Oleg S Pokrovsky
email: oleg.pokrovsky@get.omp.eu

Liudmila S Shirokova

Svetlana A Zabelina

Guntram Jordan

Pascale Bénézeth

Prepared for submission to npj Materials Degradation as

Assessment of the microbial impact on mineral dissolution is crucial for a predictive understanding of basic (Ca, Mg-bearing) silicate weathering and the associated CO2 consumption, bioerosion and CO2 storage in basaltic rocks. However, there are controversies about the mechanism of microbial effect, which ranges from inhibiting via nil to accelerating. Here we studied diopside interaction with the heterotrophic bacterium Pseudomonas reactants and the soil fungus Chaetomium brasiliense using a combination of mixed-flow and batch reactors and in-situ (AFM) and ex-situ (SEM) microscopy. The results provide new nano-level insights into the degree to which microorganisms modify silicate dissolution. Taking into account negligible effects of organic ligands on diopside dissolution as reported earlier, we conclude that the microbial effect on Ca-Mg silicates is weak and the acceleration of dissolution of "basic" silicate rocks in the presence of soil biota is solely due to pH decrease in porewaters.

INTRODUCTION

Chemical weathering of basic (Ca-and Mg-bearing) silicates is a primary factor regulating the climate of our planet via a long-term (million year scale) CO2 uptake from the atmosphere [START_REF] Berner | Weathering, plants and the long-term carbon cycle[END_REF] . Both direct biological (enzymatic action of bacteria, fungi, and plant roots) and indirect physico-chemical (soil porewater pH, extracellular organic ligands and biomass degradation products) mechanisms have been identified for chemical weathering of primary silicate minerals in soil environments [START_REF] Hutchens | The role of heterotrophic bacteria in feldspar dissolutionan experimental approach[END_REF][START_REF] Hutchens | Microbial selectivity on mineral surfaces: possible implications for weathering processes[END_REF][START_REF] Li | Specificity of low molecular weight organic acids on the release of elements from lizardite during fungal weathering[END_REF] . Among the "aqueous" factors, controlling the intensity of this process, solution pH, pCO2 and organic compounds produced during enzymatic degradation of vegetation litter or the exudates of soil bacteria and plant roots are believed to be the most important [START_REF] Barker | Geomicrobiology: Interactions Between Microbes and Minerals[END_REF] . The organic compounds capable to affect mineral dissolution rate include (but are not limited to) extracellular acid and neutral polysaccharides, simple carboxylic acids, uronic acids (galacturonic, guluronic), peptides and aminoacids [START_REF] Ullman | Laboratory evidence for microbially mediated silicate mineral dissolution in nature[END_REF][START_REF] Welch | Microbial extracellular polysaccharides and plagioclase dissolution[END_REF] , as well as various lichen polyphenolic compounds [START_REF] Adamo | Weathering of rocks and neogenesis of minerals associated with lichen activity[END_REF] .

Positive effects of bacteria and organic ligands on alumosilicate dissolution are fairly well established [START_REF] Barker | Experimental observations of the effects of bacteria on aluminosilicate weathering[END_REF][START_REF] Welch | The effect of microbial glucose metabolism on bytownite feldspar dissolution rates between 5° and 35°C[END_REF][START_REF] Bennett | Silicates, silicate weathering, and microbial ecology[END_REF][START_REF] Van Hees | Dissolution of microcline and labradorite in a forest O horizon extract: the effect of naturally occurring organic acids[END_REF][START_REF] Neaman | Effects of organic ligands on granite dissolution in batch experiments at pH 6[END_REF][START_REF] Ganor | Organics in Water-Rock Interactions: In: Reviews in Mineralogy & Geochemistry, Thermodynamics and Kinetics of Water-Rock Interaction[END_REF][START_REF] Ahmed | Microbe-mineral interactions: The impact of surface attachement on mineral weathering and element selectivity by microorganisms[END_REF] . These effects are related to the capacity of biota to facilitate the Al-O-Si and Fe-O-Si rate-limiting bond breaking, due mostly to strong complexation of bacterially-originated organic moieties with rate-controlling aqueous Al 3+ (aq) cation [START_REF] Oelkers | Does organic acid adsorption affect alkali-feldspar dissolution rates?[END_REF] , surface >AlOH2 + and >FeOH2 + sites [START_REF] Stumm | Reactivity at the mineral-water interface: dissolution and inhibition[END_REF] , or Fe 3+ (aq) mobilization in solution [START_REF] Schalscha | Chelation as a weathering mechanism: 1. Effects of complexing agents on the solubilisation of Fe from minerals and granodiorite[END_REF][START_REF] Liermann | Role of bacterial siderophores in dissolution of hornblende[END_REF][START_REF] Bonneville | Plantdriven fungal weathering: Early stages of mineral alteration at the nanometer scale[END_REF] . The latter is known to prevent passivation of mineral surfaces by newly-formed Fe(III) hydroxides [START_REF] Gerrits | How the rock-inhabiting fungus K. petricola A95 enhances olivine dissolution through attachment[END_REF][START_REF] Gerrits | High-resolution imaging of fungal biofilm-induced olivine weathering[END_REF] . However, all the above mentioned mechanisms are not likely to operate for Fe-and Al-free, Ca and Mg dominant silicates. In fact, several experimental works performed over the past two decades unambiguously demonstrated the lack of any sizable impact of CO2 and organic ligands (at otherwise constant pH) on Caand Mg-bearing mineral dissolution at typical soil water environments. The reason for this is rather weak complexation of organic ligands with >CaOH2 + and >MgOH2 + surface sites responsible for the ratecontrolling Mg-O and Ca-O bond breaking in these minerals [START_REF] Golubev | Effect of pH and organic ligands on the kinetics of smectite dissolution at 25°C[END_REF][START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF][START_REF] Pokrovsky | Kinetics and mechanism of dolomite dissolution in neutral to alkaline solutions revisited[END_REF][START_REF] Pokrovsky | Kinetics of brucite dissolution at 25°C in the presence of organic and inorganic ligands and divalent metals[END_REF][START_REF] Pokrovsky | Effect of organic and inorganic ligands on calcite and magnesite dissolution rates at 60°C and 30 atm pCO2[END_REF][START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF][START_REF] Jordan | Organic and inorganic ligand effects on magnesite dissolution at 100°C and pH = 5 to 10[END_REF][START_REF] Oelkers | Do organic ligands effect calcite dissolution rates?[END_REF] . According to these studies, unreasonably high concentrations of naturally-relevant organic ligands (0.01 -0.1 M) are necessary to significantly enhance or inhibit mineral dissolution. As a result, the effect of extracellular organic products and in general soil biota on the weathering rate of Mg-and Ca-bearing minerals in natural settings is expected to be weak. Further, work with heterotrophic bacteria and basaltic glass [START_REF] Stockmann | Does the presence of heterotrophic bacteria Pseudomonas reactans affect basaltic glass dissolution rates?[END_REF] , wollastonite [START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF] and olivine [START_REF] Shirokova | Experimental study of the effect of heterotrophic bacterium (Pseudomonas reactans) on olivine dissolution kinetics in the context of CO2 storage in basalts[END_REF] demonstrated that even unrealistically high concentration of live biomass (several gram per liter, regardless of the presence of nutrients) are not capable of substantially accelerating the Ca, Mg or Si release rate from the mineral. As a result, despite the dominant paradigm on the importance of microbes in chemical weathering of Ca-Mgsilicates in soil environments, the impact of biota on dissolution rates of these silicates might be lower than generally thought. This, in turn, may require revisiting the current understanding of biotic control on feedbacks in atmospheric CO2 consumption vs. silicate weathering processes. To further address the role of soil biota on mineral weathering process and to extend the previous results to other alkaline-earth bearing minerals (e.g., crystalline mixed calcium and magnesium silicates), in the present study we measured the dissolution rate of typical basic silicate diopside (CaMgSi2O6) in the presence of aquatic and soil microorganisms. For these experiments, we were able to separate the effect of pH from that of bacterial presence using a mixed-flow and batch reactor systems coupled with an in-situ and ex-situ microscopic examination of reacted surfaces.

To provide a better understanding of the bacteria-mineral interaction in the context of chemical weathering in soils and CO2 geological storage, we selected a common soil and underground bacterium, Pseudomonas reactans. Bacterial strains of the genera Pseudomonas, thanks to their relatively easy culturing, have been frequently used as a model organisms in studying mineral weathering in the past [START_REF] Webley | Plate method for studying the breakdown of synthetic and natural silicates by soil bacteria[END_REF][START_REF] Duff | Solubilization of minerals and related materials by 2-ketogluconic acid-producing bacteria[END_REF] and recently [START_REF] Ahmed | Microbe-mineral interactions: The impact of surface attachement on mineral weathering and element selectivity by microorganisms[END_REF][START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF][START_REF] Aouad | Microbially-mediated glass dissolution and sorption of metals by Pseudomonas aeruginosa cells and biofilm[END_REF] . Diopside was chosen as a typical model mineral for basic silicates, frequently used as a representative of silicate dissolution at the Earth surface [START_REF] Brantley | Chemical weathering rates of pyroxenes and amphiboles[END_REF] . Indeed, because hydrolysis of Ca-O and Mg-O bonds is often the rate-limiting step for Ca-and Mg-bearing mineral dissolution [START_REF] Schott | The link between mineral dissolution/precipitation kinetics and solution chemistry[END_REF] , understanding of microorganism interaction with diopside may help to predict the reactivity of other basic silicates in the presence of microbes. In the present study, the experiments were designed in batch and flow-through reactors under constant biomass concentration, in the presence of both live and dead cells in both nutrient-free and nutrient-bearing media. We measured forward far from equilibrium dissolution rates and we microscopically examined reacted surfaces with respect to microorganism attachment, etch pits and corrosion features. The ensemble of obtained results demonstrated rather weak impact of both bacterium and fungus on diopside reactivity, regardless of the presence of nutrients and live status of microorganisms. Moreover, the passivation of mineral surfaces via formation of an exopolysaccharide layer (in case of bacteria) may act as protection mechanism in natural settings. Globally, these new findings challenge the dominant paradigm on dominant biotic control of basic silicates weathering at the Earth surface conditions. This may require revising the quantitative modeling of chemical weathering as main factor of biotically driven long term CO2 consumption (coupled to Ca and Mg release from silicates). Further, the results call for a need of in-situ assessment of soil porewater pH at high spatial and temporal resolution as a sole factor controlling the rate of Ca, and Mg bearing silicate weathering.

RESULTS

Diopside interaction with P. reactans

In most experiments performed at pH of 6 to 9, stoichiometric release of Si, Ca and Mg was observed. In bacteria-free experiments, diopside dissolution rates decreased by ca. 0.5-1 order of magnitude during the pH decrease from 6 to 9 (Fig. 1, Table 1). The DOC concentration remained fairly stable in the course of given bacterial experiment and generally ranged from 7 to 25 mg L -1 being the highest at highest biomass concentration. Considering certain scatter in the rates obtained in different experiments, the presence of bacteria in either nutrient-free or nutrient-rich media did not exhibit any significant (at p < 0.05) effect on Si release rate from diopside, or even led to a decrease of dissolution rates (Fig. 2). In fact, the effect of bacterial presence did not exceed 0.2 log RSi units, whereas the reproducibility of rates measured in individual reactors at otherwise similar aqueous solution composition ranged from 0.1 to 0.3 log RSi units. Thus it can be concluded that, within the resolution of our experiments, the impact of bacteria on diopside Si dissolution rate could not be quantified or it was below 0.2 log R unit. Furthermore, there was a net inhibiting effect of bacteria on Ca and Mg release rate from the diopside in nutrient solution at pH = 6.1 (Fig. 2 A). In more alkaline solutions (pH = 8.5 to 9.0), the presence of bacteria (with or without nutrients) did not considerably (> 0.2 log R unit) impact the release rate of Si, Ca and Mg (Fig. 2 B).

The second remarkable result of BMFR experiments is that live bacteria with (D2-2, D2-3, D4-2, D4-3) and without (D1-2, D1-3, D3-2, D3-3) nutrients exhibited a negligible effect of biomass concentration (between 0.54 and 3.85 gwet/L) on Mg, Ca and Si release rates because there was no substantial (> 0.2 log R unit) change in rate during biomass increase by a factor of 7 (Table 1).

The third important observation is that there was a significant, by a factor of 3 to 5, decrease in Ca, Mg, Si concentrations and release rates between the beginning (exp. D1-1, D2-1, D3-1 and D4-1) and the end of bacteria-free experiments (exp. D1-4, D2-4, D3-4 and D4-4). This is illustrated in Supplementary Figure 1, where the Si concentration in the outlet solution corresponding to the sequence of inlet solutions "bacteria-free 0.1 M NaCl → 0.54 gwet/L bacteria → 3.85 gwet/L bacteria → bacteria-free 0.1 M NaCl", at a constant flow rate, demonstrated a 3-to 5-fold decrease between the initial and the final bacteria-free experiment. Such a decrease could be related to passivation of mineral surface by bacterial cells and their exometabolites as evidenced from in-situ microscopic observations (see section on the AFM observations below).

Ca, Mg and Si release in the presence of fungus

Results of Ca, Mg and Si release from diopside in 0.1 M NaCl and in nutrient media in the presence of live and dead Chaetomium brasiliense demonstrated that concentrations of all mineral constituents increase quasi-linearly with time in all types of experiments (illustrated for Si in Supplementary Figure 2). The linear regression coefficients varied from 0.9 to 0.99 for Si and from 0.8 to 0.95 for Ca and Mg. The pH remained generally constant (±0.2 unit) or slightly increased (within 0.2 -0.3 units) over the 19 days of exposure. In batch experiments with fungus, the DOC increased from 10 to 30 mg/L over the first 100 h of reaction and then remained constant over the following 300 h. Taking into account that 1) all solutions are strongly undersaturated with respect to diopside, all other possible Ca and Mg silicates and amorphous silica and 2) there is no effect of aqueous Ca, Mg and Si concentration on diopside forward dissolution rates in neutral solution [START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF][START_REF] Knauss | Diopside dissolution kinetics as a function of pH, CO2, temperature, and time[END_REF][START_REF] Golubev | Experimental determination of the effect of dissolved CO2 on the dissolution kinetics of Mg and Ca silicates at 25°[END_REF] , one can quantify the far-from equilibrium dissolution rate (R) for each individual experiment using the following equation which can be applied over the whole duration of the experiment:

R = (d[Ca, Mg, Si/2]tot /dt)/ s (1)
where t (s) designates the elapsed time, [Ca, Mg, Si]tot (mol/L) stands for the concentration of calcium, magnesium or silica released from the solid, and s (cm²/L) is the powder B.E.T. surface area. Results are listed in Table 2. The uncertainties on these rates stem from duplicate experiments and range between ±10 and 30%. The rates measured in the presence of dead or live fungi generally agree (within ± 0.2 log units) with those of abiotic and biotic experiments in mixed-flow reactor (Fig. 1). The Si release rates were a factor of 1.5 higher in experiments with live fungus (with or without nutrients) compared to experiments with dead fungus (Fig. 3). However, there was no difference in Mg release rate between different treatments, whereas the Ca release rate, although more variable among replicates, exhibited a maximum in nutrient-free experiments with live fungus (Fig. 3). The element release rate in the presence of fungus was mostly stoichiometric (Table 2). The disagreement between Si and Mg release rate ranged from 0.1 to 0.05 log R unit except in one experiment with dead biomass (0.2 units). Calcium release rate was in agreement with that of Si within 0.01-0.19 log R units, except in one experiment with live biomass (0.24 log R lower).

AFM observations in the presence of P. reactans

Freshly reacted diopside surfaces (2-5 h in sterile NaCl solution) largely preserved their cleavage plane morphology (Fig. 4 A). Occasionally, some etch pits and local surface roughness were observable (Fig. 4 B). After 36 h reaction in bacteria-free nutrient solution, rounding of edges occurred (Fig. 4 C). After 1 to 2 days of reaction with nutrient solution at pH = 8.5 or 6.5, we have observed a strong colonization of diopside surfaces by a biofilm-like bacterial assemblage (Fig. 4 D). This extracellular polymeric substances (EPS) coverage included individual elongated spheres and generally convex forms, less than 0.5 µm in longest axis, although their size distribution was not uniform (Figs. 4 D,E,F). Groups of objects occasionally exhibited a distinct internally preferred orientation, which was, however, quite variable within a relatively small area (~50 µm²). The obtained images are consistent with earlier reported bacterial EPS via AFM [START_REF] Van Der Aa | In situ characterization of bacteria extracellular polymeric substances by AFM[END_REF][START_REF] Beech | The use of atomic force microscopy for studying interactions of bacterial biofilms with surfaces[END_REF] . Extensive production of exopolymeric substances precluded imaging of individual bacterial cells within this thick biofilm coverage. In contrast, mineral interaction with bacteria in nutrient-free neutral electrolyte allowed recognizing individual cells attached to the diopside surface (Fig. 4 G); however, no site of preferential interactions such as steps or edges could be identified.

SEM imaging of diopside reacted with fungus

The diopside grains reacted with dead fungus in 0.1 M NaCl demonstrated sharp borders and no clear dissolution features (Fig. 5 A). After reaction with live fungus in nutrient-free media, there was an appearance of specific dissolution features such as etching and rounding of edges (Fig. 5 B). The rounded edges were also observed in experiments with live fungus; these features were distinctly different from cleavage (Fig. 5 C). The diopside crystals were also imaged in the presence of fungal mycelium: the hyphae were strongly attached to the grain surfaces and not lost during sample preparation for SEM (Fig. 5 D). Some grains appeared significantly (> 50%) covered by or even embedded into network of fungal hyphae. There were no distinct sites of hyphae location at the diopside surface, and overall, the interaction appeared to be quite inert, non-specific and site-indifferent. With increasing the resolution, some organic spherical entities, 2 to 3 µm in diameter, were identified at the extremities of the hyphae (Fig. 5 D).

DISCUSSION

Results of the present study demonstrated a weak (or non-measurable) impact of bacterium and fungus on Ca, Mg and Si release rate from diopside. Biologically accelerated weathering of silicates is thought to be controlled by microbial production of extracellular enzymes, chelates, simple and complex organic acids and exopolysaccharides [START_REF] Barker | Geomicrobiology: Interactions Between Microbes and Minerals[END_REF][START_REF] Bennett | Silicates, silicate weathering, and microbial ecology[END_REF][START_REF] Chen | Weathering of rocks induced by lichen colonization -a review[END_REF][START_REF] Buss | Etch pit formation on iron silicate surfaces during siderophorepromoted dissolution[END_REF][START_REF] Uroz | Mineral weathering by bacteria: ecology, actors and mechanisms[END_REF] . However, there are serious controversies, whether such an effect is largely direct (metabolically-driven nutrient mobilization and specific ligand production) or indirect (passive pH lowering and cation complexation via EPS excretion), given that it is largely dependent on the nature of mineral and the identity of microorganism [START_REF] Barker | Experimental observations of the effects of bacteria on aluminosilicate weathering[END_REF][START_REF] Vandevivere | Enhanced dissolution of silicate minerals by bacteria at near-neutral pH[END_REF][START_REF] Welch | Effect of microbial and other naturally occurring polymers on mineral dissolution[END_REF][START_REF] Roberts | Inhibition and enhancement of microbial surface colonization: the role of silicate composition[END_REF][START_REF] Tourney | The role of bacterial extracellular polymeric substances in geomicrobiology[END_REF] . Furthermore, considering basic silicates, some studies reported nil or inhibiting effect of bacteria on olivine [START_REF] Shirokova | Experimental study of the effect of heterotrophic bacterium (Pseudomonas reactans) on olivine dissolution kinetics in the context of CO2 storage in basalts[END_REF][START_REF] Oelkers | The efficient longterm inhibition of forsterite dissolution by common soil bacteria and fungi at Earth surface conditions[END_REF][START_REF] Garcia | An experimental model approach of biologically-assisted silicate dissolution with olivine and Escherichia coli -Impact on chemical weathering of mafic rocks and atmospheric CO2 drawdown[END_REF] or wollastonite [START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF] dissolution, whereas other claimed strong accelerating effect of fungi on olivine [START_REF] Gerrits | How the rock-inhabiting fungus K. petricola A95 enhances olivine dissolution through attachment[END_REF][START_REF] Gerrits | High-resolution imaging of fungal biofilm-induced olivine weathering[END_REF][START_REF] Pokharel | Mechanisms of olivine dissolution by rock-inhabiting fungi explored using magnesium stable isotopes[END_REF] . However, in the latter works, all the effects of microorganisms were limited to their ability to sequester rate-inhibiting Fe(III) thus preventing the slowdown of dissolution, rather than directly promoting the breaking of structural bonds in the mineral lattice. Overall, in contrast to large number of works on microbial impact on alumosilicate dissolution [START_REF] Wu | Characterization of elemental release during microbebasalt interactions at T = 28°C[END_REF][START_REF] Wu | Characterization of elemental release during microbe-granite interactions at T=28 degrees C. Geochim[END_REF][START_REF] Hausrath | Elemental release rates from dissolving basalt and granite with and without organic ligands[END_REF][START_REF] Daval | Carbon dioxide sequestration through silicate degradation and carbon mineralisation: promises and uncertainties[END_REF][START_REF] Voelz | Quantitative dissolution of environmentally accessible iron residing in iron-rich minerals: A review[END_REF] , the interaction of aquatic microorganisms and relevant organic ligands with Ca or Mg-bearing silicates remained insufficiently characterized.

In the present study, the global rate uncertainty in the studied pH range (6 to 9) was between 20 and 50%. This uncertainty stems from both the pH variation (±0.2-0.3 units) during the course of an experiment and the reproducibility of steady state concentrations of diopside constituents in mixed-flow reactors with bacteria-free systems or among duplicates in batch reactors with dead fungus (±20%). Therefore, we conclude that the difference between abiotic (bacteria-free or dead cells) and live bacteria experiments is within the experimental uncertainty. As such, the effect of selected live microorganisms on Ca, Mg and Si release rate from diopside is not significant, or cannot be resolved using the experimental design employed in the present study.

In this regard, the present result corroborates the former works of our group which demonstrated weak inhibiting or negligible impact of cultivable heterotrophic bacteria P. aureofaciens on wollastonite [START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF] and P. reactans on basaltic glass [START_REF] Stockmann | Does the presence of heterotrophic bacteria Pseudomonas reactans affect basaltic glass dissolution rates?[END_REF] and olivine [START_REF] Shirokova | Experimental study of the effect of heterotrophic bacterium (Pseudomonas reactans) on olivine dissolution kinetics in the context of CO2 storage in basalts[END_REF] , in agreement with other studies [START_REF] Ahmed | Microbe-mineral interactions: The impact of surface attachement on mineral weathering and element selectivity by microorganisms[END_REF][START_REF] Roberts | Inhibition and enhancement of microbial surface colonization: the role of silicate composition[END_REF][START_REF] Oelkers | The efficient longterm inhibition of forsterite dissolution by common soil bacteria and fungi at Earth surface conditions[END_REF] . The broad meaning of these results is that microbial impact on basic silicate mineral dissolution is limited by i) pH decrease, thus accelerating proton-promoted dissolution, which is relevant to all basic silicates; ii) release of Fe(III)complexing organic ligands including siderophores. The latter is pertinent solely to minerals whose dissolution is limited by Fe(OH)3 passivation of the surface. In all other cases of microbially-affected basic silicate dissolution, the concentrations of excreted organic ligands are not sufficient to sizably complex Mg and Ca at the surface or in the aqueous solution which is necessary for breaking of rate-controlling Mg-O or Ca-O bonds at the mineral surface. For example, gluconic acid and its derivatives are believed to be the main exometabolite of heterotrophic bacteria that use glucose as a substrate [START_REF] Welch | The effect of microbial glucose metabolism on bytownite feldspar dissolution rates between 5° and 35°C[END_REF][START_REF] Duff | Solubilization of minerals and related materials by 2-ketogluconic acid-producing bacteria[END_REF] . Gluconic acid can favor olivine dissolution in neutral waters [START_REF] Shirokova | Experimental study of the effect of heterotrophic bacterium (Pseudomonas reactans) on olivine dissolution kinetics in the context of CO2 storage in basalts[END_REF] ; however, this effect occurs at concentrations > 1 mM, totally irrelevant to natural environments. Another example comes from a concerted study of ~40 various ligand impact on wollastonite dissolution [START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF] . These authors calculated, based on the results in mixed-flow reactors, the threshold concentration of ligands necessary to triple the rates of wollastonite at pH around 7. For most ligands, very high concentration (2 to 3 orders of magnitude higher than those encountered in natural settings) were necessary to appreciably affect the rates. Finally, Golubev and Pokrovsky (2006) [START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF] demonstrated that very high concentrations (0.01-0.1 M) of organic ligands originated from enzymatic degradation of organic matter or bacterial metabolic activity, are necessary to appreciably affect diopside dissolution. Considering these results and by analogy with olivine and wollastonite, we hypothesize that possible impacts of studied heterotrophic bacteria on the diopside dissolution via gluconic acid or other soluble exometabolite products are not detectable due to insufficient concentration of these compounds generated via metabolism of P. reactans in experimental reactors.

Below we discuss the mechanisms of diopside interaction with microbial cells and natural implications. Reactive surfaces in both bacterial and fungal experiments demonstrated lack of specific sites of dissolution, etch pit formation, surface amorphisation or new mineral occurrences. Rather, our in-situ (AFM) and ex-situ (SEM) microscopic observations suggested "passivation" of reacted surfaces by microbial metabolic products. We therefore hypothesize that the biofilm coverage either limited aqueous solution contact with mineral surface or merely did not produce sufficient amount of organic ligands capable to impact the diopside dissolution.

Microbial biofilm formation, dominated by exopolysaccharides (EPS), is known to protect the mineral surface from interaction with bulk fluid [START_REF] Chen | Weathering of rocks induced by lichen colonization -a review[END_REF][START_REF] Welch | Effect of microbial and other naturally occurring polymers on mineral dissolution[END_REF][START_REF] Roberts | Inhibition and enhancement of microbial surface colonization: the role of silicate composition[END_REF][START_REF] Lee | Experimental study of the effects of Bacillus subtilis on gibbsite dissolution rates under near-neutral pH and nutrient-poor conditions[END_REF] thus inhibiting overall dissolution rate [START_REF] Hutchens | The role of heterotrophic bacteria in feldspar dissolutionan experimental approach[END_REF][START_REF] Hutchens | Microbial selectivity on mineral surfaces: possible implications for weathering processes[END_REF][START_REF] Ullman | Laboratory evidence for microbially mediated silicate mineral dissolution in nature[END_REF][START_REF] Welch | Microbial extracellular polysaccharides and plagioclase dissolution[END_REF][START_REF] Aouad | Microbially-mediated glass dissolution and sorption of metals by Pseudomonas aeruginosa cells and biofilm[END_REF][START_REF] Lüttge | Direct observation of microbial inhibition of calcite dissolution[END_REF] . In the present experiments with diopside, the biofilm formation was also evidenced from indirect assessment of dissolution rate: the rates decreased by a factor of 2 to 3 after ~500 h treatment with bacteria; this decrease persisted even after changing back the inlet solution to sterile bacteria-free background electrolyte (see Supplementary Figure 1). Noteworthy that such an irreversible rate decrease was also observed after reaction with live bacteria in nutrient-free media and suggested substantial and long-lasting interaction of live P. reactans with diopside surface in both neutral (pH = 6) and slightly alkaline (pH = 9) solutions. At the same time, no decrease in diopside dissolution rate was observed over 400-800 h of experiments in the abiotic inorganic systems [START_REF] Golubev | Effect of pH and organic ligands on the kinetics of smectite dissolution at 25°C[END_REF][START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF] .

As a result of lack of measurable rate increase in the presence of either bacteria or fungus in this range of pH, nutrient and biomass concentration, we suggest, in agreement with previous studies, that, regardless of nutrient regime, biomass concentration and vital status of microorganism, solely the solution pH can act as the main governing factor of mineral dissolution in the presence of aquatic microorganisms. A similar conclusion has been recently achieved for olivine dissolution in the presence of complex natural microbial communities [START_REF] Wild | Early stages of bacterial community adaptation to silicate aging[END_REF] . Therefore, implementation of mineral-microorganism interaction in chemical weathering codes requires solely the information on porewater solution chemistry that can be directly assessed from macroscopic measurements of interstitial soil solutions.

Moreover, extremely high concentrations of organic ligands, necessary to affect the rates of basic silicate dissolution in laboratory experiments [START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF][START_REF] Pokrovsky | Effect of organic ligands and heterotrophic bacteria on wollastonite dissolution kinetics[END_REF][START_REF] Shirokova | Experimental study of the effect of heterotrophic bacterium (Pseudomonas reactans) on olivine dissolution kinetics in the context of CO2 storage in basalts[END_REF] , together with high bacterial and fungi concentrations, comparable with those used in the present study, can be encountered exclusively in surface (litter) soil horizons, and unlikely to ever occur in deep (mineral) soil horizons. Exceptions are specific microenvironments around fungal hyphae, or near decaying organic matter. For example, a strong acidification was reported with hyphae bound to the biotite surface [START_REF] Bonneville | Tree-mycorrhiza symbiosis accelerate mineral weathering: Evidences from nanometer-scale elemental fluxes at the hypna-mineral interface[END_REF] . To quantify these effects on a broader scale, high-resolution in-situ quantitative analysis of chemical composition of solution, bacteria and organic ligand concentration in soil pore water microenvironments are needed.

At the same time, the role of microorganisms under natural environment, especially in the nonsaturated zone, might be more important than that in aqueous environment of mixed-flow experiments. It is generally understood that a lack of "wetted" mineral surface area, or mineral surfaces being exposed to the reactive fluid, could be a partial explanation for slower reaction rates observed in the field compared to those measured in the laboratory with unlimited water [START_REF] Yokoyama | Role of water film in weathering of porous rhyolite under water unsaturated condition[END_REF][START_REF] Harrison | Influence of surface passivation and water content on mineral reactions in unsaturated porous media: Implications for brucite carbonation and CO2 sequestration[END_REF] . For example, via exerting hydrated exopolysaccharides, microbial cells are capable of prolonging the degree of mineral surfaces hydration during natural wetting/drying cycles. Therefore, although our experiments, performed under nutrient-free conditions with starved cells, are most similar to natural environments under short-term scale, there is sizable uncertainty in extrapolating these short-term laboratory results to long-term field conditions. The latter are known to exhibit one to three orders of magnitude lower weathering rates, due to different time scale of observation, diversity of microorganisms, and nutritional conditions.

For more general natural implications of the obtained results, we consider below all Ca, Mg-bearing 'basic' silicates including Ca alumosilicates. Several lines of evidences allow us to suggest rather weak effect of dissolved organics on (Ca, Mg, Al)-bearing silicate dissolution in natural settings. First, the concentration range of various carboxylic ligands (oxalic, citric, malonic, succinic, lactic, formic, acetic), detected in soil solutions, spans from 10 -6 to 10 -4 M [START_REF] Hongve | Dissolved components in precipitation water percolated through forest litter[END_REF] . Laboratory experiments suggest that these concentrations are still not sufficient to appreciably affect feldspars dissolution rates. Second, the effect of organic ligands on Al release and thus on overall dissolution rates of feldspars and basaltic glass is absent or becomes negligible at pH ≥ 7 [START_REF] Oelkers | Does organic acid adsorption affect alkali-feldspar dissolution rates?[END_REF][START_REF] Oelkers | The mechanism, rates and consequences of basaltic glass dissolution: I. An experimental study of the dissolution rates of basaltic glass as a function of aqueous Al, Si and oxalic acid concentration at 25°C and pH = 3 and 11[END_REF] . The pH of rivers and streams draining 'basic' silicate rocks is between 7 and 8 [START_REF] Pokrovsky | Basalt weathering in Central Siberia under permafrost conditions[END_REF][START_REF] Dessert | Fluxes of high-versus low-temperature water-rock interactions in aerial volcanic areas: the example of the Kamchatka Peninsula, Russia[END_REF] whereas the pH of soil solutions in organic rich soils typically ranges from 4 to 7 [START_REF] Berner | Global environment: Water, Air, and Geochemical Cycles[END_REF] . However, surficial acid-and organic-rich soil horizons rarely contain primary silicates, whereas in deep (C) soil horizons, the interstitial soil solution pH is always above 7 (see ref. 65). At these conditions, the effect of organic ligands on Ca, Mg, Al and Si release rate from primary silicates should be negligible which corroborates the conclusion of Drever (1994) [START_REF] Drever | The effect of land plants on weathering rates of silicate minerals[END_REF] that vegetation should cause an increase in weathering rate through the pH effect only where the pH is below 4-5. As a results, the sole effect of soil organics on both acidic and felsic rock weathering seems to be in decreasing the aqueous activity of Al 3+ thus preventing formation of secondary minerals without significant modification of Ca and Mg ions release. It has been widely argued that the presence of soluble minerals in trace amounts in the rocks (i.e., carbonates [START_REF] White | The role of disseminated calcite in the chemical weathering of granitoid rocks[END_REF] ; phosphates 70 may strongly affect the intensity of chemical weathering and export fluxes of major ions. The effect of biological activity in this case is expected to be solely due to pH decrease in the vicinity of microorganisms or plant roots. Indeed, the data for dolomite [START_REF] Pokrovsky | Kinetics and mechanism of dolomite dissolution in neutral to alkaline solutions revisited[END_REF] , calcite and magnesite [START_REF] Pokrovsky | Effect of organic and inorganic ligands on calcite and magnesite dissolution rates at 60°C and 30 atm pCO2[END_REF][START_REF] Jordan | Organic and inorganic ligand effects on magnesite dissolution at 100°C and pH = 5 to 10[END_REF] , brucite [START_REF] Pokrovsky | Kinetics of brucite dissolution at 25°C in the presence of organic and inorganic ligands and divalent metals[END_REF] , and calcite [START_REF] Oelkers | Do organic ligands effect calcite dissolution rates?[END_REF] dissolution in the presence of various organics demonstrate a negligible effect of organic ligands on Ca and Mg release from carbonates and oxides at environmentally-relevant conditions. The effect of soil and atmospheric pCO2 is always considered to be an important factor accelerating rock weathering at the Earth surface. In this regard, we would like to underline that this effect stems solely from the decrease of soil solution pH since there is no chemical effect of CO2(aq) or dissolved H2CO3 molecules on the main rock-forming silicates dissolution rate [START_REF] Golubev | Experimental determination of the effect of dissolved CO2 on the dissolution kinetics of Mg and Ca silicates at 25°[END_REF] , and in neutral and alkaline solutions the pCO2 effect becomes even inhibiting [START_REF] Schott | Formation, growth and transformation of leached layers during silicate minerals dissolution: The example of wollastonite[END_REF] .

Overall, this work allows a better understanding of microbially-affected basic silicate dissolution and provides microscopic insights for quantifying the degree to which the heterotrophic bacteria and fungi are capable of modifying the dissolution rate of rock-forming silicates. Using bacterial-mixed flow and batch reactors combined with in-situ atomic force microscopy of bacteria-diopside surfaces and experiments on diopside dissolution in the presence of fungus combined with microscopic examination of reacted surfaces, we demonstrated a rather weak impact of microorganisms on Ca, Mg and Si release rate. Regardless of the presence or absence of nutrients, in a wide range of pH (from 6 to 9) and despite very high biomass concentration (up to 4-5 gwet/L), the rates were not considerably affected by microbial presence. Moreover, there was a detectable (ca. by a factor of 3) decrease in reactivity of diopside after its prolonged contact with live heterotrophic bacteria. This 'passivation' of the mineral surface was evidenced by extensive production of exopolymeric substances yielding a biofilm-like coverage already after 1-2 days of interaction in nutrientrich media. Despite tight contact between fungus and diopside surfaces, no site of preferential interactions such as steps, kinks or edges, etch pits or surface amorphisation was detected. The obtained results are fully consistent with large body of evidences that any possible microbially produced organic ligands at concentrations relevant to live biotic systems or natural settings are not capable of noticeably impacting the reactivity of Mg-O and Ca-O sites on diopside surface. As such, this work challenges the dominant paradigm of an overwhelming role of biota on silicate mineral weathering in soils. At the same time, it is in line with a controversial opinion of Sverdrup (2009) [START_REF] Sverdrup | Chemical weathering of soil minerals and the role of biological processes[END_REF] that physical and chemical conditions of forest soils do not allow any significant direct surface actions on minerals by microorganisms or tree roots. While we do not contest the importance of microbial organic matter in Fe and Al complexation and mobilization from silicate structure, the role of biota on basic (Ca and Mg) silicates, playing a pivotal role in atmospheric CO2 sequestration via chemical weathering, may be much lower than traditionally thought. In fact, only drastic pH decrease (i.e., over several pH units), probably occurring in the microniches adjacent to plant roots, is capable of sizably (> a factor of 2 to 3) increase mineral dissolution rate. It is not excluded however that this promoting effect will be partially attenuated due to biofilm formation and passivation of silicate surfaces by microbial cells. Detecting such 'active' or 'passive' microniches within the complex plant-microbe-mineral systems and more importantly, characterizing chemical environment of the fluids with spatial resolution on the level of µm to mm may turn out to be a bottleneck for quantitative mechanistic modeling of chemical weathering. Currently, these gradients are not taken into account even in most sophisticated modeling approaches [START_REF] Godderis | Reactive transport models of weathering[END_REF][START_REF] Maher | Tracking diverse minerals, hungry organisms, and dangerous contaminants using reactive transport models[END_REF] . Further experimental and field studies are thus needed to answer the question whether a quantitative modelling of basic silicate mineral weathering and related CO2 consumption require an explicit prediction for microbial activity or it can be merely approximated by the pH of bulk soil porewaters.

METHODS

Mineral, bacterial and fungal culture

Natural diopside (Ca0.99Mg0.98Fe0.02Cr0.01Si2O6) crystals characterized in previous works [START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF][START_REF] Golubev | Experimental determination of the effect of dissolved CO2 on the dissolution kinetics of Mg and Ca silicates at 25°[END_REF] were used in this study. Ultrasonically-cleaned 100 to 200 µm size fraction having a specific surface area of 1045 ± 50 cm²/g was selected for dissolution rate measurements.

Pseudomonas reactans is a common rod-shape groundwater and soil bacteria averaging 2 μm in size (see further description in ref. 31, 32). For experiments, cells were first cultured to a stationary stage on a shaker at 25°C in the darkness using 10% nutrient broth with the following composition: 0.1 g/L glucose, 1.5 g/L peptone, 0.6 g/L NaCl and 0.3 g/L yeast extract. Cells were allowed starving for 2 days in nutrient-free media, prior the macroscopic dissolution and microscopic (AFM) experiments. For this, freshly-grown cell suspension (at the stationary stage) was rinsed by sterile 0.6 g L -1 NaCl solution via centrifugation, and then placed in 0.6 g L -1 NaCl without nutrients for 2 days. Before the experiment, this bacterial suspension was rinsed again with sterile 0.6 g L -1 NaCl to remove, as possible, all cellular exometabolites.

Active bacteria number counts (colony forming-units, CFU/mL) were performed using Petri dish inoculation on a nutrient agar (0.1, 0.2, and 0.5 mL of sampled solution in three replicates) in a laminar hood box. Inoculation of blanks was routinely performed to assure the absence of external contamination. The biomass of live bacteria suspensions was also quantified by measuring wet (after it was centrifuged 15 min at 10,000 rpm) and freeze-dried weights in duplicates. Before the experiments, cells were rinsed twice in either the appropriate fresh culture media or a sterile 0.1 M NaCl solution using centrifugation with ~ 500 mL of solution for 1 g of wet biomass, to remove adsorbed metals and cell exudates from the surface. The conversion ratio wet/freeze-dried weight of P. reactans was equal to 8.4±0.5. The conversion factor of optical density (600 nm, 10 mm path) and wet biomass (gwet/L) to the cell number (CFU/mL) was equal to (5±1)×10 8 and (8.0±1.5)×10 [START_REF] Welch | Microbial extracellular polysaccharides and plagioclase dissolution[END_REF] , respectively, as determined by triplicate measurements. Typical live biomass concentration during experiments was 0.54 and 3.85 gwet/L.

Experiments on fungus were performed with Chaetomium brasilience, belonging to the largest genus of saprophytic ascomycetes, inhabiting soils worldwide. The environmental habitats of genus Chaetomium include deserts, salterns, agricultural, mangrove, living plants and animals, air, decayed wood and stones [START_REF] Abdel-Azeem | Taxonomy and biodiversity of the genus Chaetomium in different habitats[END_REF] . Chaetomium brasilience Bat. Et Pontual 1948, stumm F-3649 was obtained from All-Russian Collection of Microorganisms -VKM (http://www.vkm.ru/Collections.htm). Fungal biomass was produced on liquid Czapek medium (3 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L KCl; 0.5 g/L MgSO4×7H2O, 0.01 g/L FeSO4×7H2O, 30 g/L saccharose at pH = 6.0). The biomass was centrifuged at 5000 rpm during 15 min, rinsed 4 times in 0.1 M NaCl and allowed starving during 2 days in 0.1 M NaCl prior the experiment. Dead (heat-killed) cells were produced via autoclaving 1 h at 132°C the freshly grown fungal biomass followed by thorough rinsing in sterile 0.1 M NaCl. Cell concentration in the sample was analyzed by measuring optical density (OD) at 580 nm using a spectrophotometer, with the culture medium used as a reference. Diopside dissolution experiments were performed in duplicates, in 0.1 M NaCl with dead biomass, and live biomass with and without 1% nutrient solution.

Bacterial mixed-flow reactor

The Bacterial Mixed-Flow Reactor (BMFR) system used in this work was similar to the one used for basaltic glass dissolution in the presence of bacteria [START_REF] Stockmann | Does the presence of heterotrophic bacteria Pseudomonas reactans affect basaltic glass dissolution rates?[END_REF] . The Teflon-made 25 mL reactor with a suspended Teflon-coated stirbar, immersed in a water bath at 25 ± 0.5 °C, was fitted with 20 μm poresize Magna Millipore Nylon outlet filters (45 mm diameter) to allow bacteria to pass while retaining the 100-200 µm mineral powder in the reactor. This system therefore maintained a constant biomass concentration in the reactor, equal to that of the inlet fluid. The input fluids were kept in 1L polypropylene bottles closed with Biosilico® ventilated caps. These fluids were stirred continuously during the experiments and were changed typically each 7 days. This allowed maintenance of a constant and stable stationary phase bacterial culture in the bacteria-bearing inlet fluid. Bacteria concentration was verified by periodical sampling of inlet and outlet fluids for cell optical density (total biomass) and live cell numbers (via agar plate counting). Prior to each experiment, all reactor system parts, including tubing, were sterilized at 130°C for 30 min and rinsed with sterile MilliQ water.

Steady-state dissolution rates (Ri, mol/cm²/s) were computed from measured solution composition using:

Ri = -q 1/n [i(aq)]tot / s (2)
where q (L/s) designates the fluid flow rate, [i(aq)]tot (mol/L) stands for the difference between the input and output solution concentration of i-th element, n is the stoichiometric coefficient of i-th element in diopside formula equaled to 2, 1, and 1 for Si, Mg and Ca, respectively, and s (cm²) refers to the total mineral surface area present in the reactor. The surface area used to calculate the rates was that measured on the fresh (unreacted) diopside powder. Note that during 600 h of reaction time, no systematic variation of the surface area for powder reacted at 5 < pH < 10 in bacteria-free solutions was observed [START_REF] Golubev | Experimental study of the effect of organic ligands on diopside dissolution kinetics[END_REF] .

Experiments were performed in series at constant flow rate between 0.018 and 0.022 mL/min. This corresponded to a mechanical steady-state achievement (x 5 reactor volume renewal) after 4 to 5 days. At the beginning of each series, a quantity of fresh diopside powder was added to the reactor system. For each experimental series, a sterile bacteria-free 0.1 M NaCl with relevant buffer (10 mM NaMES (2morpholinoethanesulfonic acid) or NaHCO3) was first prepared and injected into the reactor until a steadystate outlet fluid composition was attained (typically 2 weeks, D1 series). Once steady state was attained, live bacteria (0.54 gwet/L) with or without nutrients were added to this inlet solution (D2 series). The next series comprised higher concentration of live bacteria in the inlet fluid (3.85 gwet/L). The 2nd and 3rd series lasted 7-10 days each. After that, the inlet solution was replaced by fresh sterile bacteria-free fluid; this experiment, which lasted > 3 weeks, was destined to assess the recovery of the mineral reactivity and to test the consequences of passivation of mineral surface by a bacterial biofilm.

The inlet solutions were composed of 0.1 M NaCl and 0.01 M NaMES (D1 and D2 series) and 0.1 M NaCl with 0.01 M NaHCO3 (D3 and D4 series). The 1:10 diluted Aldrich nutrient broth (0.1 g/L glucose, 1.5 g/L peptone, 0.6 g/L NaCl and 0.3 g/L yeast extract) was added to selected live bacteria experiments. These were compared to live bacteria experiments performed without nutrients. Overall, the experiments conducted in this study can be divided into three main categories: 1) bacteria-and nutrient-free, 2) live bacteria in nutrient-free media, and 3) live bacteria in 10% growth media.

All outlet sample fluids were additionally filtered through 0.45 μm Millipore acetate cellulose filters and acidified with ultrapure 2% HNO3 before chemical analysis. The inlet fluids were routinely analyzed at the beginning, in the middle and at the end of each experiment and were filtered and processed using exactly the same protocol as the outlet fluids.

Mechanical steady-state was achieved after 24 h of reaction. For each steady-state condition, minimum 4 independent measurements of Mg, Ca and Si concentrations, bacterial optical density, pH and flow rate were performed (reproducibility of ± 10%) and used for calculating the dissolution rate. The cell count (CFU) on nutrient agar plates was performed at the beginning, in the middle and at the end of each experimental run and did not vary significantly throughout all experiments.

Batch experiments on diopside dissolution in the presence of fungus

The aim of these experiments was to compare, under identical solution conditions, the Ca, Mg and Si release rate from diopside placed in contact with dead and live fungus cultures. The batch reactors used to study diopside-fungus interaction consisted of sterile 250 mL polypropylene culture flasks with vented caps (Biosilico). All manipulations were conducted in a sterile laminar hood box (class 100). For experiments, 1.3 g of sterilized 100-200 µm diopside were placed in 250 mL sterile solution. We conducted six experiments in duplicates, four involving live cultures, with and without 1% nutrient solution, and two with dead (autoclaved) biomass (in nutrient-free 0.1 M NaCl).

The reactors were inoculated with 4.2-5.6 gwet/L of fresh Chaetomium brasiliense culture or the equivalent amount of dead biomass and placed on rotative shaker (120 rpm) at 25 ± 1°C for 19 days. Sterile controls were routinely run both for nutrient media and 0.1 M NaCl solutions and did not demonstrate any fungal contamination. Periodically, 10 mL aliquots of homogeneous mineral suspension + fungus were collected using sterile serological pipettes and transferred in polystyrene vials for Ca, Mg, Si, pH and cell biomass measurements. The mineral/fluid ratio remained constant during experiments and the concentration of fungus was not affected by the sampling. Calcium, Mg and Si concentrations were analyzed in 0.45 µm filtrates and pH and cell biomass were measured in unfiltered samples. Analyses All input and output solutions were analyzed for Mg, Ca, Si, optical density and pH as a function of time. NIST buffers (pH = 4.008, 6.865, and 9.180 at 25°C) were used for calibration of a combination pHelectrode (Schott Geräte H62). Magnesium and Ca concentrations were measured by flame atomic absorption spectroscopy with an uncertainty of 1 and 2 % and a detection limit of 0.1 and 0.2 µM, respectively. Total silica concentration was determined by a Technicon automatic analyzer using the molybdate blue method with an uncertainty of 2% and a detection limit of 0.3 µM. The concentration of soluble organic exometabolites of bacteria and fungi was measured as total dissolved organic carbon (DOC) using a TOC Shimadzu VSCN analyzer with an uncertainty of 5% and a detection limit of 0.1 mg Corg L -1 . All experiments were performed at far from equilibrium conditions with respect to diopside as calculated via vMinteq program [START_REF] Gustafsson | [END_REF] with relevant solubility parameters 77 .

Figure 1 .

 1 Figure 1. Calcium (a), Mg (b) and Si (c) release rates from diopside measured in the mixed-flow reactor with and without bacteria and 10% nutrient or nutrient-free solution.

Figure 2 .

 2 Figure 2. Synthesis of obtained steady-state Ca, Mg and Si release rates (average ± s.d.) from diopside at pH 6 (a) and 8.5-9.0 (b) with and without Pseudomonas reactans and in the presence or not of nutrients. The error bars represent average of 2 to 4 duplicates, each comprising 3 to 4 independent sampling at the steady state.

Figure 3 .

 3 Figure3. Synthesis of obtained Ca, Mg and Si release rates (average ± s.d., mol cm -2 s -1 ) from diopside at pH 6.0-6.5 with and without Chaetomium brasiliense fungus (4.5-5.6gwet/L) and in the presence or not of nutrients (1% Czapek medium). The error bars represent average of 2 reactors, each comprising 9 to 10 independent sampling as a function of time over 19 days of exposure.

Figure 4 .

 4 Figure 4. A-C: In-situ AFM images of diopside surfaces immersed within sterile electrolyte media for up to 5 hours (A: 70 x 70 µm², deflection mode; B: 20 x 20 µm², deflection mode; C: 12 x 12 µm², height mode -with the color scale spanning a total of 238 nm). D-G: In-situ AFM images of diopside surfaces immersed in nutrient media (D: 8.6 x 8.6 µm², deflection mode; E: 10 x 10 µm², deflection mode; F: 5.5 x 5.5 µm², deflection mode; G: 1.5 x 1.5 µm², deflection mode.

Figure 5 . 1 M

 51 Figure 5. SEM images of reacted diopside surfaces in the sterile electrolyte (a), live fungus in 0.1 M Na Cl (b, d) and in 1% nutrient media (c, d).

Table 1 .

 1 Results of Bacterial MFR experiment. R units are mol cm -2 s -1 .

Table 2 .

 2 Results of fungus batch experiments (0.1 M NaCl). R units are mol cm -2 s -1 .

Microscopic observations

An AFM study using a biological instrument (JPK NanoWizard) allowed in-situ high-resolution characterization of the status of diopside surfaces in the presence of live bacteria with and without nutrients. Diopside cleavage planes were obtained by cleaving optically transparent crystals 5-10 mm in size in a laminar hood box (A100). Fresh cleavage surfaces were sterilized by alcohol and fixed within petri dishes for imaging with the AFM. For long-term (> 5 h) exposure, diopside cleavages were reacted under sterile conditions with 1 gwet/L of bacterial biomass in separate petri dishes. The petri dishes were mounted into the AFM for in-situ inspection of the diopside surfaces directly immersed within the bacteria containing medium, removed from the microscope for further storage, and re-investigated with the microscope whenever appropriate. Depending on the specific aim of the scanning process (attempt of potentially imaging all adhering matter on the surface vs. attempt of removing matter adhering to the surface), images were acquired with both intermittent-contact mode and contact mode using uncoated Si-cantilevers with integrated tips.

The diopside grains reacted with fungus were examined by Scanning Electron Microscopy (SEM). Prior to characterization by SEM, samples were centrifuged (3000 g, 30 min) and the organic matter was removed from the solid phase by rinsing them in nutrient-free sterile 0.1 M NaCl solution. The remaining solids were rinsed three times with MilliQ water and freeze-dried at -60 °C. They were observed after carbon film coated on the sample surface with a JEOL JSM 6360LV SEM coupled with a SDD PGT Sahara EDS analyzer operating at 30 kV.

DATA AVAILABILITY

Supplementary material is available. All measured dissolution rates are presented in Tables 1 and2. Other relevant data are also available from the corresponding author of this paper upon a reasonable request.

ACKNOWLEDGEMENTS:

Support from grants BIOCASTRO of MITI (CNRS) and CaBioCa of Défi (Action conjointe, MITI CNRS) are acknowledged.

AUTHOR CONTRIBUTIONS

O.S.P. developed the concept of the study. L.S.S., S.A.Z. and O.S.P. performed dissolution experiments. G.J. performed AFM study. P.B. participated in laboratory experiments and SEM study. O.S.P. wrote the paper, with input from L.S.S., P.B., G.J. and S.A.Z. All the co-authors discussed the final version of the paper.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Antoine Wystrach
email: antoine.wystrach@univ-tlse3.fr

Insect navigation: Bumblebees walk the walk

published or not. The documents may come

Starting from scratch, early neuroanatomists had no choice but to observe the brain of dead insects. The development of microscopes in the nineteenth century enabled them to distinguish various regions within insects' brain, and make some inference about their function by comparing the regions' volume across species. For instance, the brain area now called the mushroom body was assumed to be the seat of insect intelligence because it was more developed in supposedly smart insects such as hymenopterans (wasps, ants, bees) compared to orthopterans (grasshoppers, locusts and crickets), or in navigating worker ants compared to sequestered queens [START_REF] Kenyon | The meaning and structure of the so-called" mushroom bodies" of the hexapod brain[END_REF] . This was not a bad bet, as we now know that this brain region enables insects to form long-term memories of multi-modal sensory experience, and is key for complex navigational tasks such as visual route following [START_REF] Webb | Neural mechanisms of insect navigation[END_REF] .

A few decades later, the development of Golgi staining techniques enabled scientists to observe individual neurons, and to make the first the first functional interpretations about the inferred neural circuits [START_REF] López-Muñoz | Neuron theory, the cornerstone of neuroscience, on the centenary of the Nobel Prize award to Santiago Ramón y Cajal[END_REF] . Staining the brains of flies, bees, locusts, dragonflies and ants led to remarkably accurate descriptions of various circuits, including beautiful drawings of the crystallised organisation of neurons in the region now called the 'central complex' [START_REF] Wehner | Early ant trajectories: spatial behaviour before behaviourism[END_REF] , which has since been shown to be the primary brain region for the insects' representation of space.

By the mid-twentieth century, behavioural experimentalists had shed light on the strategies underlying insect navigation, such as the reliance on landmark memories, the ability for path integration, and the use of the polarised skylight for compass orientation [START_REF] Wehner | Early ant trajectories: spatial behaviour before behaviourism[END_REF] . In parallel, the dream of stimulating and recording the brain of living insects became a reality with advances in electrophysiology: the key was to use hardy beasts such as crickets or cockroaches, which could survive the requisite operation [START_REF] Huber | Untersuchungen über die Funktion des Zentralnervensystems und insbesondere des Gehirnes bei der Fortbewegung und der Lauterzeugung der Grillen[END_REF] .

Experiments involving stimulating the brain demonstrated the role of the central complex for motor control, while recording neural activity in this region disclosed the existence of 'compass' neurons [START_REF] Pfeiffer | Organization and functional roles of the central complex in the insect brain[END_REF] .

In parallel, a much smaller and more fragile insect had come the fore: the geneticists favourite Drosophila melanogaster. The availability of neurogenetic tools in this species makes it possible to silence, stimulate or image targeted populations of neurons, even while the animal is behaving. It then became possible to witness the insect compass in action: as the fly rotates, a bump of neural activity was observed to rotate along the circular structure of the central complex; quite literally keeping track of the insect's current heading [START_REF] Seelig | Neural dynamics for landmark orientation and angular path integration[END_REF] . What is more, experiments involving targeted activation of this area showed how surrounding terrestrial cues can be quickly memorised, through synaptic plasticity, to participate in maintaining this sense of direction [START_REF] Kim | Generation of stable heading representations in diverse visual scenes[END_REF] .

Drosophila may not, however, be the best subject when it comes to studying navigational skills.

Ecologically, they have no particular reason to learn routes, and current research is now trying to develop assays using reinforcers to incite these small flies to disclose their ability for path integration [START_REF] Behbahani | Drosophila re-zero their path integrator at the center of a fictive food patch[END_REF] . In contrast, champion navigators such as wasps, bees or ants can be motivated with remarkable ease to learn routes and/or display stunningly accurate path integration [START_REF] Collett | Spatial memory in insect navigation[END_REF] . There is therefore still an incentive to try to probe the brain of different species. But this requires the development of appropriate new techniques, and this endeavour has led to a panel of unorthodox approaches perhaps as diverse as the insects' navigational behaviours themselves. Some researchers decided to leave ants free to roam in the field but recorded their ability to navigate home after injection into a brain region of some pharmaceutical compound. This approach lacks neural precision, but it did show that visual route memories are indeed encoded within the mushroom bodies [START_REF] Kamhi | Vertical lobes of the mushroom bodies are essential for view-based navigation in Australian Myrmecia ants[END_REF] . Others decided to tether their favourite insects -whether by walking on a spherical treadmill [START_REF] Dahmen | Naturalistic path integration of Cataglyphis desert ants on an air-cushioned lightweight spherical treadmill[END_REF] or just flying while glued to a stick [START_REF] Beetz | Flight-induced compass representation in the monarch butterfly heading network[END_REF] -and observe them expressing long-range navigational task while staying on a same spot. In this manner, researchers recently managed to record central complex neurons of flying monarch butterflies, a migratory species, and realised that compass neuron responses are dependent on the animal activity state: they found that the cells behave as compass neurons only during active flight and not while the animal is resting [START_REF] Beetz | Flight-induced compass representation in the monarch butterfly heading network[END_REF] . Some scientists have built insect-robot interfaces, enabling for instance a moth or a cricket to drive their vehicle on-board using smell or sound [START_REF] Ando | Insect-machine hybrid robot[END_REF] . Most daring, others tackled the technological challenge of tethering a bee on a quadcopter, and used electrophysiology to probe the brain of the bee while she was being carried at 20 km/h above natural landscape [START_REF] Paffhausen | A flying platform to investigate neuronal correlates of navigation in the honey bee (Apis mellifera)[END_REF] .

In their new work, Patel et al. [START_REF] Patel | Vector navigation in walking bumblebees[END_REF] explored another promising method. They showed that bumblebees can display most aspects of path integration as observed in the wild, while walking over a short distance in the lab. They plugged a bumblebee nest-box to the floor of a 1.5 m diameter arena, and let the foraging bumblebees spontaneously explore the arena to find a food source (Figure 1). Once fed, bumblebees that were transferred to a replica of the arena, to get rid of odour cues, spontaneously walked in the correct direction and started to search where the nest entrance should have been. This demonstrates that the insect has encoded both the direction and distance of their nest entrance: that is, a proper path integration vector. What is more, rotating the visual cues provided in the arena, such as polarised light or a green-light spot, triggered an equivalent rotation of the insect homing behaviour, demonstrating that the external compass cues can be tightly controlled in this set up. The recipe is simple, and crucially, the bumblebees are tough creatures: the team demonstrated that these insects had no problem in remembering their learnt vector and home properly after a sham surgical brain operation. Together, this opens a novel avenue to study how the brain of insects can integrate and memorise direction and distance.

It is only through a diversity of approaches that we can hope to understand the richness and complexity of insect navigational behaviours [START_REF] Moël | Towards a multi-level understanding in insect navigation[END_REF] . The last decades have brought a remarkable variety of ingenious methods to probe insects' brain, so we can be confident that the field is well on route and will carry on walking. In the new work of Patel et al. [START_REF] Patel | Vector navigation in walking bumblebees[END_REF] , a bumblebee hive is directly connected to a circular arena of 150 cm diameter where visual cues such as polarised light or bright green spots can be fully controlled. Foraging bumblebees are free to explore the arena and eventually find the food location (purple trajectory). Once fed, the insect can then be transferred to a twin arena, where its return path can be recorded (green trajectory). Under these conditions, walking bumblebees exhibit the hallmarks of vector navigation: they head to where the nest should be (based on the visual cues provided) and begin searching for their nest entrance at the correct distance. This ability persists even after brain surgery, making this protocol a promising way to study the neural basis of the insect representation of distance and direction.

Figure 1 .

 1 Figure 1. A new set-up to study path integration in walking bumblebees.

Declaration of Interests

The authors declare no competing interests.


Ms Joëlle Bizeau

Mr Alexandre Adam

Dr Sylvie Bégin

Dr Damien Mertz
email: damien.mertz@ipcms.unistra.fr

Serum Albumin Antifouling Effects of Hydroxypropylcellulose and Pluronic F127 Adsorbed on Isobutyramidegrafted Stellate Silica Nanoparticles

Limiting the serum protein fouling is a major challenge in the design of nanoparticles (NPs) for nanomedicine applications. Suitable chemical surface modification strategies allow to limit the interactions with adsorbing proteins. In this communication, we address the potential of isobutyramide (IBAM) groups grafted on stellate silica nanoparticles (STMS) for the immobilization of two biocompatible polymers renown for biomedical and low fouling applications: Hydroxypropylcellulose (HPC) and Pluronic F127 (PF127). We report that both polymers can be loaded on STMS@IBAM NPs surface with a maximum loading content close to 10 wt %. Regarding their antifouling properties, we report that the coatings of such HPC or PF127 polymers allow to reduce significantly the human serum albumin (HSA) adsorption in average by 70 % as compared to the surface of the free polymer STMS@IBAM. These results highlight the antifouling potential of these polymer pretreatments on IBAM-modified STMS NPs surface.

Introduction

An important challenge in medical research is to design specific delivery systems that can target the injured site in a human body and release their therapeutic cargo in a controlled way. Recently, in our team, we have developed an interface based on isobutyramide (IBAM) groups grafted on the surface of mesoporous silica (MS) nanoparticles (NPs) or MS shelled iron oxide or carbon nanotubes which allow to adsorb a whole range of bioactive (antitumor) molecules (Doxorubicin, curcumin, camptothecin [1,2] ) and biopolymers including proteins [3,4] , polysaccharides [5] , DNA/RNA [6,7] . The IBAM groups were grafted in two sequential steps: one step with aminosilane (APTES) and then conversion of amine groups in IBAM through reaction with isobutyrylchloride (IBC). As suitable MS substrate for this binders, we used large pore MS silica named stellate MS (STMS) which display high surface area (ca. 500 m 2 .g -1 ) and pore size of ca. 10-15 nm whose initial synthesis was described by Zhang et al [8] . Such STMS were recently functionalized with various inorganic NPs (quantum dots, iron oxide) [9] for bimodal imaging combined to magnetic hyperthermia or grafted with high affinity ligand for iron removal in brain mimicking fluid [10] .

However, given the strong affinity of IBAM surface observed with many proteins, and in particular serum albumin, it is anticipated that a protein corona will form around these NPs when used for biomedical application. Indeed, it is now well-known that when NPs are introduced in a biological fluid, they interact with biomolecules and a protein corona ultimately forms at its surface. This protein corona gives a new biological identity to the NPs, and thus has an impact on its interactions with the other biomolecules and cells. Unfortunately, the protein bounding on the NPs surface can lead to the conformational change of these proteins, which then may lead to harmful side effects, for example due to the activation of an undesired cell signaling pathway [11][12][13][14][15] . Limiting the protein corona formation is thus a major challenge in the surface design of NPs for nanomedicine applications.

Aside features such as size, shape and charge, the chemical surface modification of an NP is a key aspect which determines the interactions of adsorbing proteins, and thus can be used to control it [13][14][15] . Among existing chemical strategies, there are efficient approaches to ensure antifouling properties involving mainly the pegylation (and dendronization) or the use of zwitterion polymers which are probably the main reported ways to date [15][16][17] . The introduction of such moieties can be afforded essentially by covalent grafting at the surface of various types of NPs such as quantum dots [18] or iron oxide NPs [19] . One of the main limitations of the covalent grafting is that it usually involves a multi-step synthesis procedure either at the NP surface or in the preformed polymer. To avoid the use of complex procedures or even the introduction of potentially harmful molecules at NPs surface, other methods used the direct coating/adsorption of polymer but the coating instability may preclude the antifouling effect once the polymer is desorbed.

As said previously, the IBAM group allows the high adsorption of a variety of biomolecules on NPs, which will highly probably lead to the formation of a protein corona. But this high adsorbing property could also be exploited to coat the NPs with antifouling polymers by simple adsorption, without the need to chemically modify the polymers, expanding considerably the range of potential functional polymers for this coating. In addition, this would be an extremely simple and tunable way to enable stealth properties without the need for surface linkers that could be harmful for the different potential cargos (drug, ligand, probes etc..).

Among the potential polymers, Hydroxypropylcellulose (HPC) and Pluronic F127 (PF127) appear as two polymers of interest for several reasons: they are both biocompatible, inexpensive and thermoresponsive, this latter being an interesting property that can be used to deliver active principles by hyperthermia. Indeed, HPC displays a Lower Critical Solution Temperature (LCST) at 41°C [20][21][START_REF] Gaharwar | Magnetic Nanoparticles Encapsulated Within a Thermoresponsive Polymer[END_REF] . This LCST ensures HPC to be in its expanded, hydrophilic state at physiological temperature, limiting the protein adsorption [16,[START_REF] Bordawekar | [END_REF]24] . Regarding PF127, its LCST at 37°C has been exploited to form in situ gel at physiological pH [20,25,26] and its antifouling properties due to the hydrophilicity of its polyethylene oxide groups were well studied and used to improve membrane low fouling [27][28][29] .

Herein, we report for the first time the adsorption study of HPC and PF127 polymers on STMS@IBAM NPs surface. The amount adsorbed in aqueous solution of each polymer is investigated as a function of their concentration (or feed weight ratio (FWR)) by thermogravimetric analysis (TGA). Next, with the aim to assess the antifouling potential of these polymer-coated NPs, the adsorption of human serum albumin (HSA) in the presence or in the absence of the polymer layer are evaluated and compared. The HSA adsorption study is achieved by spectrofluorimetry using intrinsic fluorescence of the HSA tryptophan. HSA is known to adsorb with a very rapid kinetics and with a very high loading capacity (LC) at the surface of STMS@IBAM (ca. 442 µg.mg -1 ). We address herein the effects of the pre-adsorbed polymers at the STMS@IBAM surface to significantly limit the final amount of adsorbed HSA. Such an effect, which was never reported up to date, may allow to develop new antifouling properties towards HSA or other serum proteins. Figure 1 represents a global view of the study.

Results and discussion

The first objective of this work was to investigate the adsorption of the two chosen polymers: HPC and PF127 individually on the surface of STMS@IBAM NPs. The chemical functionalization of STMS with APTES and IBAM groups was done following the usual protocol described in the experimental section in Supporting information. First, the STMS was functionalized with APTES to add amines on the particle surface, and then these amines reacted with IBC to obtain the IBAM groups. TGA analysis was used to quantify the amount of grafted APTES and IBAM as it gives access to the total amount of organic material per mass of inorganic material (STMS). Thus the quantity of organic material present at the surface of STMS was calculated as given in Eq. 1. The amount of the last molecule grafted or adsorbed was then calculated by subtracting the quantity of organic material from the previous layer. A more detailed explanation of the calculations is also presented in Figure S1.

Results (Figure S2) indicate that APTES grafting accounts for 93.5 μg.mg -1 (1.9 /nm 2 ) and IBAM moieties account for 131.1 μg.mg -1 (2.3 /nm 2 ) which is in agreement with previous works [5,10] .

Then, the adsorption of each polymer was performed with several FWRs (Eq. 2), which was defined as:

TGA was also used to quantify the amount of adsorbed polymer, with typical representative curves presented in Figure 2.A (HPC) and Figure 2.B (PF127), and the same calculations than for APTES and IBAM were applied to obtain the LC in polymer. The LC is defined in µg of polymer per mg of unmodified STMS. The averaged results are presented in Figure 2.C for HPC and Figure 2.D for PF127. They show that, for both polymers, we could modulate the LC by playing on the FWR. A maximum LC of 97 µg.mg -1 was reached for HPC from a FWR of 25 % while a minimum FWR of 75 % was necessary to reach the maximum LC of PF127, which was 90 µg.mg -1 . The binding interaction of these polymer coatings is hypothesized to occur through hydrogen bonding between the IBAM (amide) groups and the HPC and PF127 polymers containing respectively OH or ether groups as reported previously [7] . Indeed, aside the coating of proteins, IBAM groups were reported to non-covalently bind various polysaccharides such as dextran yielding to selfsupported microcapsules [7] or chitosan to form protective nanocoating towards heavy metal diffusion [5] . Figure 2: A) and B): TGA curves of bare STMS (dark lines), STMS@IBAM (red lines), STMS@IBAM@25polymer (bleu lines), STMS@IBAM@50polymer (green lines), STMS@IBAM@75polymer (purple lines) and STMS@IBAM@100polymer (orange lines) with "polymer" being HPC in A) and PF127 in B) and the number being the FWR in polymer. C) and D): LC in C) HPC and D) PF127 after adsorption on STMS@IBAM with different FWR in polymer (red lines show the general tendency).

Then, the adsorption of HSA on these two polymer-coated STMS@IBAM was studied in order to evaluate their antifouling properties. To do so, the FWR in HSA was fixed at 40 % for all experiments as it leads to a high LC on STMS@IBAM and thus offers a good control sample to investigate the low fouling effect. The LC in HSA was evaluated indirectly by exploiting the intrinsic fluorescence of HSA due to its tryptophan residue (λ exc / λ em =295/337 nm). Fluorescence spectroscopy was thus used to measure the HSA concentration in the supernatants collected after adsorption. A typical fluorescence curve and the calibration curve are presented in Figure 3.A and B respectively. The adsorption results are presented in Figure 3.C (HPC) and Figure 3.D (PF127). In this case, the LC in HSA was calculated with Eq 3: with m HSA the mass of adsorbed HSA determined by fluorescence, and m STMS the mass of unmodified STMS. As expected, the LC in HSA, which is ca. 442 μg.mg -1 when adsorbed on STMS@IBAM, was highly reduced in the presence of both HPC and PF127, without any correlation between the amount of polymer and the final LC in HSA. Indeed, the lowest amount of HPC led to a reduction of the LC of HSA of ca. 66 % while the highest amount led to a reduction of ca. 82%. In the range of the LC used, the presence of HPC reduced the adsorption of HSA by ca. 71 % in average. Regarding PF127, the lowest amount of polymer reduced the adsorption of HSA by ca. 72 % and the highest amount by ca. 71 %. In the range of the LC used, the average HSA reduced adsorption was ca. 69% for this polymer. These results show that the coating of STMS@IBAM with HPC or PF127 can effectively reduce the adsorbing property of these particles toward HSA into an antifouling property, even with a low quantity of polymer.

Figure 3: A) Representing fluorescence spectra of supernatant collected after the adsorption of HSA on STMS@IBAM@xpolymer. B) Calibration curve of HSA in water representing the maximum fluorescence of tryptophan residues at 337 nm in function of HSA concentration. C) and D): LC in HSA obtained with a constant FWR of 40% in HSA on C) STMS@IBAM@xHPC with x being the LC in HPC and on D) STMS@IBAM@yPF127 with y being the LC in PF127 TGA was also performed after the HSA adsorption to confirm the coating with a direct analysis. Surprisingly, TGA curves after HSA adsorption showed a total weight loss that was not higher than before HSA adsorption, as expected, but was slightly lower as it can be seen in Figure 4. These results indicate that for both polymers, a loss of material occurred during this HSA adsorption step. If we examine the difference between the final % wt and the % wt after elimination of all the remaining solvents, we can see than the difference is quite similar between the samples before and after HSA adsorption: ca. 26.2 % before adsorption versus ca. 25.3 % after for HPC and ca. 27.9 % before versus ca. 23.4% after for PF127. This means that the total quantity of organic material is similar before and after adsorption. Furthermore, the previous experiments showed a maximum polymer loading of ca. 97 and 90 µg.mg -1 for HPC and PF127 respectively obtained by TGA, and a final amount of adsorbed HSA of ca. 127 and 136 µg.mg -1 in average, respectively, obtained by fluorimetry. As these amounts are relatively close, and taking into consideration that the polymers are adsorbed on the STMS@IBAM and not covalently attached to it, we suggest that the loss of material observed with the TGA curves of Figure 4 corresponds to the removal of the polymer from the particles surface and its replacement by HSA during the HSA adsorption.

As a control experiment, and to validate that any polymer release does not influence our method for the HSA fluorimetric quantification, we checked that the fluorescence of HSA could not be influenced by the presence of polymer in the supernatants, as the tryptophan fluorescence is sensitive to its environment. This was checked by measuring the fluorescence of HSA in presence of polymer in the same ratio than when adsorbing HSA on STMS@IBAM@xpolymer. The results (presented in Figure S3) showed no effect on the fluorescence, which allowed us to still validate our previous HSA LC results. Thus, we can speak about a pre-treatment of the particles surface with polymers leading to the modification of its adsorbing properties toward HSA.

Figure 4:

TGA curves of STMS@IBAM@xpolymer (black lines) and STMS@IBAM@xpolymer@40HSA (red lines) with x being in A) the FWR in HPC, here 15%, and in B) the FWR in PF127, here 75%.

The colloidal stability at pH 7.4 of the particles before and after HSA adsorption has been checked for each polymer with an FWR leading to the highest LC in polymer: 25% for HPC and 100% for PF127. The DLS results are presented in Figure 5 and the zeta potential is reported in Table 1. As it can be seen, the particles coated with HPC are overall well dispersed however they tend to form some slight aggregates of several micron size, which may be explained by the zeta potential, not high enough in absolute value to provide sufficient electrostatic repulsion between particles. Thereafter, the adsorption of HSA tends to stabilize the particles as there is less aggregates. In any way, it does not decrease the colloidal stability. Regarding the particles coated with PF127, they are very stable before and after HSA adsorption, with a small increase of the hydrodynamic diameter after adsorption. This study has also been conducted with lower FWRs (5% for HPC and 25% for PF127). The results reported in Figure S4 and Table S1 with lower FWRs gave similar results as described above. These results suggest that PF127 is a better choice as it shows a better colloidal stability.

Figure 5: DLS measurements in triplicate for A) STMS@IBAM@25HPC, B) STMS@IBAM@25HPC@HSA, C) STMS@IBAM@100PF127 and D) STMS@IBAM@100PF127@HSA In this work, we have investigated the adsorption of HPC and PF127, two wellestablished polymers renown for biomedical applications, on IBAM-modified STMS NPs surface. We achieved adsorption curves as a function of their FWR and showed that both polymers can be loaded with a maximum loading content close to ca. 10% wt (LC of ca. 97 and 90 µg.mg -1 STMS for HPC and PF127, respectively).

Regarding HSA adsorption studies, we showed that such HPC or PF127 polymer pre-coatings, even with few deposited amounts, were able to reduce significantly the HSA adsorption in average by ca. 71 % for HPC and 69 % for PF127, as compared to the high loading content at the surface of the free polymer STMS@IBAM (442 µg.mg -1 ). Regarding the mechanism involved at NPs surfaces, TGA experiments before and after HSA adsorption suggests removal of the polymers after HSA adsorption.

Overall, these results emphasize the antifouling potential of these polymer-pretreatment on IBAM-modified STMS NPs surface to significantly limit the final amount of adsorbed HSA. Such strategies may be useful in the future as an alternative to current antifouling strategy to limit the fouling of serum proteins involved in the protein corona formation.

Figure 1 :

 1 Figure 1: Representative scheme of the HSA adsorption on STMS@IBAM without and with a polymer pre-treatment

Table 1 :

 1 Zeta potential of the polymer-coated STMS@IBAM before and after HSA adsorption

	Surface	25HPC	25HPC+HSA	100PF127	100PF127+HSA
	Zeta potential	7.21	-6.56	11.63	-18.70
	STD (mV)	±0.34	±0.66	±0.57	±1.11

Acknolwledgements. D.M. acknowledges the Materials Institute Carnot Alsace (project ProtRemote), and the Agence Nationale de la Recherche (grant ANR-19-CE09-0004-Corelmag) for financial supports. The spectroscopy and the transmission electronic microscopy platforms of the IPCMS are acknowledged for technical supports. D. Burger, B. Li, L. Kelhetter and G. Cotin are thanked for their helps and discussions with TGA. D. Cianfarani is thanked for his help with fluorimeter. Keywords: Biopolymers, Mesoporous materials, Nanoparticles, Protein corona, Serum protein antifouling/Antifouling