CE43 - Bioéconomie : chimie, biotechnologie, procédés et approches système, de la biomasse aux usages

PhotobiofilmExplorer – PhotobiofilmExplorer

Exploring and understanding the secrets of photosynthetic biofilms

It is crucial to identify new drugs to deal with pathogens that are resistant to current antibiotics. Most of the microalgae species cultivated in biofilms by the start-up Inalve are capable of forming biofilms that are highly resistant to biological contamination thanks to the synthesis of allelopathic compounds. <br />The objective of the project is to characterise and produce these new bio-compounds that can be used for human and animal health.

Microalgal biofilms produce antiviral and antimicrobial compounds

The first objective of this project is to validate our hypothesis of resistance associated with the production of allelopathic substances. The second is to characterise and produce these new bio-compounds that can be used for human and animal health. Our main targets are antibiotics, but other activities will be tested, in particular antiviral activities. The discovery of these natural molecules will have a considerable impact in terms of valorisation, particularly on the protection of aquaculture fish and shellfish (Inalve's main market), and in pharmacology thanks to the discovery of antibiotics and antiviral compounds.

Photobiofilm Explorer is organised into six workpackages. Biofilms of two marine microalgae species with high potential for the production of allelochemicals are grown and studied together with their bacterial phycosphere. We consider the effect of nitrogen stress on the production of bioactive compounds. We examine the role of bacteria associated with microalgae. Microscopy tools are being developed to assess the 3D structure of biofilms and their relationship with the production of allelopathic compounds. The antimicrobial activity, concentration and nature of allelochemicals are studied in conjunction with a large screening of viruses and bacteria pathogenic to humans and fish, inhibited by these molecules. The conditions favouring allelochemical production, once optimised using simulation tools, are tested with Inalve's pilot processes.

The consortium brings together teams recognised for their expertise in algal-bacterial interactions (MARBEC), microalgal physiology (LOV), multi-specific 3D characterisation of biofilms (INRAE & CS), mathematical modelling (INRIA & CS), bioprocess engineering (CS), and industrial biofilm culture (Inalve).

Microalgal biofilms were grown under various conditions from different experiments from laboratory systems to pilot biofilm processes (LOV and Inalve). Different fractions and extracts were produced. Protocols for the extraction of different fractions and activity tests were developed. Solvents of varying polarity were tested to assess potential antibacterial activities of various biomass fractions.

Several extracts demonstrated bacteriostatic and antiviral activities on various aquaculture pathogens (salmon and shrimp). Tests were also carried out, and bacterial antibiofilm activities were demonstrated.

An imaging toolbox for the characterisation, at different scales (cell, micro and mesoscale), of the structure of photosynthetic biofilms developed on different types of support (i.e. cotton and glass): the joint use of CLSM and longitudinal section techniques used in histology for thick biofilms and the complementary use of OCT, CLSM and light sheet microscopy for finer biofilms.

Numerical models of biofilms were developed, in particular with the aim of simulating the structure in relation to simultaneous algal-bacterial development. The 1D model has been implemented in matlab, and a 2D version is under development.

A pilot cultivation system was set up in a greenhouse, and it was adapted to the cultivation of Cylindrotheca closterium. For the first time, several kg of Cylindrotheca biofilm paste were produced under pilot conditions.

A thinner characterization of the biofilm activities for different target is expected. More work will be needed to identify the molecules involved in these activities, and also to environmental conditions maximizing the synthesis of allelopathic compounds.

These conditions will be reproduced and amplified in the outdoor pilot reactor presently producing Cylindrotheca closterium under a biofilm form. Optimization will be guided by the development of numerical models.

Exploiting these molecules and finding markets will then be the final step, to propose alternative natural approaches for protecting fishes and shrimps.

1. Polizzi, Bastien, Andrea Fanesi, Filipa Lopes, Magali Ribot, and Olivier Bernard. «Understanding photosynthetic biofilm productivity and structure through 2D simulation.« PLoS computational biology 18, no. 4 (2022): e1009904.
2. Fanesi, Andrea, Martin, Thierry, Breton, Cyril, Bernard, Olivier, Briandet, Romain and Lopes, Filipa «The architecture and metabolic traits of monospecific photosynthetic biofilms studied in a custom flow-through system.« Biotechnology and Bioengineering (2022).
3. Caillau, Jean-Baptiste, Walid Djema, Jean-Luc Gouzé, Sofya Maslovskaya, and Jean-Baptiste Pomet. «Turnpike property in optimal microbial metabolite production.« Journal of Optimization Theory and Applications (2022): 1-33.
4. Morales, Marjorie, Claude Aflalo, and Olivier Bernard. «Microalgal lipids: A review of lipids potential and quantification for 95 phytoplankton species.« Biomass and Bioenergy 150 (2021): 106108.

There is an urgent need to identify new drugs to deal with major health challenges such as the control of serious pathogens which have gained resistance against current antibiotics. Microalgae constitute a source of eukaryotic organisms to be explored for the production of more efficient and biologically active compounds. They have gained interest lately for innovative solutions in various applications and they are now recognized as a valuable and sustainable source for food and feed. They are classically cultivated in photobioreactors or raceways as liquid suspension. To further enhance productivity at reduced environmental cost an innovative approach has been developed and patented by three of the project partners, consisting in growing microalgae in a biofilm on a moving conveyer belt. The Inalve startup was launched to exploit this technology and won several prices. A biofilm is an assemblage of microorganisms associated to a surface and embedded in a secreted matrix of extracellular polymeric substances. Surprisingly, most of the tested species by Inalve are able to form biofilms extremely resistant to biological contamination. Even when adding glucose, the cellular ratio bacteria/microalgae in the biofilm remains strongly controlled to a very low value suggesting that some compounds released by microalgae (and/or associated bacteria within the biofilm) are able to inhibit invaders and play a significant role in the biofilm resilience and stability. These compounds are known to reach concentrations high enough to also impact planktonic microorganisms in the neighbourhood of the biofilm. A vast range of allelopathic substances have been identified as active over other microorganisms. They possess unique bioactivities now exploited in animal and human health.

The first objective of the project is to explore the activity of the produced molecules explaining the resistance of the biofilm. The second objective is to identify, characterize and produce novel biocompounds with benefits for human or animal health. Our main target is antibiotics, but other activities will be tested, especially antiviral activities. The seek for such innovative natural molecules will have a deep impact in term of valorization including protection of fishes and shellfishes in aquaculture (main Inalve market) and in pharmaceutics through the discovery of novel antibiotics and antiviral compounds.

Photobiofilm Explorer is organized as six technical workpackages. Biofilms from four marine microalgal species with strong potential to produce allelochemical compounds will be grown and studied along with their bacterial phycosphere. WP1 focuses on the bioactive compounds production under nitrogen stress, while WP2 will consider the role of the microalgae-associated bacteria. Microscopic tools for assessing biofilm 3D structure and its relation to allelopathic compounds production will be developed in WP3. Measurement of the antimicrobial activity, the associated allelochemical concentration and chemical identification will be carried out in WP4 with a broad screening of pathogenic bacteria, harmful for animal and human health, and viruses. The conditions leading to the highest allelochemical production, once optimized using the simulation tools of WP5 will be implemented in the Inalve biofilm pilot processes in WP6.

The consortium gathers excellent teams recognized for their expertise in algae-bacteria interaction (MARBEC), physiological study of microalgae (LOV), 3D multispecific biofilm characterization (INRAE, CS), advanced mathematical modelling (INRIA, CS), bioprocess engineering (CS), and innovative industrial biofilm cultivation (Inalve). The actors have a long track record in efficient and successful collaborations. The project is coordinated by O. Bernard, Senior Researcher, PhD in Biological oceanography, worldwide-acknowledged specialist of microalgae and bioprocess optimisation, with a long track record of successful project coordination.

Project coordinator

Monsieur Olivier Bernard (Centre de Recherche Inria Sophia Antipolis - Méditerranée)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LGPM LABORATOIRE DE GENIE DES PROCEDES ET MATERIAUX
LOV Laboratoire d'océanographie de Villefranche
Inria Centre de Recherche Inria Sophia Antipolis - Méditerranée
MARBEC Centre pour la biodiversité marine, l'exploitation et la conservation
INALVE
MICALIS MICrobiologie de l'ALImentation au service de la Santé

Help of the ANR 604,870 euros
Beginning and duration of the scientific project: - 36 Months

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