Identifying the diversity of carbon sequestering processes and their dynamics is more than ever fundamental to identify carbon sinks and predict how climate change will affect ecosystems functioning. In this context, the environmental formation of CaCO3 by bacteria has recently received increasing attention because of its impact on both C and Ca cycles, its ability to cement/stabilize sediments and soils or to bioremediate sites polluted by alkaline earth elements (AEE) (e.g., 90Sr, 226Ra). Usually, such calcification processes have been considered as extracellular and most of the times occur in solutions that are already supersaturated with CaCO3 mineral phases. However, some studies, including by our consortium, have shown that several species of Cyanobacteria and a giant uncultured sulfoxidizing gammaproteobacterium of the genus Achromatium form intracellular amorphous CaCO3 (iACC). This biomineralization can occur in undersaturated solutions, where CaCO3 precipitation would not occur otherwise. However, while intracellular carbonatogenesis by bacteria appears as a geochemically interesting process, its phylogenetic and environmental distribution, the involved geochemical mechanisms and its quantitative importance in geochemical cycles remains poorly explored. The CarboMagnet stems from our very recent discovery of diverse magnetotactic bacteria (MTB) forming intracellular carbonates at the oxic-anoxic boudary (OAB) of aquatic environments, where strong chemical gradients (e.g., fO2, [H2S]…) prevail. We will test several hypotheses: (i) these bacteria are an significant reservoir of inorganic carbon and alkaline-earth elements (AEE), and participate to their biogeochemical cycling; (ii) the dynamics of this reservoir depends on varying concentrations of of AEE and/or other chemical parameters; (iii) this sequestration process is associated with a repertoire of specific genes and metabolisms linked, in particular, with carbon and sulfur; (iv) these bacteria preferentially incorporate heavy AEEs and fractionate Ca and/or Mg isotopes specifically; (v) the carbonate phases are partly conserved in the sediments upon cell death by crystallization and/or protection within cell membranes. The molecular and geochemical processes of bacterial biomineralization will be studied by a combination of approaches in isotope and aqueous geochemistry, mineralogy, genomics and microbial ecology, from the single cell to the community level and from the natural environment to the laboratory. Together, CarboMagnet deliverables will provide new microbial models for efficient inorganic carbon / AEE sequestration and decipher the contribution of this reservoir to the cycling of C and AEE at the OAB. The CarboMagnet project sets on strong foundations to answer the different questions that are raised, providing us a unique positioning on this topic. First, we will study two, relatively close-by and easily accessible, field sites (i.e. Lake Pavin, Carry-le-Rouet) hosting abundant magnetotactc bacteria forming intracellular carbonates. These sites are environmentally contrasted (freshwater vs marine) and they are already well characterized geochemically and microbiologically. Second, we have demonstrated the possibility to use magnetic sorting procedures providing a unique advantage to efficiently study/enrich/concentrate these iACC-forming bacteria (compared with e.g., other iACC-forming bacteria). Third, we are bringing together a multidisciplinary consortium who has been working together for several years and includes all the complementary and crucial expertise needed for this project: the microbiological and genomic study of MTB; the mineralogical and microscopy characterization of iACC and derived crystalline products; isotope tracing and characterization of physicochemical conditions prevailing where these MTB thrive.

Most life forms depend on photosynthesis. Photosynthesis evolved in bacteria, its spreading and ecological dominance on earth and in the ocean were favorised by eukaryotic acquisition through one primary and many independent secondary photosymbiosis events. However, the molecular basis for photosymbiosis establishment are still unknown. The key originality of the PHOCEE project is to develop a new system to genetically track cellular events underlying the first steps of symbiont acquisition. This new system will also allow to test several current hypothesis about the selective pressures favorising photosymbiosis, mimicking natural environments at the laboratory scale. Thanks to the progress of comparative genomics of different groups of photosymbiotic eucaryotes, this project will provide a molecular framework to better understand a process of key importance for life on the planet.

Bacteria are generally thought as isolated cells growing in well-stirred culture media and we tend to forget that they attach to surfaces and form organized structures. This multicellular life is accompanied by challenges such as the acquisition and sharing of micronutrients such as iron or zinc. These micronutrients are essential for cell activity, and acquisition and expulsion mechanisms are active to finely regulate their intracellular concentration. We propose to explore these issues by focusing on essential metals and using Actinosynnema mirum as a model. This soil bacterium forms synnemata (a compact group of hyphae about 0.3 mm high) as well as colonies up to 1.3 mm high. Our questions focus on the distribution of metals in these structures, their uptake from the growth substrate and the transport mechanisms to the top of these structures. To answer these questions we will use complementary approaches of microbiology, metallomics (metabolomics focused on the study of small molecules in complex with metals), confocal imaging and X-ray fluorescence tomography, coupled with targeted approaches of biochemistry and structural biology. These approaches should reveal the diversity of metallophores produced by this bacterium as well as the distribution of micronutrients and the strength of zinc deficiency felt within these large structures. We have already discovered that A. mirum is able to synthesize a methylated form of staphylopine, a metallophore recently described in some pathogenic bacteria and necessary for zinc acquisition in metal scarce conditions. The genomic organization of the operon encoding the biosynthesis and transport of this methylated form of staphylopine, as well as the hydrophobic nature of the gangue surrounding the synnemata, have led us to hypothesize that these metallophores participate in the long-distance transport of zinc (and possibly other metals) within large structures such as synnemata. On the other hand, the enzyme responsible for this methylation is not yet described and we will continue its molecular and structural characterization while exploring its role in the transport of metals within the structures formed by A. mirum.

Coccolithophore microalgae (CM) are one of largest producers of CaCO3 (coccolith shells) in our oceans. They are involved in several major biogeochemical cycles and are one of the main drivers of the global carbon pump that removes atmospheric CO2. The threat of ocean acidification (OA) has generated concern for how CM biomineralization will be affected, with some studies that assess the impact showing contradictory results. The lack of predictive power may stem from our limited knowledge of CM biomineralization itself. This proposal aims to probe CaCO3 biomineralization in situ by combining microfluidic devices to control media conditions and monitor individual cells with X-ray microscopy measurements capable of nanoscale elemental mapping of hydrated samples. With this approach, intracellular and extracellular chemical information related to ion uptake, storage and biomineral formation will advance our understanding of CM biomineralization and test the impact of OA conditions.

Climate projections indicate higher precipitation variability along this century with more frequent drought extremes, which would have strong influence on forest biodiversity due to impacts on ecosystem functioning, tree ecophysiology and microbial communities. Forest restoration with native trees has been considered an effective strategy of climate change mitigation, but its success is hindered by the high mortality of tree seedlings in the field and the difficulty in restoring native soil microbiota, which are amplified by drought events. Thus, it is of great interest to improve seedling production practices in nurseries, with the induction of mechanisms which increase drought tolerance. In this study, we aim at evaluating simultaneously the responses of trees and associated soil microbiota to drought stress in three different forest types (Brazilian Seasonal Semideciduous Atlantic Forest, French Mediterranean Oak Forest, and German Mesic Temperate Forest), allowing to search for unifying patterns among geographically distant sites, across gradients, and by the use of experimental treatments. Further, we will test the application of different nature-based solutions as innovative strategies for improving tree seedling production and soil microbiome functioning/structuring. Associative microorganisms from tree species of the three ecosystems will be isolated and characterized, to obtain beneficial microbial strains that can be used as bioinputs for seedling production. Moreover, biodegradable and biocompatible nano/micro particles and composite materials produced from natural sources will be used as carrier systems for plant growth regulators and microbial living cells, to improve their delivery to the plants. The efficiency of these nature-based solutions in inducing the tolerance of tree seedlings to drought stress and the corresponding effects on soil microbiota diversity and functioning will be evaluated using different approaches, including greenhouse cultivation, nursery seedling production, and field trials. The economical balance and social acceptance of the proposed solutions will be evaluated in order to check their cost-effectiveness, with the engagement of stakeholders (as nursery-owners, farmers, conservation unit managers, and local authorities) in the course of the project. Thus, in addition to contribute to the basic knowledge of the mechanisms of drought response of trees and soil microbiota, this proposal strongly seeks applicability for improving the success of reforestation programs, with important environmental, economic and social impacts. The success of the project is based on an international multidisciplinary consortium (plant ecophysiologists, soil and rhizosphere microbiologists, microbial ecologists, chemists, engineers, economists) that will collaborate on a range of nature-based solutions.