Interactions of three soil bacteria species with phyllosilicate surfaces in hybrid silica gels

Authors


Abstract

To simulate iron consumption in soils, iron leaching from silicate minerals due to three heterotrophic bacterial strains and a chemical treatment was studied using hybrid silica gel (HSG) doped with two phyllosilicates, nontronite (NAu-2) or low-iron-content montmorillonite (SWy-2). HSG methodology, a novel way of separating bacteria cells from a colloidal mineral source, consisted in embedding colloidal mineral particles into an amorphous porous silica matrix using a classical sol-gel procedure. Pantoae agglomerans PA1 and Rahnella aquatilis RA1 were isolated from silicate-rich soils, that is, beech and wheat rhizospheres (Vosges, France); Burkholderia sp. G5 was selected from acidic and nutrient-poor podzol soils (Vosges, France). Fe release from clay minerals and production of bacterial metabolites, that is, low molecular weight organic acids (LMWOA) and siderophores, were monitored. Two LMWOA profiles were observed with major gluconate production (> 9000 μM) for Burkholderia sp. G5 and moderate production of lactate, acetate, propionate, formate, oxalate, citrate, and succinate (< 300 μM) for R. aquatilis RA1 and P. agglomerans PA1. HSG demonstrated its usefulness in revealing clay mineral–microorganisms interactions. The effect of bacterial exsudates was clearly separated from physical contact effect.

Introduction

In natural soils and sediments, water fills soil pores or forms film around minerals providing local anaerobic conditions until water evaporation and drainage restore aerobic conditions. Heterotrophic bacteria switch from anaerobic to aerobic metabolism and contribute to iron redox cycles of clay (Stucki, 2011). Under anaerobic conditions, production of exogeneous reductant or cell-associated reducing ability caused extensive dissolution of Fe(II) from clays (Jaisi et al., 2005, 2007abc, 2008). Under aerobic conditions, Fe solubility in soil is insufficient to fulfill nutritional needs of trees and microorganisms. Therefore, bacteria adopt various strategies for iron uptake, that is, colonization of mineral surfaces, production of low molecular weight organic acids (LMWOA) and siderophores (Hiebert & Bennett, 1992; Barker et al., 1997; Bennett et al., 2001; Rogers & Bennett, 2004; Sandy & Butler, 2009; Hider & Kong, 2010). Proton-promoted and ligand-promoted dissolution mechanisms are the two main biogeochemical processes explaining bacteria–mineral interactions in oxic conditions (Berthelin, 1983; Banfield et al., 1999). Proton-promoted dissolution is related to protonation of iron ions coordinative partners at mineral surface. Ligand-promoted dissolution is proposed to follow a three-step process (Furrer & Stumm, 1986): (1) ligand exchange for fast surface complex formation, (2) slow surface release of metal ion, and (3) ligand fast regeneration. Rate of ligand-promoted dissolution is dependent of pH, surface coverage and adsorption of organic and inorganic ligands (Dong et al., 2009; Dong & Lu, 2012). Most laboratory studies on mineral bioweathering have been carried out using natural particles in suspension. Flocculation and precipitation or adhesion to bacteria cells were observed depending on mineral particle size. For colloidal clay particles like nontronite, these events occurred and participated in bioweathering (Jaisi et al., 2008). To focus on how metabolite diffusion influences mineral weathering, a new method was recently developed. It consisted of using new synthetic hybrid material with mineral colloidal particles well dispersed in a porous silica matrix. Hybrid silica gels (HSGs) were produced according to a conventional sol-gel pathway (Grybos et al., 2010). HSG provided mineral particles embedded in highly hydrated silica matrix where only diffusing small molecules, that is, LMWOA and siderophores, were able to penetrate and alter clay minerals (Oulkadi et al., 2014). Pore diameter of wet silica gels was less than 10 nm, whereas bacterial cells are about 1–2 μm length (Dickson & Ely, 2013). Bacteria were excluded from HSG as demonstrated for GFP-tagged derivatives of Pseudomonas putida KT2440 in interaction with HSG (Grybos et al., 2011). HSG prevented bacteria adhesion to colloidal mineral and subsequent flocculation and precipitation. Only metabolites from microorganisms or plant root exsudates were able to diffuse through HSG and dissolve minerals. Figure 1 illustrates what was expected to occur when bacteria were incubated with HSG containing colloidal mineral particles.

Figure 1.

Schematic representation of a bacterial cell in interaction with HSG. Bacterial metabolites and protons diffuse through HSG micropores and mesopores to mineral colloidal particles. Iron solubilization occurs, and then, Fe(III) bound to ligands (LMWOA and siderophores) diffuse back to bacteria cell.

The aim of the present work was to use HSG doped with colloidal clay minerals for quantifying and comparing bioweathering processes due to metabolite diffusion. Nontronite (NAu-2) and montmorillonite (Wyoming, Swy-2) were chosen for their different iron pool content, which is high- and low-iron contents, respectively, to contrast the weathering conditions. On this basis, three soil bacteria species were selected for their ability to dissolve minerals in subsurface zones of accumulation of clays and iron, which is rhizosphere, or strongly leached soil that is podzol: Pantoae agglomerans PA1, Rahnella aquatilis RA1 and Burkholderia sp. G5.

Materials and methods

Mineral source

Nontronite (NAu-2), purchased from the Source Clays Minerals repository at Purdue University, was defined by the structural formula [Si7.55Al0.16Fe0.29] [Al0.34Fe3.54Mg0.05] O20(OH)4Na0.72 (Gates et al., 2002). Montmorillonite (Wyoming, SWy-2) from Iko Erbslöh (Germany) was described by the structural formula [Si7.74Al0.26] [Al3.06Mg0.48FeII0.03FeIII0.42] O20(OH)4 Na0.77 (Vantelon et al., 2003). Purified Nau-2 and SWy-2 size fractions were obtained according to the procedure previously described by Michot et al. (2008) and briefly summarized hereafter. Clay suspensions were exchanged three times with 1 M NaCl and then washed by centrifugation and dialysis against Milli-Q water until a conductivity of < 5 μS was reached. The suspensions were left for 24 h in Imhoff cones. After discarding the bottom of the cones, size fractionation was performed by centrifuging suspensions from 7000 to 35 000 g (Michot et al., 2008). The size fraction selected from a 17 000 g gravitational field was chosen for this study. The morphological properties of NAu-2 particles were determined using transmission electron microscopy on a Philips Microscope. The average size values for the fraction we used were 705 nm in length, 138 nm in width and 1.05 nm in thickness, which proves that the layers were exfoliated in suspension (Michot et al., 2008).

HSG Synthesis

HSGs were produced through an acid catalyzed sol-gel procedure using tetraethoxysilane (TEOS, Fluka; Grybos et al., 2010). A clear solution of TEOS, Milli-Q H2O, and HCl (0.1 M), defined by the molar ratio 1 : 2.45 : 0.003, was prepared by sonication (Ultrasonic cleaner, Branson 2410 DTH) at ambient temperature. Noble Agar (0.15 g L−1) was added to the solution at 12% (v/v) in order to get better mechanical properties. Adjustment of pH from pH 2.0 to 4.0 with NaOH (0.1 M) prevented NAu-2 dissolution when added in the silica sol. Mineral colloidal suspension was mixed with the sol to give a final concentration equaled to 0.5 ng μL−1 HSG. The pH was further increased up to pH 5.0 (NaOH, 0.1 M) to accelerate self-condensation and sol-gel transition. Hybrid silica solution (250 μL) was distributed into 96-well microplates (Eppendorf Deepwell) and left at room temperature for gelation that occurred within minutes. HSGs pieces were then sterilized twice by autoclaving in Milli-Q water for 30 min at 110 °C and stored in Milli-Q water at ambient temperature until use. The hydrothermal treatment discarded ethanol produced during preparation and stabilized the gel structure (SAXS measurements not shown). HSGs mineral-free (controls) were made according to the same procedure by replacing mineral suspension with Milli-Q water. All chemicals used were of extra pure grade.

Weathering experiments

Biotic experiments

Pantoae agglomerans PA1 and R. aquatilis RA1 were isolated from the rhizosphere of beech and wheat, respectively. Burkholderia sp. G5 was isolated from podzol soil (Vosges, France; Balland et al., 2010; Grybos et al., 2011). Selected clones were grown overnight in 2 mL sterilized Luria–Bertani broth at 28 °C under constant shaking at 150 r.p.m. (Stuart Scientific, orbital incubator SI50). The cell suspensions were harvested by centrifugation (Multifuge 1 S-R, Heraeus) at 4500 g for 10 min at 10 °C, and the pellets were washed three times in a Fe–Mg-deficient Bushnell–Hass medium (BHm) adjusted at pH 6.5 (Balland et al., 2010; Grybos et al., 2010). BHm medium was prepared with analytical reagents and double deionized water (Milli-Q) and contained per liter: 22.5 mg Na2HPO4·2H2O, 20 mg NaH2PO4·2H2O, 20 mg KCl, 100 mg KNO3, 65 mg (NH4)2 SO4, 20 mg CaCl2·2H2O. Prior to experiments, BHm was autoclaved for 30 min at 110 °C and supplemented with sterile-filtered (0.22 mm pore size membrane; Millipore) glucose solution to a final concentration of 3.3 g L−1. Preliminary experiments on BIOLOG® demonstrated that glucose was used by the three bacteria. Two milliliters BHm was used to inoculate each assay containing four pieces of 250 μL HSGs doped with NAu-2 or SWy-2 particles. At the start of the experiment, bacterial density was around 107 cells μL−1 (0.06 absorbance units at 600 nm). pH, Fe, glucose and organic acids were analyzed after 6 and 12 days incubation.

Abiotic experiments

To simulate proton-promoted dissolution, abiotic weathering experiments were performed in BHm medium adjusted with HNO3 (1 M) to pH ranging from 1.5 to 5.0. HSGs doped with NAu-2 or SWy-2 particles were added in pH-adjusted BHm in a volume ratio 1 : 2. After 6 and 12 days, Fe and pH measurements were determined.

Biotic and abiotic experiments were performed with HSG mineral-free for Fe measurements controls. All weathering experiments were performed in 14-mL sterile tube (Falcon™) in triplicate at 28 °C under shaking at 150 r.p.m. (Stuart Scientific, orbital incubator SI50).

Elemental analyses

All solutions from weathering experiments were filtered through 0.22-μm pore size filter (Millipore). Elemental analyses were performed on fresh filtered solutions or, after storage at −20 °C, for organic acids analysis. The pH was measured using standard pH meter electrode (MeterLab®, pHM210). The colorimetric analysis of glucose and Fe was performed in clear bottom 96-well microplates using a FLX Xenius spectrofluorometer (SAFAS, Monaco). Glucose consumption was determined by measuring the glucose remaining in 3 μL of filtrate aliquot after addition of 300 μL of GOD-PAP (enzymatic Kit; BioLabo). After 20 min in dark, the glucose concentration was calculated through absorbance measurement at 505 nm.

As Fe can precipitate and/or adsorb onto the silica and mineral surfaces, one volume HSG was treated with two volumes of potassium chloride and hydroxylamine (KCl 1 M; NH2OH 0.2%) to extract amorphous Fe at the end of each experiment After 1 h at 28 °C, amorphous Fe extracted from HSG with potassium chloride and hydroxylamine solution and soluble Fe in weathering solutions were quantified by absorbance measurement at 565 nm after addition of 20 μL of Ferrospectral® dye indicator to 200 μL of filtered solutions (Balland et al., 2010; Grybos et al., 2010; Oulkadi et al., 2014). The detection limit was found as low as 20 μg L−1 under our conditions. The total amount of clay-dissociated Fe was calculated as the sum of soluble Fe and Fe released from HSG after KCL-Hydroxylamine treatment.

Organic acids produced from biotic experiments were quantified using Ion Chromatograph (ICS 3000; Dionex Corp.) equipped with an online hydroxide eluent generator, an ion suppressor, and a conductivity detector. The separation was realized using an Ion Pac® column (AS 11 HC; Dionex Corp.) with a KOH gradient of 0.9–60 mM over 50 min at a flow rate of 1.3 mL min−1. Standard solutions (0.1 mM) used were sodium acetate, sodium citrate, sodium formate, sodium gluconate, sodium D, l-malate, sodium oxalate, sodium propionate, sodium succinate (Sigma-Aldrich), sodium lactate, and sodium malonate (Fluka). Quantification measurement uncertainty was better than 0.5% for all organic acids, and the detection limit was close to 0.1 μg mL−1.

Assay for siderophore production

The quantification of siderophores produced by R. aquatilis RA1, P. agglomerans PA1, and Burkholderia sp. G5 was performed according to the Chrome Azurol S (CAS) liquid assay (Schwyn & Neilands, 1987) after 6 days incubation at 28 °C in liquid MM9 medium. The MM9 growth medium was derived from M9 medium by decreasing the phosphates level to 0.03% KH2PO4, and it is composed of (per liter) 6.0 g Na2HPO4, 0.3 g KH2PO4, 0.5 g NaCl, 1.0 g NH4Cl, and 6.06 g Tris base (50 mM). Each liter of salt solution was supplemented with sterilized filtered solutions of 10% (w/v) Chelex 100-treated Casamino acids (Difco Laboratories) 20 mL, MgSO4 (1 M) 2 mL, 20% (w/v) glucose 10 mL, and CaCl2 (1 M) 0.1 mL (Kalinowski et al., 2000). Siderophore concentration was determined at 630 nm according to a calibration curve made with desferrioxamine mesylate (DFOM; Sigma-Aldrich; Payne, 1994; Oulkadi et al., 2014).

Data analysis

For data multiple comparisons, one-way analysis of variance (anova) was performed followed by Tukey's HSD tests using xlstat Version 2011.4.02 (P < 0.05).

Bacterial strains characterization

The genus of the three bacterial strains selected for this work was determined by amplification of 16S rRNA genes and sequencing. Polymerase chain reactions were performed using an universal set of primers in a total reaction volume of 50 μL containing 1× PCR mastermix (Eppendorf ®), 0.1 μM of primers (966 and 1401 F) and 1 μL cell extract (Felske et al., 1998). Before being sequenced by MWG biotech Company (Courtaboeuf, France), the PCR products (PCR product size: 433 bp) were purified and concentrated using mini-columns (High Pure™ PCR product Purification Kit; Roche diagnostic). A blast program was used to compare sequences with those of GenBank databases (http://www.ncbi.nlm.nih.gov/blast). The following identification was made with a maximum identification of about 99%: R. aquatilis RA1 (ENA accession number HG325834), P. agglomerans PA1 (ENA accession number HG325835), Burkholderia sp. G5 (ENA accession number HG325836).

Results

Swy-2 and NAu-2 mineral weathering was performed in a Fe–Mg-deprived medium (BHm) supplemented with glucose as sole carbon source. Burkholderia sp. G5 consumed all the glucose available after 6 days incubation, while P. agglomerans PA1 and R. aquatilis RA1 consumed < 50% glucose after 12 days (Table 1). In relation with high glucose uptake by Burkholderia sp. G5 after 6 days incubation, the pH values dropped from 6.5 to 2.91 and 2.93 in NAu-2 and SWy-2 treatments, respectively, and then stabilized until 12 days (Table 1). After 12 days incubation, R. aquatilis RA1 acidified down to pH 3.74 and pH 4.01 for the SWy-2 and NAu-2 samples, respectively. For all the experiments performed with P. agglomerans PA1, the pH values dropped from 6.5 to around pH 3.6 after 6 days up to 12 days (Table 1).

Table 1. General composition of medium containing HSGs doped with minerals (NAu-2, SWy-2) after 6 and 12 days incubation with three bacteria strains (Rahnella aquatilis RA1, Pantoae agglomerans PA1, and Burkholderia sp. G5) including glucose remaining in solution, pH, soluble and immobilized Fe
MineralBacteriaTime (days)pHGlucosea remainingFeb solubleFeb immobilized
pHSD pHGlucoseSD glucoseFeSD FeFeSD Fe
  1. Means not followed by the same letter are significantly different according to Tukey's HSD test (< 0.05), = 36 observations.

  2. a

    Glucose average concentration remaining in solution expressed in g L−1.

  3. b

    Fe average concentration expressed in mg g−1 mineral, standard deviation: ‘SD’ calculated from triplicate.

  4. –, Below detection limit.

NAu-2 RA1 64.33a0.042.16a0.260.58e0.150.53e0.06
124.01b0.151.81a,b0.152.07c0.571.36d0.40
G5 62.91d0.012.65b0.052.60b,c0.31
122.94d0.014.31a0.343.84a0.19
PA163.61c0.041.97a,b0.281.38d0.131.96c0.44
123.62c0.072.15a0.072.52b,c0.232.73b0.26
SWy-2 RA1 63.67c0.041.79a,b0.070.42e0.04
123.74c0.181.63b0.520.07e,f0.040.55e0.04
G5 62.93d0.010.14e,f0.030.84d,e0.06
122.95d0.010.14e,f0.050.49e0.03
PA1 63.66c0.031.96a,b0.090.43e0.03
123.56c0.031.90a,b0.180.08e,f0.060.60e0.03

As a consequence of bacterial metabolic activity, Fe mineral dissolution was observed in all treatments (Table 1). 8.15 mg g−1 of total Fe was dissociated from NAu-2 particles after 12 days incubation with Burkholderia sp. G5, whereas 5.25 and 3.43 mg g−1 were released by P. agglomerans PA1 and R. aquatilis RA1, respectively (Table 1). Fe leached from low-iron montmorillonite (SWy-2) was in the range 0.62–0.98 mg g−1 after 12 days (Table 1). To take into account the eightfold Fe level in NAu-2 compared with SWy-2, Fe leached was normalized to total Fe (Fig. 2). An average Fe release value of 2.0% was obtained after 12 days bioweathering except for NAu-2 incubated with Burkholderia sp. G5 and P. agglomerans PA1 that released 3.25% and 1.35% Fe, respectively. Lower soluble Fe values were systematically obtained during SWy-2 bioweathering (Fig. 2). According to abiotic experiments (Fig. 3a and b), it appeared that, except for SWy-2 bioweathering by Burkholderia sp. G5, all biotic treatments were above the proton-promoted dissolution line released in BHm medium adjusted with HNO3 (1 M) to pH ranging from 1.5 to 5.0 which indicated the effect of ligand-promoted dissolution. Strong Fe chelatants, that is siderophores, were measured in MM9 treatments for R. aquatilis RA1 and Burkholderia sp. G5 giving 52 ± 0.3 and 45 ± 1.2 mM (equivalent DFOM), respectively, whereas P. agglomerans PA1 produced 16 ± 1.5 mM (equivalent DFOM). Determination of other ligands, that is LMWOA (Table 3), gave for R. aquatilis RA1 and P. agglomerans PA1 high amount of lactic and acetic acids (< 300 mM) and moderate levels of other monodentate acids, that is, succinic, propionic and formic acids. Di and tri-dentate, that is oxalic and citric acids, were produced up to 200 mM for P. agglomerans PA1 after 12d incubation with NAu-2 (Table 2). High concentration of gluconic acid (> 9000 mM) was produced by Burkholderia sp. G5 in NAu-2 and SWy-2 weathering experiments with some acetic, formic, propionic and oxalic acids in micromolar concentrations. No lactic, citric, pyruvic acids were produced by G5 after 12 days. The metabolized carbon was calculated from carbon produced in organic acids normalized to carbon consumed in glucose (Table 3). In NAu-2 and SWy-2 bioweathering experiments, metabolized C after 12 days represented more than 60% for Burkholderia sp. G5 and <4% and 6% for R. aquatilis RA1 and P. agglomerans PA1, respectively.

Table 2. Major organic acids excreted by bacteria strains Rahnella aquatilis RA1, Pantoae agglomerans PA1, and Burkholderia sp. G5) after 6 and 12 days incubation of HSG doped with minerals (NAu-2 and SWy-2)
MineralBacteriaTime (days)Gluconic acidaCitric acidaSuccinic acidaLactic acidaAcetic acidaPropionic acidaOxalic acidaFormic acidaPyruvic acida
  1. Means not followed by the same letter are significantly different according to Tukey's HSD test (P < 0.05), N = 36 observations. Standard deviations calculated from triplicate are indicated in italic below each mean value.

  2. a

    Organic acid average concentration expressed in micromolar (n = 3).

  3. –, Below detection limit.

NAu-2 RA1 647.9b11.3b,c51.4d,e76.6c10.8b28.9e,f8.0e
7.3 2.7 9.4 2.9 3.3 1.9 1.2
1284.6a46.6a,b,c55.8d,e141.6a,b,c14.4b41.1d,e13.03c,d,e
32.1 18.0 3.4 3.4 1.0 4.8 2.2
G5 611633a80.5c9.9b3.3 g10.9d,e
396.5 10.0 2.0 0.5 2.1
1211733a2.2c116b,c3.8b10f,g21.03c,d,e
970.1 2.0 18.0 2.3 1.0 2.5
PA1 637.7d39.4b,c92.2a,b182.3a,b146.5a,b,c9.4b152.2b92.0b
10.2 2.7 14.6 15.9 14.5 2.3 16.8 12.7
1235.5c55.4a,b99.3a200a,b298.3a5.8b199a128.3a11.3a
6.3 4.7 15.0 18.8 37.2 2.4 16.3 9.5 9.1
SWy-2 RA1 694.6a68.9a,b,c92.2c,d68.3c12.5b36.3d,e7.2e
13.7 14.0 18.3 2.9 2.9 4.2 1.2
1281.6a,b20.3a,b,c153.6b,c285.3a12.1b41.1d,e8.7e23.8a
33.5 10.0 39.0 39.2 4.9 4.8 2.2 10.8
G5 69526b60.5c32.4a10f,g11.6d,e
584.9 1.9 16.3 1.1 3.3
1211300a5.7c83.3c12.1b3.3 g10.2e
519.4 1.5 2.1 2.7 0.5 1.2
PA1 670.3a,b117.33a144b,c110b,c6.25b57.0c,d25.3c,d
9.7 31.7 34.8 6.0 1.5 7.1 3.3
1278.4a,b80.5a,b,c258a258.3a,b8.1b70.7c27.5c59.4a
16.9 17.5 55.3 59.3 1.4 4.5 2.5 29.4
Table 3. Metabolized carbon (%) by Rahnella aquatilis RA1, Pantoae agglomerans PA1, and Burkholderia sp. G5 during NAu-2 and SWy-2 bioweathering
 Metabolized Carbon (C in %) during bioweathering
NAu-2 HSG experimentsSWy-2 HSG experiments
C (%)SDC (%)SD
  1. The percentage values were calculated from carbon produced in organic acids normalized to carbon consumed in glucose after 12 days at 28 °C.

Rahnella aquatilis RA1 2.640.043.230.17
Burkholderia sp. G564.0561.86
Pantoae agglomerans PA1 5.680.085.260.11
Figure 2.

Soluble Fe (in white) and immobilized Fe (in gray; in g 100 g−1 Fe) released by Rahnella aquatilis RA1, Pantoae agglomerans PA1, and Burkholderia sp. G5 from colloidal minerals trapped in HSGs after 12 days incubation at 28 °C. Means not followed by the same letter are significantly different according to Tukey's HSD test (P < 0.05), N = 36 observations.

Figure 3.

Log–Log plots of Fe released in solution (mol g−1 mineral) versus pH from HSGs containing SWy-2 (a) and NAu-2 (b) after 12 days of incubation at 28 °C with Rahnella aquatilis RA1 (black square), Pantoae agglomerans PA1 (black diamond), and Burkholderia sp. G5 (black triangle). The proton-promoted dissolution from HNO3 experiments (empty square) is indicated by dashed line.

Discussion

The HSG methodology permitted to evaluate bacterial Fe leaching from two colloidal phyllosilicates (Fig. 2). Ligand-promoted dissolution was observed in all cases except for SWy-2 bioweathering with Burkholderia sp. G5 that followed proton-promoted dissolution processing (Fig. 3a and b). The acidifying bacterial activity resulted from organic acids production but also from CO2 release that was not measured in this study (Welch & Ullman, 1993; Balland et al., 2010). Two distinct organic acids profiles were observed as a function of bacteria ecological origin. From acidic podzol type soil, Burkholderia sp. G5 metabolized more than 60% carbon and produced mainly gluconate (10 000 μM), while R. aquatilis RA1 and P. agglomerans PA1, isolated from silicate-rich rhizosphere, metabolized < 6% carbon and produced moderate levels (< 300 μM) of mono, di- and tri-dentate organic acids. Previous studies showed that bacteria such as Burkholderia sp. efficiently decreased pH (pH < 3) in poor-nutrients conditions to acquire metals from minerals by glucose oxidation and gluconic acid production (Vandevivere et al., 1994; Lin et al., 2006; Wu et al., 2007; Song et al., 2008; Balland et al., 2010; Grybos et al., 2011; Balland-Bolou-Bi & Poszwa, 2012). Vandevivere et al. (1994) used the term gluconate-promoted dissolution to describe the central role of gluconic acid on silicate mineral weathering as also previously reported in other works (Welch & Ullman, 1999; Lin et al., 2006; Mailloux et al., 2009). The di- and tri-dentate organic acids, produced by R. aquatilis RA1 and P. agglomerans PA1, were also found efficient in silicate dissolution in conditions of pH ranging from 3.6 to 4.3 (Welch & Ullman, 1993). Citric and oxalic acids are characterized by Fe(III)-binding affinity constants of 10+24.8 and 10+7.6, respectively (Neilands, 1981; Silva et al., 2009). Other chelating agents, which is siderophores, were produced in micromolar concentrations by R. aquatilis RA1, Burkholderia sp. G5 and P. agglomerans PA1. Siderophores have a high coordination affinity with ferric ions and a binding constant varying between 10+23 and 10+52 (Ams et al., 2002; Sandy & Butler, 2009). Organic acids and siderophores can also bind to other elements of minerals than Fe. Adsorption of siderophores molecules on clay mineral surfaces entrapped in HSG and formation of stable and soluble metal-organic complexes might act efficiently in our weathering experiments (Welch & Ullman, 1999; Ullman & Welch, 2002; Cama & Ganor, 2006; Haack et al., 2008). Under aerated conditions at pH5.0, inorganic iron is extremely insoluble and its concentration is less than optimal for microbial growth. To overcome this limitation, Burkholderia sp. G5, R. aquatilis RA1 and P. agglomerans PA1 were expected to produce early siderophores that have a high coordination affinity with ferric ions. In addition, synergistic mechanisms with LMWOA could occur at the mineral surface, as demonstrated in experiments dealing with Fe(III) and oxalate and DFOM (Cheah, 2003; Reichard et al., 2007). Acker and Bricker (1992) suggested that mineral ‘controlled’ the organic acids production in relation to Fe localization in octahedral or tetrahedral sheet. Fe is essentially found in octahedral sheet for Swy-2, while NAu-2 contains mainly octahedral Fe and some tetrahedral Fe (Gates et al., 2002; Vantelon et al., 2003; Michot et al., 2008). Grybos et al. (2010) showed that in the case of NAu-2, proton-promoted dissolution involved the leaching of octahedral Fe from edge surfaces and tetrahedral Fe from basal surfaces. The average percentage of total Fe release from both minerals in our experiments revealed the efficiency of the two distinct bacterial metabolic profiles representative of two soil origins, that is, poor acidic and organic-rich soils.

Conclusion

Hybrid silica gel methodology was a novel way of separating cells from a colloidal mineral source. The ligands diffuse through HSG and dissolve the minerals. In this work, P. agglomerans PA1 and R. aquatilis RA1 showed clearly ligand-promoted dissolution type behavior by producing moderate acidification and efficient ligands that is di-, tri-dentate organic acids, and siderophores. Burkholderia sp. G5, selected from acidic podzolic soils, acidified the medium down to pH < 3.0 by producing large amounts of gluconic acid. The nature and concentration of organic acids produced in our experiments reflected the distinct ecological origin of bacteria, which is rhizosphere or podzolic soils that usually contains from 0.1 to 1000 mM LMWOA. Rhizospheric soils are silicate-rich soils where microorganism metabolites and root exsudates contribute to bioweathering by acidifying, changing redox conditions, or chelating nutrients. HSG permitted to prevent cell attachment and biofilm formation on mineral surfaces and facilitate the quantification of biogeochemical processes. Studying the influence of root exsudate addition on the bioweathering efficiency of rhizospheric bacteria should be relevant in further HSG experiments. Further investigations are also necessary to get details about a size exclusion function for organic ligands. Changing silica precursors nature (i.e., MTMS: methyltrimethoxysilane precursor) should provide variations in silica surface hydrophobicity and ligands partition. Screening different classes of organic acids as a function of size or polarity should be of great interest to delineate the complex bioweathering processes.

Acknowledgements

This study was supported by grants from the Agence Nationale de la Recherche (Program HÆSPRI ANR-09-BLAN-0336), Region Lorraine and Université de Lorraine. All of these are gratefully acknowledged.

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