Description du sujet de thèse

The monitoring of in-situ biological parameters is at the heart of the new challenges of augmented observation of coastal waters, whether to respond to pathogen biomonitoring, diversity monitoring or for the monitoring of specific taxa. It is currently only possible through sampling followeb by culture-based laboratory analysis techniques that require several days before the diagnosis of contamination, making it impossible to monitor at high spatial and temporal resolution, but also to use any mitigation strategy in real time. They also fail to detect cells in a viable but non-culturable state induced by environmental changes (1, 2, 3).
Thus in an ideal scenario and to support augmented biomonitoring of coastal waters, environmental and bacteriological data should be collected, analyzed, interpreted and communicated remotely in near real time in order to detect or even better predict the presence of pathogens. With this project, we propose a multidisciplinary approach aimed at developing a measuring instrument for the continuous in-situ monitoring of the abundances of specific bacterial taxa using an automated biosensor. This sensor, currently being studied in the laboratory, makes it possible to detect molecular markers associating cellular activity and taxonomic information.

This project is based on two fundamental pillars:
i. A molecular procedure for the genetic identification of a bacterial taxon in the environment involving three steps: 1) pre-analytical step: water sampling, concentration and cell lysis; 2) analytical step: recognition of the molecular target by sandwich-type hybridization coupled with an immunological assay and 3) signal detection by colorimetry. This procedure will be evaluated on two bacterial models, on the one hand members belonging to the genus Vibrio spp. grouping together a great diversity of pathogenic species for humans and marine fauna and on the other hand the species Escherichia coli, an indicator species, regulated, witness to faecal pollution.

ii. The development of an automated microfluidic platform equipped with a new type of dispenser allowing the delivery of calibrated micro-volumes of several reagents in parallel using a single electromechanical actuator, coupled to a micro piezoelectric membrane pump allowing the implementation of an automated analytical system based on FIA (Flow Injection Analysis), the integration of 3D micromixers, and an absorption measurement cell equipped with a low-cost multispectral detector.
The combination of these two pillars makes it possible to envisage the creation of a measuring instrument that can be deployed in-situ in the water column for continuous monitoring of bacterial taxa of interest, with a low cost price and ease of replication by the technologies used. Due to its relatively generic nature, the development of this microfluidic platform can be applied, subject to adaptation, to a large number of colorimetric and/or bioluminescence methods, making it possible to address a wide spectrum of parameters (nutrients, pH, ATP, etc.) .

Goals
The main objective of the thesis will be to develop and fine-tune the analysis platform to demonstrate in the field the possibility of carrying out continuous monitoring of the presence of specific bacteria in coastal waters. The scientific questions will lie both at the level of the transfer of the molecular identification method into micro-volumes, and in the technological realization of a microfluidic platform capable of implementing all the necessary analytical steps in an automated and repeatable. The thesis will de facto involve a very close collaboration between the two host laboratories, in order to cross the constraints resulting from the colorimetric method chosen, and the technological constraints related to the microfluidic system.

Contexte de travail

The thesis will take place at the Laboratory of Analysis and Architecture of Systems (LAAS) in Toulouse, with regular missions at the Laboratory of Biodiversity and Microbial Biotechnology (LBBM) in Banyuls-sur-Mer. Accommodation solutions for Banyuls will be made available to the candidate. He/she will be able to rely on the expertise and the technological and human resources of LAAS, via its Micro-nanotechnology, MultiFAB characterization and additive manufacturing platforms, on the expertise and experimental platforms in microbiology and (bio )environmental chemistry of the LBBM, via the BIO2MAR molecular biology and BIOPIC cytometry platforms, as well as the in-situ observation and experimentation means of the Observatoire Océanologique sur Mer (BOSS, ReMiMed, ). The financing of the thesis has been acquired, and the project will rely on additional financial resources already acquired, in particular via Equipex+ Terra Forma.

https://www.laas.fr/public/fr/plates-formes
https://www.laas.fr/projects/MultiFAB/
https://www.obs-banyuls.fr/fr/observer.html
https://www.obs-banyuls.fr/fr/observer/remimed.html
https://terra-forma.cnrs.fr/

Le poste se situe dans un secteur relevant de la protection du potentiel scientifique et technique (PPST), et nécessite donc, conformément à la réglementation, que votre arrivée soit autorisée par l'autorité compétente du MESR.

Contraintes et risques

As the research theme is at the interface of microfluidics and marine and molecular biology, the candidate must demonstrate scientific curiosity, a taste for multidisciplinary approaches, and be motivated by this subject with high societal challenges. The student should have knowledge of microfluidics with an openness to applications in marine and molecular biology or vice versa.

Informations complémentaires

Nature of the funding:
OMER Research Group (Oceans and SEAs), Characterization and diagnostics of marine systems axis, “Digital Ocean” and “New Ocean Observation Tools” working groups

Management:
Vincent Raimbault (LAAS) - vincent.raimbault@laas.fr
Julia Baudart (LBBM) - baudart@obs-banyuls.fr

Bibliography:
1. Oliver J. D. (2010). FEMS Microbiol. Rev. 34, 415–425. doi: 10.1111/j.1574-6976.2009.00200.x
2. Girard L., Peuchet S., Servais P, Henry A., Charni-Ben-Tabassi N, Baudart J. 2017. Spatiotemporal dynamics of total viable Vibrio spp. in NW Mediterranean coastal area. Microbes Environ. 32 (3) 210-218. doi: 10.1264: jsme2.ME17028.
3. Schauer S, Jakwerth S, Bliem R, Baudart J, Lebaron P, Huhulescu S, Kundi M, Herzig A, Farnleitner AH, Sommer R, Kirschner A. (2015). Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry. Environ Microbiol. 17(11):4366-78. doi: 10.1111/1462-2920.12861.
4. Paniel N., and Baudart J. (2013). Colorimetric and electrochemical genosensors for the detection of Escherichia coli DNA without amplification in seawater. Talanta. 115: 133-142.
5. Da-Silva E, Barthelmebs L., Baudart J. (2017). Development of a PCR-free DNA-Based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA.Environmental Science and Pollution Research. 24(6):5690-5700. doi: 10.1007/s11356-016-8193-9.
6. Accardo A, Courson R, Riesco R, Raimbault V, and Malaquin L. (2018) Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution. Additive Manufacturing, 22:440 – 446, doi: 10.1016/j.addma.2018.04.027. URL https://hal.laas.fr/hal- 01875364
7. Lace, A, Byrne, A, Bluett, S, Malaquin, L, Raimbault, V, Courson, R, Hayat, Z, Moore, B, Murray, E. (2022) Ion chromatograph with three-dimensional printed absorbance detector for indirect ultraviolet absorbance detection of phosphate in effluent and natural waters. J Sep Sci. 45: 1042– 1050. https://doi.org/10.1002/jssc.202100897
8. Sanchez, R.; Groc, M.; Vuillemin, R.; Pujo-Pay, M.; Raimbault, V. Development of a Frugal, In Situ Sensor Implementing a Ratiometric Method for Continuous Monitoring of Turbidity in Natural Waters. (2023) Sensors, 23, 1897. https://doi.org/10.3390/s23041897