MinerAl forMation in BActerial biofilms – MAMBA

Given the high complexity of natural mineral-biofilm interfaces that partly impairs an accurate determination of the biomineralization mechanisms at the fundamental level, this proposal is based on a laboratory approach conducted on ideal systems (mono-species biofilm, controlled metal supply and pH).

WP 1: BIOFILM GROWTH AND METAL EXPOSURE
6 different biofilm structures will be tested, with mutants that produce biofilms of various densities and chemical compositions. The biofilms will be grown (Task 1) and exposed to solutions containing metals (Mn(II), Cr(III) and a combination of both (Task 2).

WP 2: BIOFILMS STRUCTURE AND MICRO-ENVIRONMENTS
The main goals of this WP2 are to localize and characterize the microenvironments physico-chemical properties in the biofilm thickness. The general spatial arrangement will be investigated (Task 3). A chemical labelling will be performed, giving access to the 3-D organization of bacteria cells and EPS (Task 4), and the physico-chemical gradients and elements partitioning within the biofilm thickness will be investigated (Task 5).

WP 3: DYNAMICS OF PRECIPITATE FORMATION
WP3 focuses on the understanding of biomineralization processes and the definition of local physico-chemical parameters leading to mineral precipitation. Determination of mineralization hotspots will be investigated at the biofilm scale (Task 6), and at the molecular scale (Task 7). Thermodynamic modeling approaches will be developed to help determine the local physico-chemical conditions during biomineralization (Task 8).

WP 4: NUCLEATION SITES IDENTIFICATION
WP4 provides strong constraints on biomineralization processes by focusing on the dynamic aspects of mineral precipitation at the molecular scale using liquid-TEM. This will be done by investigating real time mineralization in synthetic structures (Task 9). Experiments will be conducted on biofilms directly grown in liquid cells (Task 10) to access a real time idendification of nucleation sites.

WP1 :
- A development of growth protocols in phosphate-deficient medium (which strongly interacts with metals in solution and creates artifacts during their precipitation) has been carried out.
- Growth of bacterial biofilms in micro-slides (Ibidi cells). The development was rapid. These biofilms grow under controlled conditions, including limiting phosphate inputs.
WP2 :
- Observations in spatially resolved ATR-FTIR spectroscopy show strong spatial heterogeneity in functional group types and exopolymers, at the scale of several hundred of microns.
- First measurements in confocal laser scanning microscopy indicate pH gradients (varying slightly over 0.2 pH units over a few micrometers), and show also an influence of Mn on these pH gradients.
WP3 :
- Observations of Mn precipitates on natural samples collected from mining waste rock in New Caledonia indicate oxidation of Mn by microorganisms. Metagenomic analyses are being interpreted to determine which strains are responsible for this oxidation.
WP4 :
- Observation of Mn mineralization on different mutants of bacteria. This mineralization is controlled by the type of surface exopolymers.
- Development of the observation conditions of Mn mineralization on bacteria in liquid-TEM on a new microscope (improvement work having been carried out during one year on the main microscope).
- Observations of Mn mineralization in liquid-TEM on functionalized polystyrene microbeads (naked, carboxyl, amines, sulfonates, C18, protein A). It was observed that Mn mineralization occurs mainly at low electron dose (rather oxidizing regime) and is strongly dependent on the type and density of functional sites.

- Industrial impact of mining waste storage : The assessment of these mining residues reactivity has direct environmental, economical and societal impacts. Particularly, the accurate assessment of biofilms role in these mining systems remains poorly documented in the literature despite the high reactivity associated to these bacteria colonies. The most immediate outcome is an estimation of the potential environmental release of metal from mining tailings in mining areas, thus able to impact the surrounding ecosystems even if the geochemical background is supposed to be high. This project has then important societal implications for populations leaving in vicinity of these systems if they are proved to generate metal pollution. Finally, this work will also help define if depollution or pollution stabilization operations need to be initiated for the slags, which implies important economical consequences.
- Development of scientific tools for environmental science: Due to analytical challenges, liquid cell electron microscopy remains poorly developed elsewhere in the world despite the expected high-impact carried by this kind of applications . As such, by being innovative, our proposal provides a strong basis for the development of this tool for the environmental sciences community, and positions this proposal in an extremely good situation in the International competition for liquid cells microscopy applications.
- Technical applications: Biology-driven nanomaterial synthesis is developing rapidly and allows to access organized structures hardly reachable by classic chemistry. These original synthesis routes are currently under exploration by many laboratories and industrial groups. The results gathered here may be the starting point for the development of original routes for nanomaterial synthesis by biofilms. This work could also be used to establish new bacteria-mediated remediation strategies.

Couasnon T., Alloyeau D., Menez B., Guyot F., Ghigo J.M., Gelabert A. 2020 “In situ monitoring of exopolymer-dependent Mn mineralization on bacterial surfaces”, Science Advances, 6(27), eaaz3125, DOI: 10.1126/sciadv.aaz3125

Desmau M., Carboni A., Le Bars M., Doelsch E., Benedetti M., Auffan M., Levard C., Gelabert A. 2020 “How microbial biofilms control the environmental fate of engineered nanoparticles?”, Frontiers in Environmental Science, 8:882, DOI: 10.3389/fenvs.2020.00082

Davranche M., Gelabert A., Benedetti M.F. 2020 “Electron Transfer Drives Metal Cycling in the Critical Zone”, Elements, 16(3), 185-190, DOI: 10.2138/gselements.16.3.185

Microbes in nature are organized in biofilms structures, composed of extracellular polymers encasing cells, and where the diffusion of elements is limited due to the low porosity. Virtually present in all environments, these structures maintain micro-environments of highly specific physico-chemical properties, and are reported to impact mineral weathering rates. They can also create local oversaturations relative to mineral phases in their microenvironments and are thus able to induce biomineralization. However, our understanding of the associated processes in biofilm structures remains limited whereas the mechanisms they drive (metal release vs. mineralization) are central to geochemical, environmental, and depollution domains. For instance, the chromium mining site of Sukinda Valley, India, generates annually ~7.6 million tons of solid wastes, and a highly toxic Cr(VI) flux outside the mine estimated at 11.73 tons annually. Given the difficulty for oxidizing Cr(III) originally present in ore, the occurrence of bacteria identified on this site is likely to control the Cr(VI) toxic release, by promoting Mn(II) oxidation into MnO2, a highly oxidant mineral, able in turn to oxidize Cr(III) into Cr(VI). However, given the biofilms complex nature, the dynamics of Cr(VI) release from these metal-rich systems remain poorly understood, impairing an efficient positive action for limiting the surroundings pollution.

In literature, an intensive set of microbial biomineralization studies exist, but only few of them consider the specific properties of biofilms fine structure. As a result, numerous questions regarding mineralization processes in these structures remain open. We propose here to test two hypotheses to better define the Mn and Cr (found in mining areas) mineralization mechanisms in biofilms: i) biomineralization is strongly impacted by the extracellular polymers types and 3D organization, ii) biomineralization is dependent on the presence of nucleation sites and not only on local oversaturation in microenvironments.

The recent advances made by our group allow to circumvent most limitations attached to the study of these fragile 3D biological structures, and this highly innovative project is centered on a combination of state-of-the-art techniques benefiting from the collaboration of earth sciences and material physics domains, with mostly synchrotron-related techniques, Raman microanalysis, and highly innovative electron microscopy approaches.
We propose to conduct a laboratory study based on axenic biofilm cultures, including E. coli and Pseudomonas mutants with specific biofilm 3D organizations (WP1). After studying the specific 3D organization of biofilms developed by each strain, the physico-chemical nature of chemical gradients and microenvironments in these structures will be investigated (WP2). Then the dynamics of biomineralization processes at the biofilm and molecular scale, in link with the biofilm particular structure, will be studied and will allow to access the local physico-chemical conditions of precipitation in the microenvironments (WP3). Finally, the real-time observation of mineralization processes in liquid environments and at the molecular scale will be possible through the recent developments of liquid cell TEM (WP4). Monitoring directly the different steps of biomineralization, starting by the nucleation processes in liquid environment, is expected to allow significant progresses in our understanding of the processes controlling biomineralization.

Being strongly related to metal contaminant dynamics in metal-rich systems, our project is part of the Defi 1-Axe 1-sous-axe 1.1 and is expected to provide critical information on mineral resources exploitation, where the evaluation of mining activities impacts remains a major challenge. Also, the analytical strategies and tools developed here (liquid cell TEM) will initiate new research strategies and will benefit to the whole Earth sciences community.