# Weak impact of microorganisms on Ca, Mg-bearing silicate weathering

Oleg Pokrovsky, Liudmila Shirokova, Svetlana Zabelina, Guntram Jordan,

Pascale Bénézeth

► To cite this version:

Oleg Pokrovsky, Liudmila Shirokova, Svetlana Zabelina, Guntram Jordan, Pascale Bénézeth. Weak impact of microorganisms on Ca, Mg-bearing silicate weathering. npj Materials Degradation, 2021, 5 (1), 10.1038/s41529-021-00199-w. hal-03403423

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Weak impact of microorganisms on Ca, Mg-bearing silicate weathering

Oleg S. Pokrovsky<sup>1,2*</sup>, Liudmila S. Shirokova<sup>1,3</sup>, Svetlana A. Zabelina<sup>3</sup>, Guntram Jordan<sup>4</sup> & Pascale Bénézeth<sup>1</sup>

<sup>1</sup>Geosciences and Environment Toulouse, UMR 5563 CNRS, Toulouse, 31400, France

<sup>2</sup>BIO-GEO-CLIM Laboratory, Tomsk State University, Tomsk, 634050, Russia

<sup>3</sup>N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, 163000, Russia

<sup>4</sup>Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, München, Theresienstr, 41, 80333 München, Germany

Correspondence and requests for materials should be addressed to O.S.P.

(email: oleg.pokrovsky@get.omp.eu)

Abstract

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. Towards resolving these controversies, we studied diopside interaction with the heterotrophic bacterium *Pseudomonas reactants* and the soil fungus *Chaetomium brasiliense*. Using a mixed-flow reactor, diopside dissolution rates were measured in the presence of live bacteria with and without nutrients. Several grams of bacterial wet biomass per liter produced no sizable effect. Atomic Force Microscopy allowed in-situ characterization of diopside in the presence of bacteria. Colonization by a biofilm-like assemblage was observed in nutrient-rich media. However, extensive production of exopolymeric substances precluded imaging individual cells. In nutrient-free solution, individual cell attachment to the diopside was recognized, but no specific attachment site was identified. Using batch experiments, the effect of live and dead fungus was studied with and without nutrients. Neither case affected considerably the diopside dissolution rates. SEM imaging of reacted grains did not evidence any specific fungus-mineral interaction. 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<sup>1,2</sup>. 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
42 chemical weathering of primary silicate minerals in soil environments³⁻⁸. Among the “aqueous” factors,
43 controlling the intensity of this process, solution pH, pCO₂ and organic compounds produced during
44 enzymatic degradation of vegetation litter or the exudates of soil bacteria and plant roots are believed to be
45 the most important⁹. The organic compounds capable to affect mineral dissolution rate include (but are not
46 limited to) extracellular acid and neutral polysaccharides, simple carboxylic acids, uronic acids (galacturonic,
47 guluronic), peptides and aminoacids¹⁰,¹¹, as well as various lichen polyphenolic compounds¹².

48 A positive effect of bacteria and organic ligands on alumosilicate dissolution is incontestable¹³⁻²⁸.

49 Specifically, this effect is related to the capacity of biota to facilitate the Al-O-Si and Fe-O-Si rate-limiting
50 bond breaking, mostly due to strong complexation of bacterially-originated organic moieties with rate-
51 controlling aqueous Al³⁺(aq) cation²⁹, surface >AlOH₂⁺ and >FeOH₂⁺ sites³⁰,³¹, or Fe³⁺(aq) mobilization in
52 solution³²⁻³⁷ thus preventing passivation of mineral surfaces by newly-formed Fe(III) hydroxides³⁸,³⁹.

53 However, all the above mentioned mechanisms are not likely to operate for Fe- and Al-free, Ca and Mg
54 dominant silicates. In fact, several experimental works performed over the past two decades unambiguously
55 demonstrated the lack of any sizable impact of CO₂ and organic ligands (at otherwise constant pH) on Ca-
56 and Mg-bearing mineral dissolution at typical soil water environments. The reason for this is rather weak
57 complexation of organic ligands with >CaOH₂⁺ and >MgOH₂⁺ surface sites responsible for the rate-
58 controlling Mg-O and Ca-O bond breaking in these minerals⁴⁰⁻⁴⁷. According to these studies, unreasonably
59 high concentrations of naturally-relevant organic ligands (0.01 – 0.1 M) are necessary to significantly
60 enhance or inhibit mineral dissolution. As a result, the effect of extracellular organic products and in general
61 soil biota on the weathering rate of Mg- and Ca-bearing minerals in natural settings is expected to be weak.
62 Further, works with heterotrophic bacteria and basaltic glass⁴⁸, wollastonite⁴⁵ and olivine⁴⁹ demonstrated that
63 even unrealistically high concentration of live biomass (several gram per liter, regardless of the presence of
64 nutrients) are not capable of substantially accelerating the Ca, Mg or Si release rate from the mineral. As a
65 result, despite the dominant paradigm on the importance of microbes in chemical weathering of Ca-Mg-
66 silicates in soil environments, the impact of biota on dissolution rates of these silicates might be lower than
67 generally thought. This, in turn, may require revisiting the current understanding of biotic control on
68 feedbacks in atmospheric CO₂ consumption vs. silicate weathering processes. To resolve these possible
69 contradictions and to extend the previous results to other alkaline-earth bearing minerals (e.g., crystalline
70 mixed calcium and magnesium silicates), we attempted, in the present study, a rigorous measurements of the
71 dissolution rate of the typical basic silicate diopside (CaMgSi₂O₆) in the presence of aquatic and soil
72 microorganisms. For these experiments, we were able to separate the effect of pH from that of bacterial
73 presence using a mixed-flow and batch reactor systems coupled with an in-situ and ex-situ microscopic
74 examination of reacted surfaces.

75 To provide a better understanding of the bacteria-mineral interaction in the context of chemical weathering
76 in soils and CO₂ geological storage, we selected a common soil and underground bacterium, Pseudomonas
77 reactans. Bacterial strains of the genera Pseudomonas, thanks to their relatively easy culturing, have been
78 frequently used as a model organisms in studying mineral weathering in the past⁵⁰,⁵¹ and recently²⁸,⁴⁵,⁵².

79 Diopside was chosen as a typical model mineral for basic silicates, frequently used as a representative of
80 silicate dissolution at the Earth surface⁵³. Indeed, because hydrolysis of Ca-O and Mg-O bonds is often the
81 rate-limiting step for Ca- and Mg-bearing mineral dissolution⁵⁴, understanding of microorganism interaction
82 with diopside may help to predict the reactivity of other basic silicates in the presence of microbes. In the
83 present study, the experiments were designed in batch and flow-through reactors under constant biomass
84 concentration, in the presence of both live and dead cells in both nutrient-free and nutrient-bearing media.
85 We measured forward far from equilibrium dissolution rates and we microscopically examined reacted
86 surfaces with respect to microorganism attachment, etch pits and corrosion features. Ensemble of obtained
87 results demonstrated rather weak impact of both bacterium and fungus on diopside reactivity, regardless of
88 the presence of nutrients and live status of microorganisms. Moreover, the passivation of mineral surfaces
89 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.

95

## 96 RESULTS

### 97 Bacterial Mixed-Flow Reactor (BMFR) with heterotrophic bacterium 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). 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. It can be then 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 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).

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The 2nd 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 g_wet/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).

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The 3rd 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 Fig. S1, where the Si concentration in the outlet solution corresponding to the sequence of inlet solutions “bacteria-free 0.1 M NaCl → 0.54 g_wet/L bacteria → 3.85 g_wet/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).

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### 123 Calcium, magnesium and silica release rate in batch reactor 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 Fig. S2 of the Supplement).

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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. 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<sup>41,55,56</sup>, one can quantify the far-from equilibrium dissolution rates for each individual experiment using the following equation which can be applied over the whole duration of the experiment:

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$$R = (d[Ca, Mg, Si/2]_{tot} / dt)/ s \quad (2)$$
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 rates 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).

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145 AFM observation of diopside in the presence of bacteria

146 Freshly reacted diopside surfaces (2-5 h in sterile NaCl solution) largely preserved their cleavage
147 plane morphology (Fig. 4 A). Occasionally some etch pits and local surface roughness was observable (Fig.
148 4 B). After 36 h reaction in bacteria-free nutrient solution, rounding of edges occurred (Fig. 4 C). After 1 to 2
149 days of reaction with nutrient solution at pH = 8.5 or 6.5, we have observed a strong colonization of diopside
150 surfaces by a biofilm-like bacterial assemblage (Fig. 4 D). This EPS coverage included individual elongated
151 spheres and generally convex forms, less than 0.5 µm in longest axis, although their size distribution was not
152 uniform (Figs. 4 D, E, F). Groups of objects occasionally exhibited a distinct internally preferred orientation,
153 which was, however, quite variable within a relatively small area (~50 µm²). The obtained images are
154 consistent with earlier reported bacterial extracellular polymeric substances (EPS) via AFM⁵⁷,⁵⁸. Extensive
155 production of exopolymeric substances precluded imaging of individual bacterial cells within this thick
156 biofilm coverage. In contrast, mineral interaction with bacteria in nutrient-free neutral electrolyte allowed
157 recognizing individual cells attached to the diopside surface (Fig. 4 G); however, no site of preferential
158 interactions such as steps or edges could be identified.

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160 SEM imaging of diopside reacted with fungus

161 The diopside grains reacted with dead fungus in 0.1 M NaCl demonstrated sharp borders and no
162 clear dissolution features (Fig. 5 A). After reaction with live fungus in nutrient-free media, there was an
163 appearance of specific dissolution features such as etching and rounding of edges (Fig. 5 B). The rounded
164 edges were also observed in experiments with live fungus; these features were distinctly different from
165 cleavage (Fig. 5 C). The diopside crystals were also imaged in the presence of fungal mycelium: the hyphae
166 were strongly attached to the grain surfaces and not lost during sample preparation for SEM (Fig. 5 D). Some
167 grains appeared significantly (> 50%) covered by or even embedded into network of fungal hyphae. There
168 were no distinct sites of hyphae location at the diopside surface and overall, the interaction appeared to be
169 quite inert, non-specific and site-indifferent. With increasing the resolution, some organic spherical entities,
170 2 to 3 µm in diameter, were identified at the extremities of the hyphae (Fig. 5 D).

171

172 DISCUSSION

173 Weak (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 exopolysaccharides⁹,²⁴,⁵⁹,⁶⁰,⁶¹.
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
179 of microorganism<sup>22,62-67</sup>. Furthermore, considering basic silicates, some studies reported nil or inhibiting 180 effect of bacteria on olivine<sup>49,68,69</sup> or wollastonite<sup>45</sup> dissolution, whereas other claimed strong accelerating 181 effect of fungi on olivine<sup>38,39,70</sup>. However, in the latter works, all the effects of microorganisms were limited 182 to their ability to sequester rate-inhibiting Fe(III) thus preventing the slowdown of dissolution, rather than 183 directly promoting the breaking of structural bonds in the mineral lattice. Overall, in contrast to large number 184 of works on microbial impact on alumosilicate dissolution<sup>71-79</sup>, the interaction of aquatic microorganisms and 185 relevant organic ligands with Ca or Mg-bearing silicates remained insufficiently characterized.

186 In the present study, the global rate uncertainty in the studied pH range (6 to 9) was between 20 and 187 50%. This uncertainty stems from both the pH variation (±0.2-0.3 units) during the course of an experiment 188 and the reproducibility of steady state concentrations of diopside constituents in mixed-flow reactors with 189 bacteria-free systems or among duplicates in batch reactors with dead fungus (±20%). Therefore, we 190 conclude that the difference between abiotic (bacteria-free or dead cells) and live bacteria experiments is 191 within the experimental uncertainty. As such, the effect of selected live microorganisms on Ca, Mg and Si 192 release rate from diopside is not significant, or cannot be resolved using the experimental design employed in 193 the present study.

194 In this regard, the present result corroborates the former works of our group which demonstrated 195 weak inhibiting or negligible impact of cultivable heterotrophic bacteria P. aureofaciens on wollastonite<sup>45</sup> 196 and P. reactans on basaltic glass<sup>48</sup> and olivine<sup>49</sup>, in agreement with other studies<sup>28,64,68</sup>. The broad meaning of 197 these results is that microbial impact on basic silicate mineral dissolution is limited by i) pH decrease, thus 198 accelerating proton-promoted dissolution, which is relevant to all basic silicates; ii) release of Fe(III)- 199 complexing organic ligands including siderophores. The latter is pertinent solely to minerals whose 200 dissolution is limited by Fe(OH)<sub>3</sub> passivation of the surface. In all other cases of microbially-affected basic 201 silicate dissolution, the concentrations of excreted organic ligands are not sufficient to sizably complex Mg 202 and Ca at the surface or in the aqueous solution which is necessary for breaking of rate-controlling Mg-O or 203 Ca-O bonds at the mineral surface. For example, gluconic acid and its derivatives are believed to be the main 204 exometabolite of heterotrophic bacteria that use glucose as substrate<sup>23,51</sup>. Gluconic acid can favor olivine 205 dissolution in neutral waters<sup>49</sup>; however, this effect occurs at concentrations > 1 mM, totally irrelevant to 206 natural environments. Another example comes from a concerted study of ~40 various ligand impact on 207 wollastonite dissolution<sup>45</sup>. These authors calculated, based on results in mixed-flow reactors, the threshold 208 concentrations of ligands necessary to triple the rates of wollastonite at pH around 7. For most ligands, very 209 high concentration (2 to 3 orders of magnitude higher than those encountered in natural settings) were 210 necessary to appreciably affect the rates. Finally, Golubev and Pokrovsky (2006)<sup>41</sup> demonstrated that very 211 high concentrations (0.01-0.1 M) of organic ligands originated from enzymatic degradation of organic matter 212 or bacterial metabolic activity, are necessary to appreciably affect diopside dissolution. Considering these 213 results and by analogy with olivine and wollastonite, we hypothesize that possible impact of studied 214 heterotrophic bacteria on the diopside dissolution via gluconic acid or other soluble exometabolite 215 production is not detectable due to insufficient concentration of this acid generated via metabolism of P. 216 reactans in experimental reactors.

217

## Mechanisms of diopside interaction with microbial cells and natural implications.

219 Reactive surfaces in both bacterial and fungal experiments demonstrated lack of specific sites of 220 dissolution, etch pit formation, surface amorphisation or new mineral occurrences. Rather, our in-situ (AFM) 221 and ex-situ (SEM) microscopic observations suggested "passivation" of reacted surfaces by microbial 222 metabolic products. We therefore hypothesize that the biofilm coverage either limited aqueous solution 223 contact with mineral surface or merely did not produce sufficient amount of organic ligands capable to 224 impact the diopside dissolution.

225 Microbial biofilm formation, dominated by exopolysaccharides (EPS), is known to protect the 226 mineral surface from interaction with bulk fluid<sup>59,63,64,80</sup> thus inhibiting overall dissolution rate<sup>4,7,10,11,52,81,82</sup>. 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 Fig. S1). 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.

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. 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<sup>41,45,49</sup>, 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<sup>83</sup>. 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.

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<sup>-6</sup> to 10<sup>-4</sup> M<sup>84,85</sup>. 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<sup>29,86</sup>. The pH of rivers and streams draining ‘basic’ silicate rocks is between 7 and 8<sup>87-89</sup> whereas the pH of soil solutions in organic rich soils typically ranges from 4 to 7<sup>90</sup>. 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. 88). 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)<sup>91</sup> 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<sup>3+</sup> 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<sup>92,93</sup>; phosphates<sup>94</sup> 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<sup>42</sup>, calcite and magnesite<sup>44,46</sup>, brucite<sup>43</sup>, and calcite<sup>47</sup> 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 pCO<sub>2</sub> 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 CO<sub>2</sub>(aq) or dissolved H<sub>2</sub>CO<sub>3</sub> molecules on the main rock-forming silicates dissolution rate<sup>56</sup>, and in neutral and alkaline solutions the pCO<sub>2</sub> effect becomes even inhibiting<sup>95</sup>.

275
276 **CONCLUDING REMARKS**

277 Overall, this work allows a better understanding of microbially-affected basic silicate dissolution and
278 provides new microscopic insights for quantifying the degree to which the heterotrophic bacteria and fungi
279 are capable of modifying the dissolution rate of rock-forming silicates. Using bacterial-mixed flow and batch
280 reactors combined with in-situ atomic force microscopy of bacteria-diopside surfaces and experiments on
281 diopside dissolution in the presence of fungus combined with microscopic examination of reacted surfaces,
282 we demonstrated a rather weak impact of microorganisms on Ca, Mg and Si release rate. Regardless of the
283 presence or absence of nutrients, in a wide range of pH (from 6 to 9) and despite very high biomass
284 concentration (up to 4-5 g<sub>wet</sub>/L), the rates were not considerably affected by microbial presence. Moreover,
285 there was a detectable (ca. by a factor of 3) decrease in reactivity of diopside after its prolonged contact with
286 live heterotrophic bacteria. This 'passivation' of the mineral surface was evidenced by extensive production
287 of exopolymeric substances yielding a biofilm-like coverage already after 1-2 days of interaction in nutrient-
288 rich media. Despite tight contact between fungus and diopside surfaces, no site of preferential interactions
289 such as steps, kinks or edges, etch pits or surface amorphisation was detected. The obtained results are fully
290 consistent with large body of evidences that any possible microbially produced organic ligands at
291 concentrations relevant to live biotic systems or natural settings are not capable of noticeably impacting the
292 reactivity of Mg-O and Ca-O sites on diopside surface. As such, this work challenges the dominant paradigm
293 of an overwhelming role of biota on silicate mineral weathering in soils. At the same time, it is in line with a
294 controversial opinion of Sverdrup (2009)⁹⁶ that physical and chemical conditions of forest soils do not allow
295 any significant direct surface actions on minerals by microorganisms or tree roots. While we do not contest
296 the importance of microbial organic matter in Fe and Al complexation and mobilization from silicate
297 structure, the role of biota on basic (Ca and Mg) silicates, playing a pivotal role in atmospheric CO₂
298 sequestration via chemical weathering, may be much lower than traditionally thought. In fact, only drastic
299 pH decrease (i.e., over several pH units), probably occurring in the microniches adjacent to plant roots, is
300 capable of sizably (> a factor of 2 to 3) increase mineral dissolution rate. It is not excluded however that this
301 promoting effect will be partially attenuated due to biofilm formation and passivation of silicate surfaces by
302 microbial cells. Detecting such 'active' or 'passive' microniches within the complex plant-microbe-mineral
303 systems and more importantly, characterizing chemical environment of the fluids with spatial resolution on
304 the level of µm to mm may turn out to be a bottleneck for quantitative mechanistic modeling of chemical
305 weathering. Currently, these gradients are not taken into account even in most sophisticated modeling
306 approaches⁹⁷,⁹⁸. Further experimental and field studies are thus needed to answer the question whether a
307 quantitative modelling of basic silicate mineral weathering and related CO₂ consumption require an explicit
308 prediction for microbial activity or it can be merely approximated by the pH of bulk soil porewaters.
309

310 **MATERIALS AND METHODS**
311

Mineral, bacterial and fungal culture

312 Natural diopside (Ca<sub>0.99</sub>Mg<sub>0.98</sub>Fe<sub>0.02</sub>Cr<sub>0.01</sub>Si<sub>2</sub>O<sub>6</sub>) crystals characterized in previous works<sup>41,56</sup> were
313 used in this study. Ultrasonically-cleaned 100 to 200 µm size fraction having a specific surface area of 1045
314 ± 50 cm<sup>2</sup>/g was selected for dissolution rate measurements.

315 *Pseudomonas reactans* is a common rod-shape groundwater and soil bacteria averaging 2 µm in size 316 (see further description in ref. 48, 49). For experiments, cells were first cultured to a stationary stage on a 317 shaker at 25°C in the darkness using 10% nutrient broth with the following composition: 0.1 g/L glucose, 1.5 318 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 319 media, prior the macroscopic dissolution and microscopic (AFM) experiments. 320

320 Active bacteria number counts (colony forming-units, CFU/mL) were performed using Petri dish
321 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) ×10⁸ and (8.0±1.5)×10⁷, respectively as determined by triplicate measurements. Typical live biomass concentration during experiments was 0.54 and 3.85 g_wet/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 stones⁹⁹. *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 nutrients.

344

345 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 bacteria⁴⁸. 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 ($R_i$, mol/cm²/s) were computed from measured solution composition using:

$$R_i = -q \cdot 1/n \cdot \Delta[i(aq)]_{\text{tot}} / s \quad (1)$$

where *q* (L/s) designates the fluid flow rate, Δ[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 equal 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 hrs of reaction time, no systematic variation of the surface area for powder reacted at 5 < pH < 10 in bacteria-free solutions was observed (Golubev and Pokrovsky, 2006).
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 (2-morpholinoethanesulfonic acid) or NaHCO₃) was first prepared and injected into the reactor until a steady-state outlet fluid composition was attained (typically 2 weeks, D1 series). Once steady state was attained, live bacteria (0.54 g_wet/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 g_wet/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 nutrient solution, and two with dead (autoclaved) biomass (in nutrient-free 0.1 M NaCl).

The reactors were inoculated with 4.2-5.6 g_wet/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 pH-electrode (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 C<sub>org</sub> L<sup>-1</sup>.
All experiments were performed at far from equilibrium conditions with respect to diopside as calculated via
vMinteq program<sup>100</sup> with relevant solubility parameters<sup>101</sup>.

423 Microscopic observations

A detailed 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 g<sub>wet</sub>/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.

442

443 DATA AVAILABILITY

Supplementary material is available. All measured dissolution rates are presented in Tables 1 and 2. Other
relevant data are also available from the corresponding author of this paper upon a reasonable request.

446

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674 AUTHOR CONTRIBUTIONS

675 O.S.P. developed the concept of the study. L.S.S., S.A.Z. and O.S.P. performed dissolution experiments. G.J.

676 performed AFM study. P.B. participated in laboratory experiments and SEM study. O.S.P. wrote the paper,

677 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.

678

679 COMPETING INTERESTS

680 The authors declare no competing interests.

681

682 ADDITIONAL INFORMATION

683 **Supplementary information** The online version contains supplementary material available at

684 https://doi.org/XXXXX.

685 **Correspondence** and requests for materials should be addressed to O.S.P.

686 **Reprints and permission information** is available at http://www.nature.com/reprints

687 **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and

688 institutional affiliations.

689
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<caption>**Fig. 1.** Calcium (A), Mg (B) and Si (C) release rate from diopside measured in the mixed-flow reactor with and without bacteria and 10% nutrient or nutrient-free solution.</caption>

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<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Synthesis of obtained steady-state Ca, Mg and Si release rates (average ± s.d.) from diopside at pH 6 (A) and 9 (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.</figcaption></figure>

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<figure><img src="image_6.png" /><figcaption>**Fig. 3.** 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 (4.5-5.6g_wet/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.</figcaption></figure>

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<figure><img src="image_7.png" /><figcaption>**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).</figcaption></figure>

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<figure><img src="image_8.png" /><figcaption>**Figure 5.** SEM images of reacted diopside surfaces in the sterile electrolyte (A), live fungus in 0.1 M NaCl (B, D) and 1% nutrient media (C, D).</figcaption></figure>
<table><caption>**Table 1.** Results of Bacterial MFR experiment. *R* units are mol cm⁻² s⁻¹.</caption><thead><tr><th>Experiment</th><th>pH</th><th>log R<sub>Ca</sub></th><th>log R<sub>Mg</sub></th><th>log R<sub>Si</sub></th></tr></thead><tbody><tr><td>D1-1 0.1 M NaCl + 0.01 M MES, no bugs, no nutrients</td><td>6.18</td><td>-14.34±0.055</td><td>-14.35±0.10</td><td>-14.33±0.050</td></tr><tr><td>D2-1 0.1 M NaCl + 0.01 M MES, no bugs, no nutrients</td><td>6.17</td><td>-14.47±0.110</td><td>-14.44±0.075</td><td>-14.42±0.061</td></tr><tr><td>D2-4 0.1 M NaCl + 0.01 M MES, no bacteria, no nutrients</td><td>6.05</td><td>-14.81±0.10</td><td>-14.86±0.055</td><td>-14.81±0.085</td></tr><tr><td>D1-4 0.1 M NaCl + 0.01 M MES, no bacteria, no nutrients</td><td>6.30</td><td>-14.52±0.22</td><td>-14.81±0.065</td><td>-14.54±0.045</td></tr><tr><td>D3-4 0.1 M NaCl + 0.01 M NaHCO3, no bacteria, no nutrients</td><td>9.01</td><td>-15.04±0.075</td><td>-14.98±0.15</td><td>-15.14±0.056</td></tr><tr><td>D3-1 0.1 M NaCl + 0.01 M NaHCO3, no bugs, no nutrients</td><td>9.06</td><td>-15.18±0.078</td><td>-15.12±0.085</td><td>-15.02±0.065</td></tr><tr><td>D4-1 0.1 M NaCl + 0.01 M NaHCO3, no bugs, no nutrients</td><td>9.07</td><td>-15.12±0.055</td><td>-15.46±0.10</td><td>-15.43±0.15</td></tr><tr><td>D4-4 0.1 M NaCl + 0.01 M NaHCO3, no bacteria, no nutrients</td><td>9.01</td><td>-14.82±0.11</td><td>-14.95±0.075</td><td>-14.91±0.065</td></tr><tr><td>D1-2 0.1 M NaCl + 0.01 M MES, 0.54 g/l bacteria, no nutrients</td><td>6.25</td><td>-14.67±0.089</td><td>-14.57±0.15</td><td>-14.65±0.052</td></tr><tr><td>D1-3 0.1 M NaCl + 0.01 M MES, 3.85 g/l bacteria, no nutrients</td><td>6.20</td><td>-14.51±0.078</td><td>-14.63±0.082</td><td>-14.57±0.065</td></tr><tr><td>D3-2 0.1 M NaCl + 0.01 M NaHCO3, 0.54 g/l bacteria, no nutrients</td><td>8.60</td><td>-15.34±0.055</td><td>-15.23±0.10</td><td>-15.13±0.05</td></tr><tr><td>D3-3 0.1 M NaCl + 0.01 M NaHCO3, 3.85 g/l bacteria, no nutrients</td><td>8.32</td><td>-15.29±0.11</td><td>-15.07±0.075</td><td>-15.15±0.065</td></tr><tr><td>D2-2 0.1 M NaCl + 0.01 M MES, 0.54 g/l bacteria, 10% NB</td><td>7.80</td><td>-14.70±0.058</td><td>-14.80±0.065</td><td>-14.58±0.075</td></tr><tr><td>D2-3 0.1 M NaCl + 0.01 M MES, 3.85 g/l bacteria, 10% NB</td><td>8.05</td><td>-14.80±0.089</td><td>-14.97±0.15</td><td>-14.73±0.055</td></tr><tr><td>D4-2 0.1 M NaCl + 0.01 M NaHCO3, 0.54 g/l bacteria, 10% NB</td><td>8.75</td><td>-15.18±0.078</td><td>-15.09±0.085</td><td>-15.30±0.065</td></tr><tr><td>D4-3 0.1 M NaCl + 0.01 M NaHCO3, 3.85 g/l bacteria, 10% NB</td><td>8.67</td><td>-15.19±0.12</td><td>-15.23±0.11</td><td>-15.26±0.072</td></tr></tbody></table>
<table><caption>**Table 2.** Results of fungus batch experiments (0.1 M NaCl). R units are mol cm⁻² s⁻¹.</caption><thead><tr><th>Experiment</th><th>pH</th><th>log R<sub>Mg</sub></th><th>log R<sub>Ca</sub></th><th>log R<sub>Si</sub></th></tr></thead><tbody><tr><td>Dead biomass, 4.46 g/L</td><td>6.18</td><td>-14.55±0.10</td><td>-14.44±0.054</td><td>-14.63±0.051</td></tr><tr><td>Dead biomass, 4.95 g/L</td><td>6.13</td><td>-14.48±0.074</td><td>-14.71±0.11</td><td>-14.70±0.065</td></tr><tr><td>Live biomass, 4.83 g/L, no nutrient</td><td>6.35</td><td>-14.62±0.055</td><td>-14.39±0.10</td><td>-14.56±0.085</td></tr><tr><td>Live biomass, 5.1 g/L, no nutrient</td><td>6.45</td><td>-14.39±0.065</td><td>-14.25±0.058</td><td>-14.43±0.045</td></tr><tr><td>Live biomass, 5.6 g/L, live, 1% nutrient</td><td>6.50</td><td>-14.44±0.055</td><td>-14.73±0.045</td><td>-14.49±0.054</td></tr><tr><td>Live biomass, 5.1 g/L, live, 1% nutrient</td><td>6.40</td><td>-14.53±0.085</td><td>-14.52±0.078</td><td>-14.54±0.067</td></tr></tbody></table>
<figure><img src="image_9.png" /><figcaption>**Fig. S1.** Outlet Si concentration in the mixed-flow reactor at pH 6 (A) and 9 (B) in the sequence of nutrient-inlet solutions “bacteria-free 0.1 M NaCl → 0.54 g<sub>wet</sub>/L bacteria, nutrient → 3.85 g<sub>wet</sub>/L bacteria, nutrient → bacteria-free 0.1 M NaCl”. C and D: Outlet Si concentration in the mixed-flow reactor at pH 6 (C) and 9 (D) in the sequence of nutrient-free inlet solutions “bacteria-free 0.1 M NaCl → 0.54 g<sub>wet</sub>/L bacteria, 0.1 M NaCl → 3.85 g<sub>wet</sub>/L bacteria, 0.1 M NaCl → bacteria-free 0.1 M NaCl”.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. S2.** Example of Si concentration evolution in batch reactor with dead, live without nutrient and live with 1% nutrients fungus (4.5, 4.2 and 5.6 g<sub>wet</sub>/L, respectively).</figcaption></figure>