Khaoula Kouraiss
email: kouraiss.khaoula2@gmail.com

Khadija El Hariri
email: k.elhariri@uca.ac.ma

Abderrazak El Albani
email: abder.albani@univ-poitiers.fr

Abdelfattah Azizi
email: aziziabdelfattah@gmail.com

Arnaud Mazurier
email: arnaud.mazurier@univ-poitiers.fr

Bertrand Lefebvre
email: bertrand.lefebvre@univ-lyon1.fr

Digitization of fossils from the Fezouata Biota (Lower Ordovician, Morocco): Evaluating computed tomography and photogrammetry in collections enhancement

Palaeontological collections housing material from the Fezouata Shale Lagerstätte (Lower Ordovician, Morocco) are of a high scientific interest as they testify to the existence of Burgess Shale-type taxa in one of the most critical Palaeozoic period: the Cambrian-Ordovician transition. The preservation of this unique patrimony can benefit from the emergence of imaging methodologies that have provided innovative ways in three-dimensional (3D) digitization. Computed tomography and photogrammetry were applied in order to create 3D models of fossils from the Fezouata Biota. Tomographic results show the exciting potential of these techniques in internal investigation of fossils, while photogrammetric method enables surface reconstructions with great accuracy in terms of texture, color and morphology, and can be convenient when internal exploration is not required. Three-dimensional digitization techniques thus seem to be reliable methods suited to highlight the potential of palaeontological data housed in museums and make easier the scientific dissemination of information.

Introduction

Morocco has a vastly varied landscape, as well as numerous fossiliferous sites constituting a very rich and diversified palaeontological heritage worldwide. In the central Anti-Atlas, the Fezouata Shale (Zagora region, Fig. 1) is one of the most fascinating illustrations of this heritage; it encloses an extraordinary record of marine life in the Early Ordovician yielding thousands of spectacular, exceptionally well-preserved specimens (e.g. [START_REF] Van Roy | Ordovician faunas of Burgess Shale type[END_REF][START_REF] Goswami | Phenome10K: a free online repository for 3-D scans of biological and palaeontological specimens[END_REF][START_REF] Lefebvre | Soft-part preservation in a solutan echinoderm from the Fezouata Biota (Lower Ordovician, Morocco)[END_REF][START_REF] Lefebvre | Préservation exceptionnelle de parties molles chez des échinodermes stylophores de l'Ordovicien inférieur de la région de Zagora (Anti-Atlas central, Maroc)[END_REF][START_REF] Lefebvre | Exceptionally preserved soft pars in fossils from the Lower Ordovician of Morocco clarify stylophoran affinities within basal deuterostomes[END_REF][START_REF] Gutiérrez-Marco | Digestive and appendicular soft-parts, with behavioural implications, in a large Ordovician trilobite from the Fezouata Lagerstätte, Morocco[END_REF][START_REF] Vinther | Ancestral morphology of crown-group molluscs revealed by a new Ordovician stem aculiferan[END_REF] . This material is housed in natural history collections and holds a great scientific and authentic heritage value.

Palaeontological collections are often deemed to be a dry and dusty cultural heritage, with limited access to fossil specimens [START_REF] Cunningham | A virtual world of paleontology[END_REF]. However, in recent years, the discipline benefits from the emergence of powerful 3D digitization methods, making easier the communication of findings (Milory et al., 2015) as well as enabling to build flexible databases to enrich heritage inventories [START_REF] Goswami | Phenome10K: a free online repository for 3-D scans of biological and palaeontological specimens[END_REF][START_REF] De Paor | Virtual Rocks[END_REF][START_REF] Boyer | Morphosource: archiving and sharing 3-D digital specimen data[END_REF][START_REF] Lebrun | Morphomuseum: an online platform for publication and storage of virtual specimens[END_REF]. Indeed, 3D acquisition techniques such as photogrammetry and computed tomography (CT) are increasingly used in palaeontology. X-ray CT is a non-invasive tool based on the differential attenuation recording of an X-ray beam through a sample from different angles allowing the qualitative and quantitative analyses of the internal and external structures of an object in two-and three-dimensions without any risks for the original sample, which could be too rare or fragile for destructive investigation [START_REF] Sutton | Tomographic techniques for the study of exceptionally preserved fossils[END_REF][START_REF] Racicot | Fossils secret revealed: x-ray scanning and applications in paleontology[END_REF][START_REF] Sutton | Virtual Paleontology-an Overview[END_REF]. As for photogrammetry, which is one of the easiest and less expensive among all 3D surface digitization techniques [START_REF] Cunningham | A virtual world of paleontology[END_REF], it requires only a conventional camera and specific software to reconstruct the three-dimensional geometric properties of an object of interest [START_REF] Falkingham | Acquisition of high resolution three-dimensional models using free, open-source, photogrammetric software[END_REF][START_REF] Mallison | Photogrammetry in paleontology -A practical guide[END_REF][START_REF] Evin | The use of close-range photogrammetry in zooarchaeology: Creating accurate 3D models of wolf crania to study dog domestication[END_REF][START_REF] Wilson | Photogrammetry of the Oryctodromeus cubicularis type locality burrow and the utility of preexisting, standard field photographs for three dimensional digital reconstruction[END_REF]. The present contribution illustrates the results of 3D digital modelling of Fezouata fossils offered by both photogrammetry and CT, and evaluates their performances in the valorization of Fezouata Biota collections as well as in the exploration of its fossils.

The Fezouata Biota Database in Marrakesh

Since the original discovery of the Fezouata Biota by Mohamed 'Ou Saïd' Ben Moula in the early 2000s (Lefebvre et al., 2016a), to date, thousands of spectacular specimens were collected in situ , allowing the constitution of rich reference collections in institutions around the world, based mainly in France, Morocco, Switzerland, and the United States. The Cadi Ayyad University in Marrakesh, Morocco, owns one of the largest collections of fossils from the Fezouata Shale. Part of these collections is already in Marrakech, part is temporarily housed in the palaeontological collections of Lyon 1 University, France, pending its repatriation to Morocco (Fig. 3B), and finally, another part is on loan for scientific purposes in more than 15 institutions worldwide. The establishment of Fezouata collections at Marrakesh University was one of the primary aims of the collaboration initiated in 2002 between the Universities of Burgundy (Dijon) and Cadi-Ayyad (Marrakesh) by Bertrand Lefebvre and Khadija El Hariri (Lefebvre et al., 2016a, b). In this context, digital acquisition of Fezouata fossils was performed to better manage the collection and make it available for research. At first time, the digitization was designed using Microsoft Excel (Fig. 4B) and then this file was imported under the FileMaker Pro software (FileMaker Inc.), a cross-platform application, which allows the creation of customizable databases to suit the user requirements. Specimens are stored in the program as text records containing all the corresponding informations: inventory number, inventory status, systematics, stratigraphy, locality information, images, collection information (period, collector), loans, figurations, coordinates of the host organizations and any additional details. Data into FileMaker Pro can be visualized in three different ways as a form, as a list, and as a table (Figs. 4C-D). According to the last update in late October 2018, the full Marrakesh collection encompasses an estimated 4241 inventoried specimens, with 300 are figured and 12 are type specimens. This total includes annelids, arthropods, brachiopods, bryozoans, cnidarians, echinoderms, eldoniids, graptolites, hyolithids, lobopodians, molluscs, palaeoscolecidan worms, sponges, and other unidentified specimens (Fig. 3A). Any record that had no taxonomic classification, was assigned to the category 'incertae sedis'. The Fezouata Biota database in Marrakesh is regularly updated and improved to meet the demands of a constantly evolving research. The value of a collection depends on the contribution of its specimens to the scientific accumulation of knowledge [START_REF] Balzeau | Applications des méthodes d'imagerie en paléoanthropologie: apports en termes de préservation, gestion et développement des collections[END_REF][START_REF] Bradley | Assessing the value of natural history collections and addressing issues regarding long-term growth and care[END_REF]. This is substantially illustrated in the specific case of the Fezouata Biota, by the involvement of over 50 scientists of 13 different nationalities in its study (Lefebvre et al., 2016a, b). Accordingly, Fezouata fossils remain in increase demand for use in research projects on a national and international scale. This means that institutions that house this heritage regularly loan material to researchers, and the specimens are transported all over the world, exposing them to damage or even loss. Hence, curators are confronting a double challenging mission which consists in preserving this unique heritage as well as paving the way for its scientific prosperity. In this sense, it is necessary to discuss the possibility of increasing the utility and the sustainability of Fezouata Biota collections by combining recent advances in 3D imaging methods, illustrated herein by computed microtomography and photogrammetry.

Material and Methods:

Material

Studied material consists of five specimens from the Fezouata Shale: AA.OBZ2.OI.2, AA.OBZ2.OI.3 and AA.OBZ2.OI.4 were collected from a site located in the North West of Tinzouline, Zagora (Central Anti-Atlas, Morocco), section [Z-F5(2)] wich is a lateral equivalent of [Z-F5] Oued Beni Zoli, within the A. murrayi Zone, whereas AA-TER-OI-5, AA-TER-OI-11 are from an unknown locality.

All specimens used in this study are housed in the collections of Cadi Ayyad University (Faculty of Sciences and Techniques), Marrakesh, Morocco.

Microtomography

Two specimens have been imaged at the University of Poitiers, France, with a laboratory device, the EasyTom XL Duo (RX-Solutions), set at the PLATINA platform of the IC2MP. According to the size of specimens, a microfocus x-ray source (Hamamatsu L8121-03) has been used, coupled with a flat panel imager (Varian PaxScan 2520 DX). The acquisition parameters are as follows: AA.OBZ2.OI.3 (8.5cm x 4.5cm): 75kV voltage, 333µA current, 1mm Al filter, stack acquisition with 2880 projections in 2 turns to acquire the whole specimen, 10 frames per second, average of 18 frames per projection, acquisition time of 1h45, anti-ring shift procedure and a spatial resolution of 30.5µm; AA.OBZ2.OI.4 (16cm x 10.5cm): 90kV, 460µA, 1mm Al filter, 2976 proj. in 3 turns, 12.5 fr. p. s., average of 20 fr. p. proj., acquisition time of 1h20, anti-ring shift procedure, and spatial resolution of 41.8µm. The weight of the raw data are 16 Go for both specimens. The data reconstructions (i.e. serial slices in 16bits uncompressed tif format) were computed using the software XAct (RX Solutions) with a filtered back projection algorithm (based on the Feldkamp method for cone beam geometry with Tukey filter) and attenuation of the beam hardening artefacts through linearization with a polynomial function. The weight of the reconstructed data are 5 Go and 8 Go for AA.OBZ2.OI.3 and AA.OBZ2.OI.4, respectively. Segmentation of the data, two and three dimension elaborations (virtual sections, 3D rendering and movies, wavefront objects) were performed using the software AVIZO v.9.7 (Thermo Fisher TM -FEI).

Photogrammetry:

The software program used in this study for the 3D reconstructions is the commercial Agisoft PhotoScan Professional 1.4.4. Agisoft PhotoScan is a stand-alone 3D scanning software package, and is among the most reliable digitization programs [START_REF] Kwan | Empowering cultural preservation in China through participatory digitization[END_REF], offering an easy-touse interface that can be followed by non-experts. Photographs were taken using a Canon EOS 1300D fitted with an EF-S 18-55 mm. The number of photographs required varies with the size and the detail level of the specimen; 36 to 47 photos were taken from different angles with only the natural light of the room, keeping a constant distance between the focus point and the specimen. A significant overlap between images was obtained to ensure the best coverage of the fossil.

The process of 3D model building from a physical specimen consists firstly of loading photographs into the program after checking their accuracy and then running the alignment. At this step, the software finds common points on photographs, matching them and determines the camera location of each photograph (Fig. 5A). Based on the estimated camera positions, the program reconstructs the depth information of each photo to create a dense point cloud that is very close to the appearance of the real object (Fig. 5B). This cloud is subsequently used to build a polygonal mesh, which consists of triangles that connect the 3 coordinates X, Y, Z (Fig. 5C). The program calculates then the texture of the mesh and constructs a 3D model of the fossil surface (Fig. 5D). The models were made with the default parameters of the program, except for the quality setting of "build dense cloud" that was changed to medium.

Results

Microtomography

For each analyzed sample, the reconstructed dataset can be visualized and operated through various ways: series of parallel slices along the orthogonal planes, oblique slices, surface rendering, volume rendering with or without false color, with or without transparency (see attached movies created with Avizo in supplementary data). CT provides reconstructed images whose contrasts are related to the differential attenuation of x-rays by materials. Indeed, the gray levels intensity of a given voxel in CT data is linked to the mean absorption of the X-ray beam by the corresponding volume of matter (i.e. linear attenuation coefficient, µ) which is a function of material density, atomic number Z, and energy of the incident beam [START_REF] Kak | Principles of Computed Tomography Imaging: Philadelphia, Pennsylvania[END_REF][START_REF] Stock | MicroComputed Tomography: Methodology and Applications[END_REF]. Thereby, the gray scale allows relative comparison of densities: the denser the material is, the more strongly it attenuates the beam, and the higher the pixels intensity is in the reconstructed data. Conversely, the low density materials are the darkest in the reconstructions.

For the sample AA.OBZ2.OI.3, microtomographic images revealed a clear clustering of the rhombiferan echinoderm Macrocystella bohemica and trilobites embodied in the matrix (Figs. 6B-G). Specimens are dense and exhibit good contrast with the hosting rock, bioturbations are also present (Fig. 7A,E). Some brighter elements can correspond to the preservation of an undissolved mineralized organism (7E). A colour code has been used in the segmentation according to the taxonomic attribution (Fig. 7). In AA.OBZ2.OI.4, the orthoconic cephalopod presents less, almost no contrast with the surrounding matrix (Fig. 8E) and is difficult, even sometimes impossible, to be successfully extracted. Unfortunately, the color structures visible to the naked eye on the external surface do not show any contrast in cross sections. However, transverse sections revealed a pronounced feature in the body chamber that is not visible on the surface (Figs. 8C-D). Furthermore, CT have revealed the occurrence of dense ovoid structures with regular shape, varying in size but no granular texture is visible in their interiors. These structures may correspond to a secondary filling of voids (i.e. dense minerals).

In the two cases presented, the hidden structures/specimens have been segmented by the combination of digital image processing tools available within the software package Avizo 9.7 (e.g., Non-local mean filtering, Top-Hat filter, and manual corrections with the Segmentation Editor).

Photogrammetry

Photogrammetric process has produced manipulable 3D digital models of the aglaspidid Tremaglaspis sp, a new xiphosurid, and the trilobite Asaphellus jujuanus (Figs. 9A-C). Reconstructed models display satisfying informations about morphological details in terms of texture, color and shape. The resolution and accuracy of these models are not the best that can be obtained and may be further improved by adapting the camera and reconstruction parameters. The models were saved and exported in Wavefront (*.obj) and 3D pdf formats. (3D Pdfs are provided as supplementary files).

Discussion

Computed tomography has been increasingly employed by palaeontologists to investigate internal structures and obtain the maximum knowledge about the fossils without destroying them [START_REF] Tate | High-resolution computed tomography for the comparative study of fossil and extant bone[END_REF][START_REF] Ketcham | Acquisition, optimization and interpretation of x-ray computed tomographic imagery: Applications to the geosciences[END_REF][START_REF] Carlson | Applications of high-resolution X-ray computed tomography in petrology, meteoritics and palaeontology[END_REF][START_REF] El Albani | Large colonial organisms with coordinated growth in oxygenated environments 2.1 Gyr ago[END_REF], 2014, 2019;[START_REF] Meyer | Three-dimensional microCT analysis of the Ediacara fossil Pteridinium simplex sheds new light on its ecology and phylogenetic affinity[END_REF]. For example, [START_REF] Gaspard | X-ray computed tomography: A promising tool to investigate the brachiopod shell interior. Effects on 3D modelling and taxonomy[END_REF] demonstrated the performance of this technique in the investigation of brachiopod shell, leading to the improvement of taxonomic precision in brachiopods. Likewise, a number of authors illustrated the great possibilities offered by microtomography in the study of fossils, revealing wealth of taxonomical detail, e.g. in tetrapods, [START_REF] Anderson | New information on Lethiscus stocki (Tetrapoda: Lepospondyli: Aistopoda) from highresolution computed tomography and a phylogenetic analysis of Aistopoda[END_REF] bryozoans [START_REF] Koromyslova | New species of Pachydermopora Gordon, 2002 and Beisselina Canu, 1913 (Bryozoa: Cheilostomida) from a Campanian erratic block (Belarus) and their micro-CT investigation[END_REF][START_REF] Koromyslova | Summary of micro-CT studies on Late Cretaceous bryozoans[END_REF], or palaeoscolecids, [START_REF] Kouraiss | X-ray microtomography applied to fossils preserved in compression: Palaeoscolescid worms from the Lower Ordovician Fezouata Shale[END_REF], allowing in some cases a detailed description and formal assignment to a species level (e.g., fossils in amber, [START_REF] Dierick | Micro-CT of fossils preserved in amber[END_REF]. Likewise, 3D models of Fezouata fossils generated herein by microtomography (micro-CT) illustrate how informations that are optically blocked from view can be revealed, which added considerable scientific value to these samples. Micro-CT have gleaned the clustering within the rock of columnals, stem portions and isolated thecal plates of M. bohemica, as well as several trilobites fragments including 2 trilobites mostly complete (Figs. 6C-G; 7B-D, F). The only specimen of M. bohemica mostly articulated, with complete stem still in connection with the theca, is the one visible on the surface (Figs. 6A-B). Tinzouline section [Z-F5 (2)], is the lateral equivalent of Oued Beni Zoli section [Z-F5], which has yielded extremely abundant, exquisitely preserved specimens of the rhombiferan M. bohemica. Although they have been crushed nearly flat, most individuals are fully articulated, with complete stems and brachioles still in connection with the theca [START_REF] Lefebvre | Palaeoecological aspects of the diversification of echinoderms in the Lower Ordovician of central Anti-Atlas, Morocco[END_REF]. This remarkable preservation requires the rapid, in situ entombment of alive individuals [START_REF] Brett | Echinoderm taphonomy, taphofacies, and Lagersta¨tten[END_REF], probably by distal storm events [START_REF] Ebbestad | An unusual onychochilid mollusc from the Ordovician (Tremadocian) Fezouata Formation, Morocco[END_REF]. In contrast, some thin levels are crowded with skeletal elements (e.g., thecal plates of Macrocystella, trilobite fragments), and contain few complete specimens. Such levels can be interpreted as resulting from the downslope transport and accumulation of skeletal elements by stronger storm events [START_REF] Ebbestad | An unusual onychochilid mollusc from the Ordovician (Tremadocian) Fezouata Formation, Morocco[END_REF][START_REF] Lefebvre | Palaeoecological aspects of the diversification of echinoderms in the Lower Ordovician of central Anti-Atlas, Morocco[END_REF]. Similar preservation is illustrated herein in tomographic results which have gleaned the clustering of disarticulated specimens enduring likely, after death, some possible reworking before entombment [START_REF] Ebbestad | An unusual onychochilid mollusc from the Ordovician (Tremadocian) Fezouata Formation, Morocco[END_REF]. In the orthoconic cephalopod, micro-CT results in transparency mode, did not produce satisfying results (Fig. 8E), which would have permitted a meaningful comparison with the taxonomic characters used for species described previously from the Fezouata Biota [START_REF] Kröger | Palaeogeography and palaeoecology of early Floian(Early Ordovician) cephalopods from the Upper Fezouata Formation, Anti-Atlas, Morocco[END_REF]. In a broader context, the results illustrate the potential of micro-CT in the investigation of hidden specimens and inner structures, as well as in the creation of virtual specimens, that can be manipulated digitally and easily shared without any risk for the original, often unique, specimens. Indeed, we predict a more widespread and systematic use of the method during the coming years in the exploration of new, but also ancient, palaeontological discoveries. Despite its many advantages, the method has some limitations in palaeontological research. Some fossils cannot be investigated by (micro-) CT scanner due to a high degree of mineralization or low absorption contrast (but synchrotron radiation and phase contrast tomography could be the solution; [START_REF] Taforeau | Applications of X-ray synchrotron microtomography for non-destructive 3D studies of paleontological specimens[END_REF][START_REF] Bidola | Absorption and phase contrast X-ray imaging in paleontology using laboratory and synchrotron sources[END_REF]. The size and morphology of specimens may also be an obstacle. If the specimen is too thick and/or dense, the transmission rate of the x-ray beam will be too low and no contrast will be seen [START_REF] Stock | MicroComputed Tomography: Methodology and Applications[END_REF]. If the specimen (or the rock hosting it) is too large, the spatial resolution will be too coarse to detect the presence of thin features or small fossils due to the partial volume effect [START_REF] Mazurier | Development of a fractcture network in crystalline rocks during weathering: Study of Bischop Creek chronosequence using X-ray computed tomography and 14 C-PMMA impregnation method[END_REF]; but local tomography can partially resolve this point). For instance, in the present study, the magnification used for scanning the entire AA.OBZ2.OI.3 sample is too weak to study the hidden trilobites in detail (Fig. 6,7). Thereby, even it is a non-invasive technique, it is sometime necessary to cut samples to reach a higher magnification and a better transmission rate (Reid et al., 2019). Moreover, most of the fossil samples have a non-cylindrical morphology and a heterogeneous mineralogical composition. They are then far from the optimum conditions for computed tomography, especially with laboratory devices using conical and polychromatic x-ray beams, and so subject to many artefacts (e.g. beam hardening, cone beam, metal and streak artefacts), which can hide or deform structures of interest [START_REF] Ketcham | Acquisition, optimization and interpretation of x-ray computed tomographic imagery: Applications to the geosciences[END_REF][START_REF] Cnudde | High-resolution X-ray computed tomography in geosciences: A review of the current technology and applications[END_REF]; but computed laminography, for instance, can be a satisfactory solution for flat sample; [START_REF] Hann | A Jurassic gliding euharamiyidan mammal with an ear of five auditory bones[END_REF]. Furthermore, the cost of this technique is reasonably high, which restricts its application and requires a fine selection of fossils to be scanned.

Regarding photogrammetry, it is a three-dimensional (3D) surface digitization method. Agisoft photoscan constructs 3D models of studied fossil from a series of overlapping photographs taken at different orientations. Generated 3D models provide information about the shape, texture and color, representing original specimens with great objectivity (Figs. 9A-C) even in limited light conditions. Yet, this method is scale-less, which requires to include a scale bar on the images, or object with known dimensions that can serve as a scale [START_REF] Falkingham | Acquisition of high resolution three-dimensional models using free, open-source, photogrammetric software[END_REF]. Photogrammetric reconstruction is relatively easy as tutorials explaining the whole process are available online, along with many early studies, which have applied and discussed widely the method [START_REF] Falkingham | Acquisition of high resolution three-dimensional models using free, open-source, photogrammetric software[END_REF][START_REF] Kwan | Empowering cultural preservation in China through participatory digitization[END_REF][START_REF] Mallison | Photogrammetry in paleontology -A practical guide[END_REF][START_REF] Milroy | Digitising the collection evaluating photogrammetry as a means of producing a digital, three-dimensional model[END_REF]. Nonetheless, the photography phase is in many cases challenging. For instance, photographing large specimens without sacrificing accuracy can be complicated, because it is difficult to capture the whole sample and bringing out at the same time good details, if a high resolution of the model is required. Similarly, the small size of specimens makes them quite difficult to photograph adequately, as the closer an object is to the camera, the more it blurs, and both resolution and contrast suffer. The photography phase has an enormous impact on the final model, hence it was necessary to reshoot studied samples multiple times, until obtaining images with the best possible quality. Besides, the accuracy of the final model relies also on parameters fixed at each stage of the process (lowest, low, medium, high, and ultra-high). The construction time increases when the detail level and the image resolution are high [START_REF] Falkingham | Acquisition of high resolution three-dimensional models using free, open-source, photogrammetric software[END_REF][START_REF] Fau | Apport de la photogrammétrie à la numérisation 3D d'os de spécimens montés : potentiel et limites[END_REF]. Accordingly, the specific requirement of a digital specimen determines ultimately the appropriate parameters to use. In this respect, it is often helpful to avoid high options when a 'nice-looking'' 3D model for visualization purposes is required, while for documentation, high accuracy is necessary [START_REF] Remondino | Image-based 3D modelling: a review[END_REF].

Photogrammetry is an inexpensive, portable, flexible and easy to use approach to obtain 3D models [START_REF] Evin | The use of close-range photogrammetry in zooarchaeology: Creating accurate 3D models of wolf crania to study dog domestication[END_REF][START_REF] Fau | Apport de la photogrammétrie à la numérisation 3D d'os de spécimens montés : potentiel et limites[END_REF]. For these reasons, the method has seen a growing application among palaeontologists and archaeologists over the past decade [START_REF] Falkingham | Acquisition of high resolution three-dimensional models using free, open-source, photogrammetric software[END_REF][START_REF] Kersten | Image-Based Low-Cost Systems for Automatic 3D Recording and Modelling of Archaeological Finds and Objects[END_REF]. According to [START_REF] Petti | Digital 3D modelling of dinosaur footprints by photogrammetry and laser scanning techniques: integrated approach at the Coste dell'Anglone tracksite (Lower Jurassic, Southern Alps, Northern Italy)[END_REF], the photogrammetric 3D models of dinosaur tracks have provided accurate morphometric details, as well as several interesting information not recognizable in the field, leading to new interpretations about dinosaur walking dynamics. In the same way, photogrammetry performance was evaluated by many other authors as an accurate tool to produce reliable 3D models with satisfying levels of morphological detail in terms of texture, coloration and geometry (e.g. palaeobotanic specimens, [START_REF] Milroy | Digitising the collection evaluating photogrammetry as a means of producing a digital, three-dimensional model[END_REF]; extinct giant ratite bird, [START_REF] Fau | Apport de la photogrammétrie à la numérisation 3D d'os de spécimens montés : potentiel et limites[END_REF]wolf crania, Evin et al., 2016). Getting the high performance computer required by Agisoft Photoscan [START_REF] Li | Construction and Accuracy Test of a 3D Model of Non-Metric Camera Images Using Agisoft PhotoScan[END_REF], the main limitations met in this study are recognized in the large file sizes corresponding to the 3D models and the relatively low size of RAM, which has limited the general performance of the system and increased significantly the processing time.

In summary, digital models of specimens of the Fezouata Biota either produced through micro-CT or photogrammetry, underline the great capability of these 3D imaging techniques in research field and collection enhancement. On the one hand, generated 3D models could be stored in the Fezouata Biota database, providing a permanent digital record of palaeontological findings, as well as helping to reduce damages resulting from excessive handling of original specimens and therefore contributing to long-term conservation and preservation of more delicate specimens including type and figured ones. On the other hand, these methods facilitate the scientific communication, as they enable different researchers to exchange the virtual models of the studied specimens, and solve in some cases the problem of loans, especially if targeted specimens are required and cannot be transported because of their weight, administrative obstacles, or as they are of great value, it can be necessary to avoid any risk -even minimal-of loss or destruction. Further, such digital models make easier the dissemination of technical information to nonspecialists audience (e.g. [START_REF] Bates | Digital imaging and public engagement in palaeontology[END_REF][START_REF] Bates | Application of high-resolution laser scanning and photogrammetric techniques to data acquisition, analysis and interpretation in palaeontology[END_REF], allowing them to interact with manipulable virtual specimens as well as giving the chance to those who consider fossils as very old and dusty objects, to see them in an artistic way and discover the 'beauty' of the iconic specimens.

Finally, it should be emphasized that the direct interaction with the physical specimen cannot be entirely replaced by the virtual model, notably in the case of palaeontological researches, which depend strongly on physical samples and require in some cases many analyses of the rock. Interestingly, these digital models can constitute an alternative of the physical specimens in some studies, if only they are accurate enough and display the level of details sought by the researcher.

Conclusion

Specimens of the Fezouata Biota are the core of a wide collaborative research program in continuous evolution and we should make our best to preserve this unique heritage. The examples presented here demonstrate that the Fezouata fossils could be more exciting and may yield additional informations, if they are digitally reconstructed in 3D. However, the adoption of 3D imaging methods, such as microtomography and photogrammetry, in palaeontology is still far from ubiquitous and the full potential of these techniques remains to be further explored. We believe that the use of these digital techniques would certainly be beneficial, if it is extended to wider practices, stressing that the virtual 3D models could give an additional value to the fossil record in terms of internal investigation, scientific exchange, virtual documentation, conservation and valorization of collections. Supplementary data can be found in the online version at http://dx. doi.org

Fig. 1 .

 1 Fig. 1. A, Location of studied area in Africa. B, Map of the Anti-Atlas showing the location of Zagora and the distribution of Ordovician outcrops. C, General view of a part of the excavation site of Bou Izargane, lower part of the Fezouata Shale (late Tremadocian), about 20 km N. of Zagora.

Fig. 2 .

 2 Fig. 2. Examples of exceptionally preserved fossils from the Fezouata Biota. A, Tremaglaspis sp. aglaspidid and a xiphosurid (AA-TER-OI-16). B, Furca sp. marrellomorph (AA-TER-OI-14). C, Bavarilla sp. trilobite (AA-BIZ15-OI-16). D, Pirania auraeum demosponge (AA-TER-OI-15). E, undetermined palaeoscolecid worm (AA-BGF2-OI-1). F, Pirania auraeum demosponge (AA-JBZ-OI-115). C, E, F, from Martin et al., 2016. Scale bars: A, 20 mm; B, D, 10 mm; C, E, F, 5 mm.

Fig 3 .

 3 Fig 3. A, Distribution of fossiliferous groups from the Fezouata Shale belonging to the Marrakesh collections. B, Marrakesh collections temporarily deposited at Lyon 1 University, France.

Fig. 4 .

 4 Fig. 4. A, Excavation site of Bou Izargane, in the Fezouata Shale. B-D, Screenshots of the Fezouata Biota Database in Excel format (B) and FileMaker Pro layouts (C, D).

Fig. 5 .

 5 Fig.5. Main steps of photogrammetric reconstruction process (AA-TER-OI-5). A, Aligned images. B, Dense cloud generation, C, Polygon mesh construction. D, Textured model.

Fig. 6 .

 6 Fig. 6. Macrocystella bohemica sample (AA.OBZ2.OI.3). A, General view of the studied material. B, 3D rendering of the external morphology. C-E, Virtual transverse sections showing "hidden" specimens. F-G, 3D modelling using transparency. B-F, Microtomographic images. Scale bars: A, F, G, 10 mm; B, 5 mm; C-E, 2.5 mm.

Fig. 7 .

 7 Fig. 7. Macrocystella bohemica sample in transparency (AA.OBZ2.OI.3). A colour code has been used according the taxonomic attribution (A). Macrocystella bohemica fragments in green (B); trilobites in blue (C) and (D); Bioturbations in pink and other elements in beige (E); (F) profile view. Scale bars: A-E, 10 mm; F, 15 mm.

Fig. 8 .

 8 Fig.8. Orthoconic cephalopod (AA.OBZ2.OI.4). A, General view of the studied material. B, 3D surface rendering. C, D, Transverse sections revealing a feature in the living chamber (boxed area). E, 3D modelling using transparency. B-F, Microtomographic images. Scale bars: A, 2 cm; B-E, 10 mm.

Fig. 9 .

 9 Fig. 9. Final models of three specimens from Fezouata Biota, obtained with photogrammetry. A, Tremaglaspis sp., aglaspidid (AA-TER-OI-5). B, xiphosurid indet. (AA-TER-OI-11). C, Asaphellus jujuanus trilobite (AA.OBZ.OI.2).

Acknowledgments

We are grateful to the Académie Hassan II des Sciences et Techniques (Morocco) that largely funded this work. The authors acknowledge Dr. Fabrice Monna (University of Burgundy, Dijon) for the valuable discussion about the photogrammetric software. Dr. Abel Prieur is deeply thanked for the database software handling. Special thanks go also to Youssef Taib, Najib Akka and Fatim-zahra Ihbach for the kind help with specimens photography. Agisoft Photoscan team is also thanked to have provided us the License Key. We thank the PLATeforme INstrumentale d'Analyse of the IC2MP for the access to the microCT device of the University of Poitiers. This paper is an outcome of the cooperation program VALORIZ (2012VALORIZ ( -2015) ) funded by both the CNRST and the CNRS. Finally, the French region La Nouvelle Aquitaine and the FEDER are also thank here for their support.


F Sekli Belaïdi

L Salvagnac

S Assié Souleille

M.-C Blatché

E Bedel-Pereira

I Séguy

P Temple-Boyer

J Launay

Accurate physiological monitoring using lab-on-a-chip platform for aquatic micro-organisms growth and optimized culture

Keywords: Biosensor, electrochemical sensor, lab-on-chip, green microalgae culture, algal metabolisms

The present work was dedicated to the development of a lab-on-chip microsystem for the monitoring of microalgal photosynthetic activity. Thanks to integrated electrochemical microcells, dissolved oxygen O2 concentrations due to photosynthetic microalgal activity were measured in a continuous way and oxygen production rates of microalgae cultures were finally determined in the frame of artificial night/day cycles. Application was performed by studying the physiological metabolisms of the green microalga Chlamydomonas reinhardtii. First, the different growth dynamics of microalgal cultures were characterized, enabling the determination of the induction, "exponential growth", "declining growth rate" and stationary phases. Then, the influences of carbon-based nutrients, such as sodium bicarbonate HCO3Na and methanol CH3OH, as well as of pollutants, such as silver nitrate AgNO3, were studied, evidencing contradictory behaviors according to the competition between nutritional properties, toxicity effects and acclimation phenomena. This paves the way to the development of analysis microsystems for the understanding of microalgal metabolisms as well as for the improvement of microalgae growth processes and associated industrial production.

Introduction

The number of microalgae species is estimated at several hundreds of thousands [START_REF] Norton | Algal biodiversity[END_REF]. This vast diversity represents a strong potential for science and industry. Since 2010, various studies have shown that microalgae are a rich source of carbon compounds, which can be used in biofuels, health supplements, pharmaceuticals and cosmetics [START_REF] Grobbelaar | Algal nutrition[END_REF]. However, this fast increasing involves the cultivation of aquatic micro-organism biomass for generating high value product. This production is done either in closed photo-bio-reactors (PBR) or, more traditionally, in open systems which constitute a simple and mature technology already deployed at industrial scale, but without any integrated monitoring unit to deal with microalgal growth. In both cases, it is important to ensure that the conditions are the best to ascertain a perfect microalgae growth. This production and development require different key parameters to be measured: nutrients quantity and quality, light, pH, turbulence, salinity and temperature [START_REF] Grobbelaar | Algal nutrition[END_REF].

In general, variables to be monitored in microalgal growth processes can be categorized as physical, chemical and biological. For this last category, the monitoring process relies mostly on principles developed and validated using laboratory-scale systems because of difficulties in developing suitable online sensors. Recently, Havlik et al. reviewed some technologies commonly-used for online, in situ monitoring of process parameters associated to microalgae cultures, with a focus on the monitoring of physical (temperature, illumination), chemical (pH, pO2, pCO2, nitrogen-based or phosphorous-based nutriments) and biological (biomass composition and concentration, photosynthetic activity…) parameters [START_REF] Havlik | Monitoring of microalgal processes[END_REF]. Moreover, microalgae themselves may also be used as living biosensors since changes in photosynthetic activity and fluorescence allow to go back to the presence of pollutants in the analyzed water.

In this frame, microfluidics and lab-on-a-chip (LOC) developments propose low-cost, multiparameter, real-time, continuous monitoring methods [START_REF] Kim | A droplet microfluidics platform for rapid microalgal growth and oil production analysis[END_REF][START_REF] Bodénès | Microfluidic technique for enhancing biofuel and biorefinery industry based on microalgae[END_REF] while focusing on electrochemical [START_REF] Arduini | How cutting-edge technologies impact the design of electrochemical (bio)sensors for environmental analysis, a review[END_REF] and optical [START_REF] Wang | Optofluidic technology for water quality monitoring[END_REF] detection techniques. In order to deal with selectivity, beyond the use of several microalgae, the functional integration of electrochemical and optical methods will be required. Indeed, both detection principles are adapted to the analysis of microalgal biometabolisms (for example through the measurement of acidity/basicity, dissolved oxygen concentration, photosynthetic activity and fluorescence) but are concerned by different interferents. They can therefore cope more easily with water sample complexity in the frame of water pollution analysis. As a result, several works have proposed the development of labon-a-chip with both electrochemical and optical integrated microdevices in order to study microalgae cultures [START_REF] Novak | An integrated fluorescence detection system for lab-on-a-chip applications[END_REF][START_REF] Lafleur | Miniaturized centrifugal solid phase extraction platforms for in-field sampling, pre-concentration and spectrometric detection of organic pollutants in aqueous samples[END_REF][START_REF] Tsopela | Development of a lab-on-chip electrochemical biosensor for water quality analysis based on microalgal photosynthesis[END_REF][START_REF] Tahirbegi | Fast pesticide detection inside microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence[END_REF][START_REF] Zhou | Fabrication of a miniature multi-parameter sensor chip for water quality assessment[END_REF][START_REF] Maguire | Novel microfluidic analytical systems platform for the simultaneous detection of three algal toxins in water[END_REF][START_REF] Sekli Belaïdi | Towards integrated multi-sensor platform using dual electrochemical and optical detection for on-site pollutant detection in water[END_REF]. In this context, the use of such LOC platform will tackle off some bottlenecks associated to microalgae cultures. Among others, response times, a major parameter for improving process feedback, could be greatly improved, leading to the development of real-time analysis approach. This work describes the use of our integrated lab-on-a-chip recently developed [START_REF] Sekli Belaïdi | Towards integrated multi-sensor platform using dual electrochemical and optical detection for on-site pollutant detection in water[END_REF] to accurately track the physiological behavior of the green microalga Chlamydomonas reinhardtii (Cr). It is shown how it is possible to monitor growth rate or substrate/pollutant effect on microalgal populations in microvolumes, focusing on the physiological adaptation of aquatic micro-organisms to induced stress in solution.

Materials and methods

Chemical

The lab-on-a-chip detection platform was developed in previous works [START_REF] Tsopela | Development of a lab-on-chip electrochemical biosensor for water quality analysis based on microalgal photosynthesis[END_REF][START_REF] Sekli Belaïdi | Towards integrated multi-sensor platform using dual electrochemical and optical detection for on-site pollutant detection in water[END_REF]. Its microfabrication process is based on microelectronic mass production techniques in cleanroom. It leads to low-cost, easy to use microsystems and reproducible properties from one component to another. Specific B33 glass substrates were chosen in order to be cope with optical measurement [START_REF] Novak | An integrated fluorescence detection system for lab-on-a-chip applications[END_REF]. Indeed, in addition to the integration of electrochemical devices described in this paper, the optical properties (transparency, low absorption) of this type of support make it possible to perform observations under microscope for the counting of microorganisms, to initiate microalgae photosynthesis by optical illumination, and/or to consider alternative optical measurements required for the chosen culture. The electrochemical microdevices were fabricated according to the ElecCell (Electrochemical microcell) technological platform previously developed [START_REF] Christophe | Elaboration of integrated microelectrodes for the detection of antioxidant species[END_REF]. A double physical vapour deposition (PVD) step allowed producing fully integrated electrochemical microcells: a first titanium/platinum (Ti/Pt) thin film formed the working micro-electrode and the counter electrode as well as the sub-structure of the pseudo-reference electrode. A second silver (Ag) PVD film completed the integration of this last electrode. This layer was oxidized in a KCl solution through a post-process step to obtain a silver/silver chloride pseudo-reference electrode. A fully integrated three electrodes electrochemical microcell were therefore realized.

As described in previous works [START_REF] Vanhove | Final capping passivation layers for long-life microsensors in real fluids[END_REF][START_REF] Tsopela | Integrated electrochemical biosensor based on algal metabolism for water toxicity analysis[END_REF], a silicon nitride SiNx wafer-level passivation was performed using a low-temperature PVD process in order to define electrode surface and to ensure their long-term electrochemical stability.

Finally, the microfluidic systems were made by lamination of dry films [START_REF] Courson | Low-cost multilevel microchannel lab on chip: DF-1000 series dry film photoresist as a promising enabler[END_REF]. The so-obtained microchannels and associated microchambers make it possible to obtain displacements of fluids that do not require the use of micropumps or any other fluid actuation devices. In order to achieve a robust microsystem, the cover was made by wafer bounding using again B33 glass substrate. After dicing and individualization of the chips, the electrical connections were ensured by the welding of a flex, allowing the final realization of the lab-on-a-chip platform (Fig. 1). As a matter of fact, the LOC final analysis volume was estimated to one hundred cubic millimeters or microliters.

Our hybrid microsystem was finally completed by commercial blue-light emitting diodes (LED) (KP-3216QBC-D from Kingbright) that were chosen according to size (3.2 × 1.6 mm 2 ), appropriate wavelength (460-475 nm) and luminance (290 cd/m 2 ), as well as to a specific excitation filter (ET470/40x from ChromaTechnology Corp). This light source was close to the microalgae microchambers in order to initiate and control the photosynthetic activity.

Electrochemical apparatus

All electrochemical measurements were carried out by voltammetry with a multi-channel VMP3 potentiostat from biologic in a dark Faraday cage. In the frame of algal solutions, the electroactive species to generate the electrical signal was oxygen (O2) which is electrochemically reduced on the platinum (Pt) working microelectrode for a potential of -0.7

V versus integrated Ag/AgCl. Thus, dissolved oxygen was finally monitored electrochemically using chronoamperometry techniques.

Algae cultivation and sample preparation

The green microalga Chlamydomonas reinhardtii (Cr) CC-125 was obtained from the "Ecotoxicology of Aquatic Micro-organisms Laboratory" from the Quebecker "Département des Sciences Biologiques (GRIL -TOXEN, Université du Québec, Montréal, Canada). Cr was cultivated in 200 mL Erlenmeyer flasks at 25°C in mineral, liquid high salt medium (HSM) [START_REF] Sueoka | Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii[END_REF] with pH 6.9 ± 0.1. Media and flasks were autoclaved (120°C, 1.5 bar, 20 min) before inoculation and algal solutions are transplanted once a month under sterile conditions. The cultures were aerated with the atmospheric air containing 2.5% (v/v) of carbon dioxide CO2, which is passed through a bacteriological filter, and illuminated by a white light source with irradiance of 30 or 60 cd/m 2 according to culture experiments. Algal solutions were modified by adding the carbon concentration as a nutrient substrate for the algae: sodium bicarbonate HCO3Na and methanol CH3OH (Dow Chemicals Company). Silver nitrate AgNO3 (Dow Chemicals Company) was also used as a disturbing element of microalgal physiology.

The algal cell growth was synchronized by alternating light and dark periods (L/D -14/10 h) using the commercial blue-light LED as excitation source for photosynthesis. Two microalgae solution concentrations were studied with 1 × 10 6 and 6 × 10 6 cells/mL, noted C1 and C6 respectively, to evaluate the detection properties of the electrochemical sensor according to cell density. The C1 pristine solution study is here performed in order to demonstrate the possibility to use the sensor without any sample pre-treatment. To obtain the C6 concentration, 6 mL of initial algal suspension in the exponential growth phase C1 was centrifuged at 800G and resuspended in 1 mL of the culture medium. The cell count was carried out by the Fast Read 102  , which is a plastic slide that allows the simultaneous reading of ten chambers with a microscope [START_REF] Gunetti | Validation of analytical methods in GMP: the disposable Fast Read 102  device, an alternative practical approach for cell counting[END_REF]. Microalgae samples (volume: 1 mL) were finally analyzed by measuring the evolution of their photosynthetic activity in microvolumes while using LOC-based microsystems [START_REF] Tsopela | Development of a lab-on-chip electrochemical biosensor for water quality analysis based on microalgal photosynthesis[END_REF][START_REF] Sekli Belaïdi | Towards integrated multi-sensor platform using dual electrochemical and optical detection for on-site pollutant detection in water[END_REF].

All the experiments concerning the influence of the carbon-based substrates (sodium bicarbonate HCO3Na, methanol CH3OH) or pollutants (silver nitrate AgNO3) on the aquatic micro-organism growth were operated by dilution in fresh algal solutions that were collected the same day. Microalgae cultures in the exponential growth phase were specifically chosen to ensure their long-term viability in LOC-based microsystems [START_REF] Eu | A microfluidic perfusion platform for cultivation and screening study of motile microalgal cells[END_REF]. Different solutions (nutritive or toxic according to cases) were prepared for the growth of the aquatic micro-organisms while studying separately the influences of the different substrates/pollutants by varying concentrations. Since the sampling procedure required a five-minute duration, all the measurements were done after at least five minutes to provide the effect on bio-organisms.

Results and discussion

Using the same kind of LOC-based sensing platform, previous works concerned the results obtained when determining growth key parameters like pH, concentration of chloride (Cl -) ions (related to the medium salinity), as well as adapted luminance (290 cd/m 2 ) for optimized culture of microalgae [START_REF] Sekli Belaïdi | Towards integrated multi-sensor platform using dual electrochemical and optical detection for on-site pollutant detection in water[END_REF]. Here are developed the monitoring of the microalgae growth carried by measuring the evolution of the photosynthetic activity in microvolumes associated to the realized lab-on-a-chip. Briefly, it can be said that in the presence of light, photosynthetic activity is deduced from the rate of oxygen production. In the LOC, this rate is determined by measuring the dissolved oxygen (O2) thanks to the electrochemical microcell integrated in the tank. Figure 2 shows the evolution of the O2 reduction current obtained on the platinum working electrode during the day/night cycles (period: 60 s).

The "light off" curve slope provides information on the respiratory activity of the algae. This value is used to determine the number of live and active algae in the sample inserted in the LOC. Thanks to this measurement, the "light on" curve slopes can be normalized in order to determine the photosynthetic activities of different algal samples, whatever their concentration [START_REF] Tsopela | Development of a lab-on-chip electrochemical biosensor for water quality analysis based on microalgal photosynthesis[END_REF]. According to previous studies and experiments, the detection limit for the oxygen production rate measurements was estimated to 6 pA/s. It should also be noticed that this method, compared to conventional counting methods, makes it possible to discriminate the effectively functional algae. Thus, during the "light on" phase, the reduction negative current slope provides finally information on the photosynthetic rate and activity of the micro-organisms, enabling the real-time determination of optimal growth conditions. Finally, thanks to the small volumes in the LOC platform, the total response time is no longer than couples of seconds.

Growth dynamics

The growth of an axenic culture of microalgae is characterized by five phases. According to the handbook of microalgal culture: biotechnology and applied phycology [START_REF] Grobbelaar | Algal nutrition[END_REF], the theoretical growth curve of a population of microalgae as a function of time is represented according to figure 3, evidencing five different phases of algae growth in batch cultures experiences: 1) lag or induction phase: during this initial phase, little increase in cell density occurs: the lag is attributed to the physiological adaptation of the cell metabolism to growth.

2) exponential growth phase: during this second phase, the cell density increases as a function of time according to an exponential function, and the specific growth rate is mainly dependent on algal species, light intensity and temperature.

3) phase of declining growth rate: cell division slows down when nutrients, light, pH, carbon dioxide or other physical and chemical factors begin to limit growth. 4) stationary phase: in the fourth stage, the limiting factor and the growth rate are balanced, which results in a relatively constant cell density 5) death, "crash" or lethal phase: during the final stage, water quality deteriorates and nutrients are depleted to a level incapable of sustaining growth, and cell density decreases rapidly and the culture eventually collapses (in practice and more generally, culture crashes can be caused by a variety of reasons, including nutrients depletion, oxygen deficiency, overheating, pH disturbance or contamination).

Electrochemical sensing for tracking the speed and growth rate of algal cultures

As explained previously, in the LOC-based microsystem case, the microalgae population growth is studied by monitoring the evolution of the dissolved oxygen concentration during "light-on" phases. Each measurement was made while considering an analysis volume around 10 µL (10 mm 3 ). During the growth process, the algal solutions (concentrations C1 and C6) were sampled on several different days, and measurements of oxygen production rate were carried out in pico-amperes per second (pA/s). Values are reported on figure 4 and growth phase durations are estimated on table 1.

It should be noticed that, for the pristine solution C1, no measurement was possible until three days because oxygen production rates related to the collected microalgae were below the electrochemical detection limit (6 pA/s), preventing us for checking the induction phase.

Results demonstrate that the higher algae concentration, the higher oxygen production rate, and that the oxygen production rate is increasing with time. Nevertheless, similar growth phase durations were estimated for both C1 and C6 solutions. It is assumed that the induction and exponential phases of the microalgae culture growth were evidenced during the first two days and the following eight days. Then, the "declining growth rate" phases occurs as expected (duration: ~ 10 days), followed by the stationary phase (duration: 4 days and more). Since Chlamydomonas reinhardtii was characterized by a long stationary duration in certain culture conditions [START_REF] Kong | Culture of microalgae Chlamydomonas reinhardtii in wastewater for biomass feedstock production[END_REF], it was chosen not to study the lethal phase.

As a matter of fact, the analysis of microalgae growth metabolism was shown for measurement times around few minutes while considering pristine cultures. Such result should be associated to the use of microvolumes that allows high concentration variations for a given biological activity and/or metabolism [START_REF] Kim | A droplet microfluidics platform for rapid microalgal growth and oil production analysis[END_REF][START_REF] Eu | A microfluidic perfusion platform for cultivation and screening study of motile microalgal cells[END_REF][START_REF] Pourciel Gouzy | Development of pH-ISFET sensors for the detection of bacterial activity[END_REF]. This demonstrates that LOC-based microsystems allow the characterization of environmental samples in real conditions, as well as the determination of the different growth phases.

Effect of carbon-based substrates on algal culture growth

The composition of the culture medium in carbon-based substrate is supposed to influence on the photosynthetic activity of microalgae like Chlamydomonas reinhardtii, and therefore to be intimately linked to the algal growth rate. Here, by varying the intake concentration, effects of methanol CH3OH or sodium bicarbonate NaHCO3 supplies, which directly impact algal activity, were assessed for the pristine solution C1 (figures 5a and 5b).

Different starting points were evidenced for the oxygen production rate. Since measurement are normalized according to the microalgae concentration [START_REF] Tsopela | Development of a lab-on-chip electrochemical biosensor for water quality analysis based on microalgal photosynthesis[END_REF], this phenomenon is related to sample preparation. Indeed, even if higher illumination (60 cd/m 2 ) were used during culture in order to decrease preparation duration, the exponential phase was logically responsible for discrepancy of the initial value (about 70 and 35 pA/s for methanol and sodium bicarbonate respectively). Nevertheless, it was decided to discuss the true variations of the oxygen production rate rather than "normalized" values related to the maximal one.

In both cases, the oxygen production rate, and therefore the microalgae photosynthetic activity, is found to increase for the lowest carbon input concentrations and to decrease for the highest ones, evidencing the presence of an optimum. For methanol, this maximal value is obtained for a 75 mM concentration and is associated to a low amplification factor around 1.1. This result is in accordance with S. Stepanov and E. Zolotareva, whose works evidence a maximal amplification ratio of 1.3 for a 50 mM concentration [START_REF] Stepanov | Methanol-induced stimulation of growth, intracellular amino acids, and protein content in Chlamydomonas reinhardtii[END_REF]. For sodium bicarbonate, a higher amplification ratio (value: 1.5) was obtained for a concentration of 0.5 mM. Such amplification phenomena were already evidenced for other microalgae such as Chlorella vulgaris, Chlorococcum humicola and Desmococous olivaceum [START_REF] Jegan | Influence of different concentrations of sodium bicarbonate on growth rate and biochemical composition of micro algae[END_REF], and are here confirmed for Chlamydomonas reinhardtii. As a result, it can be concluded that sodium bicarbonate is characterized by higher growth enhancement, and therefore lower toxicity, than methanol. Thus, the measurement of the optimum supply of carbon source so as to increase the physiological activity of aquatic bio-organisms was finally evidenced for the pristine solution.

As a matter of fact, such results demonstrate the possibility of using LOC-based microsystems to determine quickly and simply the optimal amount of inputs for improving microalgae crops.

Monitoring of toxicity on algal culture growth

Finally, the influence of silver nitrate AgNO3 on Chlamydomonas reinhardtii (Cr) physiology was analyzed in order to study the alga physiological adaptation to the toxic stress induced by silver ions Ag + , well known as an anti-biofouling agent [START_REF] Swee-Li | Low temperature, rapid solution growth of antifouling silver-zeolite nanocomposite clusters[END_REF][START_REF] Kumar | Metal sorption by algal biomass: From batch to continuous system[END_REF]. Thus, the study of a 1 µM AgNO3 exposition on Cr photosynthetic activity was undertaken using a pristine solution (figure 6). A decreasing photosynthetic activity is shown during the lowest exposure durations, evidencing the toxic effect of silver nitrate. Nevertheless, after about an hour, the behavior of the curve changes drastically. At this time, the oxygen production rate starts to rise again. This can be explained by the assimilation of cellulose from dead cells by algae for photosynthesis [START_REF] Blifernez-Klassen | Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii[END_REF] and the final acclimation of Cr microalgae to the presence of pollutants for long exposure.

This phenomenon was already evidenced by B. Novicka et al. for macrovolumes (75 mL) and therefore longer exposures (4 days or about 100 hours) [START_REF] Novicka | Physiological characterization of Chlamydomonas reinhardtii acclimated to chronic stress induced by Ag, Cd, Cr, and Hg ions[END_REF]. Indeed, this duration discrepancy should be associated to the use of microvolumes in the case of a LOC-based microsystem, inducing the wide improvement in term of response times. Such low exposure durations allowed finally to go further and to finally study the toxicity of silver nitrate concentration Thus, Cr cultures were exposed at different AgNO3 concentrations levels and, according to our previous results, oxygen production rate was determined after the intrinsic five-minute exposure duration (figure 7). It was shown that the presence of silver ions Ag + in solution decreases the photosynthetic activity, evidencing a linear variation with concentration and characterizing fully the silver nitrate toxicity (sensitivity: 3.0 pA.s -1 .µM -1 ).

Conclusion

A portable lab-on-a-chip (LOC) microsystem was developed for the monitoring of microalgae photosynthetic activity while monitoring electrochemically the microalgal oxygen production rate thanks to integrated electrochemical microcells (ElecCell) in the frame of artificial night/day cycles. Demonstration was performed by studying the physiological metabolisms of the green microalga Chlamydomonas reinhardtii, dealing with growth phenomena as well as nutrient/pollutant metabolic influences. Thus, the monitoring of physiological activity based on the evolution of photosynthetic activity makes it possible to estimate and differentiate the different growth modes of microalgal cultures, enabling the determination of the induction, "exponential growth rate", "declining growth rate" and stationary phases. Moreover, the influences of carbon-based nutrients, such as sodium bicarbonate HCO3Na and methanol CH3OH, as well as of pollutants, such as silver nitrate AgNO3, were studied, evidencing contradictory behaviors according to the competition between nutritional properties, toxicity effects and acclimation phenomena. Specific attention was brought to the discrepancies associated to the use of microvolumes (around 10 µL or mm 3 ) rather than macrovolumes, evidencing lower durations for microalgal metabolic changes and therefore faster analysis. As a matter of fact, the proposed integrated lab-on-a-chip is adapted to the use of pristine cultures, small volumes of microalgae samples and small amounts of products/chemicals in order to optimize growth rate. It can therefore be concluded that the use of LOC-based analysis systems will allow to monitor in a simple and rapid way the change of parameters which can affect the microalgae cultures, making possible its optimization. Indeed, dealing with the optimization of bio-organisms culture processes (used for food or antibiotic production for example), the labon-a-chip contribution to rapid response times will be an added value in order to manage growth parameters as well as to detect toxic stresses or pollutant influences. It should be possible to check very precisely the growth stage, and to fix almost instantaneously the optimal harvest date. By monitoring growth phenomena and having a precise information of the state, it should also be possible to prevent any unsuitable behavior or any contamination of a given culture batch, giving the opportunity to operate a feedback on culture processes. This paves the way to the development of analysis microsystems for the understanding of microalgal metabolisms as well as for the improvement of microalga growth processes and associated agribusiness industrial production.

Figure captions

Table 1 :Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :

 1123456 Figure 1: LOC platform used for the algal physiology monitoring

Figure 7 : 1 :Figure 1 :Figure 2 :

 7112 Figure 7: Evolution of the oxygen production rate with silver nitrate concentration (exposure duration: five minutes, pristine solution C1) All data are mean experimental values and error bars are calculated standard deviations

Figure 3 :

 3 Figure 3: Five growth phases of microalgae cultures: 1) induction phase, 2) exponential growth phase, 3) declining growth rate phase, 4) stationary phase, 5) lethal phase

Figure 4 :

 4 Figure 4: Temporal variations of the oxygen production rate of fresh algal solutions (C1: pristine solution and C6: six-times concentrated solution, culture illumination: 30 cd/m 2 ) All data are mean experimental values and error bars are calculated standard deviations

Figure 5 :

 5 Figure 5: Evolution of the oxygen production rate with carbon nutrient concentration for the pristine solution C1: left) methanol CH3OH and right) sodium bicarbonate HCO3Na All data are mean experimental values and error bars are calculated standard deviations

Figure 6 :

 6 Figure 6: Evolution of the oxygen production rate with exposure duration to a 1 µM AgNO3 concentration solution (pristine solution C1) All data are mean experimental values and error bars are calculated standard deviations

Figure 7 :

 7 Figure 7: Evolution of the oxygen production rate with silver nitrate concentration (exposure duration: five minutes, pristine solution C1) All data are mean experimental values and error bars are calculated standard deviations

Acknowledgement

The authors would like to thank the French "Agence Nationale de la Recherche" (ANR, project DOLFIN, n° ANR-13-JS03-0005-01) for financing the project. Furthermore, microfabrication procedure was partly supported by the French RENATECH network.