Available online at www.sciencedirect.com
Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
www.elsevier.com/locate/gca
Tree-mycorrhiza symbiosis accelerate mineral
weathering: Evidences from nanometer-scale elemental fluxes
at the hypha–mineral interface
Steeve Bonneville a,⇑, Daniel J. Morgan a, Achim Schmalenberger c,1, Andrew Bray a,
Andrew Brown b, Steven A. Banwart c, Liane G. Benning a
a
Earth Surface Science Institute, School of Earth and Environment, University of Leeds, Leeds LS2 9JT, United Kingdom
Leeds Electron Microscopy and Spectroscopy Centre, Institute for Materials Research, SPEME, University of Leeds, Leeds LS2 9JT,
United Kingdom
c
Kroto Research Institute, Department of Civil and Structural Engineering, University of Sheffield, Sheffield S3 7HQ, United Kingdom
b
Received 27 April 2011; accepted in revised form 29 August 2011
Abstract
In soils, mycorrhiza (microscopic fungal hypha) living in symbiosis with plant roots are the biological interface by which
plants obtain, from rocks and organic matter, the nutrients necessary for their growth and maintenance. Despite their central
role in soils, the mechanism and kinetics of mineral alteration by mycorrhiza are poorly constrained quantitatively. Here, we
report in situ quantification of weathering rates from a mineral substrate, (0 0 1) basal plane of biotite, by a surface-bound
hypha of Paxillus involutus, grown in association with the root system of a Scots pine, Pinus sylvestris. Four thin-sections were
extracted by focused ion beam (FIB) milling along a single hypha grown over the biotite surface. Depth-profile of Si, O, K,
Mg, Fe and Al concentrations were performed at the hypha–biotite interface by scanning transmission electron microscopyenergy dispersive X-ray spectroscopy (STEM-EDX). Large removals of K (50–65%), Mg (55–75%), Fe (80–85%) and Al
(75–85%) were observed in the topmost 40 nm of biotite underneath the hypha while Si and O are preserved throughout
the depth-profile. A quantitative model of alteration at the hypha-scale was developed based on solid-state diffusion fluxes
of elements into the hypha and the break-down/mineralogical re-arrangement of biotite. A strong acidification was also
observed with hypha bound to the biotite surface reaching pH < 4.6. When consistently compared with the abiotic biotite dissolution, we conclude that the surface-bound mycorrhiza accelerate the biotite alteration kinetics between pH 3.5 and 5.8 to
0.04 lmol biotite m2 h1. Our current work reaffirms that fungal mineral alteration is a process that combines our previously documented bio-mechanical forcing with the lm-scale acidification mediated by surface-bound hypha and a subsequent
chemical element removal due to the fungal action. As such, our study presents a first kinetic framework for mycorrhizal alteration at the hypha-scale under close-to-natural experimental conditions.
Ó 2011 Elsevier Ltd. All rights reserved.
1. INTRODUCTION
⇑ Corresponding author. Present address: Unité de « Biogéochi-
mie et Modelisation du Systeme Terre », Département des Sciences
de la Terre (DSTE), Université Libre de Bruxelles (ULB), 50 Av. F.
D. Roosevelt, B-1050 Brussels, Belgium. Tel.: +32 26502204.
E-mail address: steeve.bonneville@ulb.ac.be (S. Bonneville).
1
Present address: Department of Life Sciences, University of
Limerick, Schrodinger Building, Limerick, Ireland.
0016-7037/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.gca.2011.08.041
Rock weathering of continental surfaces not only results
in soil formation, upon which the whole of the biosphere
depends, but also controls the long term chemical composition of groundwaters, rivers, lakes and oceans (Godderis
et al., 2009). Over geological time, the weathering of rocks
and, in particular of Mg- and Ca-silicates, exerts a major
control on the level of atmospheric CO2 and thus on
Tree-mycorrhiza symbiosis accelerate mineral weathering
Earth’s climate (Berner, 1995), through the formation of
carbonate-rich minerals sequestering carbon. Of particular
relevance, is the biological component of the mineral
weathering. The evolutionary development of plants during
the Phanerozoic coincides with large drops in atmospheric
CO2 levels (Crane et al., 1995; Kendrick and Crane, 1997;
Berner and Kothavala, 2001; Berner, 2003). The concept
underpinning these observations is that the emergence of
plants with root systems on land increased terrestrial
weathering significantly and enhanced carbon sequestration
rates in marine system (Berner, 1992; Derry, 2006; Kennedy
et al., 2006). This assertion has been strengthened by the
work of Moulton et al. (2000). In two catchment basins
of similar lithology and climate but differing in vegetation
cover (tree-vegetated vs. bare or partially covered by lichens
and mosses), trees accelerate four-folds the weathering rate
release of Ca and Mg into streams. Other calculations from
the same study indicate that plagioclase and pyroxene
weather 2 and 10 faster respectively in the tree-covered
catchment. Tree-roots and the way they interact with rocks
in soil are crucial to the weathering enhancement.
However, roots are far from being bare of life. The rhizophere indeed host a large biomass and diversity of soil
microorganisms (Kent and Triplett, 2002), in particular of
fungi in symbiosis with roots. Mycorrhiza is the absorptive
interface by which plants acquire nutrients (Landeweert et
al., 2001). These mycorrhizal fungi forms vast networks
of microscopic filaments (hypha – up to 1 mm long for
1–10 lm wide) that represent cumulative lengths of, on
average, 100 km of filament per kg of soil with local upper
limits of cumulative length reaching 600 km (Whitfield,
2007). Mycorrhiza receive an energy supply from their host
plant equivalent to 20–30% of the carbon fixed during photosynthesis (Ek, 1997; Hobbie and Wallander, 2006). In return, they supply a large array of elements (i.e., Mg, Fe, etc)
and nutrients (e.g., K and P) to the plant roots (Hogsberg
and Hogsberg, 2002; Smits et al., 2008). Mycorrhiza are
present at the global scale on Earth’s surface: recent surveys
found that 92% of present-day plant families, and 80% of
all known plant species are mycorrhizal (Bonfante, 2003;
Wang and Qiu, 2006). Given their ubiquitous character in
soils, their vast biomass and thus their massive surface area
available to interact with rocks, mycorrhiza are key agents
of biotic rock weathering. Yet, the mechanisms and especially the kinetics of fungal weathering, are not well
understood.
Mycorrhizal fungi can adopt various strategies to alter
minerals and gain access to nutrients. A much-emphasized
alteration pathway of mycorrhiza is the modification of key
chemical parameters such as the pH and redox status of the
soil solution (Gadd, 2007 and references therein). The production and excretion of low molecular weight organic
compounds into soil solutions acting as ligands and/or siderophores that enhance the dissolution of minerals directly
are well established (Adeyemi and Gadd, 2005; Ehrlich,
1998; Leyval et al., 1993). This purely “aqueous” viewpoint
overlooks the fact that in most soils and also in experiments, the vast majority of soil microorganisms: bacteria
(Bonneville et al., 2006; Buss et al., 2007; Amalfitano and
Fazi, 2008; Tobler et al., 2008; Pote et al., 2010) and fungi
6989
(Gadd, 2007; Bonneville et al., 2009) strongly attach themselves to mineral surfaces. This attachment of microorganisms to soil particles is central to the physical coherence of
soils and probably helps to form organic matter-mineral
aggregates (Jastrow et al., 1998; Mikutta et al., 2009).
Fungal weathering of primary mineral has been mainly
studied in pot experiments in symbiosis with plants/trees
(Mojallali and Weed, 1978; Leyval and Berthelin, 1991;
Wallander, 2000), in petri-dish mono-cultures (Paris et al.,
1995) or in liquid mono-cultures (Lian et al., 2007;
Balogh-Brunstad et al., 2008a). In all these studies, weathering resulted from multiple components of the experimental systems, e.g., water content, organic acid exudation and
other metabolic/nutritional needs of the mono-culture or of
the whole soil biota. Due to this combination of possible
alteration pathways, it is difficult to quantify the relative
contribution of fungi to the overall mineral weathering
process.
In Bonneville et al. (2009) we used a tree–fungi–mineral
continuum microcosm setup that permits sampling and
measurements of the alteration directly at and below the
interface between fungi and the mineral substrate (biotite),
while excluding all other weathering contributions. Our
experimental approach replicates (i) the fundamental biological relationship of the plant–fungi symbiotic systems
in nature, (ii) hyphal phenotypes found in nature and (iii)
the typical growth pattern encountered in soils (unsaturated
water conditions). Using a combination of FIB ion milling
to sample hypha–biotite ultrathin sections and high-resolution STEM-EDX to analyze the concentrations of K and
Si, we demonstrated that a substantial removal of K and
as well as a Fe(II) oxidation process (Bonneville, et al.
2009). Interestingly in concomitance with the chemical
alteration, selected area electron diffraction (SAED)
showed that a significant deformation of the biotite lattice
structure occurs when hypha enter in contact with its mineral substrate (Bonneville et al., 2009). Therefore, the physical interaction between mycorrhizal hypha and mineral
surfaces creates sub-lm-scale mechanical stresses in the
near surface layers of the mineral structure (Bonneville
et al., 2009). Potentially, these bio-mechanical stresses
may evolve all the way to form channels (Balogh-Brunstad
et al., 2008a) or tunnels penetrating the mineral (Jongmans
et al., 1997). Although the contact zone between mycorrhiza (and by extension soil microorganisms) and minerals
represents a small fraction of the total soil surface area
(probably <1%, Young and Crawford, 2004), the unique
capability of fungi to couple mechanical and chemical alteration processes makes the mineral–mycorrhiza interface the
hotspot of the weathering engine at work in soils.
Here, our aims were (i) to measure the changes in elemental composition (Si, O, K, Mg, Fe and Al) at the fungi/mineral interface and into the substrate in four FIB
sections sampled along a single biotite-bound hypha, (ii)
to quantify the rate of alteration of surface-bound hypha
and finally (iii) to develop a quantitative model of fungal
weathering at the hypha scale. This article also compares
consistently hyphal and abiotic biotite alteration kinetics
in order to determine the efficiency of mycorrhiza to alter
biotite in close-to-natural conditions. Thus, we measured
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S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
the pH of the micro-environment formed around hypha
using pH-sensitive probe and determined as well the abiotic
dissolution rates for the same biotite as used in the fungal
experiments. All these data are then used to compare and
discuss abiotic and mycorrhizal biotite weathering rates
and kinetics and ultimately provide quantitative evidence
that mycorrhiza bound to mineral surfaces and grown in
symbiotic association with living tree/plant roots can accelerate mineral weathering.
2. MATERIALS AND METHODS
2.1. Plant-mycorrhiza symbiosis and incubation with biotite
Our experimental approach relied on growing the ectomycorrhizal fungi, Paxillus involutus, in symbiosis with Pinus sylvestris (Scots Pine). Seeds of P. sylvestris were
surface-sterilized and then incubated aseptically in agar
plates (see Bonneville et al., 2009 for composition). In parallel, P. involutus was grown in sterile square petri dishes on
cellophane covering a modified Melin–Norkrans agar medium (see Bonneville et al., 2009 for composition). After a
month of growth, both the Scots Pine seedlings and P. involutus cultures were transferred into a new petri dish, with
the shoot of the tree protruding through a slot of the dish.
After 10 weeks of common growth, the roots of the tree
seedlings, now well-colonized by their fungal symbiont,
were transferred into a final experimental microcosm consisting of a sterile square petri dish, containing 80 mL nutrient agar (Rorison medium – Bonneville et al., 2009) and
20 mL of Noble nutrient agar separated by a sterilized cellophane layer to prevent root and mycorrhiza penetration
into the nutrient agar layer. This last transfer into the final
microcosm insures that all hypha growing in the microcosm
are mycorrhizal, i.e., grown in symbiosis with the root system of the Scots pine. Before solidification of the agar, HClwashed and autoclaved perlite, inert amorphous volcanic
Fig. 1. (A) Experimental microcosm showing the location of the
biotite flake colonized by P. involutus (Bt). (B) FEG-SEM
micrograph of the sampled hypha on the (0 0 1) basal plane of
the biotite flake studied with the location of the four FIB sections
forming a sequence of increasing exposure time from section 1 to
section 4.
glass, was sprinkled over the top agar layer. Finally, a previously autoclaved (20 min at 121 °C) biotite flake (Moen,
Norway, 0.5 1 cm) was cleaved and laid flat on the perlite grains (Fig. 1A). The tree–mycorrhiza–biotite
microcosm system was then incubated for 130 days in a climate controlled room at 15 °C day and 10 °C night temperature, with an 18 h photoperiod at a photon flux of
550 lmol m2 s1 and with the shoots exposed to 80%
humidity. During this period, the biotite flake was colonized exclusively by mycorrhizal mycelium and did not
experience any direct contact with the tree roots. The biotite flake was retrieved from the microcosm on the day of
the ultra-thin section preparation for high-resolution
micro-spectroscopic analysis.
2.2. Sampling and chemical analysis of hypha–biotite
interface
Using a Focused Ion Beam (FIB – dual-beam FEI Nova
200 NanoLab), a relatively isolated but continuous hypha
(1000 lm) firmly attached to a biotite basal plane was sectioned at four locations at 2, 135, 400 and 740 lm (sections 1 to 4, respectively) away from the hypha tip
(Fig. 1B). Full details of the FIB-sectioning protocols can
be found in Bonneville et al. (2009). The hypha–biotite
FIB sections were analyzed using a Philips/FEI CM 200
Field Emission Gun (FEG) transmission or scanning transmission electron microscope (TEM/STEM) operated at
197 kV and equipped with an ultra-thin-window Energy
Dispersive X-ray detector (EDX, Oxford Instruments)
and a Gatan Imaging Filter (GIF200). A double-tilt sample
holder was used to orient the FIB sections such that the
TEM viewing direction was normal to the biotite–hypha
interface. Elemental X-ray intensity profiles (EDX linescans) of between 400 and 1000 nm in length across each
interface were recorded in STEM mode with a minimum
electron beam diameter of 5 nm. The line-scans were recorded at a 1.5–3.9 nm step size along a line perpendicular
to the biotite–hypha interface in the [0 0 1] direction, normal
to the (0 0 1) cleavage plane (see Bonneville et al., 2009 –
Repository Data Fig. DR3). The intensities in counts s1
of the Ka X-ray lines of Si, Al, Mg, Fe, O and K were recorded as a function of probe position sequentially (dwell
time of a few msec for each probe position) and multiple
time over the acquisition time of the profile (10 min).
We have shown before (Bonneville et al., 2009) that irradiation damage to the reacted biotite was negligible for the
first 30 min of continuous exposure using the STEMEDX settings described above. Finally, the bulk chemical
composition of the freshly cleaved biotite was determined
by Electron Microprobe Analysis (EMPA – Cameca
SX50 equipped with three wavelength detectors and an Oxford Instruments INCA 250 EDX system) operated at
15 kV and 15 nA. The general EMPA-derived formula for
our biotite from an average of 25 spot measurements was
K0.917 Na0.018 (interlayer) Mg0.763 Mn0.055 Fe1.612 Cr0.002
Al0.227 Ti0.194 (octahedral sheet) Al1.263 Si2.737 (tetrahedral
sheet) (OH)2 O10). This pristine biotite composition was
used as a reference chemical composition to determine the
Tree-mycorrhiza symbiosis accelerate mineral weathering
compositional changes across the hypha–biotite interface
from the STEM-EDX line-scans.
2.3. Hyphal pH measurements
The pH around living hypha growing in the microcosm
experiments were measured by Confocal Laser Scanning
Microscopy (CLSM) with an inverted Zeiss microscope
with a Meta510 detector (Zeiss, Jena, Germany) and an argon multiphoton laser at 488 nm. The molecular probe
SNARF4F (Invitrogen, Paisley, UK) was used at 5 lM
concentration to determine the pH of the hyphal microenvironments. This molecular probe possesses two specific
characteristic fluorescence emission bands at 580 and
640 nm respectively for its acidic and basic forms. By ratioing the fluorescence at the two wavelengths, this method
becomes independent of the dye concentration, photobleaching, and changes of instrumental conditions such as
optical path length, excitation intensity, or detector sensitivity (Marcotte and Brouwer, 2005). A pH-calibration
(Fig. S1) using micro-slide wells (Ibidi, Martinsried,
Germany) was done by adding 5 lM of SNARF4F to
0.2 mL of a 50 mM phosphate buffer at pH 4.6, 5.0, 5.4,
5.8, 6.2, 6.6, 7.0, 7.4, 7.8 and by calculating the mean value
in a histogram of the 547–587 and 619–661 nm channels
(Photo-Paint 13, Corel, Ottawa, Canada) (Hunter and
Beveridge, 2005). Ratios of fluorescence emitted at those
two wavelengths recorded at the different pH values were
then fitted with a Boltzmann sigmoid fit (Fig. S1). After
calibration, reacted biotite flakes from two parallel microcosms systems colonized by P. involutus and containing
both biotite-surface attached as well as aerial (i.e., non attached) hyphae in close vicinity to the plant roots but off
the biotite were imaged and analyzed. Approximately
50 lL of a 5 lM SNARF4F solution was delivered directly
onto the colonized biotite flake or on aerial fungal hypha
immediately after being removed from the microcosm system. A cover slip was placed on top of the biotite flake or
the aerial hypha. The samples were “sandwiched” between
cover slip and glass slide and the open sides were sealed
with varnish. Confocal microscopy analysis started after
15 min of incubation at room temperature in the dark. Pixel
ratios of selected areas of interest of the CLSM images were
calculated and the pH determined using the calibration
curve (Fig. S1). The standard deviation error of our pHprobe method is 0.1 pH unit above pH 5.5 but increased
to 0.35 pH units for pH below 5.5. Typical CLSM images
have a pixel size of 0.14 0.14 lm by 0.5 lm in focal depth
and the hyphal pH measurements areas are 2 5 lm.
2.4. Abiotic dissolution of biotite
Biotite, from the same source as used in the fungal
microcosms, was powdered and sieved to obtain the 75–
150 lm size fraction. Fine particles were removed by repeated sonication and gravitational settling and the powder
subsequently dried. The specific surface area for the biotite
powder (2.63 ± 0.08 m2 g1) was determined by N2-BET
(Micromeritics Gemini V analyzer). The cleaned biotite
was imaged with a Field Emission Gun Scanning Electron
6991
Microscope (FEG-SEM, Leo/Zeiss Gemini 1530) operated
at 3 kV with sample coated with 3 nm of platinum. FEGSEM images were recorded on a 45° angle with respect to
the incident electron beam tilted samples holder. The basal
and edge dimensions of 20 representative individual particles were measured to determine the average dimensions
of the biotite particles and to define the relative contribution of basal (SAbasal) and edge (SAedge) planes to the total
surface area (SAtotal) of the particles.
Abiotic dissolution experiments were carried out in
batch and in flow-through mode, both at an ionic strength
of 0.01 m and at pH 2 and 4 (batch) and 3.3 (flow-through).
The ionic strength was fixed using CaCl2 (concentration of
0.0033 M) and the pH adjusted using HCl. For the batch
dissolution experiments, 0.5 g of biotite was reacted for
12 h with 500 mL of a 0.01 m CaCl2 solution at 25 °C under
constant shaking at 140 rpm. Over this period, 20 aliquots
of 1 mL each of the suspension were collected and filtered
through 0.2 lm filters directly into dilute 3% HNO3. The
aqueous concentrations of Mg, Fe and Al were analyzed
by Inductively Coupled Plasma-Mass Spectrometry (ICPMS, Perkin Elmer Elan DRC II). Ranges of Relative Standard Deviation (RSD) across the set of sample analyzed
were for Al: 0.21–5.53%; Mg: 0.21–3.74%; Fe: 0.29–5.81%.
Three flow-through biotite dissolution experiments were
carried out in a 300 mL titanium Parre mixed-flow reactor
with controlled temperature, pressure and stirring as well as
continuous pH monitoring (Wolff-Boenisch et al., 2004). In
brief, between 3 and 6 g of biotite powder was placed into
the stirred at 400 rpm in a reactor with 0.01 M CaCl2
solution at 25 °C and pH 3.3 (the pH did not vary more
than 0.1 pH units). The flow through system was operated
continuously for 80 h by pumping 0.01 M CaCl2 solution
through the reactor using a high-performance liquid chromatography (HPLC) pump at flow rates ranging from
0.07 to 0.13 L h1 depending on the experiment. At the system outlet, the liquid passing through a 2 lm titanium filter
was collected regularly directly into pre-acid-cleaned plastic
bottles that were pre-acidified with dilute 0.5 % HNO3. In
the flow-through experiments, only the total dissolved Si
concentration was analyzed as a proxy for biotite dissolution using by a colorimetric method (Fishman and Friedman, 1989) and a Varian Cary 50 Bio UV-Visible
spectrophotometer at 700 nm.
3. RESULTS
3.1. Hyphae–biotite interfaces in the STEM elemental
profiles
Fig. 1B shows that the hypha sampled by the FIB sectioning, grew over a smooth (0 0 1) basal plane biotite surface. Even at higher resolution, the hypha–biotite
interface is extremely well-defined and atomically sharp.
In no cases were localized penetrations or etch-pits visible
at the biotite–hyphae contact zone (see Fig. 2 in Bonneville
et al., 2009). Four STEM elemental line-scans were acquired perpendicularly to the hypha–biotite interface across
each FIB section (one example of a STEM dataset for Si, O
and K for section 4 is shown in Fig. 2A). In biotite, Si is the
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S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
Fig. 2. (A) STEM-EDX line-scan profiles of Si, O and K across the hypha–biotite interface for FIB-section 4; note that Si and O exhibit
similar profiles and the hypha–biotite interface position was set using the median Si counts (Eq. (1)) as shown in Supplementary Fig. S2. (B)
K, Fe, Al and Mg ratios to Si in the topmost 80 nm of biotite directly underneath the hypha in FIB-sections 4 (results for section 1 to 3 are
shown in Fig. S4). Note that all elemental ratios have been normalized between 0 and 1 with respect to the average bulk, non-altered, biotite
ratio far below the interface in each FIB section (see text in Section 3.2). The gray zones above the dashed lines in Fig. 2B and S4 indicate the
“altered biotite” layer, while the gray shaded zone between the dashed and dotted lines represent the “diffusive front” zone in sections 3 and 4
and below the dotted line is the “bulk non-altered biotite”. Standard deviations (in percent) for normalized K/Si, Fe/Si, Al/Si and Mg/Si ratio
were 15%, 13%, 15% and 17%, respectively.
main element and the change in Si peak intensity in the
STEM line-scans (in counts s1) served as a proxy to position the physical interface between the biotite and the hypha. This is exemplified in Fig. S2 where a sharp
transition in the Si profile between 320 and 345 nm is
clearly visible. Within this 25 nm zone, the Si peak intensity
quickly increased from an average background level of
12 ± 4 counts s1 (SiH ) before the biotite edge, to
239 ± 17 counts s1 (SiBt ) on average in the biotite. From
such data, the median Si counts (SiMed ) was calculated as
follows:
SiMed ¼
ðSiBt SiH Þ
þ SiH
2
ð1Þ
Subsequently, we placed the physical interface between
the hypha and the biotite at the SiMed intensity value in
the Si profile (for FIB section 4 illustrated in Fig. S2 the
SiMed is at 336 nm). The above procedure for Si was used
to position the physical interface within each of the four
FIB sections because there was no evidence of Si loss from
the biotite during hyphal contact (Bonneville et al., 2009).
The Si-defined interface (SiMed ) was then taken to be the origin of the biotite elemental profiles in all the FIB sections
(i.e., from here on, 0 nm depth = physical fungal–mineral
interface). This method also allowed us to determine the
experimental thickness/sharpness of the chemical interfaces
between the hypha and the biotite. Of the elemental profiles
measured across the biotite–hypha interface in each section,
we selected the datasets with the sharpest interface (shortest
Si interface, 25 nm, Fig. S2 for FIB section 4).
3.2. Chemical composition of the biotite–hypha interface
After positioning the hypha–biotite interface in each
line-scan profile, the peak intensities for K, Al, Mg and
Fe measured in each FIB section were ratioed to Si at
any given depth. Away from the interface with the hypha,
that is, in the bulk non-altered biotite (e.g., from 450 to
1000 nm in the profile in Fig. 2A), the average peak intensities for K, Al, Mg and Fe normalized to Si were very consistent from FIB section to FIB section (Fig. S3 and figure
caption for standard deviations). These results demonstrate
that: (i) the chemical composition of the bulk non-altered
biotite is essentially constant in all four FIB sections (hypha
grow over a homogenous mineral substrate); and (ii) the
STEM-EDX elemental profiles can therefore be used to
give a robust quantification of the concentrations of elements in the different FIB sections and also within a single
FIB section as a function of depth. For practical reasons,
K/Si, Fe/Si, Al/Si and Mg/Si intensity ratios in each of
the sections were normalized as described here for
potassium:
ðK=SiÞno ¼
ðK=SiÞ
ðK=SiÞBt
ð2Þ
where ðK=SiÞBt is the average intensity ratio of K relative to
Si measured in the bulk non-altered biotite and ðK=SiÞ is
the intensity ratio measured at any given depth. In doing
so, the normalized intensity ratio, ðK=SiÞno will vary with
depth from a value of 0 (i.e., complete depletion) to 1
(i.e., pristine biotite).By normalizing the whole depth pro-
Tree-mycorrhiza symbiosis accelerate mineral weathering
6993
Table 1
Release rates and standard deviations for Mg, Fe and Al from the abiotic biotite batch dissolution experiments at pH 2, 4 and the flow
through experiments at pH 3.3 at 25 °C and corresponding biotite alteration rates calculated from the molar fraction of the elements in the
biotite formula as determined with EMPA (Bonneville et al., 2009).
Abiotic bulk biotite dissolution (106 mol element m2 h1)
Mg
Fe
Al
Si
Batch: pH 2 release rate of elements
Batch: pH 4 release rate of elements
Batch: pH 2 – biotite dissolution rate (rbulk)a
Batch: pH 4 – biotite dissolution rate (rbulk)a
Flow-through steady state dissolution rate at pH 3.3 (rbulk)a
1.29 ± 0.13
0.17 ± 0.04
1.7 ± 0.14
0.22 ± 0.05
–
1.52 ± 0.12
0.20 ± 0.06
0.94 ± 0.07
0.14 ± 0.03
–
1.50 ± 0.13
0.08 ± 0.03
1.0 ± 0.08
0.05 ± 0.02
–
–
–
–
–
0.18 ± 0.01
a
Using biotite formula: K0.917 Na0.018 Mg0.763 Mn0.055 Fe1.612 Cr0.002 Al1.49 Ti0.194 Si2.737 (OH)2 O10.
file to the average chemical composition of the bulk biotite,
the progressive removal of elements from the biotite at the
interface with the hypha within each FIB section becomes
clearly visible with increasing distance from the tip (i.e.,
from FIB section 1 to FIB section 4 in Fig. 2B and S4; note
that for clarity only the first 80 nm below the interface were
plotted). In FIB sections 1 and 2 (Fig. S4), the normalized
intensity ratios were unchanged for Mg, Al and Fe from the
interface all the way into the bulk biotite with ratios all 1.
Only K showed a slight depletion, ðK=SiÞno down to 0.27, in
the first 5–7.5 nm from the interface in section 1 and a
slightly clearer removal trend in section 2, where ðK=SiÞno
is as low as 0.5 within the first 10 nm from the hypha–biotite interface. By contrast, sections 3 and 4 show significant
depletions in all elements relative to the bulk biotite composition with ðK=SiÞno , ðMg=SiÞno , ðFe=SiÞno and ðAl=SiÞno ratios dropping respectively to minima of 0.02, 0.15, 0.04 and
0.1 at depths from the interface ranging from 15 to
20 nm depending on the FIB section and the element
(dashed lines in FIB section 3 and 4 in Figs. S4 and 2B
respectively). At greater depth (>20 nm), all normalized ratios progressively increase to reach values close to 1 at a
depth of 35 to 40 nm from the interface depending on
the element (dotted lines in FIB sections 3 and 4 in Figs.
S4 and 2B). Overall, the profiles for sections 3 and 4 are
indicative of a strong elemental removal from the top section of the biotite (topmost 40 nm) in contact with the
hypha.
Within the two depth-profiles from FIB section 3 and 4,
we can clearly distinguish three zones: (i) the top 15–20 nm
where all normalized ratios are relatively constant at a minimal values (from here on referred to as “altered biotite”,
gray zone above the dashed lines in Figs. 2B and S4); (ii)
between 15–20 nm and up to 40 nm a transient, curved
trend of increasing normalized ratio with depth (‘diffusive
front’, gray shadings between the dashed and dotted lines
in Figs. 2B and S4); and (iii) the ‘bulk non-altered’ biotite
in the deeper part of the profile (Figs. 2B and S4). Using
these four datasets we quantified the elemental fluxes across
the hypha–biotite interface as well as the mycorrhizal alteration rates, and developed an empirical model to describe
our depth-profile data.
3.3. Abiotic dissolution of biotite
Abiotic dissolution experiments using the exact same
biotite were carried out, to provide abiotic elemental release
rates to compare with the mycorrhizal alteration experiments. The average SAbasal/SAtotal and SAedge/SAtotal ratios
of individual biotite particles as measured from the FEGSEM micrographs were 0.94 and 0.06 (±0.046, n = 20),
respectively. For all batch systems, the initial 1.5 h of the
experiments were characterized by fast elemental release
that we ascribed to minor amounts of remnant fine biotite
particles undergoing dissolution (see Fig. S5 for examples
for the batch experiments at pH 2 and 4). After 1.5 h, the
aqueous concentration of Mg, Fe and Al increased linearly
with time over the course of the experiments. Linear regressions of the biotite dissolution data between 1.5 and 12 h allowed us to calculate the abiotic release rates for Mg, Fe
and Al in mol m2 h1 and also to derive the abiotic biotite
dissolution rates at pH 2 and 4 (Table 1) assuming the
chemical formula measured by EMPA (see Section 2.2).
For the flow-through experiments at pH 3.3 (Fig. S5), the
first 45 h showed fast biotite dissolution rates (based on
the Si concentrations and chemical formula) that progressively slow down and reached a steady-state dissolution rate
at around 50 h. All abiotic elemental release and biotite dissolution rate values are listed in Table 1.
3.4. pH of the hypha microenvironment
pH measurements of the micro-environments around 8
living hypha growing either in intimate contact with the
biotite surface or were near the root of the Pine tree and
thus not in contact with the biotite, revealed a clear acidification when fungi were in contact with the biotite. Out of
32 measurement areas on biotite-bound hypha, 23 areas appeared to be below the detection limit of our method which
was pH < 4.6 (Fig. 3A, and pH-calibration curve in
Fig. S1), but 9 areas had pH values between 5.3 and 5.8
(Fig. 3C). For hypha not in contact with the biotite, out
of 21 measurement areas, only 2 had a pH below 6
(Fig. 3B), while in all other areas, the pH ranged from 6.1
to 6.8 with on average of 6.3 (Fig. 3D).
4. MODEL OF THE CHEMICAL WEATHERING AT
THE HYPHA–BIOTITE INTERFACE
We have developed an empirical model describing the
chemical changes measured in the biotite directly underneath the fungal hypha. The model is based on the concept
of a moving weathering front within the biotite, leading to
the development of a progressively deepening alteration
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S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
Fig. 3. CLSM images of surface-bound hypha in (A) and (C) and “aerial, near to the root” non biotite-bound hypha in (B) and (D) with their
respective pH values as derived from the 580/640 nm fluorescence ratio. All values in (A) were <4.6.
layer with increasing time (Fig. 4). The four STEM profiles
(Figs. 2B and S4) revealed that the hypha, strongly bound
to the mineral surface, were able to impose and maintain
a large chemical gradient inducing the diffusion of elements
out of the biotite and into the hypha. The loss of major constitutive elements from the biotite destabilizes its lattice
structure and leads to a mineralogical rearrangement into
a metastable, secondary or “altered biotite” (Fig. 4). As
Fig. 4. Conceptual model for the alteration at the hypha–biotite
interface. (A) Initial chemical profile (i.e., pristine biotite at the
beginning of the colonization by the hypha; t = t0;), (B) profile after
exposure to living hypha (t = t0 + dt). As alteration proceeds via
solid-state diffusion, the chemical profile develops into three zones:
the topmost part of the profile where, due to heavy depletion of
cations, the biotite is transformed into “altered biotite”; which is
separated from the “diffusive front” by a moving boundary which
migrates progressively deeper into the “bulk non-altered biotite”
zone.
the alteration and mineral transformation proceed within
the biotite, the mobilized elements diffuse though a progressively thickening layer of “altered biotite”. Thus, the thickness, the carrying capacity and the diffusive properties of
the “altered biotite” directly influence the overall rates of
diffusion and biotite breakdown. The model presented here
assumes that, during contact, the physical mineral–hypha
“interface” is immobile (i.e., a biotite solid-state transformation into a secondary phase takes place without dissolution of the Si–O structure (Bonneville et al., 2009)). This
assumption is supported by our high resolution imaging
and analyses of the hypha–biotite interface which showed
no hypha penetration, or depressions, etch pits or volumetric changes of the mineral underneath the hypha, yet revealed the development of vermiculite-type mineral
domains in the close vicinity (upper 30–50 nm) of the interface (Bonneville et al., 2009).
This moving boundary problem is analogous to the situation where ice is growing on top of an un-stirred water
body or the development of an oxidized layer (tarnish) at
the surface of elemental silver (Bennett et al., 1969; Crank,
1987). In our case, the moving boundary is located between
the “diffusive front” in the biotite and the “altered biotite”
layer, which migrates with time deeper into the biotite as
mineral breakdown proceeds (Fig. 4). Our model also
assumes mass conservation, i.e., the influx of elements into
the “altered biotite” layer from the “diffusive front” is
equivalent to the flux out of the “altered biotite” layer into
the hypha. Furthermore, the “altered biotite” layer has
fixed boundary conditions: (i) at the hypha–mineral interface, the chemical composition of the hypha is assumed
to be constant (i.e., no elemental accumulation in the hypha
Tree-mycorrhiza symbiosis accelerate mineral weathering
directly above the biotite, the elements removed are instead transferred by the hypha to the host–plant); and
(ii) at the moving boundary (Fig. 4), the breakdown of
the biotite into “altered biotite” occurs when the normalized elemental ratios drop below a fixed threshold that
reflects a biotite stability limit. Since the “altered biotite”
grows effectively from a zero thickness with constant
boundary conditions, diffusion equilibria are established
within the “altered biotite”. Thus the normalized ratios
in this “altered biotite” layer follow a linear gradient with
depth (Fig. 4B; i.e., bold striped zone). Within this layer,
at any depth, xi (in m), the normalized ratio to Si of an
element i, Ri, is:
RALT RH
Ri ¼
xi þ RH
ð3Þ
xbd
where RH is the normalized ratio of the element i at the
physical interface between hypha and the mineral surface,
and RALT is the normalized ratio of element i at the moving
boundary between the “altered biotite” and the “diffusive
front” in the biotite”. The depth at which this moving
boundary is positioned is defined as xbd. These three parameters (RH, RALT and xbd) were determined for each section
and each element (Table 2) by fitting the final model to the
STEM-EDX elemental concentration profiles. Once determined, these parameters can also be used to calculate the
flux of a given element, JALT, through the “altered biotite”
layer into the hypha:
J ALT ¼ DALT
RALT RH
xbd
ð4Þ
where DALT is the diffusion coefficient of the element i in the
“altered biotite” in m2 s1 (Table 2). Due to differential element partitioning between the “altered biotite” layer and
the biotite in the “diffusive front”, the model allows for a
compositional discontinuity at the moving boundary
(Fig. 4), as follows:
RALT ¼ RBti K P
ð5Þ
With RBti being the value of the normalized ratio of element i, at which the biotite breaks down into “altered biotite” and KP, the partition coefficient of i between “altered
biotite” and the biotite affected by the “diffusive front”
(both parameters determined from the fitting of the model
to the STEM-EDX elemental concentration profiles; Table
2). The KP term is necessary, as elemental diffusion must be
continuous in terms of chemical potential across the moving boundary; the elemental abundance however is not necessarily continuous (and indeed, is not in this case). Within
the biotite affected by the “diffusive front”, the normalized
ratios as a function of depth vary between RBti and 1 (the
breakdown composition and the pristine biotite composition, respectively) and this is modelled as a one-dimensional
error function distribution:
!
xi
ð6Þ
Ri ¼ RBti þ ð1 RBti Þerf pffiffiffiffiffiffiffiffiffiffiffi
2 DBt tf
from which, the flux of element i out of the biotite, JBt,
through the moving boundary (from the “diffusive front”
into the “altered biotite” layer) can be derived as:
J Bt ¼ DBt
ð1 RBti Þ erf
@xi
6995
2
i
pxffiffiffiffiffiffiffi
ffi
DBt tf
ð7Þ
where DBt is the diffusion coefficient of element i in the biotite in m2 s1, Ri the normalized element i ratio at depth xi
and tf is the fictive time elapsed to reach the value on the
profile for the element i. DBt was fixed to 3.8 1023 m2 s1
for K in biotite (Taylor et al., 2000).
In the model, the ratio of diffusion coefficients DBt and
DALT governs the curvature of the “diffusive front”. Therefore, by fixing DBt and fitting the modelled curves to the
depth profile data for K, an estimation of DALT but also
the active time period of the alteration, tr (see definition below) could be derived. In effect, K depth-profiles were used
to time-calibrate the model (see Section 5.3 below). Classically in diffusion, the flux of any given element is proportional to the inverse of the distance of diffusion (Crank,
1987) and therefore, in our case, the flux, JALT, is proportional to 1=xbd (Eq. (4)). The depth or distance of the diffusion is itself proportional to the square root of time, which
means that JALT (Eq. (4)) and JBt (Eq. (7)) are dependant
upon the inverse of the square root of the elapsed time t.
As shown in Fig. S6, the latter relationship can be used
to derive the fictive time, tf from:
pffiffiffiffi
tf
J Bt
¼ C St pffiffiffiffi
ð8Þ
J ALT
t1
where t1 is a time fixed to 1 second and representing the
time just after the hypha start interacting with the biotite
and Cst is a constant. Thus, Eq. (8) can be rearranged into:
2
J Bt
tf ¼ C St
ð9Þ
J ALT
Even though tf calculated via Eq. (9) yields the correct
depth profile in the “diffusive front” – i.e., a good agreement between the modelled value and the normalized ratio
for element i, it is, however, a fictive elapsed time. Indeed, as
the “altered biotite” layer thickens, the concentration curve
in the “diffusive front” is continuously “compressed” by the
downward movement of the moving boundary (Fig. 4).
Therefore, the derivation of the period of contact between
hypha and biotite (for each FIB section) only based on
the fitting of the concentration curve in the “diffusive front”
would be erroneous (in fact, an underestimation) as it
should also take into account the time required to form
the “altered biotite” layer. The period of contact between
hypha and biotite however can be estimated by introducing
a “compression factor” of the diffusion front calculated as:
Q¼
Total removal of element i
Removal of element i below boundary
ð10Þ
with Q, being the ratio between the total amount removed
of the element i (i.e., integrated throughout the entire depth
profile) and the element removal calculated below the moving boundary (i.e., only in the ‘diffusive front’ part and for
tf). Q is then used to derive the actual time period (tr in seconds, determined as discussed above and listed in Table 2)
required to reach the depth-profile shape via:
tr ¼ tf Q
ð11Þ
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Table 2
Parameters used for the modelling of the Fe, Al, K and Mg concentrations in the four FIB sections. RBti, RH, RALT, KP and xbd were obtained by fitting to the experimental data (Fig. 5). DBt for
the K profile was taken from Taylor et al. (2000) and DALT and tr were outputs from the model. Note that the same DBt, DALT, RBti, RALT, RH (except for K in section 2) and the KP values were
used consistently to fit each elemental profile across the four FIB sections.
FIB Sect.
2
3
4
Aluminium
1
2
3
4
Magnesium
1
2
3
4
Iron
1
2
3
4
a
RH (ratio/mol lm3)
RALT (ratio/mol lm3)
KP (–)
DBt (m2 s1)
DALT (m2 s1)
xBd (m)
tra (days)
0.63
3.8 1015
0.63
3.8 1015
0.63
3.8 1015
0.63
3.8 1015
0.35
2.3 1015
0.49
2.9 1015
0.35
2.3 1015
0.35
2.3 1015
0.5
3.0 1015
0.5
3.0 1015
0.5
3.0 1015
0.5
3.0 1015
0.8
3.8 1023
3.1 1022
2.5 109
0.41
0.8
3.8 1023
3.1 1022
2.8 109
13.5
0.8
3.8 1023
3.1 1022
28 109
61.4
0.8
3.8 1023
3.1 1022
30.5 109
72.8
–
–
0.49
4.8 1015
0.49
4.8 1015
–
–
0.15
1.5 1015
0.15
1.5 1015
–
–
0.49
–
–
4.4 1023
–
–
4.4 1022
–
–
18 109
–
–
61.4 (48)
0.49
4.4 1023
4.4 1022
21 109
72.8 (61)
–
–
0.6
3.0 1015
0.6
3.0 1015
–
–
0.25
1.2 1015
0.25
1.2 1015
–
–
0.75
–
–
2.6 1023
–
–
1.3 1022
–
–
17 109
–
–
61.4 (48)
0.75
2.6 1023
1.3 1022
18.5 109
72.8 (61)
–
–
0.4
4.2 1015
0.4
4.2 1015
–
–
0.15
1.6 1015
0.15
1.6 1015
–
–
0.5
–
–
5.6 1023
–
–
6.0 1022
–
–
14.5 109
–
–
61.4 (48)
0.5
5.6 1023
6.0 1022
15.5 109
72.8 (61)
0.24
2.4 1015
0.24
2.4 1015
0.45
2.25 1015
0.45
2.25 1015
0.2
2.1 1015
0.2
2.1 1015
The exposure period, tr, was derived from the K/Si depth profiles (see Section 4 for details). Note that the effective, tr, for Al, Fe and Mg are shorter because the diffusion of these elements
started only after section 2; the actual diffusion periods – in days) for these elements are indicated in brackets.
S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
Potassium
1
RBti (ratio/mol lm3)
Tree-mycorrhiza symbiosis accelerate mineral weathering
6997
Fig. 5. Chemical (symbols) and modelled (lines) concentration depth-profiles over the 150 nm below the hyphae–biotite interface for K, Al,
Fe and Mg in the four FIB sections. Note that only K is mobilized in all four sections in contrast to Al, Fe and Mg, which are only mobilized
from the biotite in sections 3 and 4.
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S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
So far, the depth profile datasets and the outputs of our
model were expressed in units of normalized ratios, varying
between 0 and 1. However, to quantify the flux of elements
out of the biotite, it is necessary to calculate from the normalized ratios, the concentration Ci (in mol lm3) of element i using:
Ci ¼
Ri ð%wti Þq
Mmi
ð12Þ
where Ri is the normalized ratio of element i (ranging between 0 and 1) at any given depth of the profile, % wti is
the weight percent of element i in the bulk biotite (as quantified by EMPA), q the density of biotite (i.e., 3.08 g cm3)
and Mmi is the molar mass of element i. The fits of the
above model to the profiles of the elemental concentration
per unit volume (converted from the as-measured STEMEDX intensity profiles using Eq. (12)) are plotted for each
FIB section for Al, Mg, K and Fe in Fig. 5. The quality
of the fits is evident and the elemental concentrations at
the onset of the altered biotite layer are consistent across
all sections and for each of the measured elements. The
overall root mean square deviation (RMSD) between modelled and experimental data was calculated to be 0.11
(RMSD of 0 would be an ideal fit). The values of the fitting
parameters (RH, RBti, KP, DALT, DBt and xbd) used in the
model and its output, tr are listed in Table 2 and discussed
in Section 5.2.
5. DISCUSSION
5.1. Mineral alteration underneath living hypha
Unlike many classical biotite dissolution studies, which
typically quantified alteration at the macroscopic level from
changes in solution chemistry, our perspective on biotite
myco-weathering is based on nanometer to sub-micrometer
scale analytical evidence of the interface between the fungi
and the mineral substrate. Below, we develop consistent
points of comparisons between our fungal and our abiotic
dissolution datasets and various other literature datasets
of abiotic and microbial biotite dissolution in order to
determine the weathering efficiency of mycorrhiza.
Our data shows that, in addition to an increasing K
mobilization with contact time, Fe, Al, Mg were also removed from the biotite substrate (Fig. 5) but with a delayed
onset of mobilization of 13 days (Fig. 6A and C, and Table 2). Interestingly, in all FIB sections, Si and O appeared
preserved throughout the depth-profile (Fig. 2A), implying
a non-stoichiometric dissolution and the formation of an
Si/O-enriched “altered biotite” layer directly underneath
the hypha. Overall, the mobilization of elements was visible
up to 40 nm deep into the mineral substrate (Figs. 2B, S4
and 5) including an “altered biotite” layer of up to 20 nm.
This “altered biotite” layer is marked by a different chemical (Figs. 2B and S7) and mineralogical composition compared to the bulk non-altered biotite (Bonneville et al.,
2009). Furthermore, this “altered biotite” layer is crucial
as it controls the elemental diffusion rate – and so the flux
of elements towards the hypha – and also the migration
of the moving boundary deeper into the biotite (Fig. 4).
The formal identification of the mineral phase that
makes up this “altered biotite” layer is however difficult.
The layer is extremely thin (20 nm maximum) and even
selected area electron diffraction (SAED) could not conclusively point to a change in diffraction pattern from the
structure of the parent-mineral, biotite. Nevertheless, in
our previous work (Bonneville et al., 2009), high-resolution
TEM images revealed, within 30 nm of the interface with
the hypha, (0 0 1) spacing domains of 14.0 Å as opposed
to 10.0 Å for bulk non-altered biotite. These larger d-spacings are indicative of a transformation towards a vermiculite-type phase (Wierzchos and Ascaso, 1998). This is not
surprising as vermiculite is the prime alteration product in
acidic dissolution experiments of biotite containing more
than 0.8 Mg per O10(OH)2 (Murakami et al., 2003), a value which is close to the Mg content of our biotite
(0.77 Mg per O10(OH)2). Other secondary phases that
may form during biotite alteration (e.g., Fe3+ (oxidized-)
biotite) are more difficult to distinguish from pristine biotite
based solely on high-resolution TEM micrographs because,
depending on the extent of oxidation, their (0 0 1) d-spacing
range from 10.1 to 10.3 Å (Jeong et al., 2006).
In this respect, the elemental removal from the
“altered biotite” layer (Al, Fe, Mg and K depletions by
75–85%, 80–85%, 55–75% and 50–65% compared to
the bulk non-altered biotite; Fig. S7) further reinforces
our previous evidence of a mineralogical transformation
of biotite underneath the hypha. It is likely that the loss
of the interlayer (K) and octahedral cations (Mg, Fe and
Al) mobilized during fungal alteration are partially compensated by the Fe(II) oxidation into Fe(III). We have
previously shown (Bonneville et al., 2009; Smits et al.,
2009), that a 200 nm layer of biotite directly underneath
the hypha was enriched in Fe(III) compared to the bulk
non-altered biotite. Considering the substantial removal
of cations from the biotite, the positive charge loss is
however unlikely to be balanced only by structural Fe(II)
oxidation alone.
More likely, proton incorporation due to a pH shift at
the surface of the biotite is occurring (Turpault and
Trotignon, 1994). Incorporated protons, i.e., positive
charges, are probably continuously “hopping” from one
interstitial position to another to charge balance the removal of cations at depth in the mineral structure (Hawthorne, 1992). The micron-scale pH measurement in the
current study using fluorescent probes confirmed that the
hypha filaments growing in contact with the biotite acidified substantially their near-environment by, at least one
pH unit (Fig. 3A and C). Acidification is a well-known fungal response characteristic due to the proton efflux resulting
from the activity of the plasma-membrane H+-ATPase and
CO2 production linked to the respiration of the hypha
(Jones et al., 2004; Gadd, 2007) and to the exudation of organic acids (Rosling et al. (2004). Even though most of the
fungal exudates are weak acids, it is possible that such organic acids may have contributed to the micron scale acidification. However, at high resolution we did not observe
any precipitates of oxalate or any other for that matter
(Bonneville et al., 2009), yet low levels of oxalic acid have
been detected by infrared analyses of non-symbiotic
Tree-mycorrhiza symbiosis accelerate mineral weathering
6999
Fig. 6. (A) Longitudinal alteration profile, expressed as elemental concentration removed from the biotite along the sampled hypha. (B)
Relationship between the distance of each FIB section from the hypha tip and the exposure time calculated from the alteration model. Line
indicates the hyphal extension rate used in the alteration kinetics calculation (i.e., 10.2 lm day1). (C) Data (symbols) and modelled
concentrations removed (solid lines) from the biotite as a function of the estimated contact time.
mycorrhiza grown over this same biotite (Schmalenberger
et al., 2010).
In general, the alteration features and the mineralogical
changes observed in the current study are in agreement with
previous biotite dissolution studies. Incongruent biotite dissolution in acidic conditions has been reported in various
experiments in batch, fluidized bed or flow-through systems
(Acker and Bricker, 1992; Turpault and Trotignon, 1994;
Kalinowski and Schweda, 1996; Malmstrom and Banwart,
1997). Turpault and Trotignon (1994) observed that a 200–
400 nm altered layer enriched in Si and O formed from the
(0 0 1) basal plane surface of biotite exposed to a pH 1
solution. In the presence of bacteria, the dissolution of biotite was also shown to be incongruent and greatly enhanced
with final Si, Fe and Al concentrations up to two orders of
magnitude higher than in abiotic controls (Barker et al.,
1998; Hopf et al., 2009). To the best of our knowledge,
for fungi grown in non-aqueous media and in symbiosis
with a tree, our results are the first to show: (a) an in situ
elemental removal from a mineral substrate; (b) the formation of an altered biotite layer with a different chemical and
mineralogical composition directly underneath a living hypha; and (c) a close spatial relationship between these alter-
ation features and a fungal-driven acidification at the
mineral surface. Combining all these observations and the
mathematical framework described above allowed us to
quantify element fluxes from a mineral substrate to a single
living hypha.
5.2. Quantification of elemental fluxes at the hypha–biotite
interface
To quantify the elemental fluxes and therefore the kinetics of fungal alteration, the exposure time between the biotite and the hypha needed to be determined. First, using the
modelled curves in Fig. 5, the amounts of K, Mg, Fe and Al
removed (in mol lm2) from the biotite were calculated for
each of the four FIB sections (Table 3). Plotted against distance from the hypha tip, the elemental removal in each
FIB section provides a longitudinal transect of alteration
underneath the hypha (Fig. 6A). As the removal of potassium starts as soon as the hypha interacts with the biotite
surface (Fig. 5), we could “time”-calibrate our model with
the K depth-profiles. Using values for DBt from Taylor
et al. (2000), we constrained DALT for K (see model description in Section 4 above) and thus derived the active period
Table 3
Calculated amounts of K, Al, Fe and Mg removed from biotite for each FIB section (standard deviations in brackets). The model data for the
integrated amounts of elements mobilized along the entire length of the hypha and the associated elemental release and alteration rates are
also listed.
Mycorrhizal alteration
K
Section 1 (1017 mol element lm2)
Section 2 (1017 mol element lm2)
Section 3 (1017 mol element lm2)
Section 4 (1017 mol element lm2)
Total amount of elements removed from the biotite
(1013 mol per hypha)
Release rate (108 mol elements m2 h1)
Biotite alteration rate (108 mol Bt m2 h1)
0.97
1.08
1.18
2.15
2.7
4.5
4.9
(0.16)
(0.12)
(0.14)
(0.24)
Mg
Fe
Al
–
–
6.41 (0.68)
6.97 (0.75)
1.5
–
–
19.0 (1.90)
21.0 (2.16)
4.6
–
–
17.9 (2.34)
20.7 (2.71)
4.4
2.4
3.2
7.2
4.5
6.8
4.6
7000
S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
of alteration (tr) for each of the four FIB sections: 0.4, 13.5,
61.4 and 72.8 days, respectively (Table 2).
Assuming constant and continual growth of the fungi
over the biotite, a distance of 740 lm between FIB sections
1 and 4, yields an average hyphal growth rate of
10.2 lm d1 (Fig. 6B). The actual hyphal growth mechanisms or rates over a biotite surface (or any other mineral
surface for that matter) are unknown. The only values
available for hyphal turnover rates in soils vary from 7 days
(Staddon et al., 2003) to 145 days (Treseder et al., 2010).
Thus, an independent check of our hyphal growth rate estimate is difficult. Nevertheless, considering the overall duration of the microcosm experiment (130 days) and that the
hypha had to grow first from the root to the biotite flake
(Fig. 1A), a weathering active hypha–biotite interaction
period of 73 days for the FIB section 4 (Table 2) and thus
the estimated hyphal growth rate (10.2 lm d1) seems reasonable. These values of hyphal growth rate and activeweathering periods were hereafter used to calculate the
mycorrhizal alteration rates.
Time quantification for the Al, Fe and Mg profiles is
more complex as their respective mobilization starts between FIB section 2 and FIB section 3. However, knowing:
(i) the depth of the “altered biotite” layer (xbd) from the Al,
Fe and Mg profiles in FIB sections 3 and 4 (Figs. S4 and 5),
(ii) the respective DBt/DAL ratios (from the fits of the “diffusion front”), (iii) the amounts of each element removed
(Table 3) in FIB sections 3 and 4 and (iv) the absolute exposure periods of FIB sections 3 and 4 (i.e., 61.4 and 72.8 days
based on the above K profile analysis), allowed us to constrain the diffusion coefficient values for Al, Mg and Fe in
the bulk non-altered biotite (DBt; Table 2). This then enabled the estimation, from the fitted model, of the concentration of elements removed (in mol lm2) from the biotite
as a function of exposure time between the hypha and the
biotite surface (Fig. 6C).
Similar to the classical dissolution scheme for biotite described by Malmstrom and Banwart (1997) and Schnoor
(1990), in our experiments K was mobilized first, creating
a K-depleted biotite layer, while Mg, Fe and Al only started
to diffuse 13 days after the initial hypha–biotite contact.
The rate of K, Mg, Fe and Al removal from the biotite decreases sharply with contact time because the thickening of
the “altered biotite” layer ultimately slows down the bulk
biotite breakdown (Fig. 6C). The relatively low removal
of K observed in FIB section 2 (Fig. 6C) compared to the
modelled K curve indicates some degree of heterogeneity
in the alteration process along the hypha possibly reflecting
the hyphal pH variations (Fig. 3) and which is also in accordance with the formation of discrete vermiculite-like domains as-seen in high resolution TEM micrographs
(Bonneville et al., 2009). Overall, our solid-state diffusion
model captures the essential features of elemental removal
within each FIB section but also provides a kinetic framework for myco-alteration along a living hypha attached
to a mineral surface.
In order to derive the hyphal alteration rate, we also had
to determine the contact surface area between hypha and
biotite. Our FEG-SEM and Environmental SEM observations in Bonneville et al. (2009) showed that surface-bound
hypha exhibited flattened shapes (although turgidity can be
observed at the tip level) with an average width of 5 lm.
Combined with a total length of 740 lm, the sampled hypha had therefore a contact surface are with the biotite of
3700 lm2. It is to be noted however, that P. involutus hypha can form an ultrathin layer that may expand laterally.
However so far there is no indication that this “biolayer”
mediates alteration itself (Saccone et al., 2009). By integrating the curves in Fig. 6C and using the above hyphal
contact surface area with biotite, the total removed K, Al,
Mg and Fe from the biotite was estimated to be 2.7, 4.4,
1.5 and 4.6 1013 mol, respectively. This corresponds to
an elemental uptake/release of 4.5, 6.8, 2.4, 7.2
108 mol m2 h1 and thus a biotite hyphal alteration rate
ranging from 3.2 to 4.9 108 mol of biotite m2 h1
(Table 3).
5.3. Biotite alteration kinetics: mycorrhizal vs. abiotic
Finally, in order to quantify the potential enhancing
effect of mycorrhiza on the dissolution of biotite, both
the fungal and the abiotic alteration kinetics need to be
consistently compared. However, to carry out such a
comparison several aspects have to be considered. Firstly,
biotite dissolution rates are impacted by pH and to a lesser extent by temperature. Secondly, the experimental
conditions for biotite weathering differ dramatically between experiments performed with mycorrhiza (either in
liquid cultures, in pots or in microcosms) and most abiotic dissolution experiments (in solution, in batch or
flow-through systems). For instance, in our microcosm
experiments, the sampled hypha had grown over and
interacted only with the basal plane (i.e., (0 0 1)) of the
biotite flake. Conversely, in a typical abiotic experiment,
the biotite particles are in constant and complete contact
with the reacting solutions inducing a simultaneous dissolution of the edge and the basal plane. This is important,
because biotite dissolution rates are highly, spatially
anisotropic. The edge dissolution rate is between 36 and
240 times faster than basal plane dissolution rates (Turpault and Trotignon, 1994). Similar edge vs. basal plane
differences in reactivity have been reported from Atomic
Force Microscopic (AFM) measurements on biotite by
(Aldushin et al., 2006) and phlogopite – a closely related
phyllosilicate (Rufe and Hochella, 1999). Thus, in order
to evaluate unambiguously the alteration “efficiency” of
mycorrhiza, it is essential to assess the abiotic basal plane
dissolution rate of the biotite used in our abiotic batch
and flow through dissolution experiments. From the data
from our abiotic experiments at pH 2, 3.3 and 4 (Fig. S5)
the bulk dissolution rates (rbulk in mol Bt m2 h1), were
calculated (Table 1). In addition, based on FEG-SEM
image analyses, the ratios of basal (SAbasal =SAtotal 0.94)
and edge (SAedge =SAtotal 0.06) areas relative to the total
geometrical surface area were determined. Thus, the biotite dissolution rates for the abiotic dissolution experiments can be defined as:
rbulk ¼
SAedge
SAbasal
redge þ
rbasal
SAtotal
SAtotal
ð13Þ
Tree-mycorrhiza symbiosis accelerate mineral weathering
Where redge and rbasal were the specific dissolution rates of
the edge and basal planes of our biotite (in
mol Bt m2 h1). Knowing that 36 < (redge =rbasal ) < 240
(Turpault and Trotignon, 1994), and having determined
experimentally the values of rbulk (Table 1), and having measured the relative proportions of basal and edge surface
areas of our biotite from the FEG-SEM images, allows
for Eq. (13) to be rearranged to obtain ranges for rbasal
and redge for all our abiotic dissolution experiments
(Table S1 in Supplementary information).
To compare our mycorrhizal and abiotic biotite dissolution data, a plot of dissolution rates vs. pH was constructed
using the bulk dissolution rate data from the literature
(open circles in Fig. 7) from which an average pH-dependency for the biotite dissolution kinetics was determined
(Eq. (S1) and (S2) in Supplementary information). This
was then applied to linearly extrapolate the range of redge
and rbasal over the pH range between 1 and 6 (slanting gray
areas bars in Fig. 7).
As expected, the calculated interval for the edge dissolution rates in our abiotic dissolution experiments is much
faster than the bulk dissolution rate from literature (open
circles) but in good agreement with the experimentally-defined edge dissolution rates data of (Bickmore, 2001) and
(Balogh-Brunstad et al., 2008a) (i.e., open and half-filled
triangles in Fig. 7). Conversely, the calculated interval for
the basal plane dissolution rate is in the low-end of the bulk
dissolution data from the literature. Only the bulk dissolution rate of (Taylor et al., 2000) – half filled circle in Fig. 7 –
falls substantially lower than the calculated range for basal
plane dissolution rates due to the use of larger particles
7001
compared to other studies and thus, a larger basal plane
contribution.
In Section 5.2, the rate of dissolution of the biotite basal plane by mycorrhiza was estimated to be 3.2–
4.9 108 mol of biotite m2 h1. Yet, to consistently
compare mycorrhizal and abiotic dissolution rates, the
pH at which mycorrhiza altered the biotite substrate
needs to be determined. This is problematic as hyphal
pH varies greatly along the hypha length and this can
reach more than one pH unit over 20 lm of hypha (for
example see Fig. 3B) and these values can also potentially
change with time. Minor pH variations along hypha
lengths (DpH < 0.1 pH units, the tip being slightly more
alkaline than the rest of the hypha) have been previously
reported by Schreurs and Harold (1988) and Limozin
et al. (1997). The difference in pH variations along hypha
between these studies and our observations is likely due
to very different experimental conditions. In addition, in
our experiments, hypha acidification was intense and substantial portions of analyzed hypha (60 lm) exhibited
pH values below our detection limit (i.e., pH 4.6;
Fig. S1). Thus, the lowest pH could potentially have been
even lower than pH 4.6 in some parts of the hypha.
Although our confocal measurements are, so far, probably the most accurate estimates of pH of living hypha
on mineral surface, further work is needed to fully quantify the pH of hypha especially in the acidic pH range
and also to follow the evolution of hyphal pH over time.
Despite the limitation of our pH-sensitive probe method,
some conclusions can still be drawn with respect to the
efficiency of hypha to alter biotite.
Fig. 7. Dissolution rates of biotite normalized to –O10(OH)2 as a function of pH. (s) denotes abiotic bulk dissolution rates from the literature
(Acker and Bricker, 1992; Kalinowski and Schweda, 1996; Malmstrom and Banwart, 1997) and ( ) from (Taylor et al., 2000). (d) are the bulk
batch abiotic control dissolution rates at pH 2 and 4 (this study) and at flow-through rates at pH 3.3 (this study). Triangles depict the edge
dissolution rate: ( ) and (4) from Balogh-Brunstad et al. (2008a,b) and Bickmore (2001), respectively. Squares represent mycorrhizal
dissolution rates: ( ) and ( ) illustrate the bulk and the basal plane dissolution rates respectively, from Balogh-Brunstad et al.’s (2008a,b)
experiments with fungi liquid monocultures. Finally, the mycorrhizal basal plane alteration rate from the current study ( ) is plotted across a
pH range measured (4.6–5.8) but with the arrow extending until pH 3.5 reflecting the lower pH boundary of acidic soils from the literature
(Finkl, 2008). N. B. All abiotic (i.e., bulk, basal and edge) dissolution rates as well as those for the fungi monoculture in Balogh-Brunstad et al.
(2008a,b) were performed at 25 °C as opposed to the 15 °C in the current microcosm experiment. All abiotic dissolution rates were therefore
temperature corrected using the Arrhenius equation and a pH-dependant energy of activation ranging from 67 to 10 kJ mol1 from pH 1 to
pH 6 (Carroll and Walther, 1990).
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S. Bonneville et al. / Geochimica et Cosmochimica Acta 75 (2011) 6988–7005
To our knowledge, only Wallander et al. (1999) measured bulk soil pH during biotite alteration experiments
with P. involutus–P. sylvestris symbiosis, in pot experiments. Their final bulk pH values varied between 4.8 and
5.2, in line with our biotite-bound hypha pH measurements.
Except in soils subjected to pyrite oxidation, where pH can
reach values as low as 2, acidic soils, such as podzols that
are common in boreal forest, exhibits average bulk soil
pH values between 3.5 and 5.5 (Finkl, 2008). Taking the latter pH of 3.5 as the low end of possible hyphal pH and pH
5.8 as the upper limit of surface-bound hypha (Fig. 3C), reveals that surface-bound hypha can substantially accelerate
the dissolution of the basal plane (0 0 1) of biotite when
compared to abiotic dissolution at equivalent pH conditions (gray hashed rectangular box and arrow in Fig. 7).
Quantifying this fungal alteration enhancement (i.e., beyond the hyphal acidification pH) is difficult as it obviously
depends heavily on the local hyphal pH conditions: if hyphal pH is lower than the minimum of 3.5, both abiotic
and mycorrhizal rates would be almost equivalent. However, in the case of hyphal pH values above 3.5, biotite
alteration underneath surface-bound hypha is consistently
faster than under abiotic conditions at equivalent pH. Here,
we hypothesize that this ‘extra’ alteration capacity of surface-bound hypha is due to the ability of fungi to simultaneously disrupt mechanically the lattice structure of the
mineral substrate on which it grows (Bonneville et al.,
2009) and that, in turn, the weakened biotite zone directly
underneath the hypha is more prone to chemical alteration.
In Fig. 7, we plotted also data from mycorrhizal monocultures in liquid media (half-filled squares) in the presence
of biotite (Balogh-Brunstad et al., 2008a). These authors
showed that basal plane alteration accounts for only 1%
of the total alteration dissolution rate and concluded that
“fungal weathering predominantly occurred not by attachment and direct transfer of nutrients via hyphae, but due to
the exuded organic acids in the bulk liquid which accelerated dissolution of the flake edges”. Our results from the
plant-mycorrhiza-biotite continuum microcosms (unsaturated conditions) do not support this purely “exudate/liquid-only” alteration pathway. In addition to the fact
that liquid mono-culture may lead to different fungal phenotypes than those observed in soils (Smith and Read,
2008), another aspect in the approach used by BaloghBrunstad et al., (2008a,b) may further explain the difference
between our respective findings. In Balogh-Brunstad et al.
(2008a), the biotite basal plane dissolution rate was determined from volume-loss calculations due to fungal channelling based on Atomic Force Microscopic (AFM)
observations after exposure of biotite with Suillus tomentosus, another common ectomycorrhiza strain, in liquid
mono-culture. Thus, any fungal-induced element removal
and thus chemical alteration at depth within the biotite basal plane, which we clearly showed in this study to be substantial, could not be considered as this removal occurs
without volume-loss. Therefore, possibly, the mycorrhizal
basal plane dissolution rate in Balogh-Brunstad et al.
(2008a) was actually highly underestimated when compared
to the present study. Nevertheless, fungal basal plane biotite dissolution is unlikely to be as fast as edge dissolution
and, under saturated hydric conditions and in a close system
(i.e., batch liquid mono-culture), the main biotite alteration
is probably edge dissolution mediated by solution acidification essentially due to fungal respiration. However, when
considering the occurrence of mycorrhiza fungi in natural
soils, they are rarely found in saturated conditions. Many
mycorrhizal species are highly hydrophobic and waterintolerant due to their aerobic respiration metabolic needs
(Slankis, 1974). Indeed, mycorrhiza do not colonize tree
roots in waterlogged soils (Theodorou, 1978) and, for instance, even very brief submersion (mins) are enough to inhibit the colonization of P. sylvestris roots by Suillus sp.
(Strenström, 1991). Therefore, substantial fungal acidification of large volumes of water relative to the hypha dimensions, particularly on a relatively short timescale (before it
percolates deeper, i.e., in an open system), is probably a
rare occurrence in soils.
All these lines of evidence indicate that our study marks
a step-change in the understanding of myco-weathering;
surface-bound hypha can alter substantially the biotite basal plane, a surface often quoted as “inert” in terms of abiotic dissolution (Rufe and Hochella, 1999). Furthermore,
we showed that surface-bound mycorrhiza can alter biotite
in a humid environment, yet with no free water present. As
such, these findings shift the way mineral weathering needs
to be conceptualized and studied, with water not being the
sole media controlling mineral alteration reactions.
6. CONCLUSIONS
In our experiments, the zone of contact between hypha
and biotite is an alteration “hot-spot” where chemical
weathering rates were greatly accelerated. This alteration
enhancement is due to the synergy between (i) a biomechanical process during which the biotite lattice structure is disrupted as the hypha grow over the surface as
shown in Bonneville et al. (2009) and (ii) a chemical alteration with hyphal lm-scale acidification, structural Fe(II)
oxidation and cation removal at depth directly underneath
the hypha. Here, we also showed that the elemental transfer
from the biotite into the hyphae can be modelled as a solidstate diffusion process inducing the breakdown of biotite
and the formation of an “altered biotite” layer. With time,
the thickening “altered biotite” layer slows down/limits the
overall mineral weathering rate and thus the release of
nutrient from the mineral substrate. The declining nutrient
flux may potentially trigger further growth of the hypha filament in order to renew/maintain the nutrient flux by interacting with a “fresh” non-altered mineral surface.
It is important to emphasize that this “interfacial”
myco-alteration takes place on the basal plane of biotite,
a low-reactivity mineral surface in terms of abiotic dissolution, yet representing the vast majority of the biotite surface
typically exposed in soils (in itself 7% of the total surface
of exposed Earth’s crust; Nesbitt and Young, 1984). In
soils, mycorrhiza also acquire nutrients from organic matter or litter degradation. However, this recycling of nutrients cannot be perfect and, thus, the maintenance of
vegetation requires prolonged and continual de novo rock
decomposition. Mycorrhiza, because of their unique ability
Tree-mycorrhiza symbiosis accelerate mineral weathering
to “sense” nutrient-rich substrates (Leake et al., 2008) and,
as demonstrated here, to “boost” nutrition acquisition from
raw rock-forming minerals, may therefore contribute largely to the long-term maintenance of vegetation.
Our approach has limitations. It is based on restricted
number of observations along a single hypha grown in an
experimental system that does not account for the whole
complexity of soil biotia involved in the alteration process
such as bacteria, metazoans or even earthworms (Suzuki
et al., 2003) nor does it take into consideration the effect
that these organisms can have on fungal alteration activity
(Balogh-Brunstad et al., 2008b), viability or growth (Rainey, 1991). Our study represents an idealized system where
fungi have no competitor and where the pine tree is fully
dependent on mycorrhiza for nutrient acquisition. Therefore, our results, although the first of their kind, are, at
beast, an upper estimate of the weathering capacity of single hypha in the field. Nevertheless, further work is needed
to incorporate and to upscale alteration patterns observed
at the hypha scale into a broader kinetic framework
describing mycorrhizal weathering activity in soils (Brantley et al., 2011).
ACKNOWLEDGEMENTS
Funding from the UK Natural Environment Research Council
“Weathering Science Consortium” NE/C004566/1 is acknowledged. The authors thank John Harrington and Rik Brydson and
the Leeds Electron Microscopy and Spectroscopy Centre for help
with the FIB sampling and discussions about STEM data interpretation. A. Duran, M. Smits and J. Leake from the Department of
Animal and Plant Sciences and Maria Romero-Gonzalez and the
Department of Civil and Structural Engineering at the U. of Sheffield are acknowledged for their help during the microcosm experiments. The authors acknowledge the travel grant from the UK
Geological Society for A. Bray to travel to the Science Institute
at the University of Iceland to carry out the abiotic flow-through
experiments under the supervision of Dominik Wolff-Boenisch.
Linda Forbes and Eric Condliffe, University of Leeds are thanked
for her help with ICP-MS and EMPA analyses respectively. M.
Krom and C. Peacock are thanked for reviewing the manuscript.
The authors also thank the three anonymous reviewers and the
associate editor C. Daughney for helpful comments during the review process.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.gca.2011.08.041.
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Associate editor: Christopher John Daughney