Anna Carla Araujo
email: annaaraujo@cefet-rj.br

Jose Luis Silveira

APPLICATION OF ACOUSTIC EMISSION ON END MILLING MODELLING

Keywords: force model, end milling, acoustic emission

Application of acoustic emission technique for on-line monitoring of various

manufacturing processes such as punch stretching, drawing, blanking, forging, machining and grinding has been reviewed and discussed. During the past several years has established the effectiveness of acoustic emission sensing methodologies for machine condition analysis and process monitoring. AE has been proposed and evaluated for a variety of sensing tasks as well as for use as a technique for quantitative studies of manufacturing processes. Acoustic emission generated during the end milling processes using a unique tool can give useful information of the detected wear and friction properties on tool/chip contact coefficients. The AE wear information can be added to the force prediction models developed for non-wear tools to include the edge force component.

INTRODUCTION

The optimization of the manufacturing process plays an important role in improving productivity and, for this, monitoring and prediction are becoming increasingly necessary in the industry. The force prediction was insistently studied and different types of models were developed. Machining is a complex process regarding the mechanical behavior of the material because it should describe a plastic deformation with a complex temperature distribution in a region with an open boundary as the tool-chip contact length is not fixed.

The mechanistic models achieved more accurate prediction with only the information of the tool and work piece geometry and materials and the machining parameters known by the operator. Those models use cutting energy parameters calibrated for a tool work piece pair of materials as a function of the velocities and chip thickness. The parameters not included on the model are implicit on the function although those functions are calibrated for a non wear tool. As the tool is being used the force will change not only because of the tool geometry but also because of the tool-chip contact. As tool wear affects finished surface, there is a need to develop tool wear monitoring systems which alert the operator to the state of the tool. Besides the wear information can also be used to optimize the force prediction during the work to change the parameters and improve tool life.

In this work, the edge cutting force is a function of the tool wear monitored by acoustic emission.

ACOUSTIC EMISSION

Acoustic emission is the class of phenomena whereby transient elastic waves are generated by rapid release of energy from a localized sources inside the material that travels through the work piece until the located sensor [START_REF] Li | A brief review: acoustic emission method for tool wear monitoring during turning[END_REF].

One advantage of acoustic emission monitoring is the signal frequency range much higher than that of the machine vibrations and environmental noises that does not interfere with the cutting operation.

Research has shown that acoustic emission has been successfully used in laboratory tests to detect tool wear and fracture in single point operations. Based on the analysis of AE signal sources [START_REF] Li | A brief review: acoustic emission method for tool wear monitoring during turning[END_REF], AE consists of continuous signals, associated with shearing in the primary zone and wear on the tool face and flank, and transient signal result from tool fracture or chip breakage.

Brief review of acoustic emission on machining modelling in last years

Dornfeld [1980] and Kasibu [1981], first related the AE to the modeling of cutting and compared the RMS signal to the fundamentals cutting parameters. Based on the orthogonal cutting a relationship between RMS voltage and the fundamental parameters was developed.

For a general case, the rate of energy dissipation is given by:

Ẇ = U σ ij ˙ ij dU (1)
where W is the energy from plastic deformation, σ ij the stress which causes plastic strain ij .

The energy used for primary shear deformation can be analytically written as a function of shear stress τ s , shear angle φ, rake angle α and cutting velocity v c :

Ẇs = btτ s cos(α) sin(φ) cos(φ -α) .v c (2)
and from secondary zone, considering sticking and sliding friction is:

Ẇf = 1 3 τ s b(l + 2l sd ) sin(α) sin(φ) cos(φ -α) .v c ( 3
)
where b is the width of cut, l the friction contact length, l sd the sliding friction length.

The RMS signal according to Kasibu [1981] is written as:

RM S = C 1 τ s bv c t cos(α) sin(φ) cos(φ -α) + l + 2l sd 3 sin(α) sin(φ) cos(φ -α) (4)
where C 1 is a constant experimentally defined. [START_REF] Rangwala E Dornfeld | A study of acoustic emission generated during orthogonal metal cutting -spectral analysis[END_REF] compared the theoretical RMS signal with the experimental signal and claimed that this relation is proportional to the contact length.

Using True Mean Square (TMS) signal Liu [1991] modeled acoustic emission for monitoring of peripheral milling process including rubbing and the three regions of energy involved on the cutting process: primary shear, secondary shear and rubbing regions. An equation similar to Equation 5were developed for this case. [START_REF] Saini | A quantitative model of acoustic emission in orthogonal cutting operations[END_REF] also modeled the energy based on the cutting force and cutting models considering primary and secondary regions. The relation between predicted and experimental signals was linear for turning operations and signal losses due to propagation were included on the model. The experiment used the oscilloscope to analyze the signal. [START_REF] Wilcox | The use of cutting force and acoustic emission signals for the monitoring of tool insert geometry during roush face milling[END_REF] monitored the face milling to identify the changes on the cutting as flank wear, rake angle and edge breakdown. The AE sensor is positioned on the piece and the dynamometer is also used for the monitoring of process. The continuous RMS value compute the tertiary zone with a constant value C 1 can be written as:

RM S = C 1 τ s bv c t cos(α) sin(φ) cos(φ -α) + l + 2l sd 3 sin(α) sin(φ) cos(φ -α) + C 2 (5)
Li [1998] monitored small diameter drills for real-time detection of the breakage and positioned the sensor on the tool holder using a magneto fluid between the main spindle and the sensor apparatus. This work used wavelet transform to analyze the AE signal.

Chungchoo [2000] created new parameter for monitoring oblique turning by AE Total energy and total entropy of force signals were modeled and analyzed thru pattern recognition. [START_REF] Shao | A cutting power model for tool wear monitoring in milling[END_REF] considered monitoring to change the cutting power model in milling. The experimental results show that there is a signifcant change in force measurement with the cutting edge wear.

RMS variation with cutting edge wear

Not considering the variation of the shear angle, the sliding lenght and the lenght of the tool-chip contact change as the cutting edge wears. It can be seen on the Equation 5. In order to quantify the variation of the RMS level with the wear the Equation 5 can be derived:

d d RM S = C 3 (l + 2l sd ) (6)
This variation must be included on the force prediction.

PREDICTING CUTTING FORCE WITH EDGE CONTRIBUTION

The instantaneous differential cutting force for one single tooth was proposed by Martelotti [START_REF] Tlusty E Macneil | Dynamics of Cutting in End Milling[END_REF] as:

d F cutting = K cutting t db ( 7
)
where t is the uncut chip thickness, db is an increment of the depth of cut and K cutting is a vector with the specific cutting force coefficients. More recently this expression has been rewritten (Armarego, 1989) by adding the edge contribution:

d F = d F edge + d F cutting (8a) d F = K edge db + K cutting t db (8b)
where K edge is called specific edge force vector. The uncut chip thickness t for end milling is written as:

t = s t sin φ (9)
where φ is the angle of the cutting element measured in relation to the normal direction of the feed per tooth s t ,

s t = v f ω N t ( 10
)
where v f , ω and N t are respectively the feed velocity, the rotation and the number of teeth. The cutting tool pieces can be calculated by

db = d 2 tan λ dφ ( 11
)
where d is the tool diameter and λ is the helix angle, as shown in Fig. 1. The angle δ is calculated by

δ = 2 b tan λ d ( 12
)
where b is the depth of cut.

The angle δ is used to classify the cutting geometry as type I or type II [START_REF] Tlusty E Macneil | Dynamics of Cutting in End Milling[END_REF] according to the angles ϕ 1 and ϕ 2 which are, respectively, the entry and exit angles. For type I, δ is less or equal ϕ 2 -ϕ 1 and for type II, δ is greater than ϕ 2 -ϕ 1 (Fig. 1) [START_REF] Araujo | Models for the Prediction of Instantaneous Cutting Forces in End Milling[END_REF].

In order to obtain the force acting in one tooth, the following integration is performed over the whole tooth length engaged in the cutting:

F = K edge db + K cutting t db (13)
In this paper the specific cutting and edge force coefficients are assumed to be constants, considering this and eliminating t and db, the force expression for a tooth is rewritten as:

F = K edge d 2 tan λ dφ + K cutting s t sin φ d 2 tan λ dφ ( 14
)
The total force acting in the tool, considering the N t teeth of the mill, is calculated by the sum:

F = N t i=1 F i (15)
A fixed point P in the periphery of the tool is chosen to describe the angular position θ of the tool and the variation of the milling force with the tool rotation ω (Fig. 2):

F (θ) = K edge h(θ) + K cutting A(θ) (16)
where h(θ) is a function of the cutting height and A(θ) is a function of the cutting area, which accumulate the contribution of the N t teeth of the tool and are, respectively, given by:

h(θ) = N t n=1 h n (θ) (17) A(θ) = Nt n=1 A n (θ) (18)
Table 1. Integration limits for each phase.

Type I Type II Phase L 1 (θ) L 2 (θ) L 1 (θ) L 2 (θ) For e 1 < θ ≤ e 2 -Phase A ϕ 1 θ ϕ 1 θ For e 2 < θ ≤ e 3 -Phase B θ -δ θ ϕ 1 ϕ 2 For e 3 < θ ≤ e 4 -Phase C θ -δ ϕ 2 θ -δ ϕ 2 Table 2. Variables values Type I Type II e 1 ϕ 1 ϕ 1 e 2 ϕ 1 + δ ϕ 2 e 3 ϕ 2 ϕ 1 + δ e 4 ϕ 2 + δ ϕ 2 + δ
The height function h n (θ), i.e., the height of a tooth (n) engaged in the cutting and the chip cross-sectional area function A n (θ) for a tooth (n) are calculated, respectively, by:

h n (θ) = L 2 (θ+ξ(n-1)) L 1 (θ+ξ(n-1)) d 2 sin λ dε (19) A n (θ) = L 2 (θ+ξ(n-1)) L 1 (θ+ξ(n-1)) s t d 2 sin λ sin ε dε (20)
where the limits L 1 and L 2 are determined for each cutting phase of θ (Table ), ξ is the angle between the teeth and the values of e 1 , e 2 , e 3 and e 4 are taken from Table .  Fig. 3 shows the chip cross-sectional area A 1 (θ) for the first tooth of an end milling tool with four teeth (ξ = 90 o ) and having type I geometry, with ϕ 1 = 30 o and ϕ 2 = π/2.

The force components are decomposed in the tool referential t, r, z (tangential, radial and axial directions) in which the specific force coefficients are assumed to be constants: To rewrite it on the machine referential x, y, z in which the force components are usually recorded in machining tests, a rotation matrix R n (θ) is written for each tooth (n):

F (θ) =   F t (θ) F r (θ) F z (θ)   = A(θ)   K ct K cr K cz   + h(θ)   K et K er K ez   (21)
R n (θ) =   cos(θ + ξ (n -1)) sin(θ + ξ (n -1)) 0 sin(θ + ξ (n -1)) -cos(θ + ξ (n -1)) 0 0 0 1   (22)
Thus the cutting force is rewritten in the more appropriate referential x, y, z as:

  F x (θ) F y (θ) F z (θ)   = A R (θ)   K ct K cr K cz   + h R (θ)   K et K er K ez   (23)
where the rotated area function A R (θ) and the rotated length function h R (θ) are, respectively, given by:

A R (θ) = N t n=1 R n (θ) A n (θ) (24) h R (θ) = N t n=1 R n (θ) h n (θ) (25)
To simplify the following calculations, S 1 (θ), S 2 (θ), S 3 (θ) and S 4 (θ) are defined:

S 1 (θ) = N t n=1 A n (θ) cos(θ + ξ (n -1)) (26a) S 2 (θ) = N t n=1 A n (θ) sin(θ + ξ (n -1)) (26b) S 3 (θ) = N t n=1 h n (θ) cos(θ + ξ (n -1)) (26c) S 4 (θ) = Nt n=1 h n (θ) sin(θ + ξ (n -1)) (26d)
Rewriting the Equation 23, the force can be expressed by:

  F x (θ) F y (θ) F z (θ)   =   S 1 (θ) S 2 (θ) 0 S 3 (θ) S 4 (θ) 0 S 2 (θ) -S 1 (θ) 0 S 4 (θ) -S 3 (θ) 0 0 0 A(θ) 0 0 h(θ)           K ct K cr K cz K et K er K ez         (27)
or in a more compact notation:

F(θ) = J(θ) K (28)
The Equation 28 can be easily calculated, as the specific pressures are constant in θ. For given experimental force components F(θ) and for each point θ, the following equation can be solved:

K = J -1 (θ)F(θ) (29)
From the results obtained for each point θ, an the average value K of the specific pressures can be obtained for the whole rotation.

RMS LEVEL INFLUENCE ON THE EDGE COEFFICIENT

Using the mechanistic approach, the calibration for the cutting pressure is done for a range of cutting velocity and spindle speed. In this method, the specific pressure is also calibrated for a range of wear using the information from the RMS signal and the information is added to the cutting edge pressure K e .

  F x (θ) F y (θ) F z (θ)   =   S 1 (θ) S 2 (θ) 0 S 3 (θ) S 4 (θ) 0 S 2 (θ) -S 1 (θ) 0 S 4 (θ) -S 3 (θ) 0 0 0 A(θ) 0 0 h(θ)           K ct K cr K cz K et ( ) K er ( ) K ez ( )         (30)
The force for a new tool, with no wear, is calculated based on the mechanistic model. The calibration is done for a tool and workpiece pair of materials. Afterwise begin the tool wear and the force changes behavior. The purpose in this paper is add wear information to the model using the RMS signal variation from initial measurements to the wear stage to predict. The wear information in the model is located at the K edge and to add the contribution to this parameter, the mean value of the RMS is divided by the inicial mean value to obtain the coefficinte K ( ) during the monitoring. K e ( ) = K .K e (31)

RESULTS AND DISCUSSIONS

The algoritm presented on [START_REF] Araujo | Analysis of the specific force on end milling[END_REF] is used to calculate the force without wear and the specific pressure is multiplied by a factor that corresponds to the wear contribution to the force.

The experimental result from Altintas [1996] (Figure ) is used to calculate the specific pressures and to apply the wear model mofication. The process was up milling using the parameters: d = 18.1mm, s t = 0.05mm/th, N f = 4, λ = 30 o , v = 11mm/min, b = 5.08mm, ϕ 2 = π and an Ti6Al4V Alloy workpiece.

Figures 5 and6 show the results adding an contribution of 10% and 30% on the edge especific pressure and comparing the data with and without the tool wear in both cases. The cutting specific pressure is taken from the experimental data as in [START_REF] Araujo | Analysis of the specific force on end milling[END_REF].

CONCLUSIONS

It is possible to compute wear in the mechanistic end milling model with edge parcel and it can justify a consider force increase. It is needed experimental force results to validate the model using the RMS signal to compare with the measured force. Acoustic emission can be applyed to other metal cutting prediction as in drilling and turning.

ACKNOLEDGEMENTS

The authors would like to gratefully acknowledge the PRODOC fellowship received from CAPES, a Brazilian council for scientific and technological development, and the Mechanical Engineering Department PEM/COPPE for the research support.

  Figure 1. Milling geometry

  Figure 2. Milling Tool Referential

Figure 3 .

 3 Figure 3. Chip Cross-sectional Area

Figure 4 .

 4 Figure 4. Experimental data from Altintas [1996]


Shi-Xia Liu
email: liu@dcb.unibe.ch

Dr Egmont J Rohwer

Yan Geng

Dr Maryam Akbarimoosavi

Latévi Max

Lawson Daku

Dr Olivier Aleveque

Dr Eric Levillain

Dr Jürg Hauser

Andrea Cannizzo

Robert Häner

Silvio Decurtins

Robert J Stanley

Thomas Feurer

Shi-Xia Liu

Prof Y Geng

Prof R Häner

Prof S Decurtins

Dr S.-X Liu

Prof A Cannizzo

Prof. T Feurer

Dr L M Lawson

Prof R J Stanley

Optically Controlled Electron Transfer in a Re(I) Complex

We demonstrate ultrafast optical control of intramolecular charge flow which paves the way for photocurrent modulation and switching with a highly wavelength-selective ON/OFF ratio. The system we explore is a fac-[Re(CO)3(TTF-DPPZ)Cl] complex, where TTF-DPPZ = 4',5'-bis(propylthio)tetrathiafulvenyl[i]dipyrido[3,2a:2',3'-c]phenazine. DFT calculations and AC-Stark spectroscopy confirm the presence of two distinct optically active charge-transfer processes, namely a metal-to-ligand charge transfer (MLCT) and an intra-ligand charge transfer (ILCT). Ultrafast transient absorption measurements show that the ILCT state decays in the ps regime. Upon excitation to the MLCT state, only a long-lived 3 MLCT state is observed after 80 ps. Remarkably, however, the bleaching of the ILCT absorption band remains as a result of the effective inhibition of the HOMO-LUMO transition.

A good understanding of how to control the charge transfer (CT) in multichromophore ensembles is essential for mimicking nature to capture and convert solar energy and eventually realizing high performance photocatalysis, photovoltaics, spintronics and optoelectronics. [1] Taking directed electron transfer as an example, if there is an increase in electron density on one part of the molecule upon excitation, then putting an acceptor near that part of the molecule will lower electrostatic barriers to photo-induced charge transfer. This rationale was reported to understand the placement of tryptophan donors used to reduce photoexcited flavins in DNA photolyase. [2] A variety of strategies have been developed particularly for facilitation of charge separation and inhibition of charge recombination in electron donor-bridge-acceptor (D-B-A) systems. Within the context of optical switches, gating of an intramolecular charge transfer (ICT) has been reported by incorporating a photochromic unit between D and A moieties. [3] In most cases, a photochromic Förster resonance energy transfer results in an ICT fluorescence quenching. In 2010, a state-selective electron transfer in an unsymmetric acceptor-Zn(II)porphyrin-acceptor triad was reported, however, the issue of repopulation of different states after photoexcitation still remains to be solved. [4] Although an example of excitation-wavelength-dependent photoinduced electron transfer in a fully p-conjugated oligomer was very recently reported, [5] optical control over multiple CT pathways in D-A systems remains a big challenge, and to the best of our knowledge, it has not been reported so far.

Here, we describe preparation, electronic properties, photocycle and ultrafast optical control of CT in a fac-[Re(CO)3(TTF-DPPZ)Cl] complex. The interest in fac-[Re(L)(CO)3(a-diimine)] n+ complexes stems from their dynamic and exceptionally rich excited states properties that are dramatically altered by structural variations in ancillary ligand L and a-diimine as well as solvents. [6] Specifically, the DPPZ ligand shows LUMO and LUMO+1 localized on phenazine (phz, blue in Figure 1) and phenanthroline (phen, purple in Figure 1) units, respectively, which are energetically close to each other (#171 and #172 in Supporting Information, Table S2). As a consequence, the interplay of two close-lying 3 MLCT states of different nature in fac-[Re(L)(CO)3(DPPZ)] n+ complexes renders them promising for development of phosphorescent labels and probes of biomolecules such as DNA and proteins. [8] To modulate the energy levels of these two molecular orbitals, electron-donating or withdrawing substitutes have been appended to the DPPZ core, leading to the formation of 3 ILp-p* or 3 ILCT along with two 3 MLCT excited states in the corresponding Re(I) complexes. [6d,8] To facilitate population of ILCT states that are well-separated from higher lying MLCT states, our strategy involves the coordination of a planar TTF-annulated DPPZ to the Re(CO)3Cl chromophore. Our previous work has demonstrated that TTF-DPPZ exhibits a strong ILCT transition with the TTF as an electron donor and the DPPZ as an electron acceptor. [9] As illustrated in Figure 1, the excitation of two distinct CT states results in a charge flow either from the peripheral TTF (red) or the Re(CO)3Cl chromophore to the central phz core, these being in opposite directions. This allows for optically gating the charge flow in D-A ensembles. In the present work, the involved CT states are identified via DFT calculations and AC-field Stark spectroscopy, and their ultrafast dynamics are investigated by femtosecond transient absorption spectroscopy (fs-TAS). The TTF-DPPZ ligand was synthesized according to published procedures. [9a] Reaction of [Re(CO)5Cl] with TTF-DPPZ in EtOH afforded the target complex fac-[Re(CO)3(TTF-DPPZ)Cl]. It is unambiguously characterized by elemental analysis, 1 H NMR, UV-Vis and IR spectroscopy. Single crystals suitable for X-ray structure analysis were obtained by slow evaporation of its solution in cyclohexane. The single-crystal structure (Figure 2) shows that the Re(I) ion is coordinated by three CO molecules, one chloride and one phen chelating unit from TTF-DPPZ in a distorted octahedral fashion. Two CO molecules and the phen subunit of TTF-DPPZ occupy the equatorial positions around the Re(I) ion. The skeleton of TTF-DPPZ is almost planar and the Re(I) ion is displaced out of a least-squares plane through all ligand atoms, except two propyl groups, by only 0.133(9) Å. The crystal and refinement data as well as a crystal packing diagram are given in Table S1 andFigure The electrochemical properties of the Re(I) complex in CH2Cl2 were investigated by cyclic voltammetry (Figure S2). It displays two reversible single electron oxidation waves (E1/2 ox1 = 0.28 V, E1/2 ox2 = 0.62 V) for successive oxidation of the TTF subunit to the radical cation and dication states and one reversible reduction wave at -1.41 V for the reduction of the phz component of the DPPZ subunit. The UV-vis spectrum of the complex in CH2Cl2 (Figure S3) shows a strong and broad absorption band centered at 580 nm. Akin to Ru(II) and Fe(II) complexes with TTF-DPPZ, [10] this lowest energy absorption band is attributed to a spin-allowed π-π* ILCT transition from the HOMO localized on the electron donor TTF to the LUMO localized on the phz component of the DPPZ subunit. As expected, the MLCT transition peaks appear at 375 nm. Importantly, the ILCT transition is well separated from the MLCT transition. Moreover, an intense absorption band approaching the UV edge of the spectrum is characteristic of several overlapping π-π* transitions of the TTF and DPPZ moieties.

The DFT calculations of the Re(I) complex (Table S2) predict a separated S0 → S1 transition in the low energy visible region (660 nm in vacuum) that consists of a pure HOMO-LUMO transition. The HOMO is localized on TTF, while the LUMO is localized on DPPZ weighted towards the phz part. The calculations predict an associated change in dipole moment of 20 D and a 10% increase in polarizability of 36 Å 3 over this ILCT transition. A transition with comparable oscillator strength is found at 384 nm and is attributed to the S0 → S14 transition. The excitation is dominated (76%) by electron transfer from a molecular orbital spread broadly over TTF to the LUMO. Despite the ILCT nature of the transition, the associated dipole moment change is only 1.2 D according to DFT. The next largest oscillator strength belongs to the S0 → S7 transition at 455 nm, which is dominated (93%) by electron transfer from a molecular orbital localized around the Re(I) ion, to the phen component of the DPPZ unit. This MLCT transition is characterized by a small dipole moment change of 1 D. Similar MLCT transitions but with weaker oscillator strengths can be found at 500 nm (S0 → S4) and at 410 nm (S0 → S9). The former is dominated by a charge migration (77%) from the same Re(I)-localized MO mentioned before to the phz-localized LUMO which in turn implies a larger dipole moment change of 9.5 D. The latter also entails charge migration from the Re(I) to DPPZ, however both molecular orbitals are more distributed across the Re(I) chromophore and entire DPPZ unit, respectively. The associated dipole moment change is correspondingly intermediate with a value of 3.8 D. We note that for all the aforementioned MLCT transitions, the change in polarizability is no longer marginal and at least one order of magnitude larger than for the HOMO-LUMO excitation. It is important to mention that higher energy ILCT transitions also absorb in this area albeit weakly, for example S0 → S6 at 466 nm with associated charge migration from TTF (HOMO) to DPPZ (distributed). Furthermore, in the UV region (sub-400 nm) the remaining transitions with significant oscillator strengths are S0 → S12 (395 nm) and S0 → S16 (360 nm). The former has a mixed MLCT and ILCT nature with all contributing MO transitions concentrating charge on DPPZ and an associated 11 D dipole moment change and marginal change in polarizability. The latter seems to be a π→ π* transition on the TTF part with a dipole moment change of 2.6 D and large change in polarizability.

Accepted Manuscript

To identify relevant transitions with large charge redistribution, the Re(I) complex was dissolved in toluene and studied with AC-Stark spectroscopy. The Stark spectrum shown in Figure 3 reveals three distinct Stark active transitions that correspond to peaks observed in the ground state absorption spectrum (Figure 3a). Liptay analysis [START_REF] Liptay | Excited States[END_REF] of the Stark spectra using simultaneous fitting of the ground state absorption spectrum and higher order derivative lineshapes therein allows us to extract the electronic properties of the three Stark-active transitions. They consist of a low energy absorption band attributed to the HOMO-LUMO transition, a mid-spectrum band and a UV band below 400 nm (above 25000 cm -1 ). The parameters extracted by Liptay analysis are the change in dipole moment (Δμ), the trace of the change in polarizability tensor (Tr(Δα)), the component of change in polarizability along the transition dipole moment (m•Δα•m) and the angle between the change in dipole moment and the transition dipole moment (ζ). These parameters are listed in Table 1. The spectrally isolated HOMO-LUMO transition in the low energy band shows a (21 ± 1) D change in dipole moment and a (40 ± 20) Å 3 change in polarizability, which is in perfect agreement with the DFT calculations for the S0 → S1 transition predicting 20 D and 36 Å 3 . The transition mid-spectrum has an associated dipole moment change of (9 ± 3) D and a change in polarizability of (170 ± 80) Å 3 , which strongly suggests that this transition is dominated by S0 → S4 with calculated values of 9.5 D and 300 Å 3 . The size of the dipole moment change indicates that the charge reaches phz (DPPZ) and is not limited to the more proximate phen (DPPZ). The UV transition with a measured (7 ± 2) D dipole moment change and a negligible polarizability change appears to be a higher energy ILCT transition. A breakdown of contributions to the overall Stark spectrum is given in the SI. To get a good understanding of effect of changes in dipole moment and polarizability is vital for engineering the molecules for certain applications. We can imagine that a larger dipole moment, for example, could be advantageous for solar energy harvesting because of greater (and longer) charge separation in the photo-excited state. A sudden large increase in polarizability after photo-excitation may be interesting for molecular electronics/switching.

Femtosecond transient absorption spectroscopy (fs-TAS) of the Re(I) complex in DMF was performed with excitation of the HOMO-LUMO transition at 560 nm and of higher-lying transitions at 400 nm (Figure 4a and 4b, respectively). When pumping the HOMO-LUMO transition at 560 nm, a negative ground state bleaching (GSB) signal is observed between 540 nm and 600 nm, which is attenuated by its overlap with the more intense excited state absorption signals (ESA) as shown in Figure 4a. Positive ESA signals are observed between 370 nm and 540 nm and from 630 nm to the edge of the spectral observation window. The latter absorbs with considerably lower cross-section. The ESA features and their rapid decay in the tens of ps regime are similar to those observed in other TTFfused donor-acceptor systems. [9a,10,12] Moreover, the spectroelectrochemical measurements show that the singly-oxidized TTF radical cation absorbs at 475 nm and 830 nm while the reduced DPPZ anion absorbs at 490 nm (Figure S4). Consequently, these ESA bands are tentatively assigned to a charge-separated state fac-[Re(CO)3(TTF .+ -DPPZ .-)Cl]. The back electron transfer to the ground state configuration occurs on the same time scale as the ESA signal decay.

The most apparent difference when exciting at 400 nm, is the persistent transient signal after 200 ps (Figure 4b). The early time dynamics seems to be similar to that in Figure 4a. In fact, if we compare a spectral slice from Figure 4b at 100 fs to the weighted sum of spectral slices from Figure 4b at 100 ps and Figure 4a at 100 fs, as shown in Figure 4c, we find excellent agreement. The comparison suggests that 400 nm excitation results in a superposition of two ESA features resulting from an ILCT (21%) and a MLCT (79%) transitions. This agrees surprisingly well with the S0 → S4 transition which according to DFT calculations has 77% MLCT and 19% ILCT character. The broadband ESA with peak at 450 nm seen at later times after excitation and subsequent ILCT dynamics, seems to be characteristic of a whole class of [Re(CO)3(a-diimine)Cl] complexes, which is associated with the formation of a long-lived triplet MLCT state via intersystem crossing as previously observed in Re(I) complexes. [6b,6c,6g,8a,8c,13] . Remarkably, the GSB signal in a range from 520 nm to 600 nm, which is assigned to the HOMO-LUMO transition with a charge flow from the TTF to DPPZ moieties, persists along with the triplet state ESA even after the back electron transfer following ILCT. This implies that the electron resides on the LUMO on the phz of DPPZ upon excitation to the MLCT state and therefore blocks the ILCT pathway.

In conclusion, using a combination of DFT calculations, AC-Stark spectroscopy and fs-TAS, we have characterized ILCT and MLCT in the complex [Re(CO)3(TTF-DPPZ)Cl], these being associated with CT from opposite ends of the molecule to the central DPPZ unit (Figure 1). The spectrally isolated ILCT transition from the TTF to the phz of the DPPZ unit occurs at 560 nm and is accompanied by a change in dipole moment of 20 D and a slight increase in polarizability. In contrast, various MLCT-dominated transitions appear at higher energies with up to 10 D change in dipole moment and significant polarizability change. The MLCT transitions with larger dipole moment change as shown by AC-Stark Spectroscopy and fs-TAS, even reach the phz subunit and block ILCT from the opposite end. This property provides an optical control of the direction of the photo-induced CT, which is of high importance to develop an optoelectronic molecular switch triggered by light of different wavelengths.

Figure 1 .

 1 Figure 1. Chemical structure of the target complex.

  S1, respectively.

Figure 2 .

 2 Figure 2. ORTEP structure of the Re(I) complex drawn with 50% ellipsoid probability and with atom numbering scheme; H-atoms given arbitrary displacement parameters for clarity.

Table 1 .Figure 3 .

 13 Figure 3. Ground state absorption at low temperature (a) used for Liptay analysis and Stark spectrum (b) of the Re(I) complex.

  This article is protected by copyright. All rights reserved.

Figure 4 .

 4 Figure 4. Femtosecond transient absorption spectra of the Re(I) complex in DMF a): lex = 560 nm and b): lex = 400 nm. In c) we compare the spectrum of b) at 100 fs to the sum of the spectra of b) at 100 ps plus 0.28 times the spectrum in a) at 100 fs.

Acknowledgements

We appreciate Prof. Oliver S. Wenger for his helpful discussion. RJS acknowledges funding from the NASA Exobiology Program (Grant 80NSSC17K0033). EJR, MA, AC and TF acknowledge funding from the Swiss NSF through the NCCR MUST "Molecular Ultrafast Science and Technology".

Keywords

: Re(I) complexes • charge-transfer transitions • fused tetrathiafulvalene ligand • femtosecond transient absorption spectroscopy • AC-Stark spectroscopy

Entry for the Table of Contents

Optical control of photoinduced electron transfer via selective excitation wavelength is for the first time achieved by incorporation of a tetrathiafulvalene-fused dipyrido[3,2-a:2',3'-c]phenazine ligand to a Re(I) chromophore. Gating of charge flow over multiple chargetransfer pathways is a prerequisite to success in photodiodes.


A Giaveno
email: agiaveno@uncoma.edu.ar

L Lavalle

Guibal Eric

E Donati

E Guibal

Biological ferrous sulfate oxidation by A. ferrooxidans immobilized on chitosan beads

Keywords: Acidithiobacillus ferrooxidans, Iron oxidation, Packed-bed bioreactor, Chitosan carrier

Biological ferrous sulfate oxidation by A. ferrooxidans immobilized on chitosan beads

Introduction

Acidithiobacillus ferrooxidans is an acidophilic bacterium capable of oxidizing different inorganic compounds, the most common of which is ferrous sulfate. A. ferrooxidans is one of the dominant microorganisms not only in the bioleaching of minerals but also in the pretreatment (biooxidation) of refractory gold. Both processes imply the oxidation of sulfide by ferric iron; the resultant ferrous iron is re-oxidized to the ferric state by the bacteria. The bioproduction of ferric iron has been also employed to desulfurize gases containing hydrogen sulfide or sulfur dioxide, in the desulfurization of coal and even in the treatment of acid mine drainages [START_REF] Rohwerder | Bioleaching review. Part A: Progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation[END_REF][START_REF] Rawlings | Biomineralization of metalcontaining ores and concentrates[END_REF][START_REF] Watling | The bioleaching of sulphide minerals with emphasis on copper sulphidesa review[END_REF][START_REF] Akcil | Role and contribution of pure and mixed cultures of mesophiles in bioleaching of a pyritic chalcopyrite concentrate[END_REF].

In all those cases, large volumes of ferric iron solutions should be generated. The use of free cells has a limited potential because biomass has low physical strength and low density causing serious problems to be separated from the solution. Cell immobilization provides several advantages in protecting the cells from hostile environments and maintaining higher cellular metabolic activities and increasing the value of dilution rate where the washout takes place.

The natural tendency of A. ferrooxidans to grow on surfaces not only on inert support but also on minerals makes it an adequate microorganism for cell immobilization and useful to increase iron(III) productivity [START_REF] Karamanev | Influence of some physicochemical parameters on bacterial activity of biofilm: ferrous iron oxidation by Thiobacillus ferrooxidans[END_REF][START_REF] Ramírez | Bacterial attachment: its role in bioleaching processes[END_REF][START_REF] Harneit | Adhesion to metal sulphide surfaces by cells of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans[END_REF]. Several supports have been employed, including the use of glass beads, activated carbon particles, sand, polystyrene, polyurethane, poly (vinyl alcohol), calcium alginate, ion-exchange resin, nickel alloy fibre, PVC and diatomaceous earth [START_REF] Grishin | Fast kinetics of Fe 2+ oxidation in packedbed reactors[END_REF][START_REF] Armentia | Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised in polyurethane foam support particles[END_REF][START_REF] Porro | Use of packed bed bioreactors. Application to ores bioleaching[END_REF][START_REF] Wakao | Immobilization of Thiobacillus ferrooxidans using various polymers as matrix[END_REF][START_REF] Mazuelos | Design variables in high efficiency reactors for the biooxidation of ferrous iron in solution[END_REF][START_REF] Gomez | Immobilization of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulphate oxidation[END_REF][START_REF] Curutchet | Development of Thiobacillus biofilm for metal recovery[END_REF][START_REF] Mesa | Mathematical model of the oxidation of ferrous iron by a biofilm of Thiobacillus ferrooxidans[END_REF][START_REF] Mesa | Biological oxidation of ferrous iron: study of bioreactor efficiency[END_REF][START_REF] Long | Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers[END_REF]Long et al., , 2004a,b;,b;[START_REF] Park | Comparison of different bioreactor systems for indirect H 2 S removal using iron-oxidizing bacteria[END_REF][START_REF] Giro | Immobilized cells of Acidithiobacillus ferrooxidans in PVC strands and sulfite removal in a pilot-scale bioreactor[END_REF][START_REF] Yujian | Immobilization of Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate[END_REF][START_REF] Mousavi | Influence of process variables on biooxidation of ferrous sulphate by an indigenous Acidithiobacillus ferrooxidans. Part II: Bioreactor experiments[END_REF][START_REF] Yujian | High-rate ferrous iron oxidation by immobilized Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate[END_REF]. In many of those cases, A. ferrooxidans cells immobilized could grow at higher dilution rates reaching high iron(III) productivity. Additionally, cells were retained in the support matrix leaving the ferric iron solution almost cell free. Some materials of biological origin exhibit very high sorption capacities. Chitosan has proved to be very efficient for the recovery of several toxic and strategic metals [START_REF] Guibal | Metal ion interactions with chitosana review[END_REF], or dyes [START_REF] Guibal | Chitosan interactions with metal ions and dyes: dissolved-state versus solid-state application[END_REF]. Chitosan results from the alkaline deacetylation of chitin, one of the most abundant polysaccharides in Nature after cellulose. It is commercially produced from shrimp and crab shells. In most cases (partial deacetylation) the biopolymer can be considered as a heteropolymer constituted of acetylglucosamine and glucosamine units. The biopolymer is characterized by its high percentage of nitrogen, present under the form of amine groups that are responsible for the binding of metal cations through chelation mechanisms. Glutaraldehyde has been frequently used to crosslink chitosan and to stabilize it in acidic solutions. The reaction occurs through a Schiff's base reaction between aldehyde groups of the cross-linker and some amine groups of chitosan. The chemical modification of the biopolymer may be used to increase the sorption efficiency but also to improve sorption selectivity and to decrease the sensitivity to environmental parameters [START_REF] Guibal | Metal ion interactions with chitosana review[END_REF]. Chitosan has been employed as support for cell immobilization during the last decade [START_REF] Tartakovsky | Immobilization of anaerobic sludge using chitosan crosslinked with lignosulfonate[END_REF][START_REF] Freeman | Site-protected fixation and immobilization of Escherichia coli displaying surface-anchored betalactamase[END_REF][START_REF] Kumar | A review of chitin and chitosan applications[END_REF][START_REF] Öztop | pH-sensitive chitosan films for baker's yeast immobilization[END_REF][START_REF] Peniche | Chitosan: an attractive biocompatible polymer for microencapsulation[END_REF][START_REF] Freeman | Immobilization of disguised yeast in chemically crosslinked chitosan beads[END_REF][START_REF] Selezneva | Immobilization and long-term culturing of mouse embryonic stem cells in collagen-chitosan gel matrix[END_REF][START_REF] Wang | Immobilization of cells with surface-displayed chitin-binding domain[END_REF][START_REF] Vignoli | The production of sorbitol by permeabilized and immobilized cells of Z. mobilis in sucrose[END_REF].

In the present study the immobilization of A. ferrooxidans cells on chitosan and cross-linked chitosan beads and the biooxidation of ferrous iron to ferric iron in a packed-bed bioreactor were studied.

Materials and methods

Microorganism and cultivation

A strain of A. ferrooxidans from Santa Rosa de Arequipa (DSM11477) was used throughout these experiments. The microorganism was grown and maintained on 9 K medium at initial pH 1.80 [START_REF] Jensen | Ferrous sulfate oxidation using Thiobacillus ferrooxidans: a review[END_REF].

Chitosan and cross-linked chitosan materials

Chitosan was supplied by France Chitine (France) as flaked material. The biopolymer was characterized by a degree of acetylation of 13% and a molecular weight of 125,000 g mol -1 . Gel beads were prepared using a two-step procedure consisting in: (a) chitosan dissolving in acetic acid solution (2.5% w/w of chitosan in acetic acid solution respecting the equimolarity between amine groups and acetic acid), followed by (b) the distribution of the viscous solution through a thin nozzle (0.8 μm, internal diameter) into a neutralizing alkaline bath (1 M NaOH solution). The alkaline solution contributed both to neutralizing the acidity of chitosan solution and to gelling the beads. A spherical shape was assumed for each particle, and the arithmetic mean of the diameters of each bead was used for the surface area and volume of the carrier calculations. The diameter of the beads was measured close to 1.8 mm (±0.2 mm). Water content was close to 96%.

Being chitosan soluble in most mineral and organic acids, with the exception of sulfuric acid, it must be cross-linked when the biopolymer is used in inappropriate acidic solutions. Several processes have been discussed in the literature; the glutaraldehyde cross-linking seems to be the most resistant to acidic environments. The reaction proceeds through a Schiff's base reaction between amine groups of chitosan and aldehyde functions of the cross-linking agent, contributing to the formation of imine linkages between polymer chains. The supplementary linkages cause polymer stabilization in acidic media. Chitosan crosslinking with glutaraldehyde was performed by mixing for 16 h a known amount of chitosan with an equimolar quantity of glutaraldehyde (50% v/v) in a large volume of demineralized water to obtain fluid slurry. The equimolar quantity of glutaraldehyde was calculated taking into account the deacetylation of chitosan: the equimolarity is based on the number of free amine groups of the biopolymer. After cross-linking treatment the solid was separated and washed several times with water to remove the unreacted glutaraldehyde. The size of the beads was not significantly changed by the cross-linking treatment. Gel beads were then dried at 50 °C for 16 h.

Bacterial growth in the presence of chitosan

200 chitosan or cross-linked chitosan beads (1.0 g of dried carrier) were added to 250 ml Erlenmeyer flasks containing 100 ml of 9 K medium (pH 1.80) inoculated with A. ferrooxidans cells. Initial bacterial population was 1.2 × 10 7 cells ml -1 . The flasks were incubated at 30 °C and 180 rpm. Inoculated controls without chitosan were also carried out.

Process of biofilm formation

The biofilm formation was carried out in a packed-bed reactor. The device was based on a glass column with an inner diameter of 2.5 cm and a high of 25.0 cm with inlets for fresh medium and air at the bottom. The outlet for effluent and exhaust air was at the top of the column. The reactor was loaded with 2500 chitosan or cross-linked chitosan beads (12.5 g of dried carrier with an estimated volume and surface area close to 10.5 cm 3 and 314.0 cm 2 respectively). Additionally, the reactor was filled with 100 ml of the 9 K medium (pH 1.80) previously inoculated with A. ferrooxidans cells. An air flow of 72.0 l h -1 was supplied to add the gaseous nutrients. The reactor working volume (V t ) considered for further calculations was 100 ml although the total volume occupied in the reactor consisted in the sum of several volumes (the 9 K fresh medium volume, the total carriers volume, the total fixed film volume and the total volume of air bubbles within the reactor).

In order to allow a fixed biofilm formed on the carrier matrix the reactor was operated in batch mode. When ferrous iron was completely oxidized, medium was replaced by fresh medium without any additional inoculation. After several of these consecutive batch cycles it was considered that the biofilm behavior was properly in place to switch the reactor to a continuous flow. In this mode of operation the flow rates for fresh media (F) were regulated with peristaltic pumps from 4 to 80 ml h -1 in order to obtain different dilution rates (D) ranged between values of 0.01 and 0.8 h -1 . Steady state conditions were used at each flow rate for estimating the kinetics of ferrous sulfate oxidation. After modifying the flow rate, we assumed that no further change in the iron oxidation rate for three residence times (τ = D -1 ) indicated that steady state had been reached. All these experiments were conducted in duplicate.

Analytical procedures

Ferrous iron concentration was measured by titration with potassium permanganate. Total soluble iron was determined by atomic absorption spectrophotometry. The bacterial populations (free in solution) were quantified using a Petroff Hausser counting chamber in a phase-contrast microscope. Both kinds of chitosan beads were analyzed by means of scanning electron microscopy (SEM) before and after cell immobilization in order to compare these materials, to detect the changes in their surfaces and study the morphological aspect of the precipitates at the end of the tests. The beads were removed from the columns; they were dried at 50 °C for 2 h and then they were glued onto a brass mount. The specimens were gold coated before SEM examination. Scanning electron micrographs of the beads were taken in a Philips 515 equipment.

Biofilm was studied by infrared spectroscopy in a Buck 500 spectrophotometer using KI pellet technique with 3 mg of the sample suspended in a transparent disc of 300 mg of KI. The pellet was formed in a dye under high pressure. Additionally, the biofilms were examined by X-ray diffraction in order to identify the precipitates formed on the bead surfaces. For XRD analysis the chitosan beads were withdrawn from the reactor at the end of the test and then the samples were air-dried, ground, and analyzed using a Rigaku DII-Max automated diffractometer between 5-70°2 θ with Cu-Kα radiation at 2°/min rate.

Results and discussion

Evaluation of chitosan beads as carrier for A. ferrooxidans inmobilization

Fig. 1 shows the results obtained during A. ferrooxidans growth in the presence of chitosan and cross-linked chitosan beads. It can be seen that ferrous iron oxidation (Fig. 1A) was not affected by the presence of chitosan beads and was slightly slower in the presence of cross-linked chitosan. On the other hand Fig. 1B shows that the highest productivity of ferric iron was observed in the culture with chitosan (200 mg l -1 h -1 ) while the culture with cross-linked chitosan reached the lowest value (170 mg l -1 h -1 ). The possible bacterial inhibition shown in the last culture could be explained by the presence of little amounts of glutaraldehyde that causes the attachment cell to cell. This is in agreement with large chains of cells observed in the culture with cross-linked chitosan and not in the others. As it can be expected, cell populations in suspension were lower in the presence of chitosan or cross-linked chitosan due to the bacterial attachment to the solids. The high biooxidation rate of ferrous iron in the presence of the biopolymer at level comparable to the control justify using gel beads as a possible support.

Ferrous iron oxidation in repeated batch culture of chitosan beads

In order to obtain a biofilm in the bioreactors containing chitosan and cross-linked chitosan, several isothermal colonization cycles were carried out using 9 K medium at pH 1.80. Fig. 2 shows the performance of the bioreactor loaded with crosslinked chitosan beads which was similar to that observed for untreated chitosan beads.

Along the different steps, both the rate of ferrous iron oxidation and the ferric iron productivity were increasing while the free cell concentration was decreasing until reaching a constant value. The time needed to oxidize all the substrate in the last batch cycle was nearly ten fold lower than in the first one. The maximum iron(II) biooxidation rate of 820 mg l -1 h -1 is comparable with most values reported in literature and even higher than many of them [START_REF] Nikolov | Properties of Thiobacillus ferrooxidans formed in rotating biological contactor[END_REF]Long et al., 2004a). The cell retention after 6 cycles was almost complete while iron precipitation increased continuously during the process of biofilm formation. These facts could suggest that iron precipitation contributes to cell immobilization, which is in agreement to the data reported by [START_REF] Pogliani | Immobilization of Thiobacillus ferrooxidans: importance of jarosite precipitation[END_REF].

Continuous oxidation of ferrous iron in packed-bed bioreactor

At this moment, it was possible to operate the reactor using a continuous flow of 9 K medium at different dilution rates. Fig. 3 shows the iron (III) productivity as a function of the dilution rate (D) for both carriers. The packed-bed reactor allowed increasing the flow rate of medium approximately two fold (untreated chitosan) and eight fold (cross-linked chitosan) without cells washout, compared to a free cell suspension reactor used as control. The advantage of continuous operation systems is reaching higher productivity values than those obtained in the batch mode. The highest ferrous iron oxidation rates were 1100 and 1500 mg l -1 h -1 for untreated chitosan and cross-linked chitosan, respectively. These maximum rates achieved using 12.5 g of support are comparable with most values reported in the literature and higher than many of them. It is possible to compare the performance of chitosan beads as carrier which was achieved in this study with regard to different packed and fluidized bed reactors with glass and resin beads and activated carbon tested by [START_REF] Grishin | Fast kinetics of Fe 2+ oxidation in packedbed reactors[END_REF]. Taking into account the total matrix surface in each reactor the specific productivity of ferrous iron oxidation reached in this study by chitosan beads was higher than the glass beads in a packed-bed reactor flow and activated carbon in a fluidized bed reactor. Only activated carbon in a packed-bed reactor showed to be more efficient than both kinds of chitosan beads. It was also observed that the higher the dilution rate, the lower the iron(III) precipitation. This fact is coincident with the behavior of both iron(II) and iron(III) concentrations in the outlet flow reactor (Fig. 4A). The total iron concentration slightly increases with the dilution rate. Additionally, in Fig. 4B it is possible to see the evolution of the cell concentration in the outlet flow reactor and to estimate the dilution rate that caused the reactor washout.

Chitosan beads and biofilm characterization

Fig. 5 shows the scanning electron micrographs of the surfaces of chitosan (A) and cross-linked chitosan beads (B). It can be seen that the surface of treated beads is more wrinkled than that of untreated beads. Probably this characteristic allowed operating the bioreactor at higher dilution rates.

Fig. 6 shows the infrared spectra of the beads before and after bacterial colonization indicating significant changes. From the chitosan spectrum before bacterial colonization, it can be found that peaks 1 to 3 are the distinctive absorption bands at 1662 cm -1 (Amide I), 1598 cm -1 (-NH 2 bending) and 1380 cm -1 (Amide III). While peaks 4-6 represent the absorption bands at 1156 cm -1 (anti-symmetric stretching of the C-O-C bridge), the peaks at 1075 and 1033 cm -1 (skeletal vibration involving the C-O stretching) are characteristics of the saccharidic (h) structure. Additionally, it is expected to find a broader band near 3300 to 3600 cm -1 (peak 7) that corresponds to H 2 O vibrations [START_REF] Peniche | Self-curing membranes of chitosan/PAA IPNs obtained by radical polymerization: preparation, characterization and interpolymer complexation[END_REF][START_REF] Brugnerotto | An infrared investigation in relation with chitin and chitosan characterization[END_REF][START_REF] Zhang | Synthesis and characterization of chitosan grafted poly(N; N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium) initiated by ceric (IV) ion[END_REF].

After colonization, it was possible to identify the most distinctive IR absorbance frequencies of jarosite (ferric iron sulfate) which are also shown in Fig. 6. From the transmittance spectra of jarosite, the band 8 is primarily due to the structural OH stretching mode and includes water modes as well, specially for synthetic samples. The peak 9, at 1648 cm -1 , is in agreement with the band recorded in jarosite spectrum in the range between 1630 and 1650 cm -1 , which was assigned to water deformation. This band was even observed after prolonged drying acetone-washed samples under vacuum at 80-100 °C. The strongest bands (peaks 10 and 11) are due to the ∨3 (SO 4 2-) stretching vibration and are observed as a doublet near 1100 and 1200 cm -1 . Other peaks were identified in this IR spectrum; one of them ( 12) is a band due to ∨ 1 (SO 4 2-) vibrations. The other peak ( 13) is observed near 1000 cm -1 for jarosite and it is corresponding to ∨ 1 (SO 4 2-) while that another strong doublet is observed near 625 and 660 due to ∨ 4 (SO 4 2-) bending vibration [START_REF] Lazaroff | Iron oxidation and precipitation of ferric hydroxysulfates by resting Thiobacillus ferrooxidans cells[END_REF][START_REF] Sasaki | Morphology of jarosite-group compounds precipitated from biologically and chemically oxidized Fe ions[END_REF][START_REF] Bishop | The visible and infrared spectral properties of jarosite and alunite[END_REF].

In bioreactors to produce ferric iron, jarosite precipitation is an unwanted phenomenon due to the diminishment of the diffusion of reactants and products through the layer of deposits and the blockage of pumps and valves. However, jarosite deposits participate in the process of biofilm formation [START_REF] Pogliani | Immobilization of Thiobacillus ferrooxidans: importance of jarosite precipitation[END_REF][START_REF] Daoud | Formation of jarosite during Fe 2+ oxidation by Acidithiobacillus ferrooxidans[END_REF]. After the first and reversible phase of cell adhesion, jarosite precipitation builds the biofilm matrix. These deposits on the surface would allow the later adsorption of cells on the pores and the formation of the biofilm. That is why the best performance for this kind of bioreactors is shown by support matrices with high initial cell adherence and/or high porosity. These supports can allow fast oxidation of ferrous iron very close to the surface increasing pH and ferric iron concentration and the subsequent jarosite precipitation. As the formation of jarosite is mainly dependent on pH, it has been reported that pH values between 1.6 and 1.8 are adequate not only to maintain high ferrous iron oxidation rates but also enough amounts of ferric precipitates for a good biofilm [START_REF] Pogliani | Immobilization of Thiobacillus ferrooxidans: importance of jarosite precipitation[END_REF].

The three SEM micrographs in Fig. 7 show the outside (A) and the inside (B and D) of the cross-linked chitosan beads collected from the reactor after the precipitation of the ferric hydroxysulfates. The shape of the precipitates shown in Fig. 7A and the occurrence of the characteristic crystals that can be distinguished in Fig. 7B and C are consistent with the data reported by several authors from jarosite precipitation carried out in acidic conditions [START_REF] Lazaroff | Iron oxidation and precipitation of ferric hydroxysulfates by resting Thiobacillus ferrooxidans cells[END_REF]Grishin and Touvinen, 1988;[START_REF] Sasaki | Morphology of jarosite-group compounds precipitated from biologically and chemically oxidized Fe ions[END_REF][START_REF] Cadena | Efecto del mineral de ganga en la síntesis de jarosita de potasio y su distribución de tamaños de partícula[END_REF]. On the other hand, the arrows drawn in Fig. 7C indicates the presence of some bacteria trapped into the biofilm.

The XRD patterns shown in Fig. 7D indicate that jarosite is the main product precipitated on the chitosan beads and there is not evidence of other crystalline by-products co-precipitated. Moreover, from the XRD spectrum obtained it is possible to identify the most representative diffraction bands belonging to jarosite mineral (Grishin and Touvinen, 1988;[START_REF] Stahl | Goethite and jarosite precipitation from ferrous sulphate solutions[END_REF][START_REF] Sasaki | Morphology of jarosite-group compounds precipitated from biologically and chemically oxidized Fe ions[END_REF][START_REF] Kawano | Geochemical modelling of bacterially induced mineralization of schwertmannite and jarosite in sulfuric acid spring water[END_REF][START_REF] Paktunc | Characterization of arsenate for sulfate substitution in synthetic jarosite using X-ray diffraction and X-ray absorption spectroscopy[END_REF].

Conclusions

A. ferrooxidans cells were immobilized successfully in chitosan and cross-linked chitosan beads. The packed-bed reactor allowed increasing the flow rate of medium approximately two fold (chitosan untreated) and eight fold (chitosan cross-linked) without cells washout, compared to a free cell suspension reactor used as control, and to reach ferric iron productivities as high as 1100 and 1500 mg l -1 h -1 respectively. Differences found in the performance of continuous bioreactor using untreated chitosan and cross-linked chitosan could be attributed to a more wrinkled bead surface in the last case. It was possible to identify the most distinctive peaks of jarosite in the infrared spectra and in the XRD patterns from the precipitates produced after colonization which could play an important role during biofilm formation.

Fig. 1 .

 1 Fig. 1. A. ferrooxidans growth in the presence of chitosan and cross-linked chitosan beads. (A) Ferrous iron concentration versus time. (B) Final cell population in suspension and ferric iron productivities.

Fig. 2 .

 2 Fig. 2. Ferrous iron evolution along each isothermal colonization cycle and final values of free cell population, ferric iron productivity and total iron precipitated over the cross-linked chitosan beads.

Fig. 4 .

 4 Fig. 4. The iron and cell concentrations in the outlet flow reactor loaded with cross-linked chitosan beads.

Fig. 7 .

 7 Fig. 7. A) Scanning electron micrograph of jarosite precipitation on cross-linked chitosan beads collected from the reactor after the test. Images B) and C) show the SEM micrographs taken from the inside of the same chitosan beads. Some characteristic jarosite crystals can be distinguished and the arrows could indicate some bacteria trapped into the biofilm. Note: Bar size = 10 μm D) X-ray diffraction pattern which allow identifying the jarosite precipitates.

Fig. 6 .

 6 Fig.6. IR spectra for chitosan beads before and after colonization. The peaks 1-7 are the distinctive absorption bands used to characterize this saccharide structure. The peaks 8-13 show the absorption bands detected after colonization most of them belong to the jarosite spectrum.

Acknowledgement

This work has been supported by Facultad de Ingeniería, Universidad Nacional del Comahue, partially by ANPCyT (PICT 25300) and CONICET (PIP 5147) from Argentina. Alfa Programme (Contract number AML/190901/06/18414/II-0548-FC-FA) is also gratefully acknowledged.