Thèse Lutter Contre les Blooms Atténuation Durable et Sélective des Proliférations de Cyanobactéries Toxiques et de leurs Toxines H/F Doctorat.Gouv.Fr

Établissement : Institut National des Sciences Appliquées de Toulouse École doctorale : MEGEP - Mécanique, Energétique, Génie civil, Procédés Laboratoire de recherche : TBI - Toulouse Biotechnology Institute, Bio & Chemical Engineering Direction de la thèse : Cécile FORMOSA ORCID 0000000286273784 Début de la thèse : 2027-09-01 Date limite de candidature : 2026-11-23T23:59:59 Les proliférations d'algues nuisibles, en particulier celles causées par des cyanobactéries productrices de toxines comme Microcystis aeruginosa, représentent une menace mondiale croissante pour les écosystèmes d'eau douce, la santé humaine et la sécurité de l'eau. Ces blooms, aggravés par l'eutrophisation, le changement climatique et l'augmentation des niveaux de CO, libèrent des cyanotoxines puissantes comme la microcystine-LR, qui présentent des risques graves par exposition directe ou bioaccumulation dans les organismes aquatiques. Les méthodes traditionnelles de lutte (physiques, chimiques ou biologiques) montrent souvent des limites en raison de leur manque de sélectivité, de perturbations écologiques ou de problèmes de mise à l'échelle. Ce projet de thèse propose une stratégie duale innovante pour cibler simultanément les cyanobactéries et leurs toxines, en garantissant sélectivité, durabilité et un impact écologique minimal. La première approche consiste à développer des coagulants peptidiques conçus pour floculer sélectivement les cellules de Microcystis tout en préservant les microalgues bénéfiques. Cela passera par une caractérisation détaillée des parois cellulaires de M. aeruginosa dans des conditions de production de toxines, suivie de la conception rationnelle de peptides exploitant des interactions de surface spécifiques. La seconde approche utilise les substances polymériques extracellulaires (EPS) issues de microalgues non toxiques pour adsorber et éliminer les toxines résiduelles de microcystine-LR. Les EPS, connues pour leur polyvalence dans la liaison des polluants, seront optimisées pour la capture des toxines via des analyses structurelles et fonctionnelles. Le projet est structuré en trois lots de travail : (1) caractérisation des parois cellulaires de M. aeruginosa et conception de peptides, (2) production, fractionnement et étude des interactions des EPS avec la microcystine-LR, et (3) validation de la stratégie duale en systèmes de co-culture, évaluant l'efficacité de floculation, l'élimination des toxines et l'impact écologique. Des techniques avancées comme l'AFM, la HPLC, la RMN et la cytométrie en flux seront employées pour garantir précision et reproductibilité. Ce projet interdisciplinaire, mené en collaboration entre le Toulouse Biotechnology Institute (France) et l'Université de Hasselt (Belgique), vise à proposer une solution écologique et évolutive pour atténuer les proliférations de cyanobactéries. En combinant coagulation sélective et adsorption par EPS, il cherche à révolutionner les pratiques de traitement de l'eau, protéger la santé publique et préserver les écosystèmes aquatiques.
Harmful algal blooms (HABs), particularly those caused by toxin-producing cyanobacteria such as Microcystis spp., represent one of the most pressing environmental and public health challenges in freshwater ecosystems [1]. In recent years, these blooms have been increasing in frequency, magnitude, and duration worldwide, with eutrophication, rising CO levels, and climate change driving their global expansion [2]. Microcystins, the most common and potent cyanotoxins, pose severe risks to human health through direct exposure via contaminated water and indirect exposure via bioaccumulation in aquatic organisms, particularly fish and shellfish. Once ingested, these toxins can cause liver damage, neurological disorders, and even fatal outcomes in extreme cases. The global prevalence of Microcystis blooms, reported in over 100 countries, underscores the urgency of developing effective mitigation strategies [3].

Traditional approaches to managing cyanobacterial blooms have primarily focused on removing either the microalgae or the toxins, but rarely both. Physical methods, such as mechanical harvesting or sonication, can remove biomass but are often non-selective, energy-intensive, and costly. Chemical treatments, including the use of algicides like copper sulfate or hydrogen peroxide, can effectively lyse cyanobacterial cells but may release toxic co-products into the water and harm non-target organisms. Biological methods, such as the introduction of algicidal bacteria or competitive non-toxic algae, show promise but often lack scalability and long-term stability [4]. These limitations highlight the need for innovative, sustainable, and selective solutions that can address both the source of the toxins (the cyanobacteria) and the toxins themselves.

Recent advances in biotechnology and chemical engineering have opened new avenues for tackling this dual challenge. Selective removal of toxin-producing microalgae has gained attention as a complementary strategy. Conventional coagulation-flocculation methods, which rely on chemical coagulants such as aluminum sulfate or ferric chloride, are widely used but suffer from low selectivity and potential secondary pollution [4]. Similarly, bio-inspired coagulants, including chitosan and plant-based flocculants, have been explored but also lack selectivity, often removing beneficial microalgae alongside harmful species, in addition to challenges like limited scalability, seasonal availability of raw materials, and increased chemical oxygen demand (COD) in treated water, which can promote cyanobacteria regrowth [5]. In this context, we propose an innovative alternative: the development of peptidic coagulants specifically tailored to Microcystis spp., which remains for the moment an unexplored frontier. Peptides offer several advantages, including high specificity, biodegradability, and low toxicity, making them ideal candidates for targeted flocculation of harmful cyanobacteria while preserving beneficial microalgae.

In parallel, extracellular polymeric substances (EPS), produced by various types of microalgae under diverse environmental conditions, have emerged as a promising tool for the removal of various pollutants, including heavy metals and microplastics [6]. Given their versatility and demonstrated efficiency in adsorbing contaminants, EPS may also hold significant potential for the removal of cyanotoxins such as microcystins. EPS are complex, high-molecular-weight polymers secreted by microalgae, composed of polysaccharides, proteins, and other biomolecules. Their unique chemical properties, including electrostatic charges, hydrophobic regions, and functional groups, suggest they could interact with cyanotoxins through mechanisms such as adsorption, complexation, or specific interactions. While EPS have been shown to bind heavy metals and other pollutants, no study to date has used EPS from microalgae to absorb microcystins from water. Thus, the mechanisms of interaction, the efficiency of toxin removal, and the potential feasibility of scaling up this approach remain to be discovered. The overarching goal of this PhD project is to develop and evaluate two complementary and innovative strategies to mitigate the environmental and health risks posed by harmful microalgae blooms in freshwaters, with a specific focus on Microcystis spp. This cyanobacterium is well-known for producing toxins, such as microcystins, which threaten human health through direct exposure to contaminated waters and/or bioaccumulation in aquatic organisms, particularly fish that are later consumed. While most existing approaches to combating harmful algal blooms focus either on removing the microalgae themselves or on eliminating the toxins they produce, this project takes an integrated approach by addressing both challenges simultaneously. To remove the microalgae, we will develop a groundbreaking coagulation-based strategy, moving beyond conventional chemical methods. Instead, we will develop a custom-made peptidic coagulant designed to selectively flocculate toxin-producing Microcystis cells while sparing beneficial microalgae, thereby ensuring both specificity and minimal ecological disruption. To further safeguard water quality, we will complement this with a second strategy that targets the removal of residual toxins. This approach leverages the adsorption properties of extracellular polymeric substances (EPS) produced by non-toxic microalgae species, enabling the efficient capture and removal of microcystins and other toxins from the water. By integrating these two strategies, peptidic coagulation for targeted microalgae removal and EPS-based adsorption for toxin elimination, this project aims to deliver a comprehensive, innovative, and eco-friendly solution to the pressing challenges posed by harmful algal blooms. This PhD project aims to develop and validate a dual, innovative strategy for the mitigation of Microcystis aeruginosa blooms and their associated microcystin-LR toxins in freshwater systems. The approach integrates peptidic coagulation for selective removal of toxin-producing cyanobacteria and EPS-based adsorption for efficient elimination of residual toxins, ensuring both specificity and sustainability.

Work Package 1 - Peptidic coagulation strategy. This WP will start with a full characterization of the M. aeruginosa cell wall under conditions that promote toxin production, particularly in competitive environments with non-toxic microalgae such as Chlorella vulgaris. To achieve this, M. aeruginosa will be cultured in co-culture systems to simulate nutrient limitation, a known trigger for microcystin-LR synthesis. The chemical composition and surface properties of the cyanobacterial cell wall will be thoroughly characterized using a multi-technique approach. Fourier-transform infrared spectroscopy (FTIR) will identify key functional groups, while X-ray photoelectron spectroscopy (XPS) will determine elemental composition and bonding states. The polysaccharide composition of the cell wall will be analyzed via high-performance liquid chromatography (HPLC), and zeta potential measurements will assess the surface charge [7]. Additionally, the hydrophobic and hydrophilic properties of the cell surface will be investigated using Atomic Force Microscopy (AFM) by probing interactions with the surface of a clean bubble, following a method previously developed in our group [8]. These data will serve as the foundation for the rational design of model peptides tailored to selectively interact with M. aeruginosa cells. The peptides will be engineered to include complementary functional groups (e.g., carbohydrate-binding motifs or hydrophobic residues) and electrostatic charges that match the surface properties of the target cyanobacterium, while ensuring structural stability in aquatic environments. To evaluate the binding affinity and specificity of the designed peptides, AFM in force spectroscopy mode will be employed. Peptides will be immobilized on AFM tips following known protocols [9], and their interaction forces with M. aeruginosa cells will be measured under controlled conditions. These experiments will also include non-toxic microalgae to assess the selectivity of the peptides. The most promising candidates will then be chemically synthesized and tested for their flocculation efficiency and specificity in M. aeruginosa co-cultures systems. Optimization will focus on peptide concentration, pH and ionic strength to determine the optimal conditions for maximal flocculation while minimizing non-specific interactions with beneficial microalgae.

Work Package 2 - EPS-based toxin removal strategy. For this, non-toxic microalgae (C. vulgaris and P. kessleri) will be cultured under optimized conditions to maximize EPS production. The EPS will be extracted from the cultures using centrifugation and fractionated using ultrafiltration. The composition and structure of the different EPS fractions will be thoroughly characterized using Nuclear Magnetic Resonance (NMR) spectroscopy to identify polysaccharide and protein components [10]. Zeta potential and contact angle measurements will further elucidate the surface properties of the EPS fractions, providing insights into their potential interactions with microcystin-LR. The interactions between EPS and microcystin-LR will be investigated using AFM force spectroscopy, with functionalized AFM tips carrying the commercially available toxin. These experiments will quantify the binding forces between EPS (or specific EPS fractions) and microcystin-LR, thereby identifying which type of EPS exhibit the strongest interactions with the toxin and, consequently, the highest potential for its removal from water. Following this, the adsorption potential of EPS will be evaluated. For that, commercial microcystin-LR toxin will be introduced at varying concentrations into model water systems, followed by the addition of EPS (or specific fractions). After incubation, the mixture will be centrifuged to separate the EPS, and the remaining toxin in the supernatant will be quantified using high-performance liquid chromatography (HPLC) to determine adsorption efficiency. Experiments will be conducted under different conditions, including EPS dosages, contact times, pH levels, and temperatures, to evaluate how these parameters affect the EPS adsorption capacity.

Work Package 3 - Validation of the dual mitigation strategy. To validate the efficiency of both strategies and their potential integration, we will assess their sequential or simultaneous application using the co-culture systems established in WP1, allowing an evaluation of the combined efficacy of tailored peptides (designed in WP1) and EPS (or specific fractions, optimized in WP2) for the removal of cyanobacteria and their toxins. For that, the peptide and EPS will be added to the co-culture systems following the conditions identified in WP1 and WP2; after mixing and incubation, the solution will be allowed to settle to assess flocculation efficiency. The mixture will then be centrifuged, and the remaining microcystin-LR in the supernatant will be quantified using HPLC. To further evaluate the flocculation efficiency and specificity of the removal process, flow cytometry will be used to quantify the two distinct populations in the co-culture before and after flocculation [11]. This approach will enable to assess the efficiency of the proposed method under lab-scale conditions (1 L of co-culture) while also evaluating its ecological impact. Specifically, we will verify the selectivity and non-toxicity toward the non-target microalgae in the co-culture, thereby demonstrating the potential of our approach to minimize ecological disruption. To evaluate scalability, we will use numerical modeling, including fluid dynamics simulations and mass balance calculations, to predict performance in larger-volume systems (e.g., tens to hundreds of liters) and optimize process parameters for real-world applications (collaborations within TIM team at TBI. Finally, the performance of this dual strategy will be compared to existing approaches from the literature, such as the use of known flocculants (e.g., chitosan) or algicides.