Dalla Gianfranco

Marta Barba

Claudia Brazzarola

Sara Barbera

Francesco Marangoni

Paolo Causin

Michel Bartolomeo

Thiebaut De Schotten

Gianfranco Dalla Barba

Marta Brazzarola

Claudia Barbera

Sara Marangoni

Francesco Causin

Paolo Bartolomeo
email: paolo.bartolomeo@gmail.comtel

Michel Thiebaut De Schotten

Different patterns of confabulation in left visuo-spatial neglect

Keywords: confabulation, neglect, implicit knowledge

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INTRODUCTION

Confabulation is a rather infrequent disorder, which consists in the production of actions and verbal statements incongruous to the subject's history, background, present and future situation (Dalla [START_REF] Barba | Confabulation: Knowledge and recollective experience[END_REF]. It has been described in several pathological conditions, including visual neglect. Patients with visual neglect may confabulate on the neglected stimuli, however, only few studies reported this phenomenon (e.g. [START_REF] Chatterjee | Cross-over, completion and confabulation in unilateral spatial neglect[END_REF][START_REF] Manning | Confabulations related to tacit awareness in visual neglect[END_REF]. Clinically, patients with visuo-spatial neglect may show signs of socalled 'implicit' or partial knowledge of otherwise unreported stimuli presented in the neglected field [START_REF] Bartolomeo | Attention disorders after right brain damage[END_REF][START_REF] Marshall | Blindsight and insight in visuo-spatial neglect[END_REF]. This partial knowledge is sometimes inaccurate and fits within the standard definition of a confabulation. This suggests that visual information may have been partially processed in the brain but did not access consciousness for these patients. Pre-conscious, pre-attentional processing of the neglected stimuli, would be similar to that of patients with 'blindsight' [START_REF] Weiskrantz | Residual vision in a scotoma. A follow-up study of 'form' discrimination[END_REF] and prosopagnosia [START_REF] De Haan | Covert and overt recognition in prosopagnosia[END_REF] but its anatomical mechanism remains largely unknown.

Drawing on a suggestion by [START_REF] Geschwind | Disconnexion syndromes in animals and man -Part II[END_REF], [START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF] proposed that confabulations for neglected items could result from poor transfer of right hemisphere-based, knowledge of left-sided visual stimuli to the left hemisphere. Hence, the verbalisation of the residual visual information by the left hemisphere would be at the basis of confabulatory responses.

Alternatively or in addition, intra-hemispheric disconnection may restrict processing of left-sided stimuli to a pre-conscious stage of processing. To access consciousness, sensory contents must compete, before one of them becomes dominant and accesses the limited capacity of our consciousness (i.e. Global Workspace theory, [START_REF] Baars | A Cognitive Theory of Consciousness[END_REF]. Information is pre-processed, at first preconsciously, within modular cerebral networks such as, for instance, those contained in the visual cortical ventral stream [START_REF] Ungerleider | Two Cortical Visual Systems[END_REF].

Attentional amplification through fronto-parietal networks can then bring information to a conscious level of processing, and make it available to verbal report. A disconnection between the visual ventral stream and fronto-parietal network might therefore lead to 'implicit' or partial knowledge of stimuli presented in the neglected field. Support for this hypothesis comes from neuroimaging studies demonstrating preserved sensory activation to undetected stimuli in brain-damaged patients [START_REF] Rees | Unconscious activation of visual cortex in the damaged right hemisphere of a parietal patient with extinction[END_REF][START_REF] Vuilleumier | Neural fate of seen and unseen faces in visuospatial neglect: a combined event-related functional MRI and event-related potential study[END_REF]. In the present context, this hypothesis would predict that the patient's verbal report would again be based on inadequate processing of information, and liable to be confabulatory (Bartolomeo, de Vito, & Seidel Malkinson, 2016). For instance, when confronted with the well-known images of two identical houses, except for flames coming from the left part of one of them, and asked which house he would like to live in [START_REF] Marshall | Blindsight and insight in visuo-spatial neglect[END_REF], the patient reported by [START_REF] Manning | Confabulations related to tacit awareness in visual neglect[END_REF] chose the non-burning house and confabulated that it had an extra fireplace. Another patient described by [START_REF] Bisiach | Break-down of perceptual awareness in unilateral neglect[END_REF] paradoxically kept choosing the ''burning'' house, considering it more ''spacious'' on the burning side, where the contour of the flames actually enlarged the shape of the house. In a group of 13 neglect patients [START_REF] Doricchi | Neural control of fast-regular saccades and antisaccades: an investigation using positron emission tomography[END_REF] described different patterns of responses motivating correct implicit choices of the ''non-burning house'': 1) ''there is no specific reason for my choice, the two houses are the same anyway,'' suggesting complete uncoupling of verbal output from implicit processing; 2) ''the house I chose is 'better', 'bigger', or 'works better','' suggesting, in this case, some approximation of verbal output to implicit processing.

In this paper we describe the very unusual case of a patient with left visuo-spatial neglect, who confabulated both for consciously undetected and for consciously detected stimuli presented in the neglected field. Advanced neuroimaging methods, including tractography were employed to test inter-and intra-hemispheric disconnection hypotheses accounting for confabulatory behaviour.

CASE REPORT

History

PC is a 70-years-old right-handed businessman with thirteen years of education. He is married and has two children. He has no history of psychiatric or previous neurologic diseases.

In October 2014 he was admitted to the Internal Medicine Department of the Padua University Hospital for sudden loss of consciousness. A CT brain-scan showed a right temporo-parietal ischemic stroke. Due to the appearance of epileptic seizures, he was transferred to an intensive care unit and then to the Neurology Department of the same hospital. Twenty days later he was referred to the Neurocognitive Rehabilitation Unit of the Centro Medico di Foniatria in Padua to undergo cognitive rehabilitation.

Neuropsychological examination.

PC was tested on various occasions between November 2014 and January 2015 (Table 1).

- -----------------------Insert Table 1 about here ---------------------------The ethical committee of the Centro Medico di Foniatria approved the experimental procedures. The patient provided written informed consent for publication of this case report.

He was fully cooperative, well oriented in time and place and had preserved general intellectual abilities. Clinically he was not amnesic. He performed normally on learning, episodic memory tests, except for the recall of the Rey's figure. The copy of the Rey's figure was affected by some omissions both on the right and on the left side, and was not informative about the patient's neglect.

Executive functions, short-term memory and working memory were normal. He performed normally on tests of oral expression, understanding of oral language and praxis. Visual perception, as measured with the VOSP Screening test and Object Decision, was unimpaired.

On clinical criteria, PC showed typical signs of left visuo-spatial neglect, both in ecological conditions and in classical tests sensitive to this disorder, such as line crossing, line bisection, shape and letter cancellation. PC was anosognosic for his neglect disorder.

Experimental investigation.

The following observations were made during five testing sessions, 8 weeks after the stroke. Partially following and expanding the procedure introduced by [START_REF] Marshall | Blindsight and insight in visuo-spatial neglect[END_REF], PC was presented with drawings of 9 pairs of objects. For each pair, vertically arranged objects were identical, except for a salient detail on the left side. The stimuli were: 1.

Two houses, in one of which flames were emerging from the left side of the house; 2. Two banknotes, in one of which the left part was missing; 3. Two vases, an empty one and one with a flower on the left side; 4. Two trees, in one of which the left part was missing; 5. Two wine glasses, one of which was broken in the left side; 6. Two Eiffel Towers, in one of which the left part was missing; 7. Two cows, in one of which the left part was missing; 8. Two ambulances, in one of which a left-sided wheel was missing; 9 Two sailing boats, one of which had a leak on the left side.

Procedure

The 9 stimuli were presented (Fig. 1) to PC in 5 sessions, one session per day for 5 consecutive days. In each session, the two houses were presented to the patient 12 times with alternate vertical order (in each session six times the burning house was above the other house). The other stimuli were presented one time per session. In our testing set there were more burning houses than other stimuli, because this type of stimulus provided significant results in the Marshall and Halligan's seminal study [START_REF] Marshall | Blindsight and insight in visuo-spatial neglect[END_REF]. The rationale for including more "burning houses" than other stimuli was to confirm, support and reproduce the Marshall & Halligan results and to see whether these results could be extended to other stimuli.

- -----------------------Insert Figure 1 about here ---------------------------For the houses, the examiner asked PC if they were "the same or different". After each presentation, the examiner asked the patient to say which house he would prefer to live in and why. For each of the other stimuli, the examiner asked PC if the two items were "the same or different", then which of the two he would chose and why.

The same stimuli were presented with the same procedure to 10 age-and educationmatched normal controls (NC).

Results

In the statistical analyses the α value was set at 0.05. The Crawford et al's method for statistical analysis of single case [START_REF] Crawford | Inferential methods for comparing two single cases[END_REF] was used where possible. The Chi Square test was used in other cases.

All the normal controls were able to identify the differences between the items for each display, and to name them correctly. They were all able to tell what the difference between the two stimuli was (e.g., by mentioning the flames in the burning house), and to choose which item they would prefer (e.g. an intact boat rather than a boat with a leak).

On the other hand, the patient, when presented with the houses, never noticed the presence of flames for any of the 60 trials (12 x 5 sessions), but responded that the houses were different and always chose to live in the house with flames. On some occasions the examiner, pointing at the flames, asked PC: "Could this be fire?" "No, it can't be fire, replied PC. Fire goes up." "But it does go up", insisted the examiner. "No, continued PC, it should come out from the above window and from the fireplace" (there was no above window). When the examiner asked why he would prefer to live in the burning house, in 50/60 trials PC confabulated giving one of the following answers: "Because there is a pergola" (18 times), "Because there are grapes" (11 times), "Because there is a little plant" (8 times), "Because it is more linear" (6 times), "Because it is cleaner" (5 times), "Because in this house they cook better food" (1 time), "Because it is more real" (1 time). In the remaining 10/60 trials PC did not provide any reason for his choice, stating that he did not know why he preferred the burning house. The difference with NC's responses was statistically significant (p< .05).

Concerning the other stimuli, PC's performance varied. For the banknotes he always said that they were the same, but always chose the entire one, saying once "Because it is more beautiful", and "I don't know" in the remaining trials. For the cows four times he said that the two items were the same, always choosing the entire one, saying, "Because it is a cow", "Because is more real", "Because it has a different profile", "Because it has a straight back".

For the remaining 6 stimuli he always said that they were different, and always chose the entire item, "Because it is complete".

Examination of confabulation

PC showed confabulatory behaviour outside the experimental context, mainly in informal conversation.

Since PC showed a marked tendency to confabulate in the experiment described in the previous section, it was decided to evaluate whether he would confabulate in other domains and in particular in the various memory domains, where confabulation is classically described. Accordingly, PC was administered the Confabulation Battery (CB). The CB involves the retrieval of various kinds of information and consist of 165 questions, 15 for each of the 11 following domains: (1) Personal Semantic Memory, (2) Episodic Memory, (3) Orientation in time and place, (4) Linguistic Semantic Memory, General Semantic Memory (5) Recent, (6) Contemporary, (7) Historical, (8) Semantic Plans, (9) Episodic Plans and questions to which the appropriate response would be 'I don't know', both (10) semantic and (11) episodic. Each domain has been described in detail elsewhere (Dalla [START_REF] Barba | Do you remember what you did on March 13, 1985?" A case study of confabulatory hypermnesia[END_REF].

Procedure

Questions from the 11 domains were randomized and presented to the patient and to 10 age-and education-matched normal controls (NC). Responses were scored as "correct, "wrong", "I don't know" and "confabulation". For episodic memory, responses were scored "correct" when they matched information obtained from the patient's relatives. Correct responses were self-evident for semantic memory questions. For "I don't know" questions, both Semantic and Episodic, a "I don't know" response was scored as correct. Because there is no sufficiently acceptable external criterion capable of defining confabulation, for its detection an arbitrary decision necessarily had to be made.

To distinguish between a wrong response and a confabulation, a clear-cut decision was adopted only for answers to questions probing orientation in time. In this case the strictest criterion was chosen: answers to questions regarding the current year, season, month, day of the month, day of the week, and hour of the day were judged to be confabulations only if erring for more than 5 years, 1 season, 2 months, 10 days, 3 days or 4 hours, respectively.

Answers to the other questions of the Confabulation Battery were independently rated as "correct", "wrong" and "confabulation" by four different raters, with 100% interrater reliability. It must be emphasised that the decision as to whether an answer was wrong or confabulatory was never puzzling, although it may have been made on an arbitrary of subjective basis. As far as questions concerning personal and semantic plans are concerned, it might be argued that any possible answer is a confabulation, since, by definition, the future is only "probable" and there is in principle no "correct" answer to questions about the future.

Yet, answers about the future can be definitely confabulatory when they show a marked discrepancy or a real contradiction with what a predicted future event might be, in view of the present situation. For example, although he was hospitalised, to the question "When will you take vacations, or when will you be travelling next time?", PC answered "Next Tuesday."

Results

PC's correct responses and confabulations are reported on Table 2.

- -------------------------Insert Table 2 about here --------------------------PC produced significantly fewer correct responses than NC, who performed at ceiling, to Episodic Memory, Orientation in Time and Place, Episodic Plans, "I don't know" Semantic and "I don't know" Episodic questions (all p < .05), and confabulated significantly more than NC answering Episodic Memory and "I don't know" Semantic and Episodic questions (all p < .05), questions which are known to elicit confabulation much more than standard episodic memory tests.

According to the criteria proposed elsewhere [START_REF] Serra | Confabulators mistake multiplicity for uniqueness[END_REF], PC was a severe confabulator since he produced 40% or more of confabulations in Episodic Memory.

Confabulations were also prominent in "I don't know" Episodic and in "I don't know" Semantic questions, whereas they were absent or sporadic in other domains of the CB.

Following the taxonomy of confabulation proposed by the Dalla Barba's group (Dalla Barba & Boisse, 2010;[START_REF] Corte | Confabulation in Alzheimer's disease and amnesia: a qualitative account and a new taxonomy[END_REF], 68% of PC's confabulations were "Habits Confabulations, ", i.e. either repeated personal events mistaken as specific, unique past and future personal episodes, or public events when semantic knowledge is concerned. This confirms previous results showing that this type of confabulations is the most frequently observed (Dalla Barba & Boisse, 2010;[START_REF] Corte | Confabulation in Alzheimer's disease and amnesia: a qualitative account and a new taxonomy[END_REF]. Other types of confabulation were: 16% Memory Confusions, 4% Memory Fabrications, 12% Misplacements.

Anatomical study 2.5.1 Acquisition parameters

We acquired diffusion-weighted imaging (DWI) which provided isotropic (2 × 2 × 2 mm) resolution and coverage of the whole head with a posterior-anterior phase of acquisition. A total of 65 near-axial slices were acquired on a Philips Ingenia 3T system equipped with a 32channel head coil. We used an echo time (TE) of [96"] msec and a repetition time (TR) of [10104"] msec. At each slice location, 1 image was acquired with no diffusion gradient applied (i.e. B0). Additionally, 32 diffusion-weighted images were acquired, in which gradient directions were uniformly distributed on the hemisphere with electrostatic repulsion.

The diffusion weighting was equal to a b-value of 1000 sec mm -2 . Finally, at each slice, diffusion-weighted data were simultaneously registered and corrected for subject motion and geometrical distortion adjusting the gradient accordingly (http://www.exploredti.com; see [START_REF] Leemans | The B-matrix must be rotated when correcting for subject motion in DTI data[END_REF].

Atlas-Based Analysis of Disconnection

The patient's lesion was first drawn on the native B0 image using MRIcron (http:// http://www.cabiatl.com/mricron) by an expert anatomist (M.T.S.) (Fig. 2a). B0 image was then normalized to a standard brain template, (MNI152, Montreal Neurological Institute link) using rigid (FLIRT) and elastic (FNIRT) deformation tools provided FSL. We calculated and applied the deformations to the whole brain except the lesion mask to avoid deformation of the lesioned tissue [START_REF] Brett | Spatial normalization of brain images with focal lesions using cost function masking[END_REF][START_REF] Volle | The functional architecture of the left posterior and lateral prefrontal cortex in humans[END_REF]. The deformation was subsequently applied to the original drawing of the lesion as illustrated in Fig. 2b. We mapped the normalized lesion onto tractography reconstructions of white matter pathways obtained from a group of healthy controls [START_REF] Rojkova | Atlasing the frontal lobe connections and their variability due to age and education: a spherical deconvolution tractography study[END_REF]. We used 50% overlap maps for the localization and quantification of the lesions (Thiebaut de Schotten, et al., 2015;[START_REF] Thiebaut De Schotten | Damage to white matter pathways in subacute and chronic spatial neglect: a group study and 2 single-case studies with complete virtual "in vivo" tractography dissection[END_REF]. We quantified the severity of the disconnection by measuring the proportion of the tract to be disconnected (Thiebaut de Schotten, et al.,

Lesion-Based Approach to Mapping Disconnection

We further capitalized on recently published methods to reveal brain areas deafferented by the lesion using a disconnectome map approach [START_REF] Foulon | Advanced lesion symptom mapping analyses and implementation as BCBtoolkit[END_REF]. The patient's lesion was registered to the tractography of a group of healthy controls [START_REF] Rojkova | Atlasing the frontal lobe connections and their variability due to age and education: a spherical deconvolution tractography study[END_REF] using affine and diffeomorphic deformations (Klein et al., 2009;Avants et al., 2011). The registered lesion was subsequently used as a seed point to track streamlines in an healthy dataset using Disconnectome maps software as part of the BCBtoolkit (http://toolkit.bcblab.com) [START_REF] Foulon | Advanced lesion symptom mapping analyses and implementation as BCBtoolkit[END_REF]. Subsequently, we created a binary visitation map of the streamlines intersecting the lesion. This map was normalized to MNI space using the inverse of the affine and diffeomorphic deformations mentioned above. Percentage maps were computed by summing at each voxel in the MNI space the normalized visitation map of each subject and projected onto the average 3D rendering of MNI152 using anatomist (http://brainvisa.info). Fig. 2d indicates a high probability of deafferentation of the posterior part of the temporal lobe and the inferior parietal lobule.

Tractography

We used tractography of diffusion-weighted imaging to confirm the disconnection identified by tractotron (i.e. damage to the long and of the posterior segments of the arcuate fasciculus, the optic radiations as well as the inferior longitudinal fasciculus).

A damped Richardson Lucy Spherical Deconvolution (Dell 'Acqua, et al., 2010) was computed to estimate multiple orientations in voxels containing different populations of crossing fibres [START_REF] Alexander | An introduction to computational diffusion MRI: the diffusion tensor and beyond[END_REF][START_REF] Anderson | Measurement of fiber orientation distributions using high angular resolution diffusion imaging[END_REF][START_REF] Tournier | Direct estimation of the fiber orientation density function from diffusion-weighted MRI data using spherical deconvolution[END_REF].

Algorithm parameters were chosen as previously described [START_REF] Dell'acqua | Can spherical deconvolution provide more information than fiber orientations? Hindrance modulated orientational anisotropy, a true-tract specific index to characterize white matter diffusion[END_REF]. A fixed-fibre response corresponding to a shape factor of α = 2 × 10-3 mm2/s was chosen [START_REF] Dell'acqua | Can spherical deconvolution provide more information than fiber orientations? Hindrance modulated orientational anisotropy, a true-tract specific index to characterize white matter diffusion[END_REF].

Whole brain tractography was performed selecting every brain voxel with at least one fibre orientation as a seed voxel. From these voxels, and for each fibre orientation, streamlines were propagated using Euler integration with a step size of 1 mm (as described in Dell 'Acqua, et al., 2013). When entering a region with crossing white matter bundles, the algorithm followed the orientation vector of least curvature (as described in [START_REF] Schmahmann | The complex history of the fronto-occipital fasciculus[END_REF]. Streamlines were halted when a voxel without fibre orientation was reached or when the curvature between two steps exceeded a threshold of 45°. Spherical deconvolution, fibre orientation vector estimations and tractography were performed using in Startrack (http://www.natbrainlab.co.uk).

We explored the integrity of the visual afferent system in the left and the right hemispheres using regions of interest drawn on a coronal section situated at the level of the occipital notch (Fig. 2e).

We further explored the perisylvian network in the left and the right hemispheres using regions of interest drawn on axial sections including voxels concerned by associative perisylvian white matter as reported in Catani et al. [START_REF] Catani | Perisylvian language networks of the human brain[END_REF] (Fig. 2f).

Unfortunately, the available 30 directions were not sufficient to reconstruct the dorsal fronto-parietal network as previously reported in (Thiebaut de [START_REF] Thiebaut De Schotten | A lateralized brain network for visuospatial attention[END_REF] Results presented in Fig. 2e and 2f solely display tracks passing by the defined regions of interest and revealed a clear-cut disconnection of the posterior and long segments of the arcuate fasciculus in the right hemisphere, partial damage of the optic radiations with a relative sparing of the inferior longitudinal fasciculus, as well as of interhemispheric connections.

DISCUSSION

We reported an extensive behavioural and anatomical study of PC, a patient with right hemisphere damage, who, at the time of testing, suffered from relatively pure left neglect, in the sense that his visuo-spatial abilities were affected out of all proportion to other cognitive functions and he was otherwise alert and responsive. In particular, he was not demented, had normal visual perception and performed normally on tasks of executive functions, short-term memory, working memory, episodic memory, semantic memory and praxis. Importantly, PC showed a severe [START_REF] Serra | Confabulators mistake multiplicity for uniqueness[END_REF] tendency to confabulate on the Confabulation Battery, a sensitive tool to detect confabulations in various memory domains.

When presented with visual items differing on their left side, PC showed three patterns of performance: 1. He gave "normal" responses (30 times), i.e. he detected the difference between the two items of the stimulus and chose the entire one. 2. He didn't detect the difference between the two items of the stimulus (9 times), made a "normal" choice of the "better" one, and confabulated on the reason of his choice (5 times) 3. He detected a difference between the two items of the burning house task (without identifying the flames as such), then chose the unexpected one (the burning house), and confabulated on the reason of his choice (49 times).

Confabulation is often considered a memory distortion associated to a frontal lobe lesion.

However, it has been shown that confabulation is not associated to a specific brain lesion (Dalla [START_REF] Barba | A longitudinal study of confabulation[END_REF][START_REF] Barba | The hippocampus, a time machine that makes errors[END_REF], 2015), but can occur in patients with lesions in more than twenty anterior and posterior brain regions.

PC was not amnesic either clinically, or in formal testing and performed normally in frontal tests. The results of the present study add evidence that confabulation may occur outside memory deficits [START_REF] Papagno | Confabulation in a dysexecutive patient: implication for models of retrieval[END_REF], and that a frontal lesion is not necessary for patients to confabulate (Dalla [START_REF] Barba | Confabulation following rupture of posterior communicating artery[END_REF].

As far as "normal" responses are concerned, as compared to other types of responses, these can be the result of fluctuations of PC's performance. It is well known that brain damaged patients rarely show a clear-cut performance. Notwithstanding the cognitive domain involved, patients with brain damage show, in variable proportion, both correct and incorrect responses when challenged in testing or in experimental situations. This pattern of performance is often observed in neglect patients, likely because of fluctuations in their level of alertness [START_REF] Bartolomeo | Attention disorders after right brain damage[END_REF]. Therefore PC's 30% of "normal" responses over the total number of responses is not surprising.

Ten per cent of PC's responses reflected the classical pattern described by [START_REF] Marshall | Blindsight and insight in visuo-spatial neglect[END_REF]: the patient claimed that the two items were identical, yet he chose the 'good' one and confabulated on the reason of his choice. These results are similar to those obtained in patients with 'blindsight' [START_REF] Weiskrantz | Residual vision in a scotoma. A follow-up study of 'form' discrimination[END_REF] and prosopagnosia [START_REF] De Haan | Covert and overt recognition in prosopagnosia[END_REF] and have been interpreted as reflecting pre-attentional, pre-conscious processing [START_REF] Manning | Confabulations related to tacit awareness in visual neglect[END_REF][START_REF] Marshall | Blindsight and insight in visuo-spatial neglect[END_REF] However, the main result of this study is the type of responses PC gave to the "burning house" stimuli. In 60/60 presentations, PC acknowledged a difference between the two houses. However, he never noticed, and actually denied, that one of them was in flame.

Although the stimuli were drawn in black and white, none of our control subjects had any doubts about the identity of the flames emerging from the left side of the burning house.

When asked which house he would prefer to live in, he always chose the burning house and confabulated about his choice. To the best of our knowledge, such a pattern was only briefly hinted about by [START_REF] Bisiach | Break-down of perceptual awareness in unilateral neglect[END_REF] in one of their patients. This rare occurrence cannot be accounted for in terms of the classical blindsight-like, pre-conscious, pre-attentional processing interpretation. According to the criteria proposed by the Dalla Barba's group, PC was a severe confabulator, as determined by his responses to the Episodic Memory questions of the CB. However, this doesn't explain PC's perceptual confabulations. PC's general tendency to confabulate may have played a role in his pattern of performance, but it is unlikely to be the key factor. The type of tasks we have proposed in this study have never been proposed to patients with amnesic-confabulatory syndromes, but these patients typically don't confabulate when describing perceptually presented stimuli.

Deficits of spatial working memory may contribute to neglect signs [START_REF] Malhotra | Spatial working memory capacity in unilateral neglect[END_REF][START_REF] Wansard | Support for distinct subcomponents of spatial working memory: a double dissociation between spatial-simultaneous and spatial-sequential performance in unilateral neglect[END_REF][START_REF] Wansard | Visual neglect: is there a relationship between impaired spatial working memory and re-cancellation?[END_REF], albeit with different weights in different patients [START_REF] Toba | Component deficits of visual neglect: "Magnetic" attraction of attention vs. impaired spatial working memory[END_REF]. However, by definition such deficits should affect items that must be remembered, because they are not directly present in the patient's view. This was not the case for the present stimuli, which could be explored at will until response and thus did not require the use of spatial working memory.

Although the pattern of brain lesions in a single patient requires confirmation to be accepted as strong evidence [START_REF] Bartolomeo | Botallo's error, or the quandaries of the universality assumption[END_REF], the building of a large database of such rare occurrences as confabulations in neglect would be hard to attain in a reasonable time span, and difficult to couple with an extended behavioural study when many patients have to be tested. Also, the excellent level of detail of anatomical study, which is now possible thanks to cutting-edge neuroimaging techniques, is difficult to obtain in large groups. Moreover, anatomical information from single cases can guide the building of testable hypotheses for future research. With these considerations in mind, we now outline possible conclusions about the relationships between PC's pattern of brain damage and his test performance. As suggested in the Introduction, two distinct patterns of white matter disconnection could be at the basis of PC's pattern of performance:

In the case of the "burning house", the right hemisphere might have correctly detected the difference between the two houses. If the left hemisphere is disconnected from the right hemisphere by a posterior callosal lesion, it would not be able to access information processed by the right hemisphere and leading to the production of confabulatory responses [START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF][START_REF] Geschwind | Disconnexion syndromes in animals and man -Part II[END_REF]. A similar observation and account was provided by classical studies from Gazzaniga and his group on callosotomized patients [START_REF] Gazzaniga | Cerebral specialization and interhemispheric communication: does the corpus callosum enable the human condition?[END_REF].

Alternatively, if the attentional networks were disconnected from the ventral visual stream within the right hemisphere, then left-sided items might not be able to be processed at a conscious level because of lack of attentional amplification. The resulting preconscious visual processing could have primed PC's choices. The patient might subsequently have produced rich confabulatory responses to explain his behaviour. PC's anatomical investigation supported our second hypothesis, because it revealed an intra-hemispheric temporo-parietal disconnection between occipito-temporal visual processing conveyed along the inferior longitudinal fasciculus and the fronto-parietal attentional networks. Hence, it is possible that preconscious visual processing of the burning house would have been conveyed along PC's inferior longitudinal fasciculus, but could not reach attentive/conscious processing due to a disconnection of the temporo-parietal segment of the arcuate fasciculus.

In conclusion, we described the rare case of a neglect patient showing confabulation for correctly identified stimuli in the neglected field, in addition to the more frequently described confabulations for undetected stimuli in the neglected field. We propose an intra-hemispheric disconnection account for these observations, stressing disconnection between the parietal and the temporal lobes of the right hemisphere. Future research is needed to assess the frequency of confabulations in neglect patients and their neural correlates.

FIGURE CAPTIONS

  2015) using Tractotron software as part of the BCBtoolkit (http://toolkit.bcblab.com). Results displayed in Fig 2c indicated that, when compared to control brains, the lesion damaged 19% of the long and 13% of the posterior segments of the arcuate fasciculus, 16% of the optic radiations and 11% of the inferior longitudinal fasciculus. The lesion damaged less than 5% of all other tracts.

Fig. 1 .

 1 Fig. 1. Stimuli used in the experimental investigation

ACKNOWLEDGEMENTS

The authors declare no conflict of interest. The research leading to these results received funding from the 'Agence Nationale de la Recherche' [grants number ANR-09-EMER-006 and number ANR-13-JSV4-0001-01]. Additional support comes from the program "Investissements d'avenir" ANR-10-IAIHU-06.


Yan Rafrafi

Nadège Durban
email: nadege.durban@gmail.com

Alexandra Bertron

Achim Albrecht

Jean-Charles Robinet

Benjamin Erable

Use of a continuous

Keywords:

Introduction

Part of the French long-lived intermediate-level radioactive waste (LLILW) results from the reprocessing of nuclear fuel by the PUREX (Plutonium Uranium Redox EXtraction) recycling procedure. This procedure involves the dissolution of the fuel rods in a hot nitric acid medium followed by selective solvent extraction [START_REF] Nikitenko | Potential applications of sonochemistry in spent nuclear fuel reprocessing: a short review[END_REF]. During the process, the wastes are significantly enriched with nitrate. In a final process, the waste is stabilized, notably in a bitumen matrix, and stored in cylindrical steel containers (primary packages). For final storage, it is proposed that the primary packages should be grouped in reinforced concrete packages and placed inside concrete waste cells. A project is underway in France to build such waste cells at a depth of around 500 m within a clay rock formation (the Callovo-Oxfordian mudstone formation), which constitutes a geological barrier [START_REF] Dossier | Tome Architecture et Gestion du stockage géologique[END_REF][START_REF] Gaucher | ANDRA underground research laboratory: interpretation of the mineralogical and geochemical data acquired in the Callovian-Oxfordian formation by investigative drilling[END_REF][START_REF] Vinsot | In situ characterization of the Callovo-Oxfordian pore water composition[END_REF]. After repository closure, water will start to resaturate the cell, and leaching of the concrete (hydroxide and cations such as Ca 2+ , Na + , K + , etc.) and waste components (oxyanions including nitrate, and organic matter) will occur [START_REF] Walczak | Quantitative and qualitative analysis of hydrosoluble organic matter in bitumen leachates[END_REF]. Hydrogen gas will form as a consequence of water radiolysis, anaerobic steel corrosion and possibly fermentative organic matter degradation. The presence of nitrate will promote oxidizing conditions in the repository, which may increase the mobility of some redox sensitive radionuclides (Se, U, Tc, Pu, Np, etc.) [START_REF] Albrecht | Microbial Catalysis of Redox Reactions in concrete cells of nuclear waste repositories: a review and introduction in cement-based materials for nuclear waste storage[END_REF]. Nitrate can be reduced by biotic and abiotic processes in the presence of electron donors, such as organic matter or dihydrogen [START_REF] Cao | Effects of pH on nitrite accumulation during wastewater denitrification[END_REF][START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF][START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Mateju | Biological water denitrification -a review[END_REF][START_REF] Truche | Abiotic nitrate reduction induced by carbon steel and hydrogen: implications for environmental processes in waste repositories[END_REF][START_REF] Libert | Molecular hydrogen: an abundant energy source for bacterial activity in nuclear waste repositories[END_REF]. Microorganisms are assumed to be principally introduced into the repository systems by ventilation and other human activities during both the construction and operational phases [START_REF] Poulain | Microbial investigations in Opalinus Clay, an argillaceous formation under evaluation as a potential host rock for a radioactive waste repository[END_REF][START_REF] Hallbeck | Principle organic materials in a repository for spent nuclear fuel[END_REF]. The activity of denitrifying bacteria could induce nitrate reduction into nitrogen gas (N 2 ) via several intermediate species: nitrite (NO 2 -), nitric oxide (NO) and nitrous oxide (N 2 O). Because of the presence of bacteria, nitrate concentrations should drop in the repository system, allowing a return to the initial reducing conditions and thus potentially leading to less mobile reduced forms of radionuclides (RN).

Nonetheless, the main question behind the scenario considered is whether bacterial denitrifying activity is really possible under the drastic pH conditions imposed by the large quantity of cementitious materials present in the concrete cell. The infiltration water in repository systems is considered a liquid media restrictive for bacterial development because of pH likely above 12 [START_REF] Albrecht | Microbial Catalysis of Redox Reactions in concrete cells of nuclear waste repositories: a review and introduction in cement-based materials for nuclear waste storage[END_REF]. Indeed, the literature on microbial denitrification mainly reports on experimental or environmental systems working at pH conditions between 6.0 and 8.5 [START_REF] Valera | Nutrition and physiology of denitrifying bacteria[END_REF][START_REF] Tiedje | Ecology of Denitrification and Dissimilatory Nitrate Reduction to Ammonium[END_REF][START_REF] Ashok | Remediation of nitrate-contaminated water by solid-phase denitrification process-a review[END_REF]. In alkaline systems, nitrate reactivity has been shown to be affected by the higher pH, and nitrite accumulation occurs mostly as a consequence of bacterial growth inhibition [START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Almeida | Competition between nitrate and nitrite reduction in denitrification by Pseudomonas-fluorescens[END_REF].

However, recent work has shown that a bacterial strain or consortia are capable of nitrate reduction at high pH [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF][START_REF] Rizoulis | The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste[END_REF][START_REF] Ersan | Nitrate reducing CaCO3 precipitating bacteria survive in mortar and inhibit steel corrosion[END_REF]. For example, Halomonas desiderata (Hd), an alkaliphilic, halotolerant and denitrifying bacterium from the Gammaproteobacteria class, can reduce nitrate at pH between 9.0 and 12.0 in cement dominated environments using acetate as electron donor, in both static (batch reactor) and dynamic conditions (continuous feeding) [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF][START_REF] Berendes | Halomonas desiderata sp. nov, a new alkaliphilic, halotolerant and denitrifying bacterium isolated from a municipal sewage works[END_REF]. Hd is also described to form biofilms on the surface of solid cementitious material [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF]. The biofilm formation on solid support could be a key strategy for the biomass to survive in hostile environments [START_REF] Flemming | The biofilm matrix[END_REF]. Rafrafi et al. [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF] have already shown that Hd was able to develop in cement leachate supplemented only by acetate and nitrate. The limiting pH for nitrate reduction by Hd in cement leachate was clearly identified [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF]. The cement leachate was used as a model aqueous solution representing the alkaline cementitious aqueous environment. The cement leachate is a poor nutritive medium for bacteria, containing only calcium, sodium and magnesium as major salts [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF][START_REF] Carde | Leaching of both calcium hydroxide and C-S-H from cement paste: modeling the mechanical behavior[END_REF][START_REF] Carde | Effect of the leaching of calcium hydroxide from cement paste on mechanical and physical properties[END_REF]. The new objective of our work is now to evaluate the impact of the cement leachate as well as the impact of the presence of solid cementitious surface on the nitrate reduction rate of Hd. The specific experimental set-up implemented by Rafrafi et al. [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF] was reused in the present work to determine denitrification rates by Hd in a multiphase system (presence or absence of cement matrices) for different feeding conditions. In a first experiment, the optimal culture medium for Hd cultivation described by Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF] was used. Then this culture medium was progressively replaced by a pure cement leachate supplemented with acetate and nitrate. All the experiments were carried out at pH 10, close to the optimal pH already defined for the growth of Hd [START_REF] Ersan | Nitrate reducing CaCO3 precipitating bacteria survive in mortar and inhibit steel corrosion[END_REF].

Materials and methods

The experimental set-up implemented consisted of a bioreactor connected to an exposure chamber (containing cement pastes) in an open circuit (see section 2.4) fed by solutions with various proportions of cement leachate and culture medium (see section 2.5).

Cementitious materials

CEM V/A 42.5 cement pastes (Airvault Calcia factory) were made with a water/cement ratio of 0.32. They were cast in cylindrical moulds (h = 50 mm, Ø = 27 mm). Cement paste specimen were kept in hermetic bags (to avoid any hydric exchanges with the exterior and to protect them against carbonation) for 28 days after demoulding. Then, they were cut into slices (h ≈ 10 mm) and sanded to create a surface roughness favourable to the development of a bacterial biofilm.

Cement leachate

Cement leachates were prepared by immersing CEM V/A 42.5 cement paste slices in 1L of deionized water (solid/liquid volume ratio: 1.03% -solid surface/liquid volume around 60 cm 2 /L) for 3 days under stirring. The average chemical composition of the cement leachate is given in Table 1.

Bacterial culture

Halomonas desiderata DSM 9502 obtained from the strain collection of DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) [START_REF] Berendes | Halomonas desiderata sp. nov, a new alkaliphilic, halotolerant and denitrifying bacterium isolated from a municipal sewage works[END_REF] was chosen as the model bacterium. It is a heterotrophic bacterium capable of using acetate as an electron donor and nitrate/nitrite as an electron acceptor in alkaline pH conditions (9 < pH < 12) [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF]. Culture medium DSM 9502 was prepared from the solutions described in Table 2 as detailed by Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF].

Overview of the experimental set-up

The experimental set-up was designed to evaluate the microbial denitrifying activity, notably the reduction rate for nitrate and nitrite in the presence of acetate in a multiphase system, while partly decoupling the microbial growth from the interactions between the various components of the system (microorganisms, cement leachate, solid cement paste) (Fig. 1).

The bioreactor, with a working volume of 2L, was the entrance component of the system. It was used to adapt the microbial culture to the batch reactor prior to the dynamic experiment. It was also used to explore the influence of device-related parameters (e.g. hydraulic retention time, temperature, etc.) on denitrification (Fig. 1). When the valve was open, the bioreactor was connected to a feeding solution tank ensuring a continuous supply at a constant flow rate of 0.66 mL/min via a peristaltic pump (Model 7554-85, 7-200 rpm, head Easy Load L/S for tube 13-18 7518-00 model). An exposure chamber having a working volume of 1L and containing 3 solid slices of cement pastes was connected downstream of the bioreactor (Fig. 1). Here, the aim was to evaluate the effect of solid cementitious matrices on the bacterial activity.

Experimental conditions, composition of feeding solutions

The bioreactor was inoculated with a preculture of Hd (2 mL of active growing culture, optical density around of 0.3 at 600 nm). Each experiment started with a 7-day batch culture period in the bioreactor, until an OD value around 0.1 was reached. 7 days was the time required to reach maximum biomass of Hd in a closed system [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF]. Subsequently, the bioreactor was fed continuously with a fresh medium and the exposure chamber was linked to the bioreactor.

Four feeding solutions (marked S1 to S4 -Table 3), were tested in order to evaluate their impact on denitrifying activity in the bioreactor and then in the exposure chamber. They consisted of mixtures of culture medium, water and cement leachate (Table 1) in different proportions. These solutions were then supplemented by sodium acetate (8.3 mM NaC 2 H 3 O 2 ) as the only electron donor, and sodium nitrate (5.9 mM NaNO 3 ) as the only electron acceptor (Table 3). The pH of the feeding solution was adjusted to 10 using NaOH (4 M).

The hydraulic retention time (HRT) was set at 50.5 h in the bioreactor, and thus 25.25 h in the exposure chamber. The bioreactor and the exposure chamber were thermostated at 37 • C, continuously deoxygenated by N 2 bubbling, and stirred (300 rpm).

Samples of solution were collected regularly at the outlet of the feeding tank (point K, Fig. 1), of the bioreactor (point L, Fig. 1) and of the chamber (point M, Fig. 1). Unfiltered samples were used for immediate measurements of optical density (OD) at 600 nm and pH. Two millilitre samples were filtered through a 0.2 m pore size filter (Minisart PES, Fisher Scientific) for ionic species analyses (nitrate, nitrite and acetate).

Analytical techniques

Bacterial growth

The bacterial growth was measured using optical density (OD) at 600 nm (JENWAY 7315 spectrophotometer). This method enables the quantity of biomass suspended in the medium to be measured directly. Both living cells and dead cells are counted by this method. The feeding solution was systematically used as a blank. Turbidity measurements to monitor bacterial growth were applicable when the liquid medium was not cloudy. Overestimation in the presence of cementitious particles could not be excluded.

Chemical analyses

Concentrations of Ca 2+ , K + , Na + , CH 3 COO -, NO 3 -and NO 2 -were measured by High Performance Ion Chromatography (Dionex ICS-2000 and ICS-3000) using analytical methods detailed by Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF] and Bertron et al. [START_REF] Bertron | Reactivity of nitrate and organic acids at the concrete-bitumen interface of a nuclear waste repository cell[END_REF].

Results

Reference experiment: 100% culture medium in the feeding tank (solution S1)

Based on batch experiments, Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF] described H. desiderata (Hd) as a model denitrifying bacterium capable of efficiently reducing nitrate in the presence of concrete (alkaline conditions). The main objective of the reference experiment presented here was to gain access to the growth rates and the denitrification rates under the optimal experimental conditions for Hd growth. The bacterial growth and both nitrate and acetate consumption rates followed similar trends. For this experiment, three time periods have been defined according to the trend of nitrate reduction rate in the bioreactor. For each time period, the nitrogen mass balance was calculated to highlight the preponderant nitrogen species (nitrate, nitrite, or nitrogen gas, i.e. NO or N 2 O or N 2 ) in the bioreactor, the chamber and the overall system (including bioreactor and chamber) (Fig. 2.c).

In the bioreactor, during the first time period ([0,54 h], Fig. 2.a), OD and both nitrate reduction and acetate oxidation efficiencies decreased, reaching the low values of 0.07, 81% and 46%, respectively, found at the beginning of period 2 ([54, 199 h], Fig. 2.a). This coincided with progressive production of nitrite in the bioreactor (Fig. 2

.c) ([NO 2

-] ≈ 1.8 mM on average, Figure A.1). Then, OD and both nitrate and acetate consumption rates in the bioreactor increased progressively to 0.10, 91% and 60%, respectively, and remained fairly stable during period 3 (] 199,388 h], Fig. 2.a).

The same trend was observed for the OD values in the exposure chamber over the three time periods, whereas no significant evolution was observed for nitrate and acetate consumption rates. The average nitrate consumption rate was 97% over the complete experiment. Neither significant nitrite production, nor inhibition or decrease of the microbial activity were observed. At the chamber outlet, after around 40 h, the proportion of nitrite relative to the total incoming nitrogen was always below 3% (Fig. 2.c) and the concentration was below 0.07 mM (Figure A.1). The fluctuation of the biomass growth in the exposure chamber resulted mainly from the fluctuations already observed in the bioreactor rather than from variations of growth in the exposure chamber.

For this reference experiment, the minimal nitrate consumption rate was above 80% in the bioreactor and 95% in the chamber (Fig. 2.c). In our design of experiment, it was assumed that the nitrogen gas produced was continuously evacuated from the aqueous phase (feeding solution) by the N 2 gas bubbling. Since the amount of nitrite measured was negligible compared to the nitrate consumed, the denitrification process could be considered as complete, with the gaseous stage being reached. The instantaneous nitrate reduction rate (RedR) (mM h -1 ) was calculated as in Eq. ( 1).

RedR = ([NO 3 -] t in -[NO 3 -] t out )/HRT (1)
where [NO 3 -] in (mM) and [NO 3 -] out (mM) are the concentrations of nitrate at the inlet and outlet, respectively, of the system under consideration (bioreactor, chamber or overall system) and HRT is the hydraulic retention time in hours. The same approach was applied to estimate the instantaneous total oxidized nitrogen (nitrate and nitrite -TON) reduction rate, and instantaneous acetate oxidation rate (OxR). The average of the OxR and RedR obtained by this calculation are reported in Table 4.

The reference experiment gave access to the nitrate reduction rate catalysed by Hd cells at pH 10 under optimal conditions of growth. The next step was to switch from a growth culture medium optimal for Hd to a simpler culture medium (less optimal) in order to approach the environment existing in packages of a radioactive waste repository.

3.2. 10% of optimal culture medium and 90% of sterilized water in the feeding tank (solution S2)

In the second step of the experiment, the feeding solution medium was made of an optimal culture medium diluted by replacing 90% with sterilized water. The aim was to assess Hd activity in minimal medium (low concentration of vitamins and minerals). Nitrogen mass balances were performed under four interesting time periods defined in accordance with the denitrification rate observed in the bioreactor for the period 1 and 2 and with the denitrification rate in the exposure chamber for the periods 3 and 4.

After a preliminary batch period of 7 days in the bioreactor containing the feeding solution with 10% culture medium, the OD value was 0.10. Immediately after the start of the continuous supply, the biomass dropped drastically, OD values reaching values below 0.04 after the first 72 h (time periods 1 and 2 in Fig. 3.a) and then remained globally low during the rest of the experiment (to 340 h). The same trends were observed for the nitrate and acetate consumption rates, which decreased from 50% and 30%, respectively, to below about 20% for both of them after 96 h of dynamic supply (period 2-]99, 200] h -Fig. 3.a -evolution of concentrations is available in Figure A.2). It would seem that the growth rate of the strain was severely reduced. Consequently, the bacterial cells were progressively washed out of the bioreactor and the exposure chamber, leading to lower acetate and nitrate consumption rates and no nitrite production.

In the exposure chamber during the first time period ([0,32 h] -Fig. 3.b), OD values, and also both nitrate and acetate consumption rates dropped drastically (to values around 0.08, 35% and 24%, respectively) and remained low during period 2 ([32, 96] -Fig. 3.bevolution of concentrations is available in Figure A.2). Solid cement paste slices were added into the exposure chamber after 132 h of culture to evaluate their impact on Hd's growth rate and denitrification rates. Less than 35 h after this addition, OD values and nitrate and acetate consumption rates had increased significantly, to 0.16, 56% and 50% respectively (time period 3]96, 200] h -Fig. 3.b). However, the positive effect of the cement paste addition on Hd's growth and denitrification activity was only temporary. At the beginning of time period 4 ([200, 336] h -Fig. 3.b), i.e. approximately 84 h after the cement paste was immersed, the nitrate and acetate consumption rates decreased very quickly, followed 30 h later by the OD values, which fell to below 0.1. The progressive loss of bacterial metabolic activity was thus observed first and then the bacterial cells were washed out of the reactors. Fig. 3.c clearly shows that the denitrification process was progressively interrupted in the bioreactor. Especially from time period 3, less than 10% of the nitrate concentration entering the bioreactor was reduced. The same trends of nitrate reduction occurred in the exposure chamber except in time period 3, where a production of 50% of nitrogen gas was noted. This sudden and shortterm increase of nitrate reduction rate was observed several hours after the addition of the cement paste in the exposure chamber. The release of soluble species induced by cement paste leaching seemed to boost the activity of the denitrifying strain Hd with solution 2. But after a few tens of hours (time period 4), some key elements required for sustainable Hd growth leading to nitrate reduction, and which had been released from the cement paste by leaching, seemed to be missing in the 10% culture medium. The overall nitrate reduction rate was estimated from Eq. ( 1). The average RedR defined for these experiments were very low: 0.023 mM N NO 3 -/h and 0.012 mM TON (TON includes NO 2 -and NO 3 -) in the bioreactor (Table 4), 0.054 mM N NO 3 -/h and 0.064 mM TON in the exposure chamber (Table 4), or 0.036 mM N NO 3 -/h and 0.034 mM TON if the period when the cement pastes had a positive impact was excluded. The use of only 10% of optimal culture medium clearly affected Hd growth and nitrate reduction. The nitrate reduction rate was divided by 5 in the bioreactor compared to the reduction rate obtained with 100% of optimal culture medium.

10% of culture medium and 90% of cement leachate in the feeding tank (solution S3)

Hd culture in the presence of low concentrations of optimal culture medium (10%) showed a lower denitrifying activity overall, both in the bioreactor and in the exposure chamber. However, for a short time following the addition of solid cement pastes in the exposure chamber, the denitrifying activity of Hd was improved under the low concentration of optimal culture medium (10%). In order to examine the impact of the leachate on Hd activity, distilled water was replaced by cement leachate. Three times periods were defined according to the trend of the nitrate reduction rate measured in the bioreactor.

Following the start-up of the constant supply of culture medium in the bioreactor, i.e. during time period 1 ([0,69] -Fig. 4.a), the OD values decreased from 0.14 to 0.06, with parallel generation of a decrease of 10% of the reduction rate of nitrate. Then, during time period 2 (] 69, 236] h -Fig. 4.a -evolution of concentrations is available in Figure A.3), the biomass growth remained quite stable whereas the removal efficiencies of nitrate and acetate continued to decrease -to 49% and 28%, respectively. The decrease of nitrate reduction efficiency was accompanied by nitrite production in the bioreactor, as in the reference experiment (Fig. 4.c; Fig. 2.c). Then, both acetate oxidation and nitrate reduction rates increased during period 3 ([236, 386] h -Fig. 4.a). One hundred percent of the nitrate was consumed whereas the OD values increased slightly to reach around 0.08. Finally, the global output flow of TON (nitrate and nitrite) from the bioreactor was below 1.1 mM except during the transient period 2, during which it reached around 3.4 mM, in particular because of nitrite accumulation.

In the exposure chamber, during the three distinct time periods (Fig. 4.b -evolution of concentrations is available in Figure A.3), the nitrate reduction efficiency was above 90%. The acetate consumption rate was 20% lower at the beginning of time period 1 and then increased following the input of a higher quantity of electron acceptor (nitrite and nitrate), notably during period 2 (Fig. 4). The acetate oxidation rate was limited by TON availability in the expo-Fig. 4. Bacterial growth and evolution of acetate and nitrate removal efficiencies in the bioreactor (a) and in the exposure chamber (b) during the continuous culture of Hd with 10% of optimal culture medium completed by 90% of cement leachate (solution S3) at pH 10. Nitrogen mass balance on the bioreactor, the chamber and the system (i.e. bioreactor + chamber) (c). sure chamber. In spite of difficulties in accurately monitoring Hd growth by measuring the OD in the exposure chamber (the measurements may have been affected by the presence of non-soluble inorganic particles coming from the cement paste leaching), the OD values were found to lie between 0.08 and 0.59 (Fig. 4.b), generally higher than the values obtained during the control experiment (Fig. 2

.b).

According to Fig. 4.c, it can be estimated that more than 80% of nitrate was reduced to nitrogen gas except during the peak of nitrite accumulation observed in both the bioreactor and the exposure chamber (from 69 h to 236 h). The OxR and RedR obtained for this experiment are reported in Table 3. The use of solution S3 (10% of optimal culture medium and 90% of cement leachate) was favourable to the cultivation of Hd. The cement leachate seemed to provide some elements essential for Hd growth, probably elements that were present in limiting concentrations in the 10% optimal culture medium.

100% cement leachate in the feeding tank (solution S4)

The experiment with 10% of optimal culture medium completed with 90% of cement leachate highlighted the positive impact of the cement leachate on Hd growth and its heterotrophic denitrifying activity. However, in the scenario scheduled for the LLIL waste repository, the aqueous environment should be similar to a cement leachate. Therefore, to consider conditions that were closer to reality, only cement leachate was used as a culture medium for Hd in the last step of this study. Three interesting times periods were defined according to the trend of nitrate reduction rate in the bioreactor.

In the bioreactor, during the first time period ([0,121] h -Fig. 5.a), the OD values first decreased from 0.09 to 0.05, remained quite stable during the second time period (] 121, 269] h) and then increased slightly to 0.07 during the 3rd period ([269, 368] h -Fig. 5.a). The nitrate reduction rate fluctuated between 37% and 62% during time periods 1 and 2 and then slowly increased to 78% during time period 3 (Fig. 5.a -evolution of concentrations is available in of OD and the nitrate and acetate oxidation rates did not follow the same trend in the bioreactor. The biomass quantity decreased when the nitrate reduction rates increased and vice versa. An average of 35% of nitrate was reduced into nitrite and not into nitrogen gas during the experiment time (Fig. 5.c.). The fraction of each nitrogen component studied was globally stable during the experiment. The proportions were approximately: 20% of nitrogen gas, 35% of nitrite and 45% of nitrate. Additionally, in contrast with the previous experiments, the pH was not stable (Table 5): it decreased from 10.2 to the minimal value of 9.2 after 293 h of culture and increased to 9.6 during time period 3.

In contrast with the bioreactor pH, that in the exposure chamber remained quite stable (Table 5). The nitrate reduction rate increased progressively during time periods 1 and 2 and reached values above 95% at the end of the 2nd time period, i.e. after 221 h of continuous culture (Fig. 5.cevolution of concentrations is available in Figure A.4). Nonetheless, the percentage of nitrite produced compared to the nitrite and nitrate input was 45%, 22% and 4% on average for periods 1, 2 and 3, respectively (Fig. 5.c). The nitrite proportion at the outlet of the chamber was higher than for previous positive experiments supplied with solutions S1 and S3. Denitrification could be considered as almost complete (at least 85% of nitrate and nitrite was reduced to nitrogen gas) only after 312 h of Hd cultivation with a dynamic supply of cement leachate (Fig. 5.b and Fig. 5.c).

Both the OD values and the acetate oxidation rate decreased in the exposure chamber during time period 1. The acetate oxidation rate reached the minimal value of 12% after 97 h of culture in continuous supply, while the nitrite production was the highest (3.5 mM) at the chamber outlet. OD reached its minimal value at 0.07 after 144 h of culture, i.e. roughly 50 h after the acetate oxidation inhibition. This was probably because the Hd growth was only inhibited before the bacterium was washed away and was not directly affected by the toxic impact of nitrite. Then, the bacterial activity inhibition seemed to dissipate: the OD increased to 0.13 and the acetate oxidation rate increased to 54% during the second time period (Fig. 5.b). Nonetheless, a decrease of biomass was observed at the end of time period 3, which was coupled with a slowdown of acetate oxidation rates and an increase of the nitrite concentration at the outlet of the chamber (about 1.8 mM on average).

In summary, under a constant supply of cement leachate supplemented by acetate and nitrate, after approximately 300 h of culture, the percentage of nitrate reduced to nitrogen gas was below 20% in the bioreactor and above 95% in the exposure chamber. The OxR and RedR obtained for this experiment are reported in Table 4. The reduction of nitrate by Hd was possible in an aqueous medium as poor as cement leachate (pH 10 and culture medium containing only cement leachate) [START_REF] Nikitenko | Potential applications of sonochemistry in spent nuclear fuel reprocessing: a short review[END_REF].

Discussion

Adaptation of H. desiderata from optimal growth medium to cement leachate

Decreasing the percentage of optimal culture medium from 100% to 10% by dilution with water (solution S2) or by cement leachate (solution S3, with 90% of cement leachate) had an impact on the Hd nitrate reduction rate. With solution S2, the nitrate reduction yield decreased and stayed below 20% in the bioreactor during the experiment whereas, with solution S3, the nitrate reduction was almost 100% after 240 h of culture in the bioreactor. The difference between results obtained with S2 and S3 could be explained by the mineral elements present in cement leachate. Table 1 shows that the major components of cement leachate were, by decreasing order of concentration: potassium, sodium, calcium, silicon, aluminium and iron. Given that Hd is a halotolerant microorganism and that its alkaliphilic growth depends strongly on the availability of ions [START_REF] Ersan | Nitrate reducing CaCO3 precipitating bacteria survive in mortar and inhibit steel corrosion[END_REF], it can be assumed that trace elements present in cement leachate can have an important influence on Hd activity.

This was supported by the results obtained for the solution S4, containing 100% of cement leachate. Even if the denitrification rates were lower, the culture of Hd with cement leachate alone was clearly observed and the nitrate reduction efficiency reached almost 100% after 200 h of culture in the exposure chamber.

Cement paste impact on H. desiderata culture and denitrifying activity

During the experiment with the 10% optimal culture medium (solution S2), a transient increase of biomass growth and of both nitrate and acetate removal efficiencies was observed a few hours after the addition of cement paste to the exposure chamber. This positive effect of the presence of cement paste on Hd denitrifying activity was thus time-limited.

Cementitious matrices release salts, notably cations such as calcium, sodium and magnesium, when in contact with aqueous media and, in particular, those under consideration in this study [START_REF] Albrecht | Microbial Catalysis of Redox Reactions in concrete cells of nuclear waste repositories: a review and introduction in cement-based materials for nuclear waste storage[END_REF][START_REF] Bertron | Understanding interactions between cementitious materials and microorganisms: a key to sustainable and safe concrete structures in various contexts[END_REF][START_REF] Savage | A review of analogues of alkaline alteration with regard to long-term barrier performance[END_REF]. The high and/or fast release of some minerals (Na + , Ca 2+ , K + , etc.) could stimulate Hd activity during approximately 30 h (period 3 -Fig. 3.b). The effect of adding solid cement paste was limited in time, probably because (i) the release kinetics of soluble species from the cement matrices progressively decreased and (ii) the reserve of bioavailable elements leached from cement paste was completely consumed after 30 h.

In the case of the experiment supplied with solution S4 (100% cement leachate)), the nitrate reduction was not complete in the bioreactor but the growth conditions in the exposure chamber enabled nitrate and nitrite reduction with better performances, i.e. 0.137 mM TON/h (Table 4). Nitrite and nitrate reduction started more quickly in the chamber. These differences between chamber and bioreactor may have resulted from the cement paste presence in the exposure chamber. In addition to the release by cement matrices of minerals that could have a positive impact on Hd activity, the cement pastes may have served as a solid support for the proliferation of Hd biofilms. Hd biofilm development could not be quantified by OD measurement because only free planktonic bacteria in the liquid culture medium are analysed by this analytical method. Nevertheless, microscopic observation of cement paste surfaces by epifluorescence microscopy (after staining cement surfaces with Syto ® 9 as a nucleic acid markerdata not shown) confirmed the ability of Hd to colonise cement pastes. This confirmed the results previously published by Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF] and Rafrafi et al. [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF]. More specifically, Rafrafi et al. [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF]. reported the presence of a biofilm of Hd on CEM V cement paste under conditions equivalent to those explored in the present paper (similar chemical composition of the feeding solution S4) but at pH 12.

According to Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF], Hd's biofilm could play an important role in the denitrification process. Inside the biofilm, conditions could be locally more favourable for bacterial growth and activity as the extracellular polymeric substance that constitutes the biofilm helps to sequester dissolved and particulate nutrients from the water phase, which can be then utilised as nutrients and energy sources [START_REF] Flemming | The biofilm matrix[END_REF]. Moreover, the bacterial phenotype can vary depending on whether bacteria are planktonic or organised in a biofilm; the gene expression can vary to adjust the physiology of the bacteria [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Mcneill | Acid tolerance response of biofilm cells of Streptococcus mutans[END_REF]. Thus, the gene expression of nitrate and/or nitrite reductase could be better adapted to the specific conditions of Hd growth. Biofilm formation is a key factor for survival in hostile environments as it helps the biomass to develop specific abilities and tolerance (to low substrate, unfavourable pH, toxins . . .) [START_REF] Flemming | The biofilm matrix[END_REF].

Stability of the pH

The pH stayed rather stable in the bioreactor and in the exposure chamber for the experiment series, except for the experiment without culture medium (solution S4) (Table 5). The culture medium contributed to the pH stabilisation. For this experiment, the pH decreased from 10.2 to 9.2 after 293 h of culture in the bioreactor (unlike solutions S1, S2 and S3, solution S4 was not buffered by carbonate). However, the pH stayed fairly stable in the exposure chamber, possibly due to the presence of solid cement pastes that contributed to pH stabilisation. For neutral pH, the reduction of nitrate into nitrite generates H + , which is generally reused in the nitrite reduction reaction. Thus, the complete denitrification contributed to a net consumption of 0.317 H + (equivalence of acidity) for each mole of nitrate reduced to nitrogen gas, so the pH could rise during the denitrification [START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF]. Whereas, for pH around 10.2 (pH of the feeding solution), the first step of denitrification (reduction of nitrate to nitrite) generates protons (Eq. ( 2)), which are partially consumed when nitrite is reduced in accordance with Eq. (3) [START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Mateju | Biological water denitrification -a review[END_REF].

NO - 3 + 0.25 CH 3 COO -→ NO - 2 + 0.25 HCO - 3 +0.25 CO 2- 3 + 0.5 H + (2)
NO - 2 + 0.375 CH 3 COO -+ 0.25 H + → 0.5 N 2 + 0.357 HCO - 3 +0.357

CO 2- 3 + 0.5 H 2 O (3)
At pH around 10.2, the reaction of CO 2 with H 2 O produces 0.25 equivalent of acidity, which makes the pH decrease. And, whatever the pH value, if the nitrite denitrifying activity is inhibited, H + accumulates and the pH decreases if the medium is not buffered. Thus, the pH has an important effect on the denitrification rate [START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF], but the denitrification reaction can also affect the pH stability, especially for a non-buffered medium.

Nitrite accumulation

An increase in the concentration of nitrite produced during nitrate reduction by Hd was observed in the bioreactor for experiments with feeding solutions containing culture medium alone (S1), 10% of culture medium diluted by cement leachate (S3) and cement leachate alone (S4) supplemented by acetate and nitrate. The nitrite accumulation was transient (S1 and S3) or permanent (S4) depending on the stability of the pH (around 10). The maximal nitrite concentrations never exceeded 2.3 mM. The increase of nitrite concentration was systematically accompanied by a decrease in the growth of Hd strain and its denitrifying activity.

Transient nitrite accumulation associated with an inhibition of the bacterial growth and activity has already been observed in previous works [START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Almeida | Competition between nitrate and nitrite reduction in denitrification by Pseudomonas-fluorescens[END_REF][START_REF] Parmentier | Enhanced heterotrophic denitrification in clay media: the role of mineral electron donors[END_REF]. A nitrite concentration of about 23 mmol/L has been reported to inhibit or have a weak toxic impact on the specific enzyme function and on bacterial growth [START_REF] Almeida | Competition between nitrate and nitrite reduction in denitrification by Pseudomonas-fluorescens[END_REF][START_REF] Ma | Effect of free nitrous acid as inhibitors on nitrate reduction by a biological nutrient removal sludge[END_REF]. The protonated species of nitrite, HNO 2 , has been reported as a strong inhibitor of the denitrification process [START_REF] Ma | Effect of free nitrous acid as inhibitors on nitrate reduction by a biological nutrient removal sludge[END_REF]. Nonetheless, the HNO 2 effects were mainly observed for pH below 8 [START_REF] Glass | Denitrification kinetics of high nitrate concentration water: pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Ma | Effect of free nitrous acid as inhibitors on nitrate reduction by a biological nutrient removal sludge[END_REF][START_REF] Jiang | The strong biocidal effect of free nitrous acid on anaerobic sewer biofilms[END_REF][START_REF] Yang | Strong influence of medium pH condition on gas-phase biofilter ammonia removal, nitrous oxide generation and microbial communities[END_REF]. According to Eqs. ( 4) and [START_REF] Walczak | Quantitative and qualitative analysis of hydrosoluble organic matter in bitumen leachates[END_REF] presented by Anthonisen at al. [START_REF] Anthonisen | Inhibition of nitrification by ammonia and nitrous-acid[END_REF], the HNO 2 concentration ([HNO 2 ]) that is pH dependent is almost negligible at pH 10 with low nitrite concentration ([NO 2 -]) (2.3 mmol/L maximum), i.e. below 2.10 -9 mM/L.

[HNO 2 ] = NO - 2 K a + 10 pH (4)
where K a is the ionisation constant of the nitrous acid equilibrium equation, which depends on temperature as follows:

K a = e -2300 273+T • C (5)
In an alkaline environment, the transient nitrite accumulation could correspond to the time necessary for the biomass to adjust to a change of the system, i.e. the transition from a batch system to a continuous system. Some time could be necessary for Hd cells to activate transcription of the genes encoding for nitrite reductase (NIR), the expression of which is notably induced by the nitrogen oxide [START_REF] Philippot | Denitrifying genes in bacterial and Archaeal genomes[END_REF][START_REF] Bergaust | Transcription and activities of NO(x) reductases in Agrobacterium tumefaciens: the influence of nitrate, nitrite and oxygen availability[END_REF]. The time required to reach the steady state is theoretically equivalent to 3 or 4 cycles of reactor volume renewal, i.e. 3 or 4 times the HRT, thus approximately 150-200 h for the bioreactor in this experiment. This corresponds to the time required for almost complete denitrification (>90%) in the experiment with 100% of optimal culture medium (solution S1) and with 10% of optimal culture medium completed by cement leachate (solution S3).

For the experiment with only cement leachate as the culture medium (solution S4), more time may be required to reach complete denitrification. Moreover, the pH variability from 10 to 9.2 could promote the permanent accumulation of nitrite in a concentration range of 2.3 ± 0.7 mM. Many factors, and especially the pH, may influence the nitrite and nitrate reduction rates and the effects of pH on the reduction rates of nitrite and nitrate can be different [START_REF] Cao | Effects of pH on nitrite accumulation during wastewater denitrification[END_REF][START_REF] Thomsen | Mass-spectrometric studies of the effect of ph on the accumulation of intermediates in denitrification by Paracoccus-Denitrificans[END_REF]. Denitrifying enzymes are relatively sensitive to pH values and there is a specific pH for each enzyme [START_REF] Cavigelli | The functional significance of denitrifier community composition in a terrestrial ecosystem[END_REF]. The bacterial enzymes involved in the reduction of nitrate are very different in their composition and location in the bacterial cells from those involved in the reduction of nitrite [START_REF] Jones | Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification[END_REF]. Thus, for the experiment with only cement leachate as the culture medium, the accumulation of nitrite was caused by the difference between the nitrate and nitrite reduction rates and the variability of the pH ( pH of 0.8 unit).

Nitrate and nitrite reduction rate by H. desiderata

Most studies on the determination of denitrification rates concern the domain of wastewater treatment or remediation, i.e. of polluted soil. In such research areas, the classical optimal pH for the denitrification process is close to neutrality, i.e. between 6 and 8.5 [START_REF] Valera | Nutrition and physiology of denitrifying bacteria[END_REF][START_REF] Tiedje | Ecology of Denitrification and Dissimilatory Nitrate Reduction to Ammonium[END_REF][START_REF] Ashok | Remediation of nitrate-contaminated water by solid-phase denitrification process-a review[END_REF]. According to the result obtained by Cao et al. [START_REF] Cao | Effects of pH on nitrite accumulation during wastewater denitrification[END_REF], the nitrate reduction rate decreased from 0.63 to 0.12 mg N/g MLVSS/min (equivalent to 5.29 and 1.01 mM/h with a Mixed Liquor Volatile Suspended Solids (MLVSS) concentration of 1.960 g/L), an increase of the pH value from 6.5 to 9.2. Nitrate reduction under alkaline conditions (pH above 9) is possible [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF][START_REF] Rizoulis | The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste[END_REF] but the RedR are lower than those obtained at neutral pH. Results reported in this study support this finding. The nitrate reduction rates in continuous supply with 100% of optimal culture medium, i.e. 0.103 mM NO 3 --N/h (Table 4), was similar to those obtained by Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF] and Rizoulis et al. [START_REF] Rizoulis | The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste[END_REF] in a batch reactor with a pH about 10. Alquier et al. [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF] obtained 0.13 mM NO 3 --N/h for the nitrate reduction by Hd with cement pastes in the reactor and Rizoulis et al. [START_REF] Rizoulis | The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste[END_REF] observed the reduction of 15 mM nitrate in one week, i.e. 0.089 mM NO 3 --N/h for nitrate reduction by a sedimentary microcosm. The denitrifying activity of Hd was not substantially impacted by the dynamic supply of opti-mal culture medium supplemented by acetate and nitrate; the HRT of 50.50 h was well suitable. When the optimal culture medium were replaced by 100% of cement leachate without HRT adjustment, the nitrate reduction rate decreased and reached the value of 0.066 mM NO 3 --N/h in the bioreactor. This rate was consistent to the Hd nitrate reduction rate defined by Rafrafi et al. [START_REF] Rafrafi | Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository[END_REF], i.e. 0.056 mM NO 3 --N/h in the bioreactor, under similar experimental set-up supplied continuously with cement leachate supplemented with nitrate and acetate at pH 10. In the case of denitrification at alkaline pH and especially with poor culture medium (100% cement leachate), high HRT was required. Hd needed a few hours to adapt to the environmental conditions and to complete the total reduction of nitrate and nitrite. In this study, the denitrification was considered as complete (above 90%) after approximately 220 h in the bioreactor (HRT of 50.5 h) for the experiment with solutions S1 and S3 and after 200 h -but only at the outlet of the exposure chamber -(HRT of 75.75 h) for the experiment with cement leachate (solution S4). The nitrogen mass balance calculated for all the experiments clearly showed the significant nitrite production due to incomplete denitrification. This production could be transitional (experiment S1and S3) or constant during the experiment (experiment S4).

Conclusion

The experimental set-up allowed the impact of cement leachate and solid cement paste on the denitrification rates of H. desiderata at pH 10 to be determined. We have highlighted that cement leachate (poor nutrient substrate) can provide some elements (certainly a complex combination -not identified) that enable H. desiderata growth and the reduction of nitrate and nitrite. Nonetheless, at the beginning of the experiment, nitrate reduction was faster than nitrite reduction. A temporary nitrite accumulation did occur which slowed down denitrification rates and bacterial growth.

According to these results and previous works, the cement paste in the exposure chamber can be colonised by a biofilm of H. desiderata that can help to improve the denitrification rates, especially when the growing conditions become unfavourable in the reaction chamber. Further ongoing works are exploring the possibility of microbial nitrate reduction by a natural consortium of bacteria and also the biodegradability of other carbon sources and/or electron donors.

Fig. 1 .

 1 Fig. 1. Schematic representation of the experimental set-up to study the impact of the feeding solution on the microbial activity with or without cement paste.

Fig. 2 .

 2 Fig. 2. Bacterial growth and evolution of acetate and nitrate consumption rates in the bioreactor (a) and in the exposure chamber (b) during the continuous culture of Hd with 100% of optimal culture medium (solution S1) at pH 10. Nitrogen mass balance in the bioreactor, in the chamber and in the global system (i.e. bioreactor + chamber) (c).

Fig. 2 .

 2 Fig.2.a and Fig.2.b show the evolution of the Hd growth with time and the evolution of the nitrate and acetate consumption rates ([X] t consumed /[X] t input where [X] t consumed = [X] t input -[X] t outlet ) in the bioreactor and in the exposure chamber, respectively. The evolution of the nitrate, nitrite and acetate concentrations of the feeding solution and in the outlet of the bioreactor and the expo-

Fig. 3 .

 3 Fig.3. Bacterial growth and evolution of acetate and nitrate consumption rates in the bioreactor (a) and in the exposure chamber (b) during the continuous culture of Hd with 10% of optimal culture medium completed by 90% of water (solution S2) at pH 10. Nitrogen mass balance on the bioreactor, the chamber and the global system (i.e. bioreactor + chamber) (c) over four interesting periods selected according to the particular trend of the OD values and the consumption rates in the exposure chamber.

Fig. 5 .

 5 Fig. 5. Bacterial growth and evolution of acetate and nitrate consumption in the bioreactor (a) and in the exposure chamber (b) during the continuous culture of Hd with 100% of cement leachate (solution S4) at pH 10. Nitrogen mass balance on the bioreactor, the chamber and the system (i.e. bioreactor + chamber) (c).

  Figure A.4). At the same time, the acetate oxidation rate increased progressively (around 0.2% per hour during the whole experiment) (Fig.5.a). In contrast with the previous experiment, the evolution

Table 1

 1 Chemical composition of the cement leachate (average).

	Concentration (mM)					pH
	Ca	K	Na	Si	Al	Fe
	0.38	2.85	0.63	0.38	0.15	< 0.1	11.58

Table 2

 2 Chemical composition of the culture medium.

	Solution 1		Solution 2
	Acetate	0.35 g	Na2CO3	5.40 g
	MgCl2, 6H2O	0.20 g	NaHCO3	4.20 g
	KH2PO4	1.00 g	Water	100 mL
	KNO3	0.62 g
	Water	900 mL

Table 3

 3 Composition and pH of the feeding solutions.

	Solution name	Composition in percentage (%)		Additional component	pH
		Culture medium	Water	Cement leachate
	S1 S2 S3 S4	100 10 10 0	0 90 0 0	0 0 90 100	8.3 mM NaC2H3O2 + 5.9 mM NaNO3	10 (adjusted by addition of NaOH)
	sure chamber are available in the supplementary material, Figure
	A.1.

Table 4

 4 Average values of acetate OxR, nitrite RedR and nitrate RedR for the four compositions of feeding solutions.

	Solution name		S1	S2	S3	S4
	Solution composition		100% CM a	10% CM a + water	10% CM a + cement leachate	100% cement leachate
		Bioreactor	0.103	0.023	0.093	0.066
	Nitrate (mM h -1 )	Chamber	0.020	0.054	0.033	0.099
		System	0.077	0.033	0.071	0.076
		Bioreactor	0.082	0.012	0.079	0.024
	TON (mM h -1 )	Chamber	0.063	0.056	0.044	0.137
		System	0.076	0.027	0.067	0.062
		Bioreactor	0.104	0.033	0.090	0.057
	Acetate (mM h -1 )	Chamber	0.064	0.057	0.054	0.084
		System	0.092	0.041	0.078	0.047

a CM: Culture medium.

Table 5

 5 Average pH in the bioreactor and in the exposure chamber.

	Solution name	Average pH (standard deviation)
		Feeding solution	Bioreactor	Chamber
	S1	10.2 (0.03)	10.1 (0.03)	10.1 (0.03)
	S2	10.1 (0.03)	10.0 (0.13)	10.0 (0.13)
	S3	10.2 (0.23)	10.1 (0.10)	10.1 (0.06)
	S4	10.1 (0.07)	9.7 (0.30)	10.2 (0.12)

Acknowledgments

This research project was financially supported by the French national radioactive waste management agency (Andrahttp:// www.andra.fr). The authors are grateful to S. Becker for reviewing the English language.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2017.05.016.