# MATERIALS AND METHODS

## Materials

**Mineral.** The initial material sampled was raw asbestos ore from sterile heaps in Dobšiná (Slovakia). X-ray diffraction and transmission electronic microscopy analyses shown that principal components of the ore were: Ni-bearing chrysotile and magnetite (iron oxide). Laboratory purification and calibration procedures were performed to obtain pure and homogeneous chrysotile samples. First, the ore was ground to small particles with an automatic grinder. Then, it was suspended in deionized water and magnetite was collected under stirring with a magnetic bar. Finally, the suspension was filtered on a mesh and calibrated between 20 and 40 µm. Total Ni concentration in chrysotile sample after purification, “Ch” was 2260 mg kg⁻¹. Its composition was 43.59 % SiO₂, 39.00 % MgO, 0.20 % NiO, 4.78 % Fe₂O₃, 1.18 % Al₂O₃, 0.12 % Cr₂O₃, 0.02 % Na₂O, 0.02 % K₂O, 0.06 % CaO, 0.02 % TiO₂, 0.03 % MnO, 11.00 % H₂O, and its exact chemical formula was (Mg₂.755 Fe₀.083 Al₀.064 Ni₀.008 Cr₀.005 Ca₀.003 Mn₀.001 K₀.001 Na₀.001 Ti₀.001) Si₂.07 O₅ (OH)₃.5.

**Plant.** Seeds of *Leptoplax emarginata* (Boiss.) O.E. Schulz (*Brassicaceae*) were collected in late July 2002 at Katara Pass (Pindus Mountains near Metzovo) in northern Greece, at an altitude of 1670 m, on serpentine soils with low nutrients (especially Ca) and low summer water contents. *L. emarginata*'s classification, distribution and growth environment have been previously reported (12). This species can contain Ni concentrations in shoots up to 34 000 mg kg⁻¹. Moreover, some specimens in the field can be almost 2 m high at flowering stage.

**Bacteria.** Two treatments with *L. emarginata* were inoculated with two different Ni-resistant bacterial strains previously known to increase Fe mobilization in ultramafic soils of New Caledonia. The first one, “B1”, was isolated from *Trisaniopsis guillainii* mycelium, and the second one, “B2”, was isolated from *Nothofagus codonandra* ectomycorrhizes. The samples were collected in Koniambo mountain in New Caledonia.

**Experimental devices.** Columns were made from sterile inert polypropylene tubes, 7 cm high and 3 cm in diameter, with a hole drilled at the bottom (Figure 1). Columns were filled with both *Ch* (3 g per column) and purified inert quartz, “*Qz*” (50-100 µm and 0.5-1 mm particle sizes). Quartz purification consisted in successive washing in oxalic acid (1.0 g L⁻¹) and HCl (0.1 M), then rinsing with deionised water and finally autoclaved (20 min at 120 °C) for sterilization. *Qz* was used for create an appropriate porosity. Each column was supplied with a
sterile nutrient solution at a constant rate with a peristaltic pump. The composition of the nutrient solution was adjusted so that it only contained elements that were essential to *L. emarginata* growth. Its composition was also representative of the soil solutions usually found in serpentine soils i.e. 2.50 mg L⁻¹ of calcium, 2.00 mg L⁻¹ of sulfur, 1.19 mg L⁻¹ of phosphorus, 1.50 mg L⁻¹ of potassium, 5.00 mg L⁻¹ of nitrogen, 15.00 µg L⁻¹ of zinc, 15.00 µg L⁻¹ of manganese. The mineral was the only source in the system of magnesium, nickel and iron for plant nutrition. Drained solution on leaving the columns was filtered through nylon fabric (1-µm porosity) fixed at the bottom of each column and collected in inert flasks.

<figure><img src="image_93.png" /><figcaption>**Figure 1:** Experimental column scheme.</figcaption></figure>

# Methods

## Culture conditions.

*First experiment.* Seeds of *L. emarginata* were allowed to germinate on pure quartz matrix in a growth chamber under controlled conditions (photoperiod of 18 h; temperature of 22 °C during daylight and 16 °C during night). Matrix moisture was maintained by daily watering with deionized water. After germination, seedlings were transplanted into 18 columns at the rate of one plant per column. Remaining seedlings, used as reference, were washed with deionized water and oven dried at 70 °C for 48 h. Six columns were inoculated with *B1* ("*P+B1*" treatment) and six others columns with *B2* ("*P+B2*" treatment). Bacteria were grown on 10 % TSA medium (3 g L⁻¹ tryptic soy broth from Disco and 15 g L⁻¹ agar) at 25 °C
for 48 h. Then, bacteria were suspended in sterile deionized water and washed three times. Inoculum's concentrations were adjusted to $4 \times 10^{7.6}$ CFU mL$^{-1}$ according to A600 standard curve calibrated by plate enumeration. The experiment was conducted for 15 weeks in a growth chamber under controlled conditions (photoperiod of 16 h; temperature of 22 °C during daylight and 15 °C during night; air moisture 60 %). Control columns without plant and bacteria inoculation were also prepared in the same conditions as the treatments with *L. emarginata*. After 15 weeks, shoots and roots of *L. emarginata* were collected, washed with deionized water and oven dried at 70 °C for 48 h. The mixture *Ch-Qz* was homogenized and oven dried at 50 °C for one week. Bacteria quantification was realized after one week at 28 °C on 10 % TSA medium.

**Second experiment.** This experiment was realized to evaluate the citric acid effect on chrysotile weathering. The experiment was monitored for 30 days in a dark chamber at 20 °C and was conducted in the same way as the first experiment. Three columns, *CA*, were supplied with a sterile solution containing citric acid (20 mg L$^{-1}$) added to the original nutrient solution. Three control columns, *C'*, were supplied with the original solution without citric acid and prepared in the same conditions.

## Chemical analyses

*Mg, Ni contents and pH measurements in the leachates.* The volume of all the drained solutions was measured every week. Then, Mg content was analysed by Induction-Coupled Plasma Atomic Emission Spectrometry (ICP-AES, Liberty RL, Varian). And, Ni content was analysed by Electrothermal Atomic Absorption Spectrometry (ETAAS, Zeeman 220, Varian). pH of all leachates samples was measured with a pH-meter SCHOTT CG842 (Electrod SCHOTT, Blueline 24pH).

*Calculation of Chrysotile dissolution rate.* Dissolution rate of chrysotile was calculated according to the following formula:

$$K_d = Q_{Mg} / t * AS * m$$

Where $K_d$ is the dissolution rate (in mole s$^{-1}$ m$^{-2}$), $Q_{Mg}$ is the total lixivied Mg amounts after experiment, t is the experiment period, AS is the specific area of chrysotile (in m$^2$ g$^{-1}$) and m is the mineral mass in the system.
**Balance-sheet calculation of chrysotile weathering.** Balance-sheet was realized according to Mg and Ni amounts in the leachates and in L. emarginata. After 15 weeks, plant shoots and roots were washed, oven-dried at 70°C for 48 hours, weighed and crushed. Then, samples of plant dry matter were acid-digested at 250 °C in 6 mL of concentrated HNO₃ (purity: 98%) and 6 mL of hydrogene peroxide H₂O₂ (purity: 30 %). The final solution was filtered and made up to 25 mL with deionized water and Mg and Ni contents in plant shoots and roots were measured by ICP-AES. Leachates collected for 15 weeks were mixed and homogenized. Then, Ni and Mg contents of the global solution were measured respectively by ETAAS and ICP-AES.

**Characterization of phytoavailable Ni by Isotopic Exchange Kinetics (IEK) method.**

*Principle.* The isotopic exchange kinetics (IEK) in soil suspensions allow full description of the availability of elements in soil, and the determination of the contribution of the different available pools in the solid phase to buffer the concentration of the element in the soil solution. The method quantifies the three factors that characterize the metal availability in soils (27): the intensity factor -C<sub>Ni</sub>- (i.e. the concentration of Ni in the soil solution, in µg l⁻¹), the quantity factor -E<sub>t</sub>- (i.e. the amount of Ni ions from the soil that are able to supply the soil solution according to their average time of exchange, t, in mg kg⁻¹) and the capacity factor -C<sub>s</sub>- (i.e. the buffering capacity of the soil solid phase compartments to thermodynamically maintain Ni ions in the soil solution, in L kg⁻¹).

*Experiments.* Suspensions of soil and deionized water (1:20 soil:solution ratio (w:v)) were mixed on an end-over-end shaker for 20 h until the concentration of Ni in the solution was constant. One milliliter of a <sup>63</sup>Ni<sup>2+</sup> solution was then injected into the suspension, which was kept under continuous shaking. Four aliquots of the suspension were sampled with a syringe after 1, 4, 10, and 40 min, and immediately filtered through cellulose ester filters (porosity 0.22 µm, Rotilabo®-Spritzenfilter). Radioactivity in the solution was measured in the four filtered aliquots. The concentration of free Ni (C<sub>Ni</sub>) in solution was measured in the filtered solutions (ETAAS) from the suspension after particle deposition (about one day).

*Calculation of E(t).* When radioactive tracers such <sup>63</sup>Ni<sup>2+</sup> are added to a solid phase:solution system at a thermodynamically steady state, the radioactivity in solution decreases with time t according to the following equation:

$$ \frac{r_t}{R_s} = \frac{r_1}{R_s} \times \left[ t + \left( \frac{r_1}{R} \right)^{1/n} \right]^{-n} + \frac{Ni_s}{Ni_t} \quad (1) $$
where r_t is the radioactivity in the soil solution at time t (in Bq) and R_S the total radioactivity introduced at time t=0 (in Bq). The ratio r_t/R_S is the dilution of the total radioactivity added into total Ni in soil in a time t, r_t is the radioactivity in the solution at 1 min, n is a parameter which describes the rate of transfer of the tracer from the solution to the solid phase of the soil after 1 min, Ni_S is the amount of Ni in soil solution for a 1:20 soil solution ratio, Ni_T is the total content of Ni in soil.

The quantity factor (E(t)) can be extrapolated to the experiment duration or any other duration with equation (1). This equation can be derived from equation (2) with the hypothesis that the specific radioactivity of the isotopically exchangeable Ni pool is the same as that of the soil solution over time. Therefore, equation (2) can be expressed:

$$E(t) = \frac{Ni_S \times R_S}{r_t} \qquad (2)$$

*Calculation of Ni average flow.* The average flow (F_m) describes exchanges between the solution and the different Ni exchangeable pools of the mineral.

$$F_m = Q_{Ni} * n / (r1/R)^{(1/n)} \quad (3)$$

Where F_m is the mean flow within each column (in µg Ni min⁻¹) and Q_Ni is the amount of Ni in the solution within each column.

## Statistical analyses.

Results presented in all tables and figures correspond to mean values. Significant statistical differences among values were evaluated by analyses of variance using STATBOXPRO™ program (Grimmer Logiciels Version 6.40, 1997-2002) and Newman-Keuls test at the threshold level of a P value of 0.05. Values with the same letter are not significantly different at p=0.05.

# RESULTS

## Chrysotile weathering characterization.

XRD analyses showed that there was not mineralogical difference between initial chrysotile samples and samples of all treatments collected after the experiment. Moreover, neither precipitations of new mineral phases nor transformation were found by SEM analyses. These results showed that the major process involved in chrysotile weathering certainly corresponds to dissolution.
Impact of *L. emarginata* and contribution of bacteria inoculation on chrysotile dissolution.

- Mg amounts leached from dissolved chrysotile

Mg amounts percolated from dissolved chrysotile ("$Ql_{Mg}$") were measured each week in the leachates for 14 weeks (Figure 2). $Ql_{Mg}$ values permitted to evaluate chrysotile dissolution along the experiment relatively to the different treatments. $Ql_{Mg}$ values measured for all *L. emarginata*'s treatments (i.e. P, P+B1 and P+B2) were 2-fold higher on average than those measured for control treatment without plant, especially for the first 60 days of the experiment. But, bacteria inoculation (i.e. P+B1 and P+B2 treatments) had no significant effect on $Ql_{Mg}$ values compared to P treatment without inoculation.

<figure><img src="image_94.png" /><figcaption>**Figure 2:** Quantities of leached Mg along of the experiment.</figcaption></figure>

$Ql_{Mg}$ measured for the control treatment C, appeared rather stable along the experiment, corresponding to a mean value of 0.4 mg per week. All plant treatments had the same behaviour: firstly, $Ql_{Mg}$ values were rather high (about 1.0 mg of Mg on average), then, after the 64th day, $Ql_{Mg}$ values decreased and stabilized to 0.9 mg.

- Characterization of the Ni availability by IEK

IEK technique permitted to characterize the different isotopically exchangeable pools of Ni in the mixture *Ch-Qz* (Table 1). Results showed that Ni concentration in solution was very weak, ranging from 3 to 5 µg kg⁻¹ for *C* and *P+B2*, respectively. Moreover, Ni exchangeable between 0 and 15 weeks (i.e. *E*_{(0-1min)} added to *E*_{(1min-10min)} and *E*_{(10min-15weeks)}) represented only
11 % of the total Ni content of the system. Consequently, the major part of Ni in the system was not exchangeable during the experiment period. $C_{Ni}$ and $E_{(0-1min)}$ values in the system Ch-Qz were significantly higher before experiment compared to the control treatment. On the contrary, P and P+B2 treatments significantly increased $E_{(0-1min)}$ values compared to the control without plant. But these treatments showed no significant difference with values measured before experiment. P+B1 treatment did not present significant difference compared to both *before experiment* measurements and C treatments, except for $E_{(10min-15weeks)}$. The total Ni content of the system was 2 % lower on average in all the treatments with *L. emarginata* compared to C. But, bacteria inoculation had no significant effect on the Ni availability compared to P treatment without inoculation.

<table><caption>**Table 1:** Parameters from IEK before and after 15 weeks of experiment (C, P, P+B1 and P+B2 treatments).</caption><thead><tr><th></th><th>CNiH2O</th><th>E(0-1min)</th><th>E(1min-10min)</th><th>E(10min-15 weeks)</th><th>E(&gt;15 weeks)</th><th>Total Ni</th></tr><tr><th></th><th>µg L<sup>-1</sup></th><th colspan="4">mg Kg<sup>-1</sup></th><th></th></tr></thead><tbody><tr><td>Before experiment</td><td>4.5 a</td><td>0.18 ab</td><td>0.23 ab</td><td>13.26 ab</td><td>114.33 n.s.</td><td>128.00 a</td></tr><tr><td>C</td><td>3.0 b</td><td>0.13 c</td><td>0.20 ab</td><td>19.65 a</td><td>107.91 n.s.</td><td>127.89 a</td></tr><tr><td>P</td><td>4.1 ab</td><td>0.19 ab</td><td>0.23 ab</td><td>13.76 ab</td><td>111.53 n.s.</td><td>125.71 b</td></tr><tr><td>P+B1</td><td>3.9 ab</td><td>0.16 bc</td><td>0.18 b</td><td>9.98 b</td><td>114.73 n.s.</td><td>125.04 b</td></tr><tr><td>P+B2</td><td>5.0 a</td><td>0.22 a</td><td>0.26 a</td><td>13.36 ab</td><td>112.13 n.s.</td><td>125.97 b</td></tr></tbody></table>

Ni mean flow (Table 2) calculation permitted to quantify Ni movements between the solid phase and the solution in the system Ch-Qz. Ni mean flows was very weak, i.e. about 10 µg min⁻¹ in each column. *L. emarginata* had no significant effect on Ni exchanges between solid phase and solution compared to both treatments *before experiment* and without plant, except for P+B2 where $F_m$ increased significantly compared to C.

<table><caption>**Table 2:** Ni means flows calculated from IEK parameters.</caption><thead><tr><th rowspan="2">Treatments</th><th colspan="2">F<sub>m</sub> in each column</th></tr><tr><th>µg min<sup>-1</sup></th><th></th></tr></thead><tbody><tr><td>Before culture</td><td>10.52 ab</td><td></td></tr><tr><td>C</td><td>6.15 b</td><td></td></tr><tr><td>P</td><td>12.55 ab</td><td></td></tr><tr><td>P+B1</td><td>9.32 b</td><td></td></tr><tr><td>P+B2</td><td>17.59 a</td><td></td></tr></tbody></table>
- Mass balance of Mg and Ni from chrysotile dissolution after 15 weeks

After 15 weeks, a balance-sheet of chrysotile dissolution was realized according to Mg and Ni amounts leached and absorbed by *L. emarginata*, i.e. total Mg and Ni quantities ($Q_{Mg}$ and $Q_{Ni}$) released from dissolved chrysotile (Figure 3). Results showed that $Q_{Mg}$ and $Q_{Ni}$ were significantly higher for all treatments with *L. emarginata* compared to control without plant. Effectively, the amounts of elements released from chrysotile dissolution were more than 2-fold higher with *L. emarginata* for Mg and more than 7-fold for Ni. Bacteria inoculation had no significant effect on chrysotile dissolution. Ni partitioning between the leachates and the plant was different for Mg and Ni. 91% on average of $Q_{Mg}$ for the three plant treatments were percolated compared to 12% for $Q_{Ni}$. Thus, Ni was preferentially absorbed by *L. emarginata*. The plant absorbed up to 2% of the initial total Ni content of the system.

**Figure 3:** Mass balance of total Mg (A) and Ni (B) released from dissolved chrysotile after 15 weeks of experiment with or without *L. emarginata* and inoculated bacteria.

<figure><img src="image_95.png" /></figure>

## Citric acid effect on chrysotile dissolution

- Mass balance of Mg and Ni from chrysotile dissolution after 30 days

At the end of the experiment, the balance sheet of chrysotile dissolution was realized according to the total Mg and Ni amounts leached ($Q_{Mg}$ and $Q_{Ni}$) in the solution on leaving the
columns (Figure 4). Q<sub>Mg</sub> and Q<sub>Ni</sub> were significantly higher with citric acid compared to control treatment C' without citric acid. After 30 days, 1.76 mg of Mg and 1.18 µg of Ni were released from dissolved chrysotile for C' compared to respectively 2.97 mg and 1.87 µg for CA. Q<sub>Mg</sub> released with citric acid represented 72 % of the Q<sub>Mg</sub> released after 28 days with L. emarginata (P treatment).

**Figure 4:** Quantities of Mg and Ni released from dissolved chrysotile after 30 days with citric acid (AC) or without (C'). Comparison with Mg values (C and P treatments) measured from the first experiment after 28 days.

<figure><img src="image_96.png" /></figure>

## Chrysotile dissolution rates

Chrysotile dissolution rates ("K<sub>d</sub>") were calculated from the total quantities of Mg released from chrysotile dissolution after the two experimental periods (Table 3). Control treatments of the 2 experiments have similar values, that is to say 2.5.10<sup>-13</sup> and 2.6.10<sup>-13</sup> mol s<sup>-1</sup>m<sup>-2</sup> respectively for C and C'. So, K<sub>d</sub> values estimation appeared as coherent values. That is why the comparison between treatments with L. emarginata and citric acid was possible. K<sub>d</sub> values were about 2-fold higher with L. emarginata and citric acid compared to control treatments. Bacteria inoculation had no significant effect on chrysotile dissolution rates compared to P treatment.

<table><caption>**Table 3:** Chrysotile dissolution rates (K<sub>d</sub>).</caption><thead><tr><th></th><th>K<sub>d</sub> mol s<sup>-1</sup> m<sup>-2</sup></th><th></th></tr></thead><tbody><tr><td>C</td><td>2.5E-13</td><td>b</td></tr><tr><td>P</td><td>5.7E-13</td><td>a</td></tr><tr><td>P+B1</td><td>6.2E-13</td><td>a</td></tr><tr><td>P+B2</td><td>5.8E-13</td><td>a</td></tr><tr><td>C'</td><td>2.6E-13</td><td>b'</td></tr><tr><td>AC</td><td>4.4E-13</td><td>a'</td></tr></tbody></table>
## Chrysotile dissolution stoichiometry

The analyses of the relations between total Mg and Ni amounts released from chrysotile dissolution for all treatments of the two experiments showed a highly significant positive correlation (R²=0.73) (Figure 5). Theoretical amounts of total Ni released were calculated for each amount of Mg measured, according to the exact chrysotile formula ((Mg₂,755Fe₀,083Al₀,064Ni₀,008Cr₀,005Ca₀,03) Si₂,066O₅,₀₀₀(OH)₃,475. Regression line calculated from measured points corresponds to the theoretical points (i.e. y = 0.007x). Thus, the total amounts of Mg (QMg) released from Chrysotile dissolution were about 140-fold higher than those of released Ni. QNi measured for controls C and C' and for CA treatments were lower compared to theoretical values. On the contrary, QNi measured for P+B1 treatment were higher than theoretical values.

**Figure 5:** Chrysotile dissolution stoichiometry. Theoretical values calculated from both chrysotile formula and measured QMg values (◊). Regression line (y = 0.007x) corresponds to measured Ni values partitioning as following: Control (■), P (X), P+B1 (*) and P+B2 (●).

<figure><img src="image_97.png" /></figure>

## DISCUSSION

XRD and SEM analyses did not give evidence of new mineral phase formation from the system Ch-Qz. Thus, the major process involved in this context was certainly dissolution reactions. *L. emarginata* increased more than 2-fold chrysotile dissolution, and about 7-fold Ni availability. But the inoculation of the strains B1 and B2, in this context, had no significant effect on mineral dissolution. These results may be due either to the unadapted
environmental conditions of the experiment for B1 and B2 strains (e.g. Alkaline pH, lack of soil organic matter), or to a competition between inoculated bacteria and the original microflora associated with *L. emarginata* which was observed in the three plant treatments. Indeed, bacteria counting in the uninoculated P treatment and in the two inoculated P+B1 and P+B2 treatments at the end of the experiment were not significantly different (data not shown). Control treatment was the only one showing significant lower values. Moreover, the strains observed in inoculated treatments appeared as the same as the original strains found in the natural microflora of *L. emarginata*'s rhizosphere (P treatment). The different strains associated with *L. emarginata* might possibly play an active part in chrysotile dissolution.

The quantities of Mg leached showed that, for the first weeks, chrysotile dissolution rate was significantly higher with *L. emarginata* compared to control without plant. Chrysotile dissolution rate was rather constant for all treatments for the first 64 days. After this period, chrysotile dissolution rate decreased and stabilized towards lower values, except for control treatment for which it was constant all along the experiment. The deficit of leached Mg during the last six weeks corresponded approximately to Mg uptake by *L. emarginata* at the end of the experiment. Most of the Mg uptake by plants might have occurred during this period.

The comparison of Ni availability before and after experiment by IEK techniques reinforced the hypothesis of a major dissolution in chrysotile weathering. In the control treatment C without plant, $C_{Ni}$ in solution and the labile pool $E_{(0-1min)}$ significantly decreased compared to treatment before experiment. The quantity of Ni leached after 15 weeks was 0.04 mg kg⁻¹ for C. This value approximately corresponded to the difference between $E_{(0-1min)}$ values of before experiment and control treatments after the experiment *i.e.* 0.05 mg kg⁻¹. Despite a high Ni uptake by *L. emarginata* (156 µg of Ni on average), Ni partitioning between the solution and the different exchangeable pools of the system was the same as before experiment. These results showed that the solution was not buffered by the most exchangeable pools of the system but Ni in solution was rather directly supplied from dissolved chrysotile, expressed by an apparent equilibrium of the system. Ni mean flows between solution and Ni exchangeable pools were very weak about 10 µg L⁻¹. So, to absorb sufficiently high Ni quantities, *L. emarginata* would mobilize Ni inducing chrysotile dissolution. Citric acid is an organic compound released in the rhizosphere of some hyperaccumulators to mobilize Ni and facilitate its transfer towards roots when there is a lack of bioavailable Ni (14). Citric acid increased chrysotile dissolution. After 30 days, Mg and Ni amounts released by dissolved mineral were about 1.5-fold higher with citric acid compared to control treatment without citric acid. Citric acid exudation by *L. emarginata* could be one
of the strategies used by hyperaccumulators species to mobilize Ni. Chrysotile dissolution
rates were estimated for all treatments of the two experiments. Chrysotile dissolution was
significantly faster with *L. emarginata* or with citric acid compared to control treatments
without both plant and citric acid addition. The comparison between the two experiments,
according to the total amounts of Mg released from dissolved chrysotile ($Q_{Mg}$), showed that
control treatments C and C' had similar values i.e. 2.00 mg and 1.76 mg of Mg, respectively.
But, $Q_{Mg}$ value obtained for the treatment P with *L. emarginata* after 28 days (i.e. 4.15 mg of
Mg) was higher than those obtained for AC treatment after 30 days (i.e. 2.97 mg of Mg). The
concentration of citric acid artificially provided in AC treatment, i.e. about 100 µM, was
maybe lower than the actual citric acid concentration in the rhizosphere of *L. emarginata*.

The analyses of the relations between total Mg and Ni amounts released from chrysotile dissolution ($Q_{Mg}$ and $Q_{Ni}$) for all treatments of the two experiments showed that chrysotile dissolution was stoichiometrical. Regression line calculated from measured points corresponds to the theoretical points calculated according to the precise chrysotile formula. However, when dissolution was weak (C treatment) and/or when experiment period was short (C' and AC treatments), $Q_{Ni}$ measured were lower than theoretical values according to $Q_{Mg}$ values. Preferential and fast dissolution of Ni-free external layers of Mg hydroxides would explain the massive Mg release compared to Ni release at this stage. When the dissolution was intense, $Q_{Ni}$ measured were higher than theoretical values according to $Q_{Mg}$ values (P+B1 treatment). Now, Ni was rapidly taken up by *L. emarginata* in unlimited quantities i.e. the highest the bioavailable Ni in the system, the highest the Ni uptake by the plant. In return, Mg uptake by *L. emarginata* was limited. Thus, precipitation of Mg layers around the roots could appear and so artificially decrease the expected amount of Mg leached. Roots had been washed prior to mineralization and a part of this Mg adhering to roots may have been discarded during this step. Moreover, these results showed that the unlimited Ni uptake by the hyperaccumulator species in addition to organic acid exudation might certainly activate chrysotile dissolution.

*L. emarginata* was able to extract from the system up to 2 % of the total Ni whereas chrysotile was a low-availability Ni source. Organic acids released by plant roots were certainly involved in Ni mobilization. And we suppose that the original microflora from the rhizosphere of *L. emarginata* played a role as well. That is why, it would be interesting to isolate and test rhizobacteria associated with *L. emarginata* in order to know if their stimulation could increase Ni bioavailability in soils and so Ni uptake by hyperaccumulators improving phytomining processes.
ACKNOWLEDGMENTS

We thank the French Ministry of National Education, Reasearch and Technology and
Continental Ecosphere national program (Ecotoxicology and Ecodynamics of contaminants)
for financial support for this research.

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(17) Massoura, S.T. ; Echevarria, G. ; Becquer, T. ; Ghanbaja, J. ; Leclerc-Cessac, E. ; Morel, J.L. *Geoderma*. 2006, in press, doi:10.1016/j.geoderma.2006.01.008.

(18) Shallari, S.; Echevarria, G.; Schwartz, C.; Morel, J.L. *S. Afr. J. Sci.*, 2001, 97, 568-570.

(19) Jones, D.L.; Darah, P.R.; Kochian, L.V. *Plant and Soil*.1996, 180 (1), 57 - 66

(20) Welch, S.A.; Ullman, W.J. *Geochim. Cosmochim. Acta*. 1993, 57, 2725-2736.

(21) Lee, J.; Reeves, R.D.; Brooks, R.R.; Jaffré, T. *Phytochemistry*. 1977, 16(10), 1503-1505.

(22) Yang, X. E.; Baligar, V. C.; Foster, J. G.; Martens, D. C. *Plant and Soil*. 1997, 196(2), 271-276.

(23) Calvaruso, C.; Turpault, M.P.; Frey-Klett, P. *Appl. Environ. Microbiol*. 2006, 72, 1258-1266.

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(26) Abou-Shanab, R. A.; Angle, J. S.; Delorme, T. A.; Chaney, R. L.; Van Berkum, P.; Moawad, H.; Ghanem, K.; Ghozlan, H. A. *New Phytol*. 2003, 158, 219-224.

(27) Echevarria, G.; Morel, J.L.; Fardeau, J.C.; Leclerc-Cessac, E. *Soils J. Environ. Quality*. 1998, 27, 1064-1070.
Annexe 10 : Propriétés physico-chimiques des sols de Bure, Fougères et Breuil.

Sol de Bure

<table><thead><tr><th rowspan="2">Prof. (cm)</th><th rowspan="2">pH eau</th><th colspan="5">Granulométrie %</th><th rowspan="2">C %oo</th><th rowspan="2">N %oo</th><th rowspan="2">C/N</th><th colspan="4">Cations échangeables cmol<sub>c</sub>+/kg</th><th rowspan="2">CEC cmol<sub>c</sub> /kg</th><th rowspan="2">S/T %</th></tr><tr><th>A</th><th>LF</th><th>LG</th><th>SF</th><th>SG</th><th>Ca</th><th>Mg</th><th>K</th><th>Na</th></tr></thead><tbody><tr><td>0-20</td><td>8.2</td><td>38.6</td><td>37.8</td><td>12.9</td><td>2.8</td><td>7.9</td><td>19.5</td><td>1.95</td><td>10.0</td><td>42.3</td><td>0.67</td><td>0.72</td><td>0.04</td><td>10.0</td><td>Sat.</td></tr></tbody></table>

Sol de Breuil

<table><thead><tr><th rowspan="2">Prof. (cm)</th><th rowspan="2">pH eau</th><th colspan="5">Granulométrie %</th><th rowspan="2">C %oo</th><th rowspan="2">N %oo</th><th rowspan="2">C/N</th><th colspan="4">Cations échangeables</th><th rowspan="2">CEC cmol<sub>c</sub> /kg</th><th rowspan="2">S/T</th></tr><tr><th>A</th><th>LF</th><th>LG</th><th>SF</th><th>SG</th><th>Ca</th><th>Mg</th><th>K</th><th>Na</th></tr></thead><tbody><tr><td>0-8</td><td>4.3</td><td>15.9</td><td>10</td><td>5</td><td>5</td><td>47</td><td>7.9</td><td>nd</td><td>nd</td><td>0</td><td>0.5</td><td>0.3</td><td>nd</td><td>12.2</td><td>7.7</td></tr><tr><td>10-18</td><td>4.6</td><td>13.2</td><td>19</td><td>7</td><td>8</td><td>51</td><td>1.2</td><td>nd</td><td>nd</td><td>0</td><td>0.3</td><td>0.2</td><td>nd</td><td>10.5</td><td>5</td></tr><tr><td>20-28</td><td>4.7</td><td>14.5</td><td>18</td><td>6</td><td>7</td><td>52</td><td>0.9</td><td>nd</td><td>nd</td><td>0</td><td>0.2</td><td>0.1</td><td>nd</td><td>8.9</td><td>3.5</td></tr></tbody></table>

Sol de Fougères

<table><thead><tr><th rowspan="2">Prof. (cm)</th><th rowspan="2">pH eau</th><th colspan="5">Granulométrie %</th><th rowspan="2">C %oo</th><th rowspan="2">N %oo</th><th rowspan="2">C/N</th><th colspan="4">Cations échangeables</th><th rowspan="2">CEC cmol<sub>c</sub> /kg</th><th rowspan="2">S/T</th></tr><tr><th>A</th><th>LF</th><th>LG</th><th>SF</th><th>SG</th><th>Ca</th><th>Mg</th><th>K</th><th>Na</th></tr></thead><tbody><tr><td>0-8</td><td>3.6</td><td>15.8</td><td>23.9</td><td>49.1</td><td>10.6</td><td>6</td><td>7.6</td><td>0.4</td><td>19.6</td><td>0.6</td><td>0.4</td><td>0.3</td><td>0.1</td><td>8.3</td><td>16.9</td></tr><tr><td>8-16</td><td>4.0</td><td>13.1</td><td>24.4</td><td>50.7</td><td>11.1</td><td>7</td><td>2.2</td><td>1.0</td><td>22.0</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>5.5</td><td>6.3</td></tr><tr><td>16-35</td><td>4.3</td><td>13.5</td><td>23.9</td><td>50.5</td><td>11.2</td><td>9</td><td>1.5</td><td>0.7</td><td>19.6</td><td>0.1</td><td>0</td><td>0.1</td><td>0.1</td><td>3.7</td><td>6.2</td></tr></tbody></table>

A : argiles, LF : limons fins, LG : limons grossiers, SF : sables fins, SG : sables grossiers.
# Annexe 11: Liste des publications, rapports et communications issus de ce travail de thèse.

## Publications

### • Articles publiés :

Root-associated bacteria contribute to mineral weathering and to mineral nutrition in trees : a budgeting analysis.

Calvaruso Christophe, Turpault Marie-Pierre et Frey-Klett Pascale.

Applied and Environmental Microbiology 72, 2, pp. 1258-1266.

Altération microbienne des minéraux.

Uroz Stéphane, Calvaruso Christophe, Turpault Marie-Pierre et Frey-Klett Pascale.

Biofutur 268, pp. 37-41.

### • Articles soumis :

Impact of ectomycorrhizosphere on the functional diversity of soil bacterial and fungal communities from a forest stand in relation to nutrient mobilization process.

Calvaruso Christophe, Turpault Marie-Pierre, Leclerc-Cessac Elisabeth and Frey-Klett Pascale.

Microbial Ecology, le 11 septembre 2006.

Mycorrhizosphere effect on the genotypic and metabolic diversity of the soil bacterial communities involved in mineral weathering in a forest soil.

Uroz Stéphane, Calvaruso Christophe, Turpault Marie-Pierre, Pierrat Jean-Claude, Mustin Christian et Frey-Klett Pascale.

Applied and Environmental Microbiology, le 02 octobre 2006.

### • Articles en préparation :

Rapid clay weathering in the rhizosphere of different tree species in an acid forest ecosystem.

Calvaruso Christophe, Mareschal Louis, Leclerc Elisabeth et Turpault Marie-Pierre.
Forest trees select high mineral weathering bacteria to balance low nutrient availability.

Calvaruso Christophe, Turpault Marie-Pierre, Uroz Stéphane, Leclerc Elisabeth, Ranger
Jacques, Garbaye Jean et Frey-Klett Pascale.

Alteration of chrysotile in the rhizosphere of the nickel hyperaccumulator *Leptoplax emarginata*.

Chardot Vanessa, Echevarria Guillaume, Calvaruso Christophe, Turpault Marie-Pierre et Jean-Louis Morel.

Biological, environmental and anthropogenic effects on the test-mineral dissolution in a forest ecosystem.

Turpault Marie-Pierre, Nys Claude et Calvaruso Christophe.

Communications courtes dans congrès, symposiums

TURPAULT M-P, CALVARUSO C., NOURRISSON G. (2004) Rhizosphere in forest ecosystems : a favorable interface for tree nutrition. Rhizosphere12-17 september 2004, Munich, Germany [résumé, poster].

CALVARUSO C., TURPAULT M-P, FREY-KLETT P. (2004) Effect of plants and soil bacteria on the mineral weathering. Rhizosphere12-17 september 2004, Munich, Germany [résumé, communication orale].

TURPAULT M.-P., CALVARUSO C., E. LECLERC-CESSAC, NOURRISSON G., RENAUD J.-P. AND NDIRA V. (2004) Effect of forest tree species on rhizospheric soil. Eurosoil 2004 4-12 september, Freiburg Germany [résumé, poster].

CALVARUSO C., TURPAULT M.-P., FREY-KLETT P. (2004) The effect of plants and soil bacteria on the mineral weathering. Eurosoil 2004 4-12 september, Freiburg Germany [communication orale].
CALVARUSO C., TURPAULT M.P., FREY-KLETT P. (2004) The effect of plant and soil bacteria on mineral weathering. VIèmes Journées d'Ecologie Fonctionnelle, Rennes, 10-12 mars 2004 [poster].

CALVARUSO C, TURPAULT M.P., MUSTIN C., AMELLAL N, FREY-KLETT P (2004) Structuration fonctionnelle des communautés bactériennes dans l'ectomycorhizosphère du chêne, en relation avec les processus d'altération des minéraux des sols. VIèmes Journées d'Ecologie Fonctionnelle, Rennes, 10-12 mars 2004 [résumé, poster].

CALVARUSO C., FREY-KLETT P., TURPAULT M.P. & MUSTIN C. (2003) Structuration of bacterial communities in the mycorrhizosphere of oak, in relation to mineral weathering processes. 2nd Joint Meeting of COST 631 Management Committee and Working Groups. Montpellier du 24-27 october 2003 [résumé, poster].

TURPAULT M.-P., FREY-KLET P., MUSTIN C., CALVARUSO C. & RAFIE O. (2003) Weathering rates in forest soils : the influence of plant roots and micro-organisms. 16th International Symposium on Environmental Biogeochemistry. Oirase 1-7 september. [résumé étendu, conférence invitée].

TURPAULT M.-P., BAKKER M., BOUDOT J.-P., CALVARUSO C., CHAUSSIDON M., COLIN-BELGRAND M., DAMBRINE E., FREY-KLETT P., GARBAYE J., GERARD F., HUMBERT B., LAPEYRIE F., LEYVAL C., LUAIS B., MUSTIN C., NOURRISSON G., PIERRAT J.-C. & WALCARIUS A. (2003) Quantification de l'altération biologique des minéraux du sol. Etude des interfaces plantes-microorganismes-solutions-minéraux-phases gazeuses dans des écosystèmes forestiers et en conditions contrôlées. Vèmes Journées d'Ecologie Fonctionnelle, Nancy, 12-14 mars 2003. [résumé, poster].

FREY-KLETT P., CALVARUSO C., SATRANI B., RAY P., AMELLAL N., LAPEYRIE F., GARBAYE J. & TURPAULT M.-P. (2003). Les bactéries du sol jouent-elles un rôle dans l'altération des minéraux? Vèmes Journées d'Ecologie Fonctionnelle, Nancy, 12-14 mars 2003. [résumé, poster].

## Autres supports

TURPAULT M.-P., FREY-KLET P., MUSTIN C., CALVARUSO C. & RAFIE O. (2003) Weathering rates in forest soils : the influence of plant roots and micro-
organisms. In Biochemical aspect of earth system and bioremediation of polluted environments. Ed. T. Hattori, ISEB16, Sendai, Japan.

TURPAULT M.-P., BOUDOT J.P., CALVARUSO C., CHAUSSIDON M., COLIN-BELGRAND M., FREY-KLETT P., MUSTIN C., WALCARIUS A. (2004) Quantification de l'altération biologique des minéraux du sol. Rapport Final ACI Ecologie quantitative-Contrat 1317A, 109p.

ECU UHP NANCY 1
Institut des Sciences
Rue de la Ferté-Bel-Artoise - CS 20148
54601 VILLERS LES NANCY CEDEX
Monsieur CALVARUSO Christophe

DOCTORAT de l'UNIVERSITE HENRI POINCARE, NANCY 1

en GEOSCIENCES

VU, APPROUVÉ ET PERMIS D'IMPRIMER

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Nancy, le

<figure><img src="image_99.png" /></figure>

Le Président de l'Université

<figure><img src="image_100.png" /></figure>
Résumé

L'importance du processus d'altération des minéraux pour la nutrition des arbres en sols forestiers acides est aujourd'hui admise. En revanche, la contribution du facteur biologique dans ce processus reste inconnue. Dans ce contexte, en associant des études et des expérimentations en forêt à des expériences de laboratoire, notre travail a eu pour objectif de quantifier l'effet de la rhizosphère d'arbres forestiers (racines et microorganismes associés) sur l'altération des minéraux du sol.

Nous avons mis en évidence une augmentation de l'altération des minéraux (dissolution et transformation) dans la rhizosphère des arbres en forêt, suggérant un rôle important de ce compartiment dans la dynamique des éléments minéraux et dans l'évolution des sols. Grâce à la caractérisation *in vitro* du potentiel d'altération de souches microbiennes isolées de sol forestier global et d'ectomycorhizes, nous avons pu montrer une structuration fonctionnelle des communautés bactériennes cultivables, qui suggère une sélection exercée par la symbiose ectomycorhizienne en faveur des souches améliorant la nutrition minérale. Par ailleurs, grâce à un dispositif de culture en colonne que nous avons développé, nous avons démontré lors de différentes interactions plante - bactéries - champignons que racines et bactéries augmentent de manière significative la mobilisation de certains cations (Mg, K, Fe, Ni, Cs), *via* l'altération d'un minéral test. Ce processus biologique est affecté en présence d'éléments toxiques comme l'uranium.

Au vu de l'ensemble de nos résultats, notre travail permet de proposer l'hypothèse que, dans les sols forestiers, la disponibilité en nutriments contrôlerait l'altération biologique par son impact sur l'activité des racines et des microorganismes associés. Ceci suggère une forte plasticité des écosystèmes forestiers dans le domaine de la nutrition, qui pourrait expliquer en partie la durabilité de ces écosystèmes.

Mots-clés : altération des minéraux, bactéries, champignons, études et expérimentations en forêt, expérimentations en laboratoire, racines, rhizosphère.

Summary

The impact of the mineral weathering process on tree nutrition in acidic forest soils is well known. However, the quantitative effect of different biological factors on this process remains to be clarified. In this context, the aim of the present work was to quantify the impact of the rhizosphere of forest trees (roots and microorganisms) on the weathering of soil minerals.

In forest, we observed an increase of the weathering of minerals (dissolution and transformation) in the rhizosphere of trees, suggesting an important impact of the tree rhizosphere on the dynamics of minerals as well as on soil evolution. We characterized *in vitro* the weathering potential of microorganisms which were isolated in forest from bulk soil and ectomycorrhizas. We showed that the ectomycorrhizal symbiosis significantly structures the functional diversity of culturable bacterial communities and selects strains potentially beneficial to tree nutrition. We developed a column experiment to quantify the respective impact of plant, bacteria and fungi on the weathering process. We demonstrated that tree roots and bacteria significantly increase Mg, K, Fe, Ni, Cs mobilisation *via* the weathering of a reference mineral. This process is altered in the presence of toxic elements such as uranium.

Considering the whole of our results, we can propose the following hypothesis: in forest soils, the nutrient bioavailability determines the mineral weathering due to its impact on the root and microorganism activity. This suggests an important plasticity of forest ecosystems towards nutrition, which could partly explain the sustainability of these ecosystems.

Key-words: bacteria, experiment in laboratory, fungi, mineral weathering, observations in forest, roots, rhizosphere.