Charlène Chorao

Franck Charmantray

Pascale Besse-Hoggan

Martine Sancelme

Angela Cincilei

Mounir Traïkia

Gilles Mailhot

Anne-Marie Delort

2-Aminobenzothiazole degradation by free and Ca-alginate immobilized cells of Rhodococcus rhodochrous

Keywords: Photodegradation, Biodegradation, 31 P NMR, Benzothiazole, FeNTA, Rhodococcus rhodochrous

2-Aminobenzothiazole (ABT) degradation was investigated using free and immobilized systems during photodegradation under solar light in the presence of Fe(III)-nitrilotriacetic acid (FeNTA), biodegradation by Rhodococcus rhodochrous, and during combined conditions. Ca-alginate hydrogel was chosen as a model matrix and some complementary studies were required to characterize this new system. R. rhodochrous metabolism in this type of environment was monitored by NMR spectroscopy. Neither change in intracellular pH values nor in ATP concentrations was observed by in vivo 31 P NMR, showing that no metabolic modification occurred between free and immobilized cells. 1 H NMR demonstrated that alginate was not used as carbon source by R. rhodochrous. After establishing the pre-treatment protocol by SPE to eliminate solubilised alginate, ABT adsorption on beads and degradation were studied. The same pathways of transformation were observed in suspended and immobilized cell systems. Considering the ABT adsorption phenomenon on alginate beads (8%), the efficiency of the two systems was found to be comparable although the degradation rate was slightly lower with immobilized cells. The most important result was the finding that the positive effect of FeNTA on ABT degradation with immobilized cells was similar to that observed previously with free cells. All these results show that mechanisms observed with free cells can be extrapolated to entrapped cells, i.e. under conditions much closer to those usually encountered in the environment.

Introduction

Benzothiazoles belong to an extensive family of synthetic compounds. Their skeleton is constituted by fused benzene and thiazole rings. Benzothiazoles are used in various fields e.g. in the tire industry as catalysts in vulcanization processes [START_REF] Ghosh | Sulfur vulcanization of natural rubber for benzothiazole accelerated formulations: from reaction mechanisms to a rational kinetic model[END_REF], as medicines for amyotrophic lateral sclerosis ( [START_REF] Bryson | Riluzole: a review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in amyotrophic lateral sclerosis[END_REF] and [START_REF] Lacomblez | Dose-ranging of riluzole and amyotrophic lateral sclerosis[END_REF]) and cancer chemotherapeutics [START_REF] Dubey | 2-(4-Aminophenyl)benzothiazole: a potent and selective pharmacophore with novel mechanistic action towards various tumour cell lines[END_REF], as pesticides/biocides [START_REF] Brownlee | Aquatic environmental chemistry of 2-(thiocyanomethylthio)benzothiazole and related benzothiazoles[END_REF] and in the manufacture of azo-dyes [START_REF] Gaja | The microbial degradation of benzothiazoles[END_REF]. As a consequence of their large range of applications, these compounds are often found in the aquatic environment [START_REF] Kloepfer | Occurrence of benzothiazoles in municipal wastewater and their fate in biological treatment[END_REF], [START_REF] Kloepfer | Occurrence, sources, and fate of benzothiazoles in municipal wastewater treatment plants[END_REF], [START_REF] Reemtsma | Polar pollutants entry into the water cycle by municipal wastewater: a European perspective[END_REF] and [START_REF] Wever | Comparison of sulfonated and other micropollutants removal in membrane bioreactor and conventional wastewater treatment[END_REF]). Some of these molecules show toxic effects towards microorganisms, animals or humans. Therefore, it is important to investigate the mechanisms involved in the photo and biodegradation processes of such compounds in the environment. First studies of biodegradation reported the degradation of benzothiazole-2-sulfonic acid by activated sludges [START_REF] Mainprize | The fate of benzothiazole-2-sulfonic acid in biologically treated industrial effluents[END_REF]. Some years later, several strains of Rhodococcus able to degrade certain benzothiazoles were isolated ( [START_REF] Gaja | The microbial degradation of benzothiazoles[END_REF], [START_REF] Wever | Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole[END_REF] and [START_REF] Wever | Initial transformations in the biodegradation of benzothiazoles by Rhodococcus isolates[END_REF]). Biodegradation pathways of benzothiazole, 2-hydroxybenzothiazole and 2-mercaptobenzothiazole have been elucidated with these Rhodococcus strains ( [START_REF] Besse | Long range 1 H-15 N heteronuclear shift correlation at natural abundance: a tool to study benzothiazole biodegradation by two Rhodococcus strains[END_REF], [START_REF] Haroune | Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2dioxygenase activity[END_REF] and [START_REF] Haroune | Metabolism of 2-mercaptobenzothiazole by Rhodococcus rhodochrous[END_REF]). In a recent paper, Bunescu et al. (2008a) investigated in detail the degradation pathways of 2-aminobenzothiazole (ABT), a compound used as reagent in azo-dye manufactories. They tested various conditions that could occur in the environment: (1) direct photodegradation under sunlight and photo-induced degradation in the presence of Fe(III)-nitrilotriacetic acid (FeNTA). This complex is usually used as a model of natural organic matter complexes of iron, (2) biodegradation by Rhodococcus rhodochrous cells, (3) two combined conditions, R. rhodochrous cells and FeNTA with or without irradiation. Direct photolysis under sunlight did not show any degradation of ABT since this molecule does not absorb in the solar spectrum, but the addition of FeNTA complex led to the transformation of ABT into 2-amino-6-hydroxybenzothiazole (6OH-ABT) and 2-amino-4-hydroxybenzothiazole (4OH-ABT) (Scheme 1). The biodegradation process led to the accumulation of 6OH-ABT, while the combined processes (biodegradation in the presence of FeNTA with or without exposure to sunlight) produced 6OH-ABT and 4OH-ABT. Apart from the identification of the pathways involved in these different ABT degradation processes, Bunescu et al. (2008a) showed a very important feature: the presence of FeNTA, even in the absence of light, improved dramatically the rate of ABT biodegradation. Indeed 99% of ABT was degraded after 25 h in the combined systems while 37% and 26% of ABT were photo-and biodegraded after 125 h, respectively. In order to understand better which mechanisms were responsible for this improvement of ABT degradation rate, the fate of FeNTA was investigated (Bunescu et al., 2008b). It was shown that FeNTA was transported and biotransformed inside the cell and/or phototransformed in the presence of sunlight (Bunescu et al., 2008b). The same products were obtained during the photo-and biodegradation processes of FeNTA, including IDA (iminodiacetic acid), glycine and formate, probably because they both involve oxidoreduction mechanisms. Photochemistry of FeNTA led to the production of radicals that activated the degradation of ABT. The intracellular Fe(II) resulting from the enzymatic reduction of Fe(III) was likely to activate degradation enzymes (dioxygenases) that contain iron in their active site and therefore increased the biodegradation rate of ABT. These unexpected results and mechanisms concerning the biodegradation of ABT in the presence of FeNTA (with or without irradiation) were observed with suspensions of free R. rhodochrous cells. However, microorganisms involved in biodegradation mechanisms are not usually found as free cells. They can be naturally entrapped or attached to matrices and form biofilms in natural waters because of extracellular polymers that they secrete themselves, or aggregates such as flocks in water treatment plants. The immobilization can also be artificial for various biotechnology applications [START_REF] Junter | Immobilized viable microbial cells: from the process to the proteome... Or the cart before the horse[END_REF]. It thus appeared very important to investigate transformation processes under conditions closer to environmental ones, i.e. with cells entrapped in a matrix to see whether the same mechanisms occurred as those with free cells. In this context, the main objective of this work was to compare ABT degradation by free and immobilized cell (IC) systems during various transformation processes: photodegradation in the presence of FeNTA, biodegradation by R. rhodochrous and during combined processes. More precisely, the aims were (1) to compare degradation pathways and efficiencies between free and immobilized cells, (2) to investigate whether the FeNTA complex still stimulated ABT degradation in the IC system and whether the mechanisms observed with free cells could be extrapolated to other physiological conditions involving matrix entrapped cells, much closer to environmental conditions. Ca-alginate hydrogel was chosen as a model matrix because many papers describe the entrapment of Rhodococcus cells with alginate and especially when rhodococci degrade organic pollutants (e.g. [START_REF] Pai | Continuous degradation of phenol by Rhodococcus sp. immobilized on granular activated carbon and calcium alginate[END_REF], [START_REF] Mukerjee-Dhar | Degradation of polychlorinated biphenyl by cells of Rhodococcus opacus strain TSP203 immobilized in alginate and in solution[END_REF]] and [START_REF] Naito | Long-term repeated biodesulfurization by immobilized Rhodococcus erythropolis KA2-5-1 cells[END_REF]). Complementary studies were carried out to characterize this new system: (i) NMR study of R. rhodochrous metabolism in such an environment, (ii) adsorption measurements of ABT and FeNTA on alginate beads. In addition the set-up of a pre-treatment protocol to eliminate dissolved alginate from incubation media was necessary in order to monitor ABT and the degradation products by HPLC.

Materials and methods

2.1. Chemicals 2-Aminobenzothiazole (97%), hydroxylamine hydrochloride (99%), 3-(2-pyridyl)-5,6-diphenyl-1,2,4triazine-4,4′-disulfonic acid sodium salt (ferrocine, 97%) and Fe(III) perchlorate nonahydrate (99%) were purchased from Aldrich. Nitrilotriacetic acid (>99.5%) and methylenediphosphonic acid (MDP) ( 99%) were purchased from Fluka, 1,10-phenanthroline (>99.5%) from Riedel-de-Haën, tetradeuterated sodium trimethylsilylpropionate (TSPd 4 ) from Eurisotop, sodium acetate (>99%) and ammonium acetate (>99%) from Merck, ammonia, hydrochloric acid, sulfuric acid and HEPES from Acros and alginic acid (polymannuronic acid) sodium salt (low viscosity: 40-90 mPa s in 1% aqueous solution) from Lancaster. 2-Amino-6-hydroxybenzothiazole and 2-amino-4-hydroxybenzothiazole were synthesized in the laboratory (Bunescu et al., 2008a).

Preparation of FeNTA solution

A 2 mM FeNTA solution was prepared in Volvic ® mineral water. NTA was completely dissolved in NaOH (1 M) and pH was adjusted to 4.0. A solution of iron perchlorate was then added, in stoichiometric proportions, to the NTA solution. The solution obtained was protected from light, stirred for one hour and then stocked at 4 °C. Perchlorate was used as a non-photosensitive and non-oxidant counter-anion rather than chlorate anion that might contribute to the oxidation of benzothiazoles. A UV-visible spectrum was recorded to check that the complete complexation of iron was completed (FeNTA ε 296 nm = 3300 M -1 cm -1 ). Solution was stored at pH 4.0 for better stability. pH was then adjusted to 7.0 just before the experiment.

Photobioreactor

The photobioreactor was equipped with 7 TLD Philips 15 W fluorescent tubes emitting within the wavelength range λ = 300-500 nm with a maximum emission at 360 nm which corresponds to part of the solar spectrum. The temperature was kept at 27 °C and a stirring system ensured the oxygenation and homogeneity of the media. The polychromatic photon flux was measured and controlled for each position with a Testo 545 luxmeter.

Growth conditions

R. rhodochrous OBT18 was grown in 100 mL portions of Trypcase-soy broth (bioMérieux, Marcy l'Etoile, France) in 500 mL Erlenmeyer flasks incubated at 27 °C, 200 rpm and for 24 h. 300 mL of the culture medium (≈3 g of wet-weight cells) were then centrifuged at 8000 rpm, at 4 °C and for 15 min. The bacterial pellet was first washed with NaCl (8‰) and then with Volvic ® mineral water.

Alginate bead preparation

Alginate (0.75 g) was added to 10 mL of distilled water and autoclaved (20 min at 121 °C) to make the dissolution easier. A solution containing 8 mL of 8‰ NaCl and 3 g of cells was then added to the alginate preparation and the mixture was stirred, slowly to avoid air bubbles, for 15 min. This mixture was extruded dropwise through a needle (Ø 0.8 mm) to a 0.2 M CaCl 2 solution where they gelled instantaneously giving spherical entrapment beads. To ensure better stability, the beads were stored at 4 °C for 90 min. They were washed twice with NaCl (8‰) and then stored at 4 °C in 100 mL of NaCl (8‰). The Ca-alginate beads had a diameter of 3 mm and contained ≈10 7 bacteria each. The same protocol was used for the controls without cells.

Incubation with ABT

Cells (3 g) were incubated with 100 mL of ABT (0.5 mM) and/or FeNTA (1 mM) in transparent or opaque 500 mL flasks depending on whether or not irradiation of the solution was required. Stocked solutions of ABT and FeNTA twice concentrated were prepared in Volvic ® mineral water, in order to work with water of constant composition. The pH of the FeNTA solution was adjusted to 7.0 just before the experiment. Samples (2 × 1 mL) were regularly taken from each flask, centrifuged at 12 500 rpm for 3 min and then frozen till analyses.

Incubation with alginate

Cells (3 g) were incubated with 100 mL of a 400 mg L -1 alginate solution. Samples of 5 mL were taken every 24 h and freeze-dried after centrifugation. The residue was dissolved in 500 µL of D 2 O, freezedried again and dissolved in 500 µL of D 2 O to perform 1 H NMR analyses.

Analytical techniques

All the experimental points are a mean value of three samplings. Moreover each experiment was repeated at least twice. The experimental error for the ABT and iron measurements was estimated to be less than 8%.

HPLC

Analyses were carried out with an Agilent 1100 series apparatus fitted with a DAD detector and driven by Chemstation software. Analyses were performed in isocratic mode at 25 °C with a reversed phase endcapped column (Zorbax Eclipse XDB-C18, Agilent, 250 mm × 4.6 mm, 5 µm, 80 Å). The mobile phase was composed of acetonitrile and Milli-Q water in 25/75 ratio with a flow rate of 1 mL min -1 . Spectra were recorded between λ = 200 and 400 nm, the detection wavelength was λ = 265 nm. Ten microliters of the sample were injected with a 15 min run time.

Iron analyses

Iron analyses were performed with a liquid handling robot, Freedom Evo from TECAN ® , coupled with a Safire 2 UV-visible detector. Ninety-six well plates were used for the assays.

Total iron titration

Fe(III) species were reduced in Fe(II) with hydroxylamine chlorhydrate (3 M in 30% of hydrochloric acid). Addition of ferrocine (0.02 mM in Milli-Q) led to the formation of a strong colored magenta complex with Fe(II) stable at basic pH [START_REF] Stookey | Ferrozine -a new spectrophotometric reagent for iron[END_REF]. For the titration of a sample, 10 µL of supernatant was reduced with 15 µL of hydroxylamine chlorhydrate, 150 µL of Milli-Q water was then added and the solution was stirred for 1 min at 20 Hz. The addition of 15 µL of ferrocine was followed by the addition of 30 µL of ammonium acetate buffer (5 mM in ammonia 25%) and 30 µL of Milli-Q water. Absorbance was recorded at λ = 562 nm, corresponding to the maximal absorption of the magenta complex.

SPE treatment

The incubation medium with alginate beads contained dissolved alginate which was removed by Solid Phase Extraction (SPE) treatment using Oasis ® HLB cartridges (Waters, 30 µm, 10 mg, 1 mL) (Fig. 1A). The work-up was performed with the TECAN ® liquid handling robot using 96-cartridge plates. The phase was conditioned with 350 µL of methanol and washed with 700 µL of Milli-Q water. The sample (200 µL) was loaded and the phase was washed with 700 µL of Milli-Q water. The compounds were eluted with 200 µL of acetonitrile and 600 µL of Milli-Q water were added to reproduce the chromatographic conditions (mobile phase: acetonitrile/water in 25/75 ratio).

1 H NMR spectroscopy for study of the alginate metabolism by Rhodococcus rhodochrous

The spectrometer used was a Varian 600 MHz UNITY INOVA 600 NB equipped with a 5 mm multinuclear probe with inverse detection and 5 mm 1 H-13 C-15 N-PFG Triple Res IDTG600-5 with zgradients in D 2 O using TSPd 4 as internal standard. Water resonance was suppressed by standard sequence PRESAT. Spectra were processed with Topspin version 2.0.

In vivo 31 P NMR spectroscopy

The spectrometer used was a Bruker Avance 500 equipped with a 10 mm Broadband probe. Methylene diphosphonic acid (0.25 mM) in 100 mM Hepes buffer pH 7.5, contained in an in situ capillary was used as an internal reference to calibrate chemical shifts (16.38 ppm relative to 85% H 3 PO 4 (0 ppm)).

Incubation medium was composed of only Volvic ® Water containing 10% D 2 O. Spectra were recorded with inverse-gated 1 H decoupling (WALTZ16 sequence). Acquisitions parameters: number of scans: 25 400 (corresponding to 2 h acquisition for each spectrum); 90° pulse time: 10 µs; relaxation delay: 0.05 s; acquisition time: 0.2 s; spectral window: 11160 Hz; data points: 4 K. Spectra were processed with Topspin version 2.0.

Perfusion system

31 P NMR experiments were performed in vivo using a perfusion system described in Fig. 2. R. rhodochrous cells were perfused with Volvic ® water saturated with oxygen. The perfusion rate (flow rate of 20 mL min -1 ) was controlled by a peristaltic pump. Capillary containing MDP as reference was inserted in the center of the tube. When experiments were performed with alginate immobilized bacteria, the beads were placed in the analysis area. Bacteria in suspension circulated continuously in the system.

Temperature was regulated at 27 °C.

Fig. 2. Perfusion set-up to perform in vivo 31 P NMR studies. Rhodococcus rhodochrous cells are perfused with oxygen saturated Volvic ® water from a central reservoir. The perfusion rate (flow rate of 20 mL min -1 ) is controlled by a peristaltic pump. Capillary containing MDP as reference is inserted in the center of the tube. When bacteria are immobilized in alginate beads, the beads are placed in the measurement area; when bacteria are free, they circulate continuously in the system.

Temperature is regulated at 27 °C.

Results

Entrapment of Rhodococcus rhodochrous in Ca-alginate beads 3.1.1. Metabolic status of Rhodococcus rhodochrous cells entrapped in alginate beads

The energetic metabolic status of Rhodococcus rhodochrous cells entrapped in Ca-alginate beads was compared to that of free cells by in vivo 31 P NMR [START_REF] Grivet | NMR for microbiology: in vivo and in situ applications[END_REF]. The 31 P NMR spectra collected for immobilized and free cells (Fig. 3A

Alginate degradation by Rhodococcus rhodochrous

Alginate is a potential carbon source for bacteria [START_REF] Kitamikado | Two types of bacterial alginate lyases[END_REF]; therefore the metabolism of Rhodococcus cells towards alginate was investigated. Rhodococcus cells in suspension were incubated with alginate in aqueous medium. The incubation was monitored during 9 days by in situ 1 H NMR performed directly on the incubation medium without purification. 1 H NMR signals corresponding to the sugar moieties of alginate did not change with time thus proving that R. rhodochrous does not use alginate as a carbon source (Supplementary material Fig. 1).

Removal of alginate from the incubation media

Mechanical stirring of alginate beads in water medium leads to partial dissolution of alginate in the medium with time. The HPLC column can be blocked by its presence so its removal is essential to complete further analysis. Its elimination was achieved by a pre-treatment based on Solid Phase Extraction (SPE). After several phase tests, the Oasis ® HLB cartridge (Waters) was chosen. Its solid phase is composed of two polymers: a lipophilic and a hydrophilic monomer, a balance which enables the retention of hydrophobic and non-charged compounds. The alginate removal was controlled by 1 H NMR at each step of the SPE treatment (Fig. 1B). The 1 H NMR spectrum recorded before treatment of the supernatant showed the presence of alginate signals in the range between 3.50 and 5.30 ppm (Fig. 1B, spectrum 1). The spectrum of the solution harvested after loading (Fig. 1B, spectrum 2) clearly showed the presence of alginate with a recovery rate of 80%. The spectrum of the first washing solution loading (Fig. 1B, spectrum 3) showed a recovery rate of 10% but the spectrum of the second washing solution loading (Fig. 1B, spectrum 4) did not display any alginate signals. These results show that one washing step is necessary and that a second one is preferable to ensure complete removal of the polysaccharide.

The spectrum of the solution after elution (Fig. 1B, spectrum 5) did not show any peaks corresponding to alginate. Therefore this SPE protocol is very efficient for alginate removal from the samples. To ensure a quantitative recovery of the compounds studied, the protocol was applied to pure solutions of various concentrations of ABT and its metabolite 6OH-ABT (0.10, 0.125, 0.25 and 0.5 mM): ABT and 6OH-ABT recovery rates were found to be 100% and 94%, respectively after SPE treatment.

Adsorption on alginate beads

Because alginate beads are macromolecular structures with cavities and because alginate displays alcohol and carboxylic acid functions, this material might adsorb ABT and FeNTA and therefore mislead the interpretation of quantitative data concerning degradation processes in the presence of alginate. Experiments were performed in the presence of alginate beads which did not contain bacteria, without irradiation. In order to quantify only adsorption, ABT and FeNTA adsorption were assayed in independent experiments on this material (concentrations tested: ABT, 0.5 mM and FeNTA, 1 mM). The concentration of ABT in the incubation medium was measured by HPLC after SPE treatment while total iron was measured directly by spectrophotometry. Results are reported in Table 1. ABT was rapidly adsorbed on alginate beads: 8% (0.04 mM) disappeared from the medium within 30 minutes. Equilibrium between adsorbed and free ABT was then reached under the conditions used. On the contrary, no adsorption of iron was observed. However, with free and Ca-alginate immobilized cells (IC), iron concentration decreased by about 20% in the solution. This result could be attributed to a phenomenon of FeNTA decomplexation observed at pH higher than 7.0 (Abida, 2005), which was the pH reached in the presence of cells. Under these conditions, free iron precipitates.

Comparison of the degradation processes with free and immobilized cells (IC)

The various conditions of ABT degradation studied during this work are presented in Table 1 in the Supplementary material. They correspond to different combinations of processes that can occur in the environment: bacteria alone free and immobilized, sunlight alone, presence of iron complexes, combination of various parameters.

Biodegradation process

Experiments were carried out to compare both the ABT biodegradation pathway and the efficiency of the process conducted by free and Ca-alginate immobilized R. rhodochrous cells. The effect of irradiation was also tested on these two cellular systems in order to assess the potential for protection of the microorganisms by the alginate beads with respect to light.

Table 2 shows the evolution of ABT concentrations measured by HPLC in free and IC systems with or without irradiation. In both systems, the biodegradation of ABT was more efficient in the absence of light, showing that alginate does not in fact protect the cells. Rhodococcus cells are indeed slightly sensitive to light but to a low extent because they produce certain red pigments under irradiation that naturally protect them (data not shown). By considering the adsorption effect of ABT on alginate beads, similar degradation rates were observed in both IC and free cell systems. After 100 h of incubation, the conversion percentages of ABT were very similar: in the presence of light, 14% were converted with cells in suspension and 22% (adsorption considered) with the alginate system; in the absence of light, 28% and 30% (adsorption considered), respectively. No 6OH-ABT was observed in irradiated cases. In the absence of irradiation, the concentration of 6OH-ABT was similar in both systems.

Photodegradation process (FeNTA ± light)

The effect of alginate beads (without cells) on the photodegradation process induced by FeNTA was assessed with or without irradiation. In both systems, ABT was almost not degraded in the absence of light. The slight decrease of ABT concentration resulted only from its adsorption on alginate beads. Under irradiation, evolutions of ABT concentration with time were similar in both systems. After 100 h of irradiation, 46% of ABT was photodegraded in solution versus 38% (corrected value) with alginate beads. Moreover, the presence of alginate beads did not modify the photodegradation pathway (the same intermediates were observed). This result is important for understanding combined photo-biodegradation processes conducted with Ca-alginate embedded cells.

Combined bio-and/or photodegradation process

As mentioned in the first part of the results, the light had no real effect on the combined processes and only experiments without light are presented. The combined process consisted of assays with FeNTA and R. rhodochrous cells. The most important effect was due to the presence of FeNTA which greatly accelerates the ABT degradation compared with bacteria alone. After 29 h, in the absence of light, 22% and 26% (adsorption considered) (Table 2) of ABT was converted with the biodegradation process for the suspension cell and the IC systems, respectively, and 96% and 76% (adsorption considered) with the combined process (Fig. 4A). This strong effect of FeNTA was observed previously for free cells (Bunescu et al., 2008a) and is confirmed here for embedded cells. The degradation in the free cell system with FeNTA is slightly more efficient than in alginate beads. Fig. 4B shows, for free cell system, a rapid production of 6OH-ABT, reaching 0.05 mM after 3 h, followed by a complete disappearance after 145 h. In the IC system 6OH-ABT is also produced but more slowly. Its concentration is lower; the kinetics appears to be slowed down with encapsulated cells. Fig. 4C shows the same evolution tendencies for 4OH-ABT. Concerning total iron titration presented in Fig. 4D, in both cases, a regular decrease is observed. Nevertheless much more total iron is consumed in the IC system although the ABT degradation is less efficient (0.08 mM in IC system versus 0.37 mM in free cells after 145 h of incubation). A breaking-up of alginate beads is observed with time: interactions between the units of sugar and the calcium cation responsible for the gelling are probably replaced by interactions with iron cation. This exchange of cation makes iron unavailable in alginate experiments and explains the difference of total iron concentrations between the two systems [START_REF] Sreeram | Studies on the nature of interaction of iron(III) with alginates[END_REF].

Discussion and conclusion

In this paper, the degradation of ABT by free and Ca-alginate immobilized R. rhodochrous cells was investigated, and the two systems compared. This comparison required the development of an SPE procedure to eliminate alginate from the incubation samples. Coupled with a 96-well microtiter plate, this protocol allowed large series of analyses. Because alginate is one of the most frequent matrices used to entrap cells, this analytical technique can be useful for other studies. The first important result of this work is that ABT degradation follows the same pathway in systems containing alginate beads or not, with the same intermediates: 6OH-ABT and 4OH-ABT in the photodegradation and combined processes; 6OH-ABT in the biodegradation process. Considering the small adsorption (8%) of ABT on Ca-alginate gel, the efficiency of ABT degradation in the different systems is comparable. However we noticed a slightly slower degradation rate in the presence of alginate beads. This is not due to a phenomenon of co-metabolism with alginate since we have shown that R. rhodochrous does not use alginate as a carbon source. In addition we proved, using in vivo 31 P NMR, that free and immobilized cells had a similar energetic status. Clearly the Ca-alginate matrix had no negative effect on R. rhodochrous metabolism which could have been responsible for the small decrease of degradation efficiency. We suggest that a slightly restricted diffusion of the xenobiotic through the Caalginate matrix could occur. Also, iron could partly replace Ca in the gel matrix [START_REF] Min | Arsenate sorption by Fe(III)-doped alginate gels[END_REF]] and [START_REF] Sreeram | Studies on the nature of interaction of iron(III) with alginates[END_REF]) avoiding its uptake by the bacteria and thus the activation of bacterial degradative enzymes. All these results show that mechanisms observed with free cells can be extrapolated to entrapped cells, i.e. conditions much closer to those usually encountered in the environment. Finally, the major result is that the positive effect of FeNTA on ABT degradation is observed with immobilized cells in a similar way as was shown with free cells. This feature is very important with respect to the environmental context. Indeed FeNTA is considered as a good model of iron natural matter complexes existing in surface waters. Moreover, NTA, used as a domestic detergent, is present in large quantities in water treatment plants [START_REF] Egli | Biodegradation of metal-complexing aminopolycarboxylic acids[END_REF]. Thus, FeNTA could activate bacteria which can be in the form of immobilized cells (biofilms) in this environment [START_REF] Junter | Immobilized viable microbial cells: from the process to the proteome... Or the cart before the horse[END_REF].

Fig. 1 .

 1 Fig.1. Alginate removal by SPE method. (A) Extraction scheme, (B) 1 H NMR spectra (sugar zone), 1: before SPE treatment, 2: after loading, 3: after the 1st wash, 4: after the 2nd wash, 5: after elution.2.8.5. 1 H NMR spectroscopy for study of the SPE treatmentThe spectrometer used was a Bruker Avance 500 equipped with a 5 mm triple-tuned 1 H-13 C-15 N probe fitted with a z-gradient coil. Water resonance was suppressed by low presaturation method. TSPd 4 (5 mM) dissolved in D 2 O was used as internal reference. Acquisition parameters: number of scans: 128; impulsion time: 6.3 µs; relaxation delay: 5 s; acquisition time: 4.67 s; spectral window: 7002 Hz; data points: 65 536. Spectra were treated with Topspin version 2.0. For the preparation of samples, 60 µL of TSPd 4 solution in D 2 O (5 mM) was added to 540 µL of the supernatant.

  and B, respectively) showed different phosphorylated metabolites that were identified by comparison with the literature[START_REF] Lohmeier-Vogel | In vivo 31 P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli[END_REF]): α-ATP (δ = -10.6 ppm), β-ATP (δ = -19.1 ppm), γ-ATP (δ = -5.4 ppm), β-ADP (δ no precisely determined), Pi (δ = 2.2 ppm), sugar phosphates (δ = no precisely determined), α-UDPG and NAD(P)H (δ = -11.0 ppm), β-UDPG signals (δ = -12.5 ppm). Whatever the cell system studied, these metabolic markers presented the same pattern or abundance. In particular, intracellular pHs determined from Pi chemical shifts were similar (pH 6.9) and ATP concentrations determined from the integral of β-ATP signals were the same. These experiments clearly show that encapsulation in alginate beads does not modify the energetic status of Rhodococcus cells.

Fig. 3 .

 3 Fig. 3. 31 P NMR spectra acquired in vivo on immobilized (A) and suspended (B) cell system. X unidentified metabolite.

Fig. 4 .

 4 Fig. 4. ABT degradation by the combined process (ABT: 0.5 mM, FeNTA: 1 mM, no light): comparison between free suspended cells and IC systems. ( , -) immobilized bacteria; (■, ) bacteria in suspension. Concentrations of ABT (A), the metabolite 6OH-ABT (B), the metabolite 4OH-ABT (C) and total iron (D).

Table 1 .

 1 Adsorption of ABT and iron on alginate beads (absence of light and of bacteria). Concentrations are measured in the incubation medium by HPLC (ABT) and by spectrophotometry (total iron).

		Time (h)
		T0	0.5	1	2	3	21	77	145
	No alginate beads ABT (mM)	0.50 0.48 0.48 0.49 0.49 0.48 0.49 0.50
	Alginate beads	0.50 0.46 0.45 0.44 0.44 0.45 0.44 0.44
	No alginate beads Total iron (mM)	1.00 0.82 0.76 0.76 0.76 0.83 0.85 0.79
	Alginate beads	1.00 0.90 0.89 0.88 0.87 0.75 0.78 0.81

Table 2 .

 2 Percentages of ABT degradation during biodegradation processes: comparison between IC and free cell systems. Concentrations are measured by HPLC (values between brackets: corrected values with the value of ABT adsorption on alginate beads).

				T0 (%)	0.5 h (%)	2 h (%)	3 h (%) 29 h (%) 77h (%)	100 h(%)
	Biodegradation	Free cells	Light	0	12	14	12	12	16	14
			No light	0	12	10	8	22	28	28
		Alginate beads Light	0	20 (12)	16 (8)	28 (20)	22 (14)	24 (16)	30 (22)
			No light	0	28 (20)	18 (10)	26 (18)	34 (26)	38 (30)	38 (30)

Acknowledgements

We would like to thank the INTAS for its financial support and the Région d'Auvergne for C. Chorao's PhD fellowship. We greatly acknowledge Elisabeth Gout and Richard Bligny (CEA Grenoble, France) for their help in setting-up the perfusion system for in vivo NMR experiments. We would like to thank Elodie Cano for microbiological assistance and Maria Matulova for her NMR study on alginate degradation by R. rhodochrous.


Basam A E Ben-Arfa

Stéphane Abanades

Isabel M Miranda Salvado

José M F Ferreira

Robert C Pullar
email: robertcarlyle.pullar@unive.it

Robocasting of 3D Printed and Sintered Ceria Scaffold Structures with Hierarchical Porosity for Solar Thermochemical Fuel Production from the Splitting of CO 2

We report the first ever robocast (additive manufacturing / 3D printing) sintered ceria scaffolds, and explore their use for the production of renewable fuels via solar thermochemical fuel production (STFP, water and carbon dioxide splitting using concentrated solar energy). CeO 2 catalyst scaffolds were fabricated as 50 mm diameter discs (struts and voids ~500 m), sintered at 1450 °C, with specific surface area of 1.58 m 2 /g. These scaffolds have hierarchical porosity, consisting of the macroporous scaffold structure combined with nanoscale porosity within the ceria struts, with mesopores <75 Å and an average pore size of ∼4 nm, and microporosity <2 nm with a microporous surface area of 0.29 m 2 /g. The ceria grains were ≤500 nm in diameter after sintering. STFP testing was carried out via thermogravimetric analysis (TGA) with reduction between 1050-1400 °C under argon, and oxidation at 1050 °C with 50% CO 2 , gave rapid CO production during oxidation, with high peak CO production rates (0.436 mol/g/s, 0.586 ml/g/min), for total CO yield of 78 mol/g (1.747 ml/g). 90% CO was obtained after just 10 min of oxidation, comparing well to reticulated ceria foams, this CO production rate being an order of magnitude greater than ceria powders when tested at similar temperatures.

Introduction

Cerium dioxide (ceria) is an extremely versatile oxide with a wide range of applications in catalysis, due to its ability to reduce to the nonstoichiometric form, CeO 2- , containing a mixture of Ce 4+ and Ce 3+ ions. Pure stoichiometric CeO 2 has the face centred cubic calcium fluoride (fluorite) structure, and it can tolerate a considerable degree of reduction without change of crystal phase, withstanding reduced nonstoichiometries of  up to 0.286, [1] especially at elevated temperatures. [2] Cerium can also form cerium(III) oxide, Ce 2 O 3 , but this is unstable and will easily oxidise to cerium(IV) oxide (CeO 2 ). Ceria undergoes partial and reversible reduction at high temperatures (>1000 °C) in low pO 2 (10 -5 atm) atmospheres, and it contains many oxygen vacancies within its structure, which is essential for improving oxygen mobility. [3] CeO 2 has an unusually high entropy change associated with oxygen exchange, which can result in lowered temperature swings between the reduction and oxidation steps [4] compared to other non-stoichiometric redox materials. It also features rapid reaction kinetics and oxygen diffusion rates, [5] and it is thermally stable and resistant to sintering even at high temperatures because of its high melting point of ~2400 °C. [6] This makes ceria an ideal material for the thermochemical production of renewable fuels, due to its redox capabilities and ability to maintain the fluorite structure during thermochemical cycling over the typical 900-1500 °C range of operating temperatures used. [7] Solar thermochemical fuel production (STFP) is one of the potential solutions to our current environmental and energy crisis. It enables the production of renewable fuels by utilising concentrated solar power (CSP, sunlight focused with parabolic mirrors to heat up to 2000 °C) to drive a redox reaction on a metal oxide catalyst, such as ceria. [8] This oxide is reduced in a neutral atmosphere at high temperatures (1400-1500 °C) to CeO 2- , and then cooled to around 1000 °C as either water vapour or carbon dioxide are introduced. This takes oxygen from the H 2 O or CO 2 to re-oxidise the ceria, splitting these molecules to produce H 2 or CO. [9] H 2 is a potential fuel in its own right, and CO can be used to produce other renewable synthetic fuels. This fuel production cycle is powered by solar energy, and can be used to mitigate CO 2 emissions if the CO 2 feed is taken directly from such a source. Ceria was first investigated as a material specifically for solar thermochemical water splitting in 2006 by Abanades and Flamant, [10] and for the solar thermochemical splitting of CO 2 in 2010 by Chueh et al. [11] and Haussener and Steinfeld. [START_REF] Haussener | Conference Proceedings 2010 AIChE Annual Meeting[END_REF][START_REF] Haussener | [END_REF] Since then, many studies have investigated ceria redox materials, but as we demonstrated in a recent review paper, the Please do not adjust margins Please do not adjust margins morphology of the ceria can have a great effect on its thermochemical fuel production efficiency. [14] Repeating 3-DOM porous structures such as reticulated foams [15] or cork derived ecoceramics [16,17,18] of ceria proved to be excellent materials because of their structure, rather than any intrinsic chemical or ionic enhancements or dopants. This led us to speculate that 3D printed or robocast ceria, with a regular but open network structure, similar to robocast bone scaffolds developed for biomedicine, [19] could be a potentially superior form for such STFP applications. There has been very little previous work on the 3D printing or robocasting of ceria. For the additive manufacturing of ceramics, alumina was 3D printed by freeze-form extrusion fabrication in 2007, [START_REF] Zhao | 18th Solid Freeform Fabrication Symposium[END_REF] and inkjet printing of 80 m layers in 2008, [START_REF] Kaufmann | [END_REF] followed by lithography-based ceramic manufacturing in 2014 [22] and 3D powder bed printing in 2015. [23] Alumina extruded with a polymer binder was tested for syngas reforming when coated with NiO or CeO 2 in 2017, [24] colloidal alumina gels and a hydrogel were robocast in 2012-2013 [25,26] and 2016, [27] respectively, and robocast alumina pastes were reported in 2017-2018. [28,29] Solid 20 mol% Sm-doped ceria pellets were produced by 3D direct writing from a paraffin based slurry for solid oxide fuel cell applications (and then sintered at only 700 ºC) in 2019, [30] a 10 mol% ceria-stabilised-zirconia and alumina composite biomaterial was robocast from a hydrogel in 2017, [31] and the 3D printing by stereo lithography of Al 2 O 3 with 12 mol% CeO 2 -ZrO 2 (Zr 4+ :Ce 4+ = molar ratio of 88:12) was reported in 2020. [32] The only reports of similar porous ceria structures are those made by the replication method on extruded polymer supports for CSP applications in 2019, [33] and 3D printed polymer scaffolds in 2021 [34] , ceria coated on 3D printed polymer supports / structures / scaffolds, [35,36] CuO/CeO 2 catalysts coated on 3D printed polymer scaffolds, [37] Ni/CeO 2 -ZrO 2 powder deposited on 3D printed stainless-steel honeycomb monoliths, [38] CeO 2 -ZrO 2 -La 2 O 3 nanopowder catalysts supported on robocast graphene oxide scaffolds, [39] 3D printed ceria/silica microsphere/boehmite (γ-AlO(OH)) particlestabilised foams by moulding and direct ink writing, [40] and robocast ceria coated with a nickel catalyst. [41] All such 3D printed ceramics need to be sintered after manufacture to produce the ceria ceramic, particularly if destined for high temperature use -in nearly all of the cases above, the ceria was used unsintered as a catalyst. Reference [33] does not state at what temperature the materials were heated/sintered after coating the polymer templates, just that they were thermochemically cycled in tests at 1450 °C, the polymer scaffold templated ceria in reference [34] was sintered at 1400 °C, and the ceria-based foams in [40] were sintered at 1500 °C. The robocast ceria in reference [41] was intended as a support for the 10 wt% nickel catalyst, and so was not sintered, but just calcined at 450-500 °C to maintain a porous nature. The catalytic decomposition of ammonia was carried out at a maximum of 600 °C with this material.

ARTICLE

In this paper we report the ever first synthesis and characterisation of sintered robocast pure ceria, and its use in STFP to split water and carbon dioxide. This 3D printed sintered ceria would also be suitable for other high temperature applications. The macroporous robocast ceria structures were in the form of "woodpile" type cylinders and discs around 50 mm in diameter after sintering, with 500 m diameter struts and 500 m sized voids, produced from a ceria powder printing paste/ink with 46% solids loading. They were sintered at 1450 °C, their crystal structure, microstructure, sintering behaviour and porosity characterised, and their redox properties at high temperatures investigated for potential use in renewable solar energy applications.

Experimental

Ceria powder with mean particle size ≤5 μm (99.9 % purity) was used as received without further milling. Hydroxypropyl methyl cellulose (HPMC) was used as thickener, and poly(ethyleneimine) (PEI) solution, 50% (w/v) in H 2 O was used as coagulant. All materials were supplied from Sigma-Aldrich, except DISPEX ® A40 which was supplied from BASF, and all were used as received.

Ceria suspension preparation

The ceria powder with mean particle size ≤5 μm, as shown in Fig. 1, was gradually added to the dispersing solution (0.4 wt.% DISPEX ® A40 in de-ionised water) in several consecutive small doses. After each addition, the powder and the dispersing solution were mixed in a planetary centrifugal mixer (ARE-250, Thinky Corp. Tokyo, Japan) to ensure a final consistent suspension. After completion of powder addition, and ensuring a homogeneous suspension, 2% HPMC was added as a thickening agent to provide flowability, and to add shear thinning behaviour to the paste at applied shear, followed by centrifugal mixing for 6 minutes. 0.02% PEI was added as a coagulant, to help preserve the shape after extrusion / robocasting, and the paste was then mixed for a further 10 minutes.

Robocasting of ceria

The ceria scaffolds were deposited layer-by-layer using robocasting equipment (3-D Inks, Stillwater, OK, USA). To adapt the system to extrude large scaffold sizes, a plunger actuated by air was used instead of a stepper motor. The paste was extruded through a nozzle with a diameter of 510 µm, using a printing pressure of 6 bar. The designed scaffold consisted of a 3D logcabin type structure made of 6 layers, with the next layer being rotated around 90° from the previous layer in the plane of the struts. Scaffolds were printed as discs (50 mm diameter after sintering) with 510 m diameter struts and a macropore / void size (the distance between the struts) of 500 m. The deposition was carried out in a reservoir containing paraffin oil to allow a uniform drying of the green scaffolds. Once printing was finished, the scaffolds were removed from the oil and dried at room temperature for 24 h. They were then sintered at 1450 °C for 2 h, using a heating rate of 1 °C / min to ensure a complete removal of the residual organics.

Characterisation

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The average particle size and the particle size distribution of the powder, on a volume basis, were determined using a laser diffraction particle size analyser (Coulter LS particle size analyser; Beckman Coulter, CA) with triplicate measurements. Shrinkage of the scaffold was measured on a TA Instruments DIL 801L PRO dilatometer, heated in air at a rate of 10 °C / min. Measurements were taken in both longitudinal (parallel to the direction of the struts) and vertical (perpendicular to the direction of the struts, into the surface of the disc) directions. The samples were rectangular sections with dimensions of about 10 mm x 5 mm cut from the centre of the scaffold. X-ray diffraction (XRD) patterns were measured on a PANalytical X'PERT PRO 3 instrument (Cu Kα radiation, 10-80°, 0.01° 2θ step-scan and 200 s/step) with phase identification by HighScore Plus software. Scanning electron microscopy (SEM) images were taken with a Hitachi S-4100 microscope, on samples coated with carbon. The specific surface area (SSA) & porosity were measured by the Brunauer-Emmett-Teller (BET) method on a Micrometrics Gemini VII Surface Area Analyzer (Micrometrics Instruments Corp., Norcross, GA, USA). Adsorption/desorption isotherms were measured using N 2 as the adsorbate at 77 K, and outgassing the samples at 120 °C. The geometrical density of the scaffolds was measured from their dimensions and mass. The bulk density of ceria is 7.22 g / cm 3 . The thermochemical redox capability of the sintered ceria scaffold catalyst was investigated by thermogravimetric analysis (TGA) performed in controlled atmospheres on a Setaram Setsys Evo 1750. The sample was placed in a platinum crucible hung inside the furnace chamber, the chamber evacuated to eliminate residual air, and filled with argon gas flowing at 20 mL / min (99.999% purity). Heating and cooling rates of 20 °C / min were used throughout. The reduction step was carried out at 1400 °C / 45 min under Ar, and then the temperature was lowered to 1050 °C in Ar and held for 60 min for the re-oxidation step at isothermal conditions. For this a CO 2 stream was injected from the auxiliary gas inlet and mixed with the Ar carrier gas (50% CO 2 in Ar) at a total flow rate of 20 mL / min. The mixture entered at the top of the furnace chamber towards the sample and then exited the chamber at the bottom. The mass losses during thermal reduction are converted to the mole amount of O 2 released per gram of ceria according to:

nO 2 = Δm loss /(M O2 •m ceria ) (1)
where Δm loss is the mass variation measured by TG; M O2 the molecular weight of O 2 ; and m ceria the mass of ceria used in each experiment.

The mass gain associated with CO 2 splitting results in a CO amount (mol / g) which can be calculated as:

nCO = Δm gain /(M O •m ceria ) (2)
with Δm gain , the mass gain during the re-oxidation step, and M O , the atomic weight of oxygen.

Results and discussion

Physical characterisation of the sintered robocast ceria catalyst scaffold

Photographs of the sintered robocast ceria disc are shown in Fig. 2a-b, and the surface of this disc has been polished flat to enable us to see the interior microstructure. The shrinkage of the green ceria scaffold upon heating to 1475 °C, along the longitudinal (horizontal direction, parallel to the struts) and vertical (into the surface of the disc, through the layers) directions, is shown in Fig. 2c. The small, rapid decreases in length while heating from 200 to 400 °C observed in both directions were due to failures of irregularities on the surface/edge, and the normalised plots from 400 °C onwards are shown in the insert. It can be seen that the thermal expansion / shrinkage was identical in both directions until the onset of sintering at ~1050 °C. Beyond this temperature, the shrinkage was slightly quicker and greater in the vertical direction than the longitudinal one. This relatively small difference can be attributed to two cooperative effects: (i) the mechanical friction between the surface of the scaffold and the refractory support, which tends to mitigate the shrinkage; (ii) the gravity force that commonly adds to the driving force for shrinkage in the vertical direction. Because of this, the shrinkage tends to occur more in the diameter of the struts rather than in their length in robocast scaffolds, leading to anisotropic shrinkage, with proportionately more of this total shrinkage occurring in the vertical direction, the difference here being 1.5% more total shrinkage measured in the vertical direction (9.5% shrinkage in the longitudinal direction, 11.0% in the vertical direction). The sintered scaffolds were not fragile, and very handleable.

The geometrical density of the green ceria scaffolds before sintering was 1.08 g / cm 3 , 15.0% of that of bulk ceria. The geometrical density of the ceria scaffolds after sintering at 1450 °C was 1.85 g / cm 3 , or 25.6% of that of bulk ceria, giving an increase in volume density of 71.3%. This indicated that as well as the shrinkage of the volume of the scaffolds, the ceria struts themselves had become significantly more dense, with probable loss of some meso-and microporosity. The density of the sintered ceria scaffolds indicated a scaffold macroporosity of around 75%. As expected, XRD showed that the sintered scaffolds consisted of highly crystalline CeO 2 with the cubic fluorite structure (Fig. 3a), matching JCPDS file 34-0394. SEM images show that the diameter of the struts had shrunk to ~450 m, with a noticeable narrowing to smaller diameters between the intersections where the struts cross (Fig. 3b). It was probably this increased densification between the intersections which led to the greater scaffold density than just linear shrinkage would allow for, and suggested that the struts will be more meso-/microporous near the intersections. This resulted in the formation of rhombohedral shaped voids in some cases, with dimensions up to 750 x 750 m (Fig. 3a), larger than the intended 500 m. The scaffold in Fig. 3 was polished on the surface, but the shrinkage does appear to be greater in the struts in lower layers. The internal microstructure can be seen in Fig. 3c, and it consists of Please do not adjust margins Please do not adjust margins poorly sintered submicron grains <0.5 m in diameter, with an observable intergranular porosity. The surface of the struts was also poorly sintered (Fig. 3d), and appeared to be even more porous than the interior, although the grains were slightly larger on the exterior, up to 1 m in diameter (Fig. 3e). The fact the surface and interior of the ceria scaffolds seems to be to some extent porous even when sintered at 1450 °C is a very promising feature for STFP applications, as it will allow the CO 2 or H 2 O vapour to permeate into the scaffold material (inside the struts featuring submicron grains forming an interconnected macroporous network), and O 2 to escape, during the solarpowered redox reactions.

The BET adsorption/desorption isotherm is shown in Fig. 4a, and is a type IV isotherm, indicating initial monolayer coverage, followed by multilayer coverage with the hysteresis loop suggesting that capillary condensation is taking place in mesopores (20-500 Å). The shape of the isotherm is H4, indicating particles with broad size distribution and with internal irregularly-shaped voids between them, and it is typically due to the filling of micropores and/or mesopores. The BET SSA was 1.58 m 2 /g, and the T-plot indicated that 1.29 m 2 /g was external porosity and 0.29 m 2 /g was due to internal microporosity (<20 Å). The BJH (Barrett, Joyner and Halenda) analysis of desorption and adsorption only accounts for mesoand macroporosity (>20 Å), and this gives an adsorption SSA of 1.14 m 2 /g and a desorption SSA of 1.01 m 2 /g. The cumulative BJH pore area and pore volume plots are shown in Fig. 4b-c, along with details of the pore width intervals and average pore width value for each point. The overall average BJH pore size was 39.5 Å and 42.9 Å for adsorption and desorption, respectively, with most of the SSA coming from pores being in the 20-75 Å range (Fig. 4b). Larger pores contribute relatively much greater volume, so the pore volume distribution showed significant contribution from pores with width up to 250 Å, with average pore volumes of 0.001127 cm 3 /g and 0.001079 cm 3 /g from adsorption and desorption, respectively (Fig. 4c). Once again, this observed micro-and mesoporosity still existing in a material sintered at 1450 °C should be helpful to the STFP capabilities of the ceria scaffolds.

Catalytic performance of the robocast ceria scaffold for the thermochemical production of CO from the splitting of CO 2

The TGA measurements of the sintered ceria scaffold catalyst under thermochemical reaction conditions are shown in Fig. 5. The samples were heated to 1400 °C at 20 °C / min under Ar, and then put through two thermochemical cycles with the reduction step at 1400 °C / 45 min under Ar, heating / cooling at 20 °C / min under Ar, and the oxidation step at 1050 °C / 60 min under 50% CO 2 in Ar. The entire process is shown in Fig. 5a, for a sample of the scaffold weighing 273.2 mg. The ceria began to reduce at ~1050 °C, and in the first cycle it underwent a weight loss of 0.302 mg (0.111%) during the reduction step at 1400 °C, for a total O 2 production of 35 mol/g (0.784 ml/g), which equates to a  value (oxygen nonstoichiometry) of 0.012 for CeO 2- . Upon cooling to 1050 °C there was a significant weight gain of ~0.078 mg (0.029%) as the ceria partially re-oxidised, but with the injection of CO 2 gas at 1050 °C there was an immediate and sudden weight gain over a few minutes for a total weight gain of 0.273 mg (0.100%). This was due to the ceria splitting some of the injected CO 2 to re-oxidise, producing 63 mol/g (1.411 ml/g) of CO in this first oxidation step. In the second cycle the ceria underwent greater reduction in the high temperature step, losing 0.375 mg (0.138%) at 1400 °C, producing 43 mol/g (0.963 ml/g) of O 2 ( = 0.015). This is a commonly seen feature in these thermochemical cycles, with the ceria not becoming fully reduced in the first cycle, and requiring further cycles to stabilise. [16] There was much less weight gain on cooling to 1050 °C in the second cycle, and when CO 2 was injected for the oxidation step the weight gain was 0.342 mg (0.126%), with a CO yield of 78 mol/g (1.747 ml/g). These data are also summarised in Table 1. If the reduction/oxidation process is fully efficient the weight loss and weight gain should be equal, and the quantity of CO produced should be double that of the O 2 released. In this case, we can see that in both cycles the oxidation was slightly incomplete (90.1% and 91.2%), and the ratio of CO:O 2 was 1.80 and 1.81. The oxidation step was completed well before the end of the 60 min dwell, suggesting that it is the material morphology rather than any thermodynamic/kinetic factor that is limiting the 100% re-oxidation of the ceria. It is also clear that the oxidation step is much more rapid than the reduction step for both cycles, and the derivative of mass change (dw/dt) is shown for the second cycle in Fig. 5b. In both Please do not adjust margins Please do not adjust margins cycles the reduction began as soon as the temperature increase began >1050 °C, and the peak of O 2 production was actually achieved before the 1400 °C isothermal step was reached, at 1391 °C and 1394 °C in the first and second cycles. In the second, stabilised cycle, weight loss continued throughout the 45 min 1400 °C isothermal period, albeit at a lowered rate. The peak rates of O 2 production were very similar, at 0.0555 mol/g/s (0.029 mg/min) and 0.0574 mol/g/s (0.030 mg/min), for the first and second cycles. 90% of the total weight loss was achieved after 41.75 min of the reduction step (Fig. 5b). On the other hand, the oxidation step is instantaneous and very rapid as soon as the CO 2 is injected at 1050 °C, with a sudden peak in dw/dt. The peak CO production rates were 0.2525 mol/g/s (0.066 mg/min) and 0.4361 mol/g/s (0.114 mg/min) for the first and second cycles, more than 4.5 and 7.6 times greater than the peak O 2 rates for those cycles, respectively. In the second cycle CO production is effectively over after 30 min of the oxidation step, and a 90% weight gain was achieved after only 10.10 min (Fig. 5b). This means that the oxidation step could be greatly reduced in duration, with savings in both energy and time.

Although there have been no reports on the use of sintered robocast ceria scaffolds before this, these data can be compared to other ceria STFP materials tested for CO 2 splitting. [14] The reduction/oxidation temperatures used are crucial, so comparison should only be made to results at similar redox temperatures of 1400 °C and ~1000 °C. The total CO yields of 1.411 ml/g and 1.747 ml/g are lower than those reported for ceria powders (2.28 ml/g) [42] and cork derived (3.36 ml/g) [16] and pine-derived (2.1 ml/g) [43] 3-DOM ecoceramics, and are comparable to those for reticulated ceria foams made by the replication method (1.68 ml/g). [16] However, the peak CO production rate in the second stabilised cycle of 0.5861 ml/g/min is an order of magnitude greater than that reported for the ceria powder (0.067 ml/g/min), [42] demonstrating the utility of these ordered scaffold structures. These values also compare well to the recently published results for ceria scaffolds made from coating sacrificial 3D printed polymer scaffolds with a ceria paste, which was then sintered at 1400 °C, when tested by TGA. [34] Under identical reduction and oxidation conditions, these polymer-derived scaffolds produced slightly higher peak CO production rates of 0.64 and 0.72 ml/g/min in their first and second cycles, and CO yields of 93 and 100 mol/g. This was despite them having a much lower density of only 0.27 g/cm 3 and significantly greater estimated macroporosity of 98%, compared to the values of 1.85 g/cm 3 and scaffold porosity of 75% for the pure ceria robocast structures reported here, and suggest that the capability of these robocast ceria scaffolds could be significantly increased with further refinements of the scaffold structure to increase macroporosity. The peak fuel production rates are still significantly lower than those of the biomimetic ecoceramics or reticulated foams mentioned above, but again these results could be improved upon by either varying the dimensions of the struts and voids, or by incurring a level of hierarchical porosity within the struts of the ceria scaffold itself.

The relatively low  values of 0.012-0.015 indicated that the ceria scaffold was not greatly reduced, for example being slightly lower compared to the ceria powder or cork ecoceramic. [16,42] The reduction temperature of 1400 °C was chosen because it will be the nominal operation temperature of the solar reactor during actual STFP tests of these ceria scaffolds in the future. Although a higher reduction yield could be obtained at a higher temperature, the main issues would be linked to the reactant losses by sublimation and to constraints of thermal stability for the materials of the solar reactor and the reactive material itself. Furthermore, heat losses (especially radiative) are dramatically increased when increasing the reactor temperature, which in turn downgrades the solar-tofuel energy conversion efficiency. For these reasons, measurements were not made at higher reduction temperatures.

Conclusions

50 mm diameter ceria scaffold discs were successfully fabricated by robocasting. Above 1050 °C, the shrinkage was not identical in longitudinal and vertical directions as the contact between the scaffold and the refractory support is not frictionless, tending to hinder shrinkage in the longitudinal direction, and because the gravity effect adds to the driving force exacerbating the shrinkage in the vertical direction. When heated up to 1475 °C ~12% more total shrinkage occurs in the vertical direction (strut diameter). Sintered, but still porous (SSA = 1.58 m 2 /g), scaffolds were produced which consisted of highly crystalline CeO 2 with the cubic fluorite structure. The ceria struts were shown to contain mesopores <75 Å, and have a micropore (<20 Å) surface area of 0.29 m 2 /g, after sintering. The size of the voids between the struts remained virtually unchanged ~500 μm, and the diameter of the struts shrank to ~450 μm, with a noticeable narrowing to smaller diameters between the intersections where the struts cross. The fact that the surface and interior of the ceria scaffolds seems to be to some extent porous is a very promising feature for STFP applications, as the remaining porosity will allow the CO 2 or H 2 O vapour to permeate into the scaffold material, and O 2 to escape, during solar-powered redox reactions. The catalytic redox process was assessed by TGA for STFP applications, with reduction between 1050-1400 °C under Ar, and oxidation at 1050 °C under 50% CO 2 . The reduction was a slow process controlled by heat transfer rate, as is usually seen in ceria, needing ~40 min to reach 90% of the maximum reduction. In contrast, CO production during oxidation was rapid, with a high peak CO production rate of 0.436 mol/g/s (0.586 ml/g/min) in the second stabilised cycle, ~7.75 times quicker than the O 2 production rate, for a total CO yield of 78 mol/g (1.747 ml/g) in the second cycle. 90% of the CO yield was obtained after just 10 min of oxidation. This CO yield compares well to reticulated ceria foams tested at similar temperatures, and the CO production rate is greatly superior to that obtained from ceria powders at the same temperatures. The total reduction yield achieved was low ( = 0.015), and the oxidation was slightly uncomplete (90.1% and 91.2% in cycles 1 and 2), indicating that This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins this material could be optimised and greatly improved by either varying the dimensions of the struts and voids, or by instilling a level of hierarchical porosity in the struts of the ceria scaffold.

Figure captions

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 2 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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 4 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Fig. 1

 1 Fig. 1 Particle size distribution for the ceria powder. The data points show the particle size distribution, and the dotted lines indicate the probable bimodal nature of this powder.

Fig. 2

 2 Fig. 2 Photographs of the sintered robocast ceria scaffolds (ab), and their shrinkage in the vertical and longitudinal directions with heating (c).

Fig. 3

 3 Fig. 3 XRD pattern of the ceria scaffold sintered at 1450 ºC, with the major peaks for cubic CeO 2 indexed. SEM images of a sintered and polished scaffold (b) show the interior (c) and exterior (d-e) microstructure.

Fig. 4

 4 Fig.4BET isotherm (a) and BJH cumulative pore area (b) and pore volume (c) plots for N 2 adsorption/desorption on sintered ceria scaffolds. The boxes show the pore width range intervals and pore width average data point values for adsorption (blue) and desorption (red).

Fig. 5

 5 Fig. 5 TGA plots of the redox capability of the sintered ceria scaffolds, with reduction during heating from 1050-1400 ºC under Ar, and oxidation under CO 2 at 1400 ºC. Heating and cooling at 20 ºC / min under Ar, dwell for 45 min at 1400 ºC under Ar and for 60 min at 1400 ºC under 50% CO 2 in Ar. a) Temperature profile and weight loss over time of a 273.3 mg ceria scaffold sample through two cycles. b) Weight loss and derivative of weight loss for the second cycle -dotted lines indicate the interval to reach 90% of the maximum weight loss or gain in this cycle.

Fig. 1 Fig. 2

 12 Fig. 1 Particle size distribution for the ceria powder. The data points show the particle size distribution, and the dotted lines indicate the probable bimodal nature of this powder. 220x165mm (144 x 144 DPI)

Table 1 .

 1 TGA data for the sintered ceria scaffold catalyst in two thermochemical cycles.

	273.2 mg sample	1 st Cycle 2 nd Cycle
	Weight loss (mg)	0.302	0.375
	Weight loss (%)	0.111	0.138
	Weight gain (mg)	0.273	0.342
	Weight gain (%)	0.100	0.126
	O 2 production (mol/g)	35	43
	O 2 production (ml/g)	0.784	0.963
	Peak O 2 production (mol/g/s)	0.0555	0.0574
	Peak O 2 production (ml/g/min) 0.0746	0.0771
	Peak O 2 production (mg/min)	0.029	0.030
	CO production (mol/g)	63	78
	CO production (ml/g)	1.411	1.747
	Peak CO production (mol/g/s) 0.2525	0.4361
	Peak CO production (ml/g/min) 0.3394	0.5861
	Peak CO production (mg/min)	0.066	0.114
	Peak O 2 production temp ( o C)	1391	1394
		0.012	0.015

255x128mm (150 x 150 DPI)

Acknowledgements R.C. Pullar wishes to thank the FCT ((Fundação para a Ciência e a Tecnologia, Portugal) Grant IF/00681/2015 for supporting this work. B. A. E. Ben-Arfa thanks FCT project H2CORK (PTDC/CTM-ENE/6762/2014) for supporting him during this work. This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement.

Conflicts of interest

There are no conflicts to declare.

Authors' contributions