential oils from 2 species of mint was investigated by ABTS assay. The ABTS has not been formerly tested for Mentha species. A moderate antioxidant activity was obtained to scavenge the ABTS radical cation (Table 2). M. longifolia essential oil (476.3 mg/L) was less active than M. viridis (195.1 mg/L).

The DPPH radical scavenging activity of essential oils confirms the result obtained with ABTS assay. Therefore, the DPPH activity of M. viridis essential oil (IC 50 of 3476.3 mg/L) was more important than for M. longifolia (>8000 mg/L, Table 2). IC 50 values obtained for Tunisian M. longifolia essential oil (>8000 mg/L) are comparable with Turkish M. longifolia essential oil (10700 ± 5.01 µg/mL) (Gulluce and others 2007). A previous study showed that methanol extract of M. longifolia was more able to reduce the stable free radical DPPH than that of essential oil (Gulluce and others 2007).

The essential oil of M. viridis contains more antioxidants than M. longifolia essential oil. Activity might be related to its phenolic content as reported in previous studies (Mimica-Dukic and others 1999; Gulluce and others 2007). Our study confirmed this result; in effect, the percentage of phenolics in M. viridis was 0.45% (dihydroeugenol and eugenol) relatively superior to that in M. longifolia essential oil that attained a percentage of 0.18% (dihydroeugenol).

Antimicrobial activity

Antimicrobial activity of essential oils was evaluated against 2 Gram-positive bacteria, 2 Gram-negative bacteria, 2 fungi, and 2 yeast species by zone diameter (Table 3). These microorganisms are morphologically and physiologically different. Essential oils exhibited a great potential for antimicrobial activity against 2 fungi (M. ramamnianus and A. ochraceus). In fact, inhibition zone diameters for M. ramamnianus (35 to 40 mm) and A. ochraceus (32 to 43 mm) were higher (Table 3). This is the 1st study of antimicrobial activity of M. longifolia and M. viridis essential oils against yeast species (C. albicans and S. cerevisiae). Inhibition zones were obtained by essential oils of 2 species against yeast C. albicans (19 to 21 mm) and S. cerevisiae (25 to 28 mm). These activities may be related to major compounds of the M. longifolia and M. viridis essential oils (pulegone and carvone, respectively) but there is no previous study that reported antimicrobial activity of pulegone and carvone components against yeast. The maximal inhibition zones for bacterial strains were 25 to 29 mm for M. viridis essential oil and 20 to 24 mm for M. longifolia essential oil against, respectively, K. pneumonial and L. monocytogenes. However, no antimicrobial activity of essential oils tested was noted against E. coli and S. aureus. Results obtained in this study show that mint essential oils do not have selective antimicrobial activity on the basis of the cell-wall differences of bacterial microorganisms (Gram-positive or Gram-negative bacteria) as reported previously (Cosentino and others 1999;Karaman and others 2003). Referring to the literature, the antimicrobial activity of the tested essential oils may be due to monoterpene hydrocarbons (Carlo and others 2005). M. viridis essential oil contains more terpene hydrocarbons (monoterpenes and sesquiterpenes) than M. longifolia (Table 3). This result is in agreement with the greatest diameter of inhibition obtained for about 7/8 of microorganisms tested (except S. cerevisiae) for M. viridis essential oil (Table 3). Despite the feeble percentage of the monterpene and sesquiterpene hydrocarbons for the 2 essential oils (17.31% for M. viridis and 8.35% for M. longifolia) the samples presented an interesting antimicrobial activity. Although these compounds are not abundant in essential oil, activity was important. It is necessary to signal that other compounds can contribute to improve this activity.

Conclusions

C arvone, thymol, and piperitone oxide have not been detected in Tunisian M. longifolia. Camphor is reported for the 1st time for M. viridis. Essential oils of M. viridis and M. longifolia have a medium antioxidant activity against ABTS and DPPH free radical scavenging. This activity is related to low phenolic content. Essential oils of Tunisian M. longifolia and M. viridis cultivated species exhibited a high antimicrobial activity against fungi and yeast species.

Essential oil represents a complex mixture of different chemical components. The antimicrobial effect of total essential oil cannot be reduced to major components. The results of this study showed that M. longifolia and M. viridis possess essential oils with important antimicrobial properties. Antibacterial activity could be influenced by terpene hydrocarbons (monterpenes and sesquiterpenes) but other components can contribute to this activity. In vivo assay is also necessary to confirm antimicrobial activity of M. longifolia and M. viridis. These studies can be used in pharmaceuticals and natural therapies of infectious diseases for humans, and management of plant diseases.

Separation of terpene hydrocarbons, responsible for the antimicrobial activity, would be an interesting study to identify the molecule generating the good efficacy.

  = absence of inhibition zone detected; essential oils concentration: 15 µL/well; reference standards concentration: 30 µg/well.

  Romdhane are with Laboratoire de Mod élisation Analyse et Commande des Systèmes. Ecole Natl. d'Ingénieurs de Gabès. Rue Omar elkhattab-ZRIG-6029 Gabès Tunisie. Author Bouajila is with Laboratoire de Synthèse et de Physicochimie de Molécules d'Intérêt Biologique, UMR CNRS 5068, Univ. Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse, France. Authors Lebrihi and Mathieu are with Univ. de Toulouse, LGC UMR 5503 (CNRS/INPT/UPS), ENSAT/ INPT, 1, Ave. de l

Table 1 -

 1 --Chemical composition (percent) of essential oils extracted from Mentha longifolia and Mentha viridis leaves using hydrodistillation methods. tested against a panel of microorganisms including 2 Gram-positive (S. aureus CIP7625, Listeria monocytogenes Scott A 724), 2 Gram-negative (E. coli ATCC10536, Klebseilla pneumoniae CIP8291), 2 yeasts (S. cerevisiae ATCC 4226 A and C. albicans IPA 200), and 2 fungi (M. ramamnianus ATCC 9314 and A. ochraceus NRRL 3174). The bacterial strains were cultured on nutrient broth for 48 h at 37 • C, while fungi and yeasts were propagated in CYB at 37 • C for 48 h to 3 d before use. All microorganisms were stocked at -20 • C in appropriate conditions and regenerated twice before use in the manipulations.

	Nr	KI a	Compounds	M. longifolia (%)	M. viridis (%)
	1	936	α-pinene	0.52	0.50
	2	976	sabinene	0.70	0.20
	3	985	β-myrcene	0.04	0.19
	4	990	3-octanol	---	0.54
	5	1011	δ-3-carene	0.18	0.34
	6	1025	p-cymene	0.04	0.33
	7	1028	limonene	0.12	4.87
	8	1030	1,8-cineole	7.41	9.14
	9	1057	γ -terpinene	0.63	0.78
	10	1072	linalool oxide	1.17	---
	11	1086	α-terpinolene	---	0.05
	12	1094	linalool	0.04	0.17
	13	1105	p-menth-2-en-1-ol	---	1.20
	14	1141	trans-pinocarveol	0.09	---
	15	1143	camphor	1.67	3.68
	16	1148	isopulegol	0.04	0.28
	17	1155	menthone	0.18	---
	18	1159	β-terpineol	---	0.30
	19	1164	isomenthone	12.02	1.13
	20	1166	borneol	6.85	0.74
	21	1178	terpinen-4-ol	0.06	2.97
	22	1185	α-terpineol	1.52	1.97
	23	1190	dihydrocarveol b	---	0.45
	24	1193	neodihydrocarveol b	---	0.69
	25	1202	carveol	---	0.83
	26	1205	verbenone	0.19	---
	27	1238	pulegone	54.41	1.11
	28	1241	carvone	---	50.47
	29	1285	bornyl acetate	0.37	0.64
	30	1330	1,2,4-trimethoxybenzene b	0.65	---
	31	1350	eugenol	---	0.27
	32	1352	terpinyl acetate	0.61	0.43
	33	1366	dihydroeugenol	0.18	0.18
	34	1369	piperitenone oxide	3.19	0.48
	35	1389	β-elemene	---	1.00
	36	1390	β-cubebene	---	0.12
	37	1394	cis-jasmone	0.16	1.16
	38	1406	methyl eugenol	---	0.36
	39	1415	β-caryophyllene	2.04	3.00
	40	1432	β-gurjunene	---	0.51
	41	1438	geranyl acetone	0.04	---
	42	1439	α-guaiene	---	1.01
	43	1450	α-himachalene	0.17	0.17
	44	1454	α-humulene	2.97	0.62
	45	1482	β-selinene	0.33	1.86
	46	1486	α-selinene	---	0.51
	47	1499	α-muurolene	0.34	---
	48	1512	calamenene b	---	0.22
	49	1513	γ -cadinene	0.31	1.12
	50	1524	δ-cadinene	---	0.26
	51	1534	α-cadinene	---	0.20
	52	1580	β-caryophyllene oxide	0.24	0.86
	53	1636	T-muurolol	0.26	0.51
	54	1678	cis-α-santalol	---	0.42
	55	ND	grandisol b	---	0.27
	56	ND	dimethylfuran lactone b	---	0.11
			Total	99.72	99.18
			Monoterpene hydrocarbons	2.19	6.93
			Monoterpenes oxygenated	89.18	77.22
			Sesquiterpenes hydrocarbons	6.16	10.38
			Sesquiterpenes Oxygenated	0.5	1.79
			Other	1.71	2.90
			Phenolics	0.18	0.45

a DB-5MS nonpolar column relative to C8-C24 n-alkanes. b Tentatively identified supported by good match of MS. ND = not determined.

individually

Table 2 -

 2 --Antioxidant activity of Mentha longifolia and Mentha viridis using ABTS and DPPH radical scavenging assay.

	Samples	ABTS IC 50 (mg/L)	DPPH IC 50 (mg/L)
	Mentha longifolia	476.3 ± 11.7	>8000
	Mentha viridis	195.1 ± 4.2	3476.3 ± 133
	Vitamin C	1.9 ± 0.1	4.4 ± 0.2

Table 3 -

 3 --Antimicrobial activity (diameter of the inhibition zones in mm) of Mentha longifolia and Mentha viridis essential oils.