B Gupta

B Ducharne

G Sebald

T Uchimoto

A Space Discretized Ferromagnetic Model for Non-Destructive Eddy Current Evaluation

Keywords: Eddy current testing (ECT), electromagnetic modeling, magnetic hysteresis

Accurate magnetic material laws are necessary to understand and interpret electrical signals generated by eddy current testing non-destructive control technique. Taking into account simultaneously, both microscopic and macroscopic eddy currents, a numerical resolution is obtained which leads to the global magnetic behavior that can be compared to measured quantities. The 2-D or 3-D (depending on the dimension of the test sample) finite differences space discretization is used for the resolution of the diffusion equation and dynamic hysteresis model is locally simultaneously solved for the microscopic eddy currents (domain wall movements) consideration. Local cracks defects are considered in this model as a variation in the local electrical conductivity and magnetic permeability. The numerical issues such as the proposed solutions for the implementation are described in this paper.

I. INTRODUCTION

T HE development of new electromagnetic designs, such as the improvement of already existing ones, requires precise simulation tools. Similar tools can also be used for the understanding and interpretation of non-destructive eddy current testing (ECT) and Barkhausen noise measurements' electrical signatures.

The ECT consists of setting up a magnetic flux by passing alternating current through a test coil. When this coil is brought closer to the conductive test sample, induced eddy currents are observed and the changes are interpreted in the coil impedance or the voltage drop [START_REF] Yusa | Profile reconstruction of simulated natural cracks from eddy current signals[END_REF].

Numerical simulations are of large interest in ECT domain. By coupling accurate model to experimental results one can precisely define the shape and the position of the defects and cracks in the ferromagnetic material.

Recent scientific investigations around ferromagnetic model mainly focus on coupling space discretization techniques (SDT), finite-elements method, finite differences method (DFM) extended with accurate scalar or vectorial, dynamic or static, and considering hysteresis material law. For this magnetic material law, it seems that the best results come from the extension of the quasi-static hysteresis model (Preisach's model [START_REF] Preisach | Über die magnetische nachwirkung[END_REF]- [START_REF] Chwastek | An alternative method to estimate the parameters of Jiles-Atherton model[END_REF]) to dynamic behavior as a result of the separation losses techniques as proposed by Bertotti [START_REF] Bertotti | Hysteresis in Magnetism[END_REF]. The simultaneous resolution between SDT procedures and hysteresis models can be realized by iterative techniques. One of them is the so-called fixed point scheme. This technique leads to accurate results, but numerical problems of convergence appear in particular cases [START_REF] Kuczmann | The Finite Element Method in Magnetics[END_REF]- [START_REF] Henrotte | Modeling of ferromagnetic materials in 20 finite element problems using Preisach's model[END_REF].

To correctly simulate ECT technique, the electromagnetic model must be able to provide the local and time evolution of both magnetic induction B and excitation field H . The 2-D Manuscript received June 26, 2017; revised November 7, 2017; accepted November 7, 2017. Corresponding author: B. Ducharne (e-mail: benjamin.ducharne@insa-lyon.fr).

Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. resolution gives the evolution of both magnetic fields through the cross section of the test sample, 3-D gives this local information through the whole tested sample. To overcome numerical issues due to fixed point or Newton Raphson's algorithm, solving the diffusion equation (linked to the macroscopic eddy currents) and the dynamic hysteresis model (microscopic eddy currents) simultaneously is proposed.

II. MODEL

A. Diffusion Equation-Macroscopic Eddy Current Contribution

To correctly perform the ECT simulation a coupled resolution of dynamic material law and the magnetic field diffusion equation must be effected [START_REF] Raulet | The magnetic field diffusion equation including dynamic hysteresis: A linear formulation of the problem[END_REF]- [START_REF] Ducharne | Fractional model of magnetic field penetration into a toroidal soft ferromagnetic sample[END_REF]. The magnetic diffusion equation (1) results from Maxwell's equations and the law, which describes the conductive property of the material

-→ rot -→ rot -→ H = -σ • d -→ B dt . ( 1
)
As the magnetic field is considered perpendicular to the cross section, in 2-D (1) becomes

∂ 2 H (x, t) ∂ x 2 + ∂ 2 H (y, t) ∂y 2 = -σ • d -→ B dt . ( 2
)
The diffusion equation gives precise description of the macroscopic eddy currents distribution through the cross section of the test sample.

B. Material Law-Microscopic Eddy Current Contribution

Due to the domain's wall movements, microscopic eddy currents appear through the cross section of a magnetic sample as soon as it is exposed to a varying magnetic field. Below a threshold frequency (in the decreasing direction) hysteresis loop area becomes frequency independent, which can be called as the quasi-static state. Different approaches are available in the literature for the simulation of the quasi-static hysteresis behavior [START_REF] Torre | Hysteresis modeling[END_REF]- [START_REF] Ducharne | Low frequency modelling of hysteresis behaviour and dielectric permittivity in ferroelectric ceramics under electric field[END_REF]. Among all, Preisach's model exhibits the interesting property of being easily reversible. It is indeed relatively easy to switch from H to B as input in the quasi-static hysteresis model. The material law solved in this paper required an inverse hysteresis quasi-static contributions. Preisach's quasi-static model has been used to provide this information.

Preisach's model has widely been used to describe the hysteresis phenomenon in magnetic materials [START_REF] Torre | Hysteresis modeling[END_REF], [START_REF] Torre | Parameter identification of the completemoving-hysteresis model using major loop data[END_REF]. It is based on the following assumptions: the major hysteresis loop of a given ferromagnetic sample consists of an infinite number of square hysteresis loops known as hysterons. Each hystereron has associated threshold reversal fields up α and down β as saturation magnetization switches from (+1) back to (-1) state as illustrated in Fig. 1.

The magnetization M is determined by the contribution of the set of these elementary particles having a distribution function μ(α, β) over Preisach's triangle in the (α, β)-plane (see Fig. 1)

M = Ms • α β h(α, β) • μ(α, β)dαdβ (3) with α β μ(α, β)dαdβ = 1 and -H S ≤ α ≤ H S, β ≤ α
where Ms is the magnetization value when H = HS. In order to model precisely the quasi-static magnetic material behavior, it is necessary to accurately determine the weighing distribution function μ(α, β) from experimental data. There are mainly two ways to determine this distribution function. The first way assumes that the distribution function has a particular form (Lorentzian, Gaussian) and then determines the parameters of the chosen function in order to depict the average hysteretic behavior. The second way discretizes the distribution function in a finite set of values which are determined by suitable experimental data. In this paper, the second option is chosen which is expected to provide higher accuracy.

In this paper, two techniques have been tested for the acquisition of the discretized distribution function: The centered cycle technique as described in [START_REF] Torre | Parameter identification of the completemoving-hysteresis model using major loop data[END_REF] and Biorci's et al. [START_REF] Biorci | Analytical Theory of the behaviour of ferromagnetic materials[END_REF] method. Both techniques provide relatively correct simulation results. A higher number of experimental data is required by the first technique but a correct behavior can be reached with a lower size of the discretized distribution implementation and this means a simpler and reduced memory management.

Fig. 2 shows Preisach's space discretized distribution function obtained with Biorci's method for the tested sample. If only the quasi-static contribution material law is considered in the diffusion equation, the resolution is easy but leads to inaccurate results. In this case, the dynamic effects related to the high-frequency dynamics of the wall motions are neglected. The dynamic contribution is considered in the material law by adding to the quasi-static lump model the product of a damping constant ρ to the time domain derivation of the induction field B

ρ • d B(t) dt = H dyn (t) -f -1 static (B(t)). ( 4
)
This product is homogeneous to an equivalent excitation field H .

C. Simultaneous Resolution

The idea of the simultaneous resolution comes from the material law equation (4) (microscopic eddy current dynamic contribution). We note the dB/dt term in (4) constitutes also a part of the second term in diffusion equation [START_REF] Preisach | Über die magnetische nachwirkung[END_REF]. It becomes natural to replace the dB/dt term of (2) by the second member of (4). A new formulation of the diffusion equation is obtained then

∂ 2 H (x, t) ∂ x 2 + ∂ 2 H (y, t) ∂y 2 = -σ • H dyn (t) -f -1 static (B(t)) ρ . ( 5
)
The DFM is used for the space discretization resolution. Without the permeability calculation step, we avoid a lot of numerical issues. Fig. 3(b) gives a first illustration of the simulation configuration. We assume a high section yoke of infinite permeability surrounded by a large turn's number coil supplied by a perfect current source. This experimental inductor creates a deterministic (sine waveform, triangular waveform …) surface tangent excitation field H. Fig. 3(c) shows the first simulation results. The 3-D configuration is simulated here. As illustrated Fig. 3(a), in this first simulation, we consider Dirichlet's boundaries conditions (imposed H = H surf ) on three rows of spots on the top of the sample, other Dirichlet's boundaries conditions (imposed H = 0) on the sides and on the bottom of the geometry and finally and Neuman's boundaries conditions (d H/dY = 0) on the remaining spots on the top of the geometry. Fig. 3(c) shows first the induction distribution for various levels of excitation field H . The hysteresis loop in Fig. 3(b) is related to the evolution of the average induction B (through the cross section of the sample, xy direction) versus the imposed sine waveform tangent excitation field H . The numerical result gives the evolution of both the local induction field B i and H i for each spots of the simulation.

D. Defects Taken Into Account

All metals (including ferromagnetic materials) contain defects. Different aspects of defects exist. It includes holes, cracks, segregation, inclusions, surface marks, or undesirable metallurgical changes. From a physical point of view, defects in the matter are characterized by a local variation of the physical properties (permittivity ε d , permeability μ d , conductivity σ d ). In our simulation, defects will be considered through their physical properties, i.e., a local variation of μ and σ in the finite differences resolution of (5). In the case of crack defects, as cracks are filled with air a permeability equal to the vacuum permeability μ 0 and a very weak conductivity is considered. III. SIMULATION RESULTS Fig. 4 shows numerical results obtained considering successively:

1) the macroscopic eddy current contribution; 2) the microscopic eddy current contribution;

3) both contributions. The model has been set using soft iron silicon material referenced M400P50 (Euronorm) properties and excitation frequency has been set to 200 Hz. Table I gives the manufacturer's information for this material.

The conductivity is identified to the value of 4.25 10 6 (•m) -1 and the thickness is 0.5 mm. In this simulation, B is calculated from the average value of all the local inductions through the cross section of the sample (xy plane, one spot in the z-direction).

The next numerical result displayed shows the whole simulated experimental setup, including crack defect and magnetic field B distribution through the 2-D cross section of the test sample (Fig. 5). In this simulation, the crack defect is considered as a geometrical space inside the material where permeability and conductivity are equal to the vacuum ones. B distribution considering successively: the microscopic eddy currents, the macroscopic eddy currents, both contributions. Fig. 6 shows the induction distribution, considering 2-D simulation. In Fig. 6 (left), where just the microscopic eddy currents contribution is taken into account, the induction is homogeneously distributed between the defect and the surface of the sample. In the middle, where just the macroscopic eddy currents are considered, we can observe a shielding effect which borders the magnetic induction in a very thin layer on top of the sample.

Finally, Fig. 6 (right) shows the induction distribution as both contributions are taken into account. In Fig. 6, even if the magnetic induction distribution is limited comparing to the first one, the shield effect due to the macroscopic eddy current is also limited. Fig. 7 shows the hysteresis loop derived from the measured electrical quantities (voltage and current) in both cases: with and without defect presence. After a large number of simulations, we have noticed that the differences observed between the hysteresis loops obtained with and without the defect presence is as imagined directly linked to the frequency of the surface excitation field, the size of the defect and its space position through the cross section.

IV. CONCLUSION

As a conclusion of this paper, we can say that accurate magnetic material laws are necessary to understand and interpret electrical signals generated by ECT non-destructive control technique. By taking into account simultaneously, both microscopic and macroscopic eddy currents authors succeed to obtain a very accurate numerical resolution of the local magnetic quantities (H i , B i ). The space average of these local quantities is obtained which leads to a global magnetic behavior that can be compared to measured quantities. The 2-D or 3-D (depending on the dimension of the test sample) finite differences space discretization is used for the resolution of the diffusion equation and a dynamic hysteresis model is locally simultaneously solved for the microscopic eddy currents (domain wall movements) consideration. Cracks defects are considered in the model as a local variation of the physical properties (electrical conductivity and magnetic permeability). The numerical solutions allow to give some conclusions dealing with the influence of each contributions (macroscopic and microscopic eddy currents) on the evolution of the average measurable quantities. The influence of the geographic position such as the size of the defects can also be studied. Finally, the model can also be used as a tool to precisely define the excitation waveform (frequency, amplitude) required for the control of a given sample.

Fig. 1 .

 1 Fig. 1. Fundamental switching operator-hystereon.

Fig. 2 .

 2 Fig. 2. Preisach's distribution function.

Fig. 3 .

 3 Fig. 3. (a) Simulation's boundary conditions. (b) Simulation's configuration. (c) Induction distribution.

Fig. 4 .

 4 Fig. 4. Hysteresis loop considering different losses contribution.

Fig. 5 .

 5 Fig. 5. Comparison simulation/measure for increasing frequency condition.

Fig. 7 .

 7 Fig. 7. B(H ) hysteresis loop. Defect (in red), no defect (in blue).


Nolwenn Bougon
email: nbougon@gmail.com.

Luc Aquilina
email: luc.aquilina@univ-rennes1.fr

Jérôme Molenat

Dominique Marie

Yannick R Delettre

Emilie Chancerel

Philippe Vandenkoornhuyse

Influence of depth and time on diversity of free-living microbial community in the variably saturated zone of a granitic aquifer

Keywords: hydrology, microbial community, 16S rRNA gene, T-RFLP, water table fluctuation zone

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Aquifer quality is considered as a major point in water protection and directives management.

Beside these policies, the understanding of ecosystem health is crucial [START_REF] Griebler | Ecological assessment of groundwater ecosystems -Vision or illusion?[END_REF].

If stream water quality has been investigated and monitored for a while, aquifer quality has been neglected across the world (e.g. [START_REF] Korbel | A Tiered framework for assessing groundwater ecosystem health[END_REF]. Aquifers are not anymore considered as a water resource but also as a living ecosystem which controls the water quality (e.g. [START_REF] Griebler | Ecological assessment of groundwater ecosystems -Vision or illusion?[END_REF]. Investigations of groundwater microbial diversity and ecosystem have increased in the last few years (e.g. [START_REF] Griebler | Microbial diversity in groundwater ecosystems[END_REF][START_REF] Bougon | Influence of hydrological fluxes on the structure of nitrate-reducing bacteria communities in a peatland[END_REF]).

However our understanding of the microbial diversity involved in key biogeochemical processes for groundwater functioning is still limited. The knowledge of groundwater habitats is mainly framed under the characterization of hydrological processes including fluxes, chemistry and geological hetereogeneity [START_REF] Madsen | Groundwater microbiology: subsurface ecosystem processes[END_REF].

Microbial community (diversity and abundance) in groundwater seems to be related to complex relationships between organic contaminants, nutrients and hydrogeology [START_REF] Chapelle | The significance of microbial processes in hydrogeology and geochemistry[END_REF][START_REF] Christensen | Biogeochemistry of landfill leachate plumes[END_REF][START_REF] Griebler | Microbial diversity in groundwater ecosystems[END_REF][START_REF] Pearce | Subsurface characterization of groundwater contaminated by landfill leachate using microbial community profile data and a non parametric decision-making process[END_REF]. Despite our understanding about the impact of contamination on microbial diversity (e.g. [START_REF] Röling | Relationship between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer[END_REF], little is known about the impact of physical processes as hydrologic changes on the microbial diversity in groundwater. Studies on porous media revealed that bacterial distribution was mostly affected by passive transport processes (e.g., advection, diffusion or filtration) [START_REF] Balkwill | Microbial communities in high and low recharge environments: implications for microbial transport in the vadose zone[END_REF][START_REF] Huysman | Water-facilitated transport of bacteria in unsaturated soil columns: Influence of cell surface hydrophobicity and soil properties[END_REF][START_REF] Trevors | Transport of a genetically-engineered Pseudomonas fluorescens strain through a soil microcosm[END_REF]) and several models have focused on understanding and predicting microbial transport in natural porous and high nutrient media (e.g. [START_REF] Ginn | Processes in microbial transport in the natural subsurface[END_REF][START_REF] Harvey | Role of physical heterogeneity in the interpretation of small-scale laboratory and field observations of bacteria, microbial-sized microsphere, and bromide transport through aquifer sediments[END_REF][START_REF] Murphy | Modeling microbial processes in porous media[END_REF][START_REF] Rockhold | Coupled microbial and transport processes in soil[END_REF]). Few attempts have been made to characterize microbial distribution and activities in unsaturated or variably saturated systems. More research seems to be required to better understand microbial responses to hydrological changes [START_REF] Fisk | Microbial activity and functional composition among northern peatland ecosystems[END_REF].

Within aquifers, knowledge of the physical processes occurring in the fluctuation zone is also important to our understanding of biological and biochemical processes. This zone of water table fluctuation is unique with highly variable saturation conditions controlling the availability of nutrients to microbial organisms [START_REF] Legout | Solute transfer in the unsaturated zone-groundwater continuum of a headwater catchment[END_REF]). Although considered mainly as a transfer zone it also exhibits biogeochemical reactions such as the various denitrification processes [START_REF] Dunn | Demonstration of microbial transport into the capillary fringe via advection from below the water table[END_REF][START_REF] Legout | Investigation of biogeochemical activities in the soil and unsaturated zone of weathered granite[END_REF]. Although the recharge process has been widely investigated (e.g. [START_REF] Vries | Groundwater recharge: an overview of processes and challenges[END_REF], few studies have focused on the processes governing water flow and solutes transfer in the fluctuation zone of the water table. The different processes recently identified in this zone include: rapid vertical fluxes from soil to water table [START_REF] Legout | Solute transfer in the unsaturated zone-groundwater continuum of a headwater catchment[END_REF], lateral fluxes along the hillslope below the soil [START_REF] Derby | Field-scale preferential transport of water and chloride tracer by depression-focused recharge[END_REF][START_REF] Haria | Evidence for deep subsurface flow routing in forested upland Wales: implications for contaminant transport and stream flow generation[END_REF][START_REF] Rawlins | Absence of preferential flow in the percolating waters of a coniferous forest soil[END_REF]) and cyclic chemistry control by groundwater fluctuations [START_REF] Fretwell | Redistribution of contaminants by a fluctuating water table in a micro-porous, double-porosity aquifer: Field observations and model simulations[END_REF]. Hydrological conditions in the variably saturated zone present both seasonal variations and a vertical contrast with the permanently-saturated zone. With a seasonal rhythm, conditions change from a permanentlysaturated zone in depth to a variably-saturated medium, close to the soil. The vertical contrast between the two zones is characterized by a water table and chemical characteristics seasonally close to the soil in the variably-saturated zone and more constant chemical characteristics in the deeper permanently-saturated zone. Groundwater ecosystems are generally characterized by low concentration of dissolved organic carbon and by the lack of light. These factors are amongst the most differences with the soil ecosystem (Gibert 1994, Griebler and[START_REF] Griebler | Microbial diversity in groundwater ecosystems[END_REF]. The investigation of these 2 zones presents an ecological interest by presenting differing environmental conditions but also potential seasonal connections.

In this study we (i) analyse the temporal and vertical changes in the microbial communities (i.e. distribution and structure) related to hydrological variations in the water table fluctuation zone of a granitic aquifer and immediately below. The studied aquifer is characterized by nitrate concentrations [START_REF] Legout | Investigation of biogeochemical activities in the soil and unsaturated zone of weathered granite[END_REF] exceeding regularly the upper limit of 50 mg.L -1 recommended by EU groundwater directive. [START_REF] Legout | Investigation of biogeochemical activities in the soil and unsaturated zone of weathered granite[END_REF] also demonstrated a denitrification potential in the fluctuation zone of this particular catchment. In this context, the work presented herein aims (ii) to address a possible link between the nitrate concentration in groundwater and changes in nitrate reducing community. To do so, molecular tools allowing rapid profiling of microbial community were used. To assess the 'global' diversity and 'nitrate reducing' diversity, the 16S rRNA and narG genes were used as targets respectively. The narG gene encoding a subunit of the nitrate reductase that catalyses the reduction of nitrate to nitrite is found in many bacteria (e.g. Firmicutes, Actinomycetes and Proteobacteria) and Archaea [START_REF] Chèneby | Genetic characterization of the nitrate reducing community based on narG nucleotide sequence analysis[END_REF][START_REF] Packman | Hyporheic exchange with gravel beds: basic hydrodynamic interactions and bedform-induced advective flows[END_REF]). We hereafter define 'nitrate-reducing bacteria' as those bacteria possessing the capacity, within their genome, to transform nitrate to nitrite. As a first approximation, we assumed that 'nitratereducing bacteria' could be assessed by analysing narG gene diversity, although other genes harbour the ability to perform this function.

The analysis of temporal and spatial changes in the microbial communities is assessed herein from water samplings using a pan-flute device. This device allows investigating the unsaturated zone by intercepting water at 6 m, 8m, 10 m and 15 m depth by different piezometers. The analysis of the communities of free-living bacteria was coupled with a characterization of chemical and hydrological properties over 2.4 years, during 3 hydrological cycles at each depth.

Material and methods

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273..01273.x

Site description

The Kerrien catchment covers an area of 0.12 km², is located in an agricultural site, mainly devoted to pasture and dairy cows, in south-west Brittany (47°57'N-4°8'W, 2347.35N -117.5E, see Fig. S1 in Supplementary material). The outlet is about 10 km from the sea [START_REF] Legout | Etude des mécanismes hydrologiques et biogéochimiques de la recharge des nappes à surface libre[END_REF][START_REF] Martin | Mécanismes hydrologiques et hydrochimiques impliqués dans les variations saisonnières des teneurs en nitrates dans les bassins versants agricoles[END_REF][START_REF] Martin | Seasonal and inter-annual variations of nitrate and chloride in stream waters related to spatial and temporal patterns of groundwater concentrations in agricultural catchments[END_REF][START_REF] Molénat | Etude du rôle de l'interface sol-nappe sur les processus de Transfert de solutés, d'érosion chimique et sur la diversité bactérienne dans un bassin versant SONATE)[END_REF]). This catchment serves as a national research observatory for environmental investigations (INSU-CNRS and INRA observatory, http://www.inra.fr/ore_agrhys) which has been intensively monitored for hydrochemistry for more than 10 yrs. The different compartments of the aquifer and their contribution to catchment functioning has been highlighted in previous research [START_REF] Martin | Mécanismes hydrologiques et hydrochimiques impliqués dans les variations saisonnières des teneurs en nitrates dans les bassins versants agricoles[END_REF][START_REF] Martin | Seasonal and inter-annual variations of nitrate and chloride in stream waters related to spatial and temporal patterns of groundwater concentrations in agricultural catchments[END_REF][START_REF] Molénat | Etude du rôle de l'interface sol-nappe sur les processus de Transfert de solutés, d'érosion chimique et sur la diversité bactérienne dans un bassin versant SONATE)[END_REF][START_REF] Pierson-Wickmann | High chemical weathering rates in first-order granitic catchments induced by agricultural stress[END_REF]) along with solute transfer in the unsaturated zone [START_REF] Legout | Solute transfer in the unsaturated zone-groundwater continuum of a headwater catchment[END_REF]).

The climate is oceanic with a mean annual precipitation of 1167 +/-195 mm. The bedrock is fissured, fractured granite, overlain by weathered material within which the shallow groundwater accumulates and fluctuates [START_REF] Legout | Solute transfer in the unsaturated zone-groundwater continuum of a headwater catchment[END_REF]). The weathered granite lies between 0.7 and 1.2 m below the soil surface. The soils are mainly sandy loams. The depth of the water-table ranges from 2 to 8 m below the soil surface. A plot with six piezometers at 3 to 15 m depth (3, 5, 6, 8, 10 and 15 m) was set up in the weathered material. These piezometers consisted of PVC tubes with a 1-m-thick perforated screen at one end, which allowed the collection of water from a 1-m-thick groundwater layer. Whilst only one piezometer set was investigated for microbiology, three other similar plots are located along the slope of the catchment and a further line of three other plots are located 100 m to the West as a replicate. The chemical concentrations have been monitored for 10years and variations of the vertical chemical gradients have been observed along the slope as well as spatially. This

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x monitoring (Legout et al. 1997[START_REF] Rouxel | Seasonal and spatial variation in groundwater quality along the hillslope of an agricultural research catchment (Western France)[END_REF]) provides a good characterization of the chemical variations with depth and the adequacy of the sampling design.

Analytical methods

The piezometric level in this plot in the Kerrien catchment was continuously recorded from ) by ionic chromatography (Dionex DX120) in the CAREN-Geosciences Rennes laboratory facilities.

The level of uncertainty for the major anion concentrations was below 4 %. The total dissolved carbon (TDC) and dissolved inorganic carbon (DIC) were analyzed with a Shimadzu TOC 5050A (quantification limits: 0.85 mg L -1 ; detection limits: 0.08 mg L -1 ). The dissolved organic carbon (DOC) was calculated from TDC and DIC (i.e. DOC=TDC-DIC).

The organic carbon was not measured during the 3 rd and 5 th campaigns due to technical problems.

Groundwater sampling

Groundwater samples were collected at the Kerrien Site from the piezometers at 6, 8, 10 and 15 m depth. This pan-flute device has the advantage to allow the recharge process, seasonal variation and vertical gradient to be monitored, and to allow easy water sampling. For the latter, an ISMA® sampler was used during the first two field campaigns but was replaced in subsequent campaigns by a Grundfos MP1 immersed pump to ensure better efficiency. The ISMA sampler was rinsed three times with deionized water and once with groundwater between each well sampling. The Grundfos pump was rinsed with groundwater. Two liters were collected from each piezometer. The fourth field sampling campaign was characterized by a lack of water in the 6 m depth piezometer. The microbial sampling protocol was adapted from a marine sampling strategy [START_REF] Massana | Phylogenetic and Ecological Analysis of Novel Marine Stramenopiles[END_REF]. Each 2 litre sample was subjected to cascade filtration using three thresholds i.e., 10µm (Isopore™ -TCTP, membrane filters), 5µm (Isopore™ -TMTP, membrane filters) and 0.22µm (Sterivex™ -GV, Millipore) via a peristaltic pump. This step was necessary to concentrate the microbial biomass before nucleic acid extraction and was done immediately after sampling in the field. The filters were placed in Petri dishes and stored at 0°C in a refrigerant container, and then at -20°C in the laboratory prior to DNA extraction. DNA was extracted in the 96 hours following the sampling and then stored at -80°C.

The potential effect of well structure (tube and gravel surrounding the tube), on water biochemistry and bacterial diversity, was estimated by sampling before and after purging the borehole (i.e. replicates, see below for details). This purge consisted of removing a water volume equivalent to three times the well volume i.e. a minimum of 90 liters, with a Grundfos MP1 immersed pump. The pump was made of inert materials to avoid any contamination of the pumped liquid. For a given well, we expected the local heterogenity being the highest before and after purge. Thus, for each piezometer we decided to perform 2 true-sampling replicate one before and one after purge.

Bacterial counts

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x The bacteria were counted by flow cytometry on a FACSort (Becton Dickinson). Two milliliters of water placed in a sterile cryovial were fixed with 7µL of glutaraldehyde in the field and stored in the refrigerant container during the sampling period. Back in the laboratory, the cryovials were dipped in liquid nitrogen and stored at -80°C before analyses.

This was done similarly for all the samples.

DNA Extraction

DNA was extracted from the filters (see above, 'groundwater sampling') by physical disruption of cells by bead-beating at a frequency of 30 oscillations per sec for 27s (Retsch MM400, Germany) and by using the DNeasy Plant miniKit (Qiagen) according to the manufacturer's protocol. This method was chosen from preliminary tests using different strategies as beat-beating frequencies and durations, standard CTAB extraction method and Dneasy Plant mini kit (results not shown).

Primers

A set of narG sequences of bacteria and Archaea, available from the GenBank database, were aligned and scanned for conserved regions that could provide suitable primer targets. Two degenerated primers, narGr (5'-TTTTCSTACCAGGTCGC-3') and narGf (5'-TAYGTCGGGCAGGAAAA-3'), were selected from the analysis to amplify a specific 840 bp fragment of the narG gene [START_REF] Bougon | Influence of hydrological fluxes on the structure of nitrate-reducing bacteria communities in a peatland[END_REF]).

The universal bacterial primers (Orphan et al. 2000, modified gene amplification, the cycling regime was an initial step at 94°C for 2 min 30s, followed by 35 cycles at 94°C for 30 s, 52°C for 1 min, and 72°C for 50 s. The amplification ended by a final extension step at 72°C for 10min. For the narG gene amplification, the cycling regime was an initial step at 94°C for 1 min 30 s; followed by 36 cycles at 94°C for 30 s, 51°C minus 0.1°C per cycle for 1 min 15 s, and 72°C for 1 min 30 s. The program ended by a final extension step at 72°C for 10 min. All the amplifications were done in an MJ Research PTC-100 TM thermocycler. Amplifications were checked by electrophoresis (4 µl of PCR product in 1% agarose in 0.5×TBE buffer).

Terminal-Restriction fragment length polymorphisms (T-RFLPs)

Environmental independent PCRs and independent digestions). These true replicates eliminated all inconsistent peaks and increased the number of observed T-RFs per T-RFLP profile by retaining any very small but consistently present peaks. Thus there was no need to apply a minimal peak cut-off height. Background noise was determined by comparing the replicates of the profiles of the peaks [START_REF] Vandenkoornhuyse | Co-existing grass species have distinctive arbuscular mycorrhizal communities[END_REF]. By using this strategy, between 1

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j. 1574-6941.2011.01273.x to 35 TRF and 1 to 27 TRF were detected respectively using HinfI and StyI. It should also be underlined that the reproducibility was excellent in between replicates (i.e. maximum divergence observed in terms of presence/absence was 9.5 %). However, we have noticed important differences in the peak intensities. Even more problematic, we observed differences in peak intensities even for identical samples ran in different capillaries. Thus, only presence/absence of a given T-RF was analysed.

Clone library construction and sequencing

The purified PCR products from 16S rRNA gene amplifications were cloned. The first step was to add a non-complemented A to the amplicons using an A-overhanging postamplification procedure. One unit of Taq DNA polymerase (Invitrogen), 1x concentrated Taq DNA polymerase buffer (Invitrogen) and 0.1 mM of dATP was added to the purified products, and incubated for 15 min at 72°C. The ligation mixture containing 1.5 µL of 3'Atailed PCR products, 1.0 µL of pGEM-T vector, 5.0 µL of rapid pGEM-T buffer and 1 µL of T4-DNA ligase (pGEM-T cloning kit, Promega) in a final volume of 10 µL was incubated overnight at 4°C. Transformation of E. coli DH5α competent cells (Invitrogen) was carried out following the manufacturer's instructions. The positive clones were screened by PCR amplifications targeting sequences of the vector with the primers T-7 and SP-6. Cloned DNA fragments (871 bp) were sequenced for the 2 strands on an automated sequencer ABI Prism® 3130xl DNA analyser (Applied Biosystems). This cloned library was built to highlight whether or not an original microbial diversity exists in the studied aquifers. The purpose of this analysis was thus not to compare bacterial communities among samples.

Sequence data analyses

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol. All the contigs were produced using Sequencher 4.7 (GeneCodes) and all the nucleotides for each sequence were checked by the software and corrected manually when required. After cloning and sequencing, chimeras were eliminated by analyzing the sequences with the chimera check program on the Ribosomal RNA Database website. A multiple alignment of the checked 16S rRNA gene sequences was performed using ClustalX 1.81 [START_REF] Thompson | The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]) and refined by eye. Each sequence was compared with the NCBI sequences using the Basic Local Alignment Search Tool (BLASTn).

Three techniques were used to calculate the phylogenetic relationships between 48 known bacterial sequences (i.e. the highest BLAST hits, closest phylogenetic neighbours and additional sequences belonging to the different bacterial phyla) and the 107 environmental sequences. These were (i) phenetic reconstruction using the Kimura 2-parameters (K2P) distance model and clustering using the neighbor joining (NJ) algorithm [START_REF] Saitou | The Neighbor-Joining method: a new method for reconstructing phylogenetic trees[END_REF] with Clustal X 1.81; (ii) maximum parsimony (MP) reconstruction [START_REF] Swofford | PAUP: Phylogenetic Analysis Using Parsimony, Version 4.0 BETA[END_REF] done by a heuristic tree search with tree bisection and reconnection (TBR) as swapping algorithm and the use of 500 replicates of random addition of sequences to find the shortest tree and (iii) maximum likelihood (ML) reconstruction using the GTR+I+G distance model, selected from modeltest analyses [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF]. The MP and ML Phylogeny were calculated using PAUP 4.0β10 (Sinauer associates). Bootstrap support at the nodes was estimated from 500 pseudo-replicates for MP, 100 for ML and 1000 replicates for the NJ phylogenies. The chosen outgroup was an uncultured Crenarchaeota (AJ347774).

Patterns of microbial community analyses

The information provided by the different T-RFs was combined by applying a multivariate approach based on principal component analysis (PCA). Non-centred PCAs were calculated because they maintain the mean differences between variables (i.e. frequency of observation of each T-RF, [START_REF] Vandenkoornhuyse | Co-existing grass species have distinctive arbuscular mycorrhizal communities[END_REF]. The statistical reliability of the data was determined from the PCAs by computing confidence ellipses and stars joining the gravity centre to each dot of the considered modality in the vectorial space [START_REF] Sokal | Biometry: the Principles and Practice of Statistics in Biological Research[END_REF].

The PCA analyses were implemented with the ADE-4 software package [START_REF] Thioulouse | ADE-4: a multivariate analysis and graphical display software[END_REF].

Nucleotide sequence accession numbers.

The 16S rRNA gene sequences recovered in this study were deposited in the GenBank nucleotide sequence database under the accession numbers FJ429673 to FJ429781.

Community comparisons

Genetic diversity (hereafter meaning species richness) was estimated using the Shannon index H'. The proportion of species i in relation to the total number of species (P i ) was calculated, and then multiplied by the logarithm of this proportion (log 2 P i ). The resulting product was summed across species, and multiplied by -1:

(1

)
where Pi is the frequency of a given T-RF. This synthetic index was used to compare the species richness of the nitrate-reducing communities in relation to the different forcing variables.

The Morisita-Horn index provides a similarity measurement based on presence-absence data only [START_REF] Magurran | Ecological diversity and its measurements[END_REF]). This index is not influenced by sample size or richness [START_REF] Wolda | Similarity indices, sample size and diversity[END_REF].

The Morisita-Horn index was calculated as follows:

(2)

CmH= 2 ∑ i= 1 n (ani×bni)( da+db) ( aN×bN ) H'=-∑ i=1 n Pi×log 2 Pi;
Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol. where CmH is the Morisita-Horn index of similarity, N is the total number of species for communities A and B; aN and bN are the total number of individuals of all species collected from community A and B, respectively; ani and bni are the number of individuals of the i th species collected from community A and B, respectively. The denominator incorporates two terms which are defined as:

(3) and This index was used to compare sample pairs such as two campaigns for a given depth and two depths for a given campaign. The index was calculated using EstimateS software [START_REF] Colwell | EstimateS: Statistical estimation of species richness and shared species from samples. Version 7.5. User's Guide and application[END_REF]. The Morisita-Horn index ranges from 0 (no congruence between samples) to 1 (full congruence). As species abundance is taken into account, the index of communities containing the same species but in different abundances will be less than 1 [START_REF] Heijs | Microbial Community Structure in Three Deep-Sea Carbonate Crusts[END_REF][START_REF] Juottonen | Methanogen communities and Bacteria along an ecohydrological gradient in a northern raised bog complex[END_REF].

Results

Hydrogeology/Hydrochemistry

Hydrogeology

The water-table fluctuation depth ranged from 2.1 to 6.1 m below the soil surface (Fig. 1) with long-term chronicles revealed that the first two monitoring years could be considered as relatively dry (results not shown).

during

Groundwater chemistry

The groundwater was characterized by constant temperature, slight acidity (Tab. 1), low total organic carbon concentrations and high nitrate concentrations (Tab. 1). For the sake of clarity, we mainly used two depth measurements as a reference to the variably-saturated zone (6 m depth) and the permanently-saturated zone (15 m depth). Two chemical trends were apparent within the data set: a relatively stable chemical composition in the permanent aquifer (15 m depth) and time-related chemical variability in the water-table fluctuation zone (6 m depth) (Fig. 1, Tab. 1). Such evolution is characteristic of this catchment. It was observed for several years along the piezometer plots replicates placed in the hillslope (Legout et al. 1997;[START_REF] Rouxel | Seasonal and spatial variation in groundwater quality along the hillslope of an agricultural research catchment (Western France)[END_REF].

Groundwater samples at 6 m depth showed variations in chloride concentrations ranging from 17.7 mgL -1 (12/20/2006) to 47.2 mgL -1 (01/27/2005) (Fig. 1). These variations were also correlated with nitrate (∆=31.2 mgL -1 ), sulphate (∆=43.2 mgL -1 ) and dissolved oxygen (∆=5 A slight vertical gradient was noted for oxygen, with relatively higher concentrations at 6 and 8 m depths (Tab. 1). The hydrological cycle was also characterized by higher oxygen concentrations at all depths during the periods of high water table (Ker3 and Ker5).

Purge effect

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol. The bacterial cell counts were estimated by flow cytometry. The number of cells ranged from 2.3 10 5 to 2.5 10 6 per mL and did not show any clear gradient with depth (Fig. S2 in Supplementary material). Comparison of the cell counts before and after purging the borehole revealed modifications related to purging i.e. a decrease in bacterial cells at 6, 8 and 10 m.

However, only slight differences were apparent between T-RFLP analyses of the community structure of free-living bacteria before and after the purges (Fig. S3 in Supplementary material). Homology in diversity signatures among sample pairs ranged 78% to 98%. Thus, ellipses drawn on the PCA vectorial space overlapped considerably indicating no significant statistical differences. Heterogenity between true replicates was so low that our results

suggested that the community of free-living bacteria analysed in each sample could be considered as representative of the groundwater community. It also confirms the reliability of the sampling design.

Phylogenetic analyses

A sequence analysis of a fragment of the 16S rRNA genes was performed to better understand the phylogenetic diversity of microorganisms in groundwater. The aim of this analysis was not to compare communities. Accordingly, the sequencing depth was not the same in all the samples. Twenty seven different phylotypes were obtained (Fig. 2). The resulting rarefaction curves (not shown) indicated that the number of phylotypes was probably under-estimated.

Strikingly, 6 of these 27 phylogenetic species did not belong to a known class or phylum (phylotypes 1, 2, 3, 6, 9, 11; Fig. 2). Most of the observed phylotypes in the Proteobacteria phylum were also new. Only 6 of the 27 phylotypes (phylotypes 5, 14, 16, 17, 20, 24; Fig. 2) displayed a high level of homology with known bacteria. The diversity at the Kerrien soilaquifer interface seemed to be original and possibly specific to this environment. This could result from (i) the use of new primers [START_REF] Vandenkoornhuyse | Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA[END_REF] date and a related bias in the knowledge , (ii) the very limited number of studies of microbial diversity published for this particular environment. The diversity of soil microorganisms is now better understood. If the detected microorganisms had colonized the aquifer habitat after percolation from the soil surface, a more classical bacterial diversity would be expected. The bacterial communities at 6 m contained mainly α-, β-, and γproteobacteria phylotypes which are closely related to Pseudomonas and to a Rickettsiella known to be abundant in soils. None of the results obtained during sub-sampling of the 16S rRNA gene sequences contradicted the hypothesis that bacterial community composition should be modified with depth or that the microbial community at 6 m would be more closely connected to the soil community than microbial communities from deeper samples.

Comparisons of microbial communities based on 16S rRNA gene diversity signatures

T-RFLP analyses provide an interesting approach for assessing the microbial diversity and dynamics of microbial communities that occur temporally or spatially in response to environmental perturbations [START_REF] Liu | Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[END_REF]. The 2 enzymes chosen to digest the 16S rRNAs amplicons generated 720 different T-RFs. This high level of detected polymorphism suggests the presence of considerable diversity. The experimental design was defined to address the hypothesis of possible differentiation of the microbial community in connection to depth and time.

Time (Fig. 3) affected the community in both the long and short term. As regards the sampling date, the bacterial community compositions of the closest samplings (i.e. Ker4/Ker5 and Ker2/Ker3) showed greater similarity than those of the more distant (Ker1/Ker5). This can be seen in the proximity of the Ker4 /Ker5 campaigns and in the strong difference between Ker1 and Ker5 at 15 m (Fig. 3) and indicates a time-dependent community structure.

This effect is also apparent in the similarity index (Tab. 2a For the Ker2 and Ker4 campaigns, the index rose to 60% similarity between the 8 m and 15 m depths (Tab. 2b). Two different communities i.e. one at 6 m and the other at 8/10/15 m could be distinguished from the similarity indexes.

narG gene diversity

The non-centred PCA did not indicate any clear structuring of the nitrate reducer community in relation to depth (Fig. 4). However, some modifications of the nitrate reducer community were independent of depth (i.e. the difference between the Ker1 & Ker2 communities and those of Ker4 & Ker5,Fig 4). These results accord with the16S rRNA gene analysis which indicated dynamic changes in the microbial community over time.

Recharge hydrological conditions

In this section, we investigated the nature of physical water transfer mechanisms and the effects of these physical hydraulic recharge processes on chemical concentrations in order to

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x provide a clear framework to the investigation of the relationships between microbial communities and hydrological conditions. Chemical composition, as regards nutrient and oxygen concentrations, was relatively homogeneous, with time and depth, throughout the entire groundwater section. No variation in the physico-chemical parameters (dissolved oxygen and pH) was detected between the water table fluctuation zone (3-8 m) and the permanently saturated zone (8-15 m) (Tab. 1). As mentioned above, we used the 6 and 15 m depth as representative of the variably-and permanently-saturated zones. Slightly lower oxygen concentrations were found at 15 m depth (Tab. 1) but the redox conditions remained relatively oxidant whatever the depth and time. The Kerrien subsurface is characterized by a homogeneous redox environment. In contrast it is apparent from the chloride concentration that hydraulic processes are important and modify the anion concentration (Fig. 1). The nitrate and sulphate concentrations also exhibit variations but these variations have no deep impact on biological activity. Nitrates are always available and no reducing conditions develop. A seasonal variation in Cl -was found at 15 m but was limited compared to that at 6 m. The high Cl -variations observed at 6 m were strongly related to water table fluctuations.

During periods when the water table was low, the chloride concentrations at 6 m were higher than at any other depth whereas during periods when the water table was high, the chloride concentrations at 6 m were lower than at any other depth (Fig. 1). The observed variations in chloride concentration can be explained by the recharge processes. Different water compartments i.e. mobile porosity and immobile porosity are mobilized during recharge [START_REF] Legout | Solute transfer in the unsaturated zone-groundwater continuum of a headwater catchment[END_REF] (ii) At 15 m depth, seasonal variation is much more limited and mainly related to vertical transfer from the variably saturated zone. Although this variation is also time-dependent, it is mainly due to vertical transfer from the variably saturated zone to the permanently saturated zone. This description provides an interesting framework to investigate the effects of physical transfer on microbial communities since the redox parameters are mainly constant from surface to depth and nitrates are always available whatever the conditions. In the 2 zones, 2 physical recharge processes occur. From diffusion to advection from soil in the variablysaturated zone, and moderate advective transfer from the variably-to the permanentlysaturated zone are the main drivers of the environmental changes in the two superposed ecosystems. The effects of seasonal changes in the hydrological conditions at 6 and 15 m depth on the microbial communities are investigated below.

DISCUSSION

Bacterial diversity characterization

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j. 1574-6941.2011.01273.x The number of bacteria in the water samples was close to other measures in groundwater environments [START_REF] Pedersen | Microbial life in deep granitic rocks[END_REF]) with values ranging from 2 10 5 to 2 10 6 cells per milliliter (see Fig. S2 in Supplementary material). These numbers are clearly less than those found in soil environments and correspond only to unattached bacteria. It has been estimated that 10 10 prokaryote microorganisms can coexist in one gram of soil [START_REF] Torsvik | High diversity in DNA of soil bacteria[END_REF] corresponding to total community of attached and pelagic bacteria. The difference observed can be explained by (i) the absence of the attached microorganisms in our samples and by (ii) the low total organic carbon content of groundwater samples, as in the Kerrien aquifer. The bacterial diversity was surprisingly high in that at least 27 different phylotypes were found (Fig. 2) which probably represented a subset of the real diversity in this environment.

Saturation was not reached on the rarefaction curves. Our results are in agreement with other studies mainly performed on porous aquifers where β and γ Proteobacteria predominated [START_REF] Bordenave | Effects of heavy fuel oil on the bacterial community structure of a pristine microbial mat[END_REF][START_REF] Detmers | FISH shows that Desulfotomaculum spp. are the dominating sulphate-reducing bacteria in a pristine aquifer[END_REF][START_REF] Dojka | Microbial Diversity in a Hydrocarbon-and Chlorinated-Solvent-Contaminated Aquifer Undergoing Intrinsic Bioremediation[END_REF][START_REF] Hess | In situ analysis of denitrifying toluene-and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column[END_REF][START_REF] Reardon | Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer[END_REF][START_REF] Rodríguez-Martínez | Microbial Diversity and Bioremediation of a Hydrocarbon-Contaminated Aquifer (Vega Baja, Puerto Rico)[END_REF]. It might be possible that Archaea were present in our environmental DNAs since Archaea have recently been shown to predominate over their better known bacterial counterparts during ammonia oxidation (the first step of nitrification) in pristine aquifers [START_REF] Leininger | Archaea predominate among ammonia-oxidizing prokaryotes in soils[END_REF]. Contrary to expectations based on the literature [START_REF] Lamontagne | Comparison of subsurface and surface soil bacterial communities in California grassland as assessed by terminal restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes[END_REF], a greater diversity of free-living bacteria was observed at 10 and 15 m than at 6 m. In fact, LaMontagne et al. ( 2003) who showed a decline in bacterial species richness with depth, compared bacterial communities in the subsurface soil and the vadose zone (1-4 m). In our study, the subsurface soil was not taken into account and the variably saturated zone (3-8 m) was compared with the permanently saturated zone (8-15 m).

Relationships between nitrate reducing community and nitrate concentrations

A heterotrophic lifestyle is mundane in micro-organisms in nature. Heterotrophic nitrate consumption was demonstrated in a similar environment in a previous study (Legout et The absence of a clear differentiation of the nitrate-reducing community with depth cannot be attributed to the sampling approach. No difference was found before and after the purge (i.e.

true replicates) of the pan-flute piezometers (Fig S3 in Supplementary material). The stability of nitrate reducing communities can be attributed to the chemical concentration of minerals, in particular the high nitrate concentration and very low amount of carbon which remained homogeneous throughout the water column. We suggest that this homogenous oligotrophic status is the main driver of this community stability. It is in agreement with other findings [START_REF] Rivett | Nitrate attenuation in groundwater: Review of biogeochemical controlling processes[END_REF]. Along with that, we observed a contrasting pattern of divergence in the nitrate reducer community between different sampling campaigns only in the first sampling at 6 m. This sample was characterized by a low NO 3 -/Cl -ratio (1.4) compared to the other campaigns (~3). Chloride is known to be a conservative element and is only influenced by physical processes such as evaporation or dilution (i.e. mixing). The absence of a concomitant decrease in chloride implied that the 'low' nitrate concentration was due to nitrate reducer activity. The T-RFLP analysis only indicates the diversity of free-living bacteria and not their activity.

Influence of seasonal variations of physical recharge processes on community structure

The water pumped from a well is not representative of all the groundwater present around the well, but only of the mobile water. Water which is more strongly bound to rock, especially within the micropores (De Marsily 1994), may constitute an active part of the groundwater, from a biological point of view, as observed in similar sites [START_REF] Ayraud | Compartmentalization of physical and chemical properties in hard-rock aquifers deduced from chemical and groundwater age analyses[END_REF][START_REF] Legout | Investigation of biogeochemical activities in the soil and unsaturated zone of weathered granite[END_REF][START_REF] Tarits | Oxidoreduction sequence related to flux variations of groundwater from a fractured basement aquifer (Ploemeur area, France)[END_REF]. The relative contribution of mobile vs immobile water from a given geological layer is dependent on the hydraulic conductivity of this latter. Consequently, the

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x bacteria in water samples from highly permeable layers may over-represent the free-living bacteria [START_REF] Goldscheider | Review: Microbial biocenoses in pristine aquifers and an assessment of investigative methods[END_REF]. However the diversity within our true replicates (i.e. water sample before and after pumping for a given piezometer) indicate that the purge had no effect on community structure (Fig. S3 in Supplementary material). From this it is concluded that the free-living bacteria in water might be a proxy of the bacteria in aquifers.

The relationships between microbial populations and their environment are often complex and non-linear [START_REF] Palumbo | Coupling of functional gene diversity and geochemical data from environmental samples[END_REF]). In the case of the Kerrien aquifer, the distribution of bacterial diversity is related to environmental characteristics expressed as seasonal variations, as shown in Figure 4. Since the physico-chemical parameters (i.e. pH, dissolved oxygen) remain stable through time especially at 15 m, the composition and structure of the bacterial community in the subsurface groundwater should thus be driven mainly by hydrological conditions, i.e. physical recharge processes described above. The effects of large seasonal variability at 6 m depth and limited variability at 15 m depth are presented below. The effect of the vertical contrast between the variably-and permanently-saturated zones is investigated in section 4.

Effect of seasonal physical processes on time-related bacterial diversity (6 m depth):

An increase in microbial diversity was observed in the fluctuation zone during high water table periods (Tab.3). During low water table periods, the decline in chloride concentration was concomitant with a decrease in diversity. The chloride concentration at 6 m depth was strongly related to diffusion/mixing and dependent on microporosity, as explained above.

Chloride diffusion might also be concomitant with bacterial diffusion. During subsurface bacterial transport, bacteria can passively migrate with the ambient groundwater but they also can attach to or detach from the solid matrix and flow or swim along a chemical gradient, [START_REF] Fenchel | The ubiquity of small species: patterns of local and global diversity[END_REF]. In oligotrophic environments, chemotaxis can be a very important transport mechanism [START_REF] Ginn | Processes in microbial transport in the natural subsurface[END_REF]. This active process is however oriented and

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x chemotaxis is generally related to food availability which could be relevant when moving from reduced conditions of micro-porosity to oxygen-rich macro-porosity. The cyclic hydrological stresses are expected to induce a seasonal variation through advective transport, which allows chemotaxis when the water content is sufficiently high to create a continuous water film [START_REF] Schaefer | Prediction and experimental validation of liquid-phase diffusion resistance in unsaturated soils[END_REF], and to limit transport conditions governed by capillary forces when the water content is low [START_REF] Huysman | Water-facilitated transport of bacteria in unsaturated soil columns: Influence of cell surface hydrophobicity and soil properties[END_REF]. This variation may explain the short-term changes in diversity apparent from the T-RFLP diversity signatures of the 16S rRNA gene amplicons. The increase in diversity during high water periods might be related to an introduction of bacteria from the soil. As explained above, during this period there is a large introduction of precipitation water which has been rapidly transferred from the soil to the variably-saturated zone.

Effect of seasonal physical processes on depth

Structuring of the microbial communities in the variably-and permanently-saturated zones (influence of depth)

We here discuss the microbial structuring in the hydrologically contrasted zones showing highly variable conditions and stable conditions. Stable conditions are generally associated with an increase in diversity, whereas disturbed environments lead to the outgrowth of dominant adapted populations [START_REF] Haack | Spatial and temporal changes in microbial community structure associated with Author-produced version of the article[END_REF][START_REF] Torsvik | Prokaryotic diversity -magnitude, dynamics and controlling factors[END_REF] The results obtained for the vadose zone are in accordance with the hypothesis of increased bacterial community diversity under stable conditions. The water table fluctuation zone displays a specific community and a lower diversity (Fig. 3; Tab. 3). A few meters lower, the communities at 15 m depth present higher diversity related to seasonal mixing with the more specific bacteria from the variably saturated zone. This compartmentalization with depth over time is apparent as a clear structuring of the bacterial communities (Fig. 3). Microbial interactions may also contribute to the community structure but little is known about the species interactions that occur within microbial communities during transport processes [START_REF] Balkwill | Microbial communities in high and low recharge environments: implications for microbial transport in the vadose zone[END_REF]. Our results do not confirm a potential interaction during transport but instead emphasize the potential 'mixing' of bacterial communities from two different environments.

The estimates of bacterial diversity and comparisons of community composition along a depth profile during three successive hydrological cycles provided clear evidence of bacterial community structuring. Despite the relative similarity of the physico-chemical conditions over time, the bacterial communities could be differentiated whatever the depth. This suggests that bacterial community structure and diversity were influenced by the physical processes of water migration. They might also be explained by local competition between microorganisms which could result in differentiation of the communities over time and a subsequent drift in community composition. The physical transport induced by the recharge process would lead to a higher diversity in the permanently saturated zone, even several meters below the zone of water table variation. Our results suggest that the physical processes of water fluxes in the aquifer play an important role in bacterial community structure.

The investigated site presents rather specific characteristics such as a water table depth which remains relatively shallow and fluctuates rapidly [START_REF] Martin | Seasonal and inter-annual variations of nitrate and chloride in stream waters related to spatial and temporal patterns of groundwater concentrations in agricultural catchments[END_REF]. Various kinds of geological formations, vadose thickness and heterogeneity would lead to various influences of physical processes and water transfer kinetics. However, the recharge mechanisms can be considered as ubiquist and the overall characteristics of the hydrological cycle in the upper part of the aquifers may concern a large number of various kinds of aquifers. More precisely, the contrast between the permanently-and the variably-saturated zone, the seasonal effects of diffusion and advection in the variably-saturated zone, and the seasonal connection of the variably-saturated zone with the soil water may constitute a major driver to microbial community structuring in many aquifers. Further studies should be however carried out to asses to which extent the observed microbial patterns extend to other aquifers. The non-centred PCAs were derived from the same database but are represented in relation to time and depth.

TABLES

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273..01273.x

  DNA extracts were amplified with fluorescently labelled primers i.e. Eub-519F (5'-[6FAM] GTTTCAGCMGCMGCCGCGGT) andEub-1390R (5'-[5Hex] GTTTGACGGGCGGTGTGT), or narGf (5'-[6FAM] TAYGTCGGGCAGGAAAA-3') and narGr (5'-[5Hex] TTTTCSTACCAGGTCGC-3'). The specificity of amplification was checked and PCR products were purified using the High Pure PCR product purification kit (Roche) according to the manufacturer's protocol. Two aliquots of the purified amplicons were digested, one with 2 U HinfI and the other with StyI restriction enzymes (Promega), atAuthor-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x 37°C for 2 h in a low adsorption microtube (Axygen) to limit the loss of T-RFs during the different steps of the protocol. The quantity of digested DNA per reaction corresponded to 1/3 of a PCR reaction. The digested products were precipitated with 2 volumes of absolute alcohol and 1/10 volume of 3M Acetate buffer pH 5.4. The mixture was then placed overnight at -20°C, and centrifuged at 16400 g for 20 min at 4°C. The pellets were washed in 200 µL of 70% alcohol, centrifuged at 16400g for 10 min, and incubated for 20 min at 37°C. The dried pellets were resuspended in 10 µL of ultrapure water and placed for 2 to 4 h in darkness on melting ice. The quantity of T-RFs to load on the sequencer was defined in preliminary tests to accurately size the fragments (no signal saturation mainly). Aliquots of the digested products (1 µl) were mixed with 9 µl of deionized formamide and ROX labelled GS400HD internal size standard. T-RFs were separated and sized using an automated sequencer ABI Prism® 3130xl DNA analyser (Applied Biosystems). The size of the T-RFs was estimated in reference to the internal standard (Genemapper version 3.7 software, Applied Biosystems) which allowed accurate sizing of the T-RF lengths. Because of the use of the GS 400HD internal standard, the accuracy of the sizing of the T-RFs was good in between 50 bp and 400 bp. Thus only the fragments within this interval were used for the data analyses. The sequencer detected all fluorescent DNA fragments and background noise. The samples/formamide mixture and partial digestions may generate signals that do not correspond to a true terminal restricting fragment (T-RF). These potential artefacts were avoided by performing 3 replicates (i.e.

  mgL -1 ) concentrations (Tab. 1), and were closely linked to the hydrological fluctuations. The highest chloride, nitrate and sulphate concentrations were observed during the low water period (the whole 2004-2005 cycle) and the lowest concentrations were observed at the beginning of the recharge period (January-February 2006 and January 2007).

  -related bacterial diversity (15 m depth):The physical processes are different in the deeper zone. The weathered granite is constantly saturated with water and therefore the bacteria are not subjected to cyclic hydrological stress.The periods of high water are characterized by increased diversity and a 'blending' of the bacterial communities along the water column. Comparisons of the communities at 6 m and 15 m during the periods of high water in the different campaigns (Ker3 and Ker5 samples) revealed greater homogeneity than during the periods of low water when discrimination between 6 m and 15 m was easier (Tab. 2b, Tab. 3). This variation in diversity can be explained by a mixing of non-native and native species. Indeed, almost the same number of bacterial cells was found at 15 m and 6 m (Fig.S2in Supplementary material). A connection between the bacterial communities at 6 m and 15 m results from the occurrence of vertical fluxes of solutes and bacteria during the recharge process. The long distance passive dispersal of bacteria is facilitated by their small size.

  , which can result in an 'epidemic' population structure […] when a successful individual arises and increases rapidly to produce an epidemic clone […] (Maynard-Smith et al. 1993). In fact, environmental changes or perturbations might lead to higher infraspecific diversity and allow the selection of more rapidly adapted genotypes, and the appearance of an 'epidemic' population structure (e.g. Vandenkoornhuyse et al. 2001).

Figure 2 :

 2 Figure 2: Phylogenetic affinities of the environmental 16S rRNA gene sequences isolated from environmental ecosystems, in a consensus tree.

Figure 3 :Figure 4 :

 34 Figure 3: 16S rRNA gene bacterial community structuring with time (A) and depth (B).The non-centred PCA from T-RFLPs data highlighted bacterial community differentiation with time for the 6 and 15 m water-samples. At 6 m depth, Ker4 is missing because no water was present at this depth on the sampling date. 1, 2, 3, 4, and 5 are the gravity centres for each modality: Ker1, Ker2, Ker3, Ker4 and Ker5

Figure 1 .Figure 4 .

 14 Figure 1.Author-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

16S rRNA and narG genes amplifications PCRs

  amplifications were performed for each 16S rRNA and narG genes. The PCRs amplifications were performed in a total volume of 50 µl containing 0.2 to 0.3 µg of purified DNA, 250 µM of each deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, dTTP; Promega, Madison WI), 1 µL of proof reading Taq DNA polymerase (5U/µl -Applied Biosystems), 1x Taq DNA polymerase buffer containing 1.5 mM magnesium chloride (Applied Biosystems) and 2 µM of Eub-519F and Eub-1390R for the 16S rRNA gene amplification or 1 µM narGr and narGf for the narG gene amplification. For the 16SrRNA

	Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113
	The original publication is available at http://onlinelibrary.wiley.com
	DOI: 10.1111/j.1574-6941.2011.01273.x
	by Vandenkoornhuyse et al.
	2007), Eub-519F (5'-GTTTCAGCMGCMGCCGCGGT) and Eub-1390R (5'-
	GTTTGACGGGCGGTGTGT) were used to amplify a 871 bp internal fragment of the 16S
	rRNA gene.

  80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

  Author-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.comDOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x

  80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

  the 2.4 years of monitoring. The hydrological year 2004/2005 was relatively dry. The highest water-table level was 4.2 m during December 2004. The recharge period during the Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

			S					S
			∑	2 i an			∑	2 i bn
	da	=	= i aN 1	2	db	=	= i bN 1	2
	following year (2005-2006), was relatively long with a slow water table increase during
	winter and a final peak during spring (highest level 2.9 m during March). In comparison, the

recharge process was greater during winter of the final hydrological year

(2006)(2007)

, the level of the high water table ranging from 3.8 to 2.1 m from January to March. Comparison

  80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

  not widely used up toAuthor-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.comDOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x

  ). Apart from some values obtainedAuthor-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x during the Ker1 campaign, this index increased over time in the Ker3/Ker4 and Ker4/Ker5 campaigns. Maximum similarity (Tab. 2a) was found between Ker4 and Ker5 at 15 m, as alsoshown in Figure3. The greatest diversity (Tab.3) was observed during the high water periods.

	The Shannon index ranged from 4.4 to 5.1 at Ker3 and from 4.3 to 5.8 at Ker5 with the
	highest values occurring at 15 m. The PCAs and the similarity indexes indicate that the
	communities evolved towards more homogeneous structures during the monitoring period.
	The diversity results indicate that short term variations in community structure occurred
	during the high water periods.
	The T-RFLP diversity signatures generated for each depth revealed different community
	structures, those from 6 m and 8 m depth showing greater similarity than those from 10 m and
	15 m. The highest similarity indexes were obtained between 8/10 and 15 m in each campaign.

  . Mobile porosity transfers solutes down to the water-table by advection through the effect of gravity in the mobile pore volume. Immobile or matrix porosity exchanges solutes with the mobile pore volume by diffusion. The mobile/immobile concept of water fluxes allows interpretations of the observed chemical variations. -produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x (i) At 6 m depth, at the beginning of the hydrological cycle, the immobile porosity contains water with high chloride concentrations brought about by evapotranspiration during summer. As the water-table rises, waters containing a high proportion of rainwater are transferred by mobile porosity. This leads to saturation of the bulk volume of the water-table fluctuation zone, and low solute concentrations. Solute mixing processes then occur by diffusion between matrix porosity and mobile porosity, producing concentrations similar to those at 15 m. The entire process is highly time-dependent. At 6 m depth, there is a seasonal variation from a diffusive-dominant process during summer to an advective-dominant process during recharge period. Whilst the first period is characteristics of matrix water, the second one is characteristic of precipitation and soil water rapidly transferred from the soil.

	Author

  al. -produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x 2007). The main electron acceptor in the Kerrien aquifer is nitrate which is available irrespective of depth and time (Tab. 1).

	Author

  Author-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.comDOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x

  Author-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.comDOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x

Table 1 :

 1 Dissolved oxygen (O 2 ), pH and temperature measurements; Nitrates, sulphates and dissolved organic carbon (DOC) concentrations in groundwater samples collected from the different wells from 2005 to 2007. No values are indicated at 6 m for the fourth campaign (Ker4) because the water table was below this level. No DOC measurements were done during the third and the fifth sampling campaigns for technical reasons. The quantification limit for carbon analysis is 0.85 mg L -1 .Author-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.comDOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273.x .01273.x

		Physico -chemical parameters
		Dissolved oxygen			pH				Temperature
	Campaign	(mg.L -1 )				(Units)				(°C)
	Samples	H6	H8	H10	H15	H6	H8	H10	H15	H6	H8	H10	H15
	Ker1	4.4	4.5	4	5	4.8	5.5	5.5	5.4	17.9	16	17.1	18.1
	Ker2	6.8	7.6	5.7	4.8	6.4	5.9	6.2	6	12.6	12.4	13	12.1
	Ker3	7.9	7.6	6.2	6.5	5.9	5.1	5.2	5.3	12.5	12.5	12.6	12.9
	Ker4	-	5.7	5.8	5.5	-	5.7	5.6	5.5	-	14.1	13.4	14.4
	Ker5	9.4	8.2	8.7	6.5	6	5.2	5.4	5.2	11.3	12.1	10.2	12.2
		Chemical parameters
		Nitrates				Sulphates			Dissolved Organic Carbon
	Campaign	(mg.L -1 )				(mg.L -1 )				(mg.L -1 )
	Samples	H6	H8	H10	H15	H6	H8	H10	H15	H6	H8	H10	H15
	Ker1	56.1	71	61.8	62.6	65.4	22.8	20.2	17.6	3.73	0.98	1.42	1.45
	Ker2	80.0	71.3	65.1	68	37.5	29.2	19.7	19.2	1.66	0.89	0.87	1.13
	Ker3	87.3	73.5	69.8	72.4	35.9	21.6	20.1	18.5	-	-	-	-
	Ker4	-	70.7	66	65.6	-	23	20.2	19.3	-	0.9	<QL	<QL
	Ker5	57.2	67.7	69.2	64.8	22.2	21.9	19.7	19.5	-	-	-	-

Table 3 :

 3 Diversity index (Shannon index) calculated from the 16S rRNA gene T-RFLP analysis.Author-produced version of the article published in FEMS MicrobiologyEcology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

	Depth	Diversity indexes -Campaign
	Samples	Ker1	Ker2	Ker3	Ker4	Ker5
	6 m	3.69	2.40	4.63	-	4.29
	8 m	3.94	4.89	4.37	3.90	4.89
	10 m	4.63	3.83	4.74	4.12	5.07
	15 m	3.15	4.96	5.08	4.32	5.76

FIGURE Captions Figure 1: Chemical concentration variation with time and depth.

  Depth of the water table vs time and chloride concentration changes in the water-table fluctuation zone (6 m depth) and the permanently saturated zone (15 m depth). The water table depth is given with the different sampling dates. Ker1 was sampled on 05/25/05, Ker2 on 01/18/06, Ker3 on 03/13/06, Ker4 on 11/22/06 and Ker5 on 01/25/07. Ker1, Ker3 and Ker5 were sampled during high-water periods and Ker2 and Ker4 during low-water periods.

Acknowledgments

This work was supported by the CNRS 'programme de recherche ECCO-PNBC' and by the ANR Biodiversité (ANR InBioProcess Project). The authors are grateful to Pierre Marmonier,

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111DOI: 10. /j.1574DOI: 10. -6941.2011.01273..01273.x

Supporting Information

Fig. S1. The Kerrien catchment is located in western France and the device used for sampling water is a pan-flute piezometer (modified from [START_REF] Molénat | Etude du rôle de l'interface sol-nappe sur les processus de Transfert de solutés, d'érosion chimique et sur la diversité bactérienne dans un bassin versant SONATE)[END_REF]. The ellipses grouped 95% of the communities (dots) projected on the vectorial space for a vine modality (i.e. before purge 'BP' vs after purge 'AP'). 'BP' and 'AP' represent the gravity centres of the ellipses. For a given piezometer water samples before purge and after purge were considered as true replicates. These 2 true replicates were expected more heterogeneous in comparison to two samples before purge or two samples after purge.

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

Figure S1. 931

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

Figure S2. 933

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j.1574-6941.2011.01273.x

Figure S3. 935

Author-produced version of the article published in FEMS Microbiology Ecology, 2012, vol.80, n° 1, pp 98-113 The original publication is available at http://onlinelibrary.wiley.com DOI: 10.1111/j. 1574-6941.2011.01273.x