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Tianjun Li

Felipe A Aguilar Sandoval

Mickael Geitner

Gianpietro Cagnoli
email: g.cagnoli@lma.in2p3.frc

Vincent Dolique

Jérôme Degallaix

Raffaele Flaminio

Danièle Forest

Massimo Granata

Christophe Michel

Nazario Morgado

Laurent Pinard

Ludovic Bellon
email: ludovic.bellon@ens-lyon.fr

Measurements of mechanical thermal noise and energy dissipation in optical dielectric coatings

1

In recent years an increasing number of devices and experiments are shown to be limited by mechanical thermal noise. In particular sub-Hertz laser frequency stabilization and gravitational wave detectors, that are able to measure fluctuations of 10 -18 m/ √ Hz or less, are being limited by thermal noise in the dielectric coatings deposited on mirrors. In this paper we present a new measurement of thermal noise in low absorption dielectric coatings deposited on micro-cantilevers and we compare it with the results obtained from the mechanical loss measurements. The coating thermal noise is measured on the widest range of frequencies with the highest signal to noise ratio ever achieved. In addition we present a novel technique to deduce the coating mechanical losses from the measurement of the mechanical quality factor which does not rely on the knowledge of the coating and substrate Young moduli.

The dielectric coatings are deposited by ion beam sputtering. The results presented here give a frequency independent loss angle of (4.7 ± 0.2) × 10 -4 with a Young's modulus of 118 GPa for annealed tantala from 10 Hz to 20 kHz. For as-deposited silica, a weak frequency dependence (∝ f -0.025 ) is observed in this frequency range, with a Young's modulus of 70 GPa and an internal damping of (6.0 ± 0.3) × 10 -4 at 16 kHz, but this value decreases by one order of magnitude after annealing and the frequency dependence disappears.

a) Present address: Universidad de Santiago de Chile, Departamento de Fìsica, Avenida Ecuador 3493, Casilla

I. INTRODUCTION

The detection of gravitational waves is based on laser interferometry used to monitor the relative displacement of suspended masses [START_REF] Pitkin | Gravitational Wave Detection by Interferometry (Ground and Space)[END_REF] . Although the first generation of GW detectors has been concluded with an extraordinary success, the event rate at the level of sensitivity of these detectors is about one per year in the most optimistic predictions and the absence of a detected signal in the 3 years of data taking is totally compatible with the event rate uncertainty. Therefore, a second generation of detectors is being built in USA (Advanced LIGO [START_REF] Harry | Advanced LIGO: the next generation of gravitational wave detectors[END_REF] ), in Europe (Advanced Virgo 3 ) and in Japan (KAGRA [START_REF] Somiya For | Detector configuration of KAGRA-the Japanese cryogenic gravitational-wave detector[END_REF] ). In these advanced detectors the sensitivity limit in displacement is about 6 × 10 -21 m/ √ Hz over a wide band centred around 200 Hz. The sensitivity limit of the detector Advanced Virgo, with its main noise components, is shown in Fig. 1. Lowering the noise level by a certain factor corresponds to increase by the same factor the maximum distance at which a source can be detected. The larger this distance, the higher the event rate because the larger the number of galaxies present in the detection range. From Fig. 1 one can see that the thermal noise coming from the mirror coatings (coating Brownian noise) is the limiting noise component in the band where the detector is most sensitive. To clearly understand that, it is important to know that during the detector designing process, in general, the noise that comes from the optical readout system (Quantum noise) is shaped to match the displacement noise level. If the coating noise gets reduced the quantum noise follows, either by a simple adjustment of optical parameters or by a major redesign of the optical readout 5 , depending on the level of noise reduction to attain. The reflective coating on the suspend masses are made by stacks of alternate layers of two transparent materials having different refractive index. After a long process of selection two materials have been chosen for the optical coating of GW detectors: silica (SiO 2 , low index) and titanium-doped tantala (Ti:Ta 2 O 5 , high index). The selection has been made on the basis of the lowest optical absorption and thermal noise level [START_REF] Harry | Titaniadoped tantala/silica coatings for gravitational-wave detection[END_REF][START_REF] Flaminio | A study of coating mechanical and optical losses in view of reducing mirror thermal noise in gravitational wave detectors[END_REF] .

Lowering coating thermal noise will be beneficial also for other precision measurements where high finesse optical cavities are used, such as in the development of optical clocks 8 as well as in the field of quantum opto-mechanics.

In amorphous materials thermal noise comes from unknown relaxation processes whose characteristics are fairly well explained by a model called Asymmetric Double Well Potential 9 .

In the case of bulk silica, a well known amorphous material, a number of measurements FIG. 1. Advanced Virgo sensitivity curve as in [START_REF]Losurdo for the[END_REF] . The noise level on the y axis is given in equivalent gravitational wave amplitude that has the same physical nature of strain. To convert the strain noise in displacement noise it's relevant to know that there are 4 sources of uncorrelated noise assumed to be of the same level and that the interferometer arms length is 3 km.

indicate that the relaxation comes from the twisting of SiO 4 tetrahedrons one respect to the nearest. For other materials like coated tantala, that has a higher noise level than coated silica, data are few in absolute terms, and measurements indicating the parts of the microscopic structure responsible of the relaxations are totally missing. Recent works

G(ω) = F (ω) d(ω) = k 1 - ω 2 ω 2 0 + iφ (1)
with ω the angular frequency, ω 0 the angular resonance frequency, k the cantilever stiffness and φ its loss angle (also named mechanical loss or damping). In general φ may be frequency dependent (in this case k is frequency dependent too), in the model shown here it is assumed to be constant : this is called structural damping. The quality factor of the resonance is

Q = 1/φ(ω 0 ).
The thermal noise at room temperature T is then simply obtained through the Fluctuation-Dissipation Theorem [START_REF] Callen | On a theorem of irreversible thermodynamics[END_REF] (FDT): the deflection thermal noise Power Spectrum

Density (PSD) S d (ω) is S d (ω) = - 2k B T πω Im[1/G(ω)] (2)
with k B the Boltzmann's constant. Far below resonance, we immediately get the expression of the noise at low frequency :

S d (ω ≪ ω 0 ) = 2k B T πω φ k (3)
The signature of a structural damping is thus a 1/f noise at low frequency. In macroscopic bodies mechanical thermal noise is too low to be directly measured by relatively simple detectors. This is the reason why the investigation on thermal noise is almost always done indirectly through the measurement of the mechanical energy loss that is always present in the phenomenon of relaxation. When the scale of samples is reduced below 1 mm two positive effects happen: i) the elastic constant k approaches 1 N/m, hence thermal noise level increases up to a level (∼ 10 -26 m 2 /Hz) attainable by relatively simple optical readout systems as the one used in this work; ii) the lowest resonant frequency is at several kHz, below that frequency the cantilever dynamics is equivalent to a massless spring and equation (3) holds. We will show in this article that using thermal noise of commercial AFM cantilevers, the coating internal dissipation can be measured down to a level of 3 × 10 -5 with a good precision on a large frequency range (10 Hz-20 kHz).

This articles is organized as follows: Section II presents the interferometer that has been used to measure directly the thermal noise on the micro cantilevers. Section III describes the micro cantilevers that have been tested and the coatings that have been deposited.

Section IV will show how the interferometric data have been analyzed to extract the relevant parameters of the oscillator. Section V is dedicated to the theory that have been applied

to work out the relation between the coating loss angle and the one related to the entire oscillator. For the first time a technique based on the frequency shifts has been applied to give a measurement of the dilution factor and of the coating loss. Finally, in Section VI, the conclusions are drawn.

Our measurements of the micro-cantilever deflection rely on a quadrature phase differential interferometer [17][START_REF] Bellon | Differential interferometry with a complex contrast[END_REF][START_REF] Paolino | Quadrature phase interferometer for high resolution force spectroscopy[END_REF] . The optical path difference is measured between the sensing and reference beams focused respectively on the free end and close to the clamping base of the cantilever (see inset of figure 2). The use of the light polarization allows extending the linear range of the instrument from the sub-wavelength range, typical of a Michelson interferometer, to several micrometers. In this way one can avoid the control of the reference mirror position to find the optimal working point (middle fringe position). Moreover, by design the interferometer is nearly common path, resulting in a low sensitivity to external vibration and a low drift, allowing precise measurements of mechanical thermal noise.

The total light intensity on the cantilever is less than 500 µW at 633 nm, resulting in a negligible heating with respect to room temperature (less than 5 K). The background noise of the instrument is measured by reflection of light on a macroscopic rigid mirror, tuning light intensity on the photodiodes to the same values measured during the thermal noise measurement on the cantilevers. As seen in figure 4, this background noise S bg is as low as 10 -27 m 2 /Hz at high frequency, it mainly results from the shot noise of the photodiodes. At lower frequency, 1/f noise from the electronics is present, with a corner frequency around 100 Hz.

More details about the instrument can be found in reference [START_REF] Paolino | Quadrature phase interferometer for high resolution force spectroscopy[END_REF] . This set-up has notably been used previously to characterize mechanical thermal noise and viscoelastic behavior of metallic coatings on micro-cantilevers [START_REF] Paolino | Frequency dependence of viscous and viscoelastic dissipation in coated micro-cantilevers from noise measurement[END_REF][START_REF] Li | Dissipation of micro-cantilevers as a function of air pressure and metallic coating[END_REF] .

III. THE SAMPLE PREPARATION AND PARAMETERS

The samples are made of tipless AFM cantilevers [START_REF] Www | Product Model AIO-TL[END_REF] with thin films of silica SiO 2 or tantala Ta 2 O 5 deposited through Ion Beam Sputtering (IBS). A cantilever used in this experiment is shown in Fig. 2.

The cross section has the shape of a isosceles trapezium with dimensions a = (31.4 ± 0.8) µm (the long side), b = (16.3 ± 0.9) µm (the short side) both measured by the electron microscope. The thickness is h Ta = (3.07 ± 0.12) µm and h Si = (3.13 ± 0.12) µm for the cantilever coated with tantala and silica respectively, as measured indirectly through the resonant frequencies (from a linear fit between the frequencies of the firsts 3 modes and the mode number squared). Before the coating deposition a side view of the cantilever with the electron microscope was not possible and the slanted lateral surfaces made the direct measurement of thickness very inaccurate. The total length of the cantilever is L = (505 ± 5) µm.

Although the uniformity of the thickness of the thin film is below 1 % over 10 cm of substrate, during the deposition the cantilever bent considerably under the coating stress and this might have an effect on the thickness uniformity. In a second imaging session, using a different microscope, a measurements of coating thickness was done on the two sides of the cantilever at different points along its length. A side view of the cantilever is shown in Fig 3 . The measurements show that the coating thickness is uniform within the reading uncertainty of 10%. In order to have a small residual curvature the thickness of the deposited coating has to be different for the two sides of the cantilever because the cross section has the form of a trapeze. With the thicknesses listed in table I the inclination of the tip was less than 0.5 • with respect to the cantilever base. The silica coating could be measured right after deposition (sample Si), and after annealing 10 h at 500 • C (sample Si A ). The cantilever used for the tantala film had a thermal oxide of 20 -30 nm grown before deposition. The parameters of the heat treatment are: heating rate of 100 • C/h up to 800 • C and then after 1 minute cooled to room temperature at the same rate. This oxide layer is necessary for the adhesion of tantala. After deposition the tantala sample Ta A was annealed for 10 h at The density of tantala and silica were measured by mass measurement on silicon wafers.

The annealing causes a decrease of coating density that correspond to an increase of coating thickness. The thicknesses reported in

IV. DATA PROCESSING OF THE INTERFEROMETER SIGNALS AND

RESULTS.

The cantilever is placed at room temperature in a vacuum chamber to reduce the viscous damping by the surrounding atmosphere (static vacuum around 3 × 10 -2 mbar). Its thermal noise driven deflection is measured directly by the quadrature phase interferometer. The data acquisition system samples the signals at a rate of 250 kHz with 24 bit resolution. The length of each data stream is 4 s. After an off-line processing of the sampled photodiodes signals a PSD calibrated in m 2 /Hz is produced. After a data cleaning process that removes all the harmonics of the power line and electronic devices, the single data stream is stored and made available for the further data analysis. Data analysis was done in three steps as described in the following sections. The cantilever loss angle is estimated at first from the shape of the resonant peak and then on a much wider frequency range using the Fluctuation-Dissipation theorem and the Kramers-Kronig relation.

A. Evaluation of the SHO parameters

On a narrow 140 Hz frequency interval around the first resonance, the measured PSD is fitted with the following function [START_REF] Saulson | Thermal noise in mechanical experiments[END_REF] , derived from equation ( 2):

S f it (ω) = 2k B T πkω φ 0 1 -(ω/ω 0 ) 2 2 + φ 2 0 + S f itbg (4)
where S f itbg , the background noise level that is constant for frequencies greater than 1 kHz, is measured directly from the spectra, while the angular resonant frequency of the mode ω 0 = 2πf 0 , the loss angle φ 0 and the elastic constant k are determined by the fitting process.

In particular, f 0 is related to the peak frequency position, φ 0 to the peak width and 1/k to the curve integral 24 . The temperature is assumed to be the room temperature T = 298 K since, given the laser power used during this measurement the cantilever heating is negligible.

In the fitting process of a linear PSD spectrum the relative weight of the few points around the resonance is enormous in comparison to the weight of the many points far from the resonance so, in order to better balance the weight of all the points, the fitting process is done on the logarithm of the PSD.

For each thermal noise measurement done in this work, at least 120 spectra were taken.

In order to deal with any potential non stationary process two kinds of average were done:

the average of each parameter that results from the fitting of each single spectrum and the fitting done on the average spectra. In all the measurements done in this work the two averages gave the same results within the experimental uncertainties. The averaged PSDs S meas and the fitting curves S f it are given in Fig. 4 for sample Si. In Table II results of the fitting process on spectra taken before and after the coating deposition for the three measured samples 24 . The effect of annealing on the silica sample is clearly evidenced by the very low internal damping measured on sample Si A . 4 for sample Si). The fit of the resonance with equation ( 4) is still valid at low frequencies, demonstrating the very weak frequency dependence of φ. The thermal noise of the annealed sample is an order of magnitude lower than that of the as-coated sample, almost reaching the limit of our detection system. Peaks around 10 kHz and high noise level below 5 Hz are residual environmental noise.

B. Extraction of the cantilever thermal noise PSD

In the thermal noise spectrum of Fig. 4 a clear 1/f noise can be seen at low frequency.

Although a contribution from the instrument background noise is present, the measured signal is much larger than this detection limit. The mechanical thermal noise contribution of the sample can be extracted by the difference between the averaged PSD of the measured signal S meas and the averaged PSD of the background noise S bg (measured on a rigid mirror).

The result of this subtraction is given in Fig. 5 and Fig. 6 for the silica and tantala coated samples respectively. At frequencies lower than 5 kHz the noise is proportional to 1/f as predicted by the structural noise model with constant loss eq.( 3). The fit reported in those figures is simply the one obtained previously from eq.( 4) (imposing S f itbg = 0 in this case): its parameters are adjusted in a 140 Hz frequency range around the resonance. We see however that the agreement at any frequency is good, demonstrating the weak dependence on f of the structural damping φ.

C. Extraction of the cantilever internal dissipation φ

Since we measure the PSD of thermal fluctuations on a wide frequency range, we can use the FDT and Kramers-Kronig's relations to rebuilt the full mechanical response G(ω) of the cantilever [START_REF] Paolino | Frequency dependence of viscous and viscoelastic dissipation in coated micro-cantilevers from noise measurement[END_REF][START_REF] Li | Dissipation of micro-cantilevers as a function of air pressure and metallic coating[END_REF] . Indeed, through the FDT we actually measure Im[1/G(ω)] (see equation ( 2)).

We then apply Kramers-Kronig's integral relations for the response function 1/G(ω) to infer its real part from the knowledge of its imaginary part, eventually getting a full measurement of G(ω). In figure 7 we plot the result of this reconstruction process with the imaginary part of the response function scaled by the cantilever stiffness: using equation (1), this should directly lead to the internal damping of the cantilever φ = Im(G)/k. We notice that the frequency dependance is very weak for the 3 samples: φ(ω) is flat between 10 Hz and 20 kHz for the annealed samples Si A and Ta A , and presents a very slight decrease for sample Si. This weak frequency dependence can be fitted for example with a power law ω α , with α = -0.025. with the structural damping model (φ 0 measured with the fitting procedure of the thermal noise at resonance through equation ( 4)). The dissipation is much weaker after annealing for the silica coating, reaching the limits of our detection system. Peaks around 10 kHz and high noise level below 5 Hz are due to the residual environmental noise.

V. FROM THE CANTILEVER DISSIPATION TO THE COATING LOSS ANGLE

In this section the relation between the loss angle of the resonator φ and the loss angle of the coating φ c will be worked out. To do that one can use the definition of quality factor knowing that at the resonant frequencies φ = 1/Q. The extention of this relation to all frequencies is assumed. Knowing that Q = 2π E/E lost , where E lost is the energy lost in one cycle, E is the total energy of the oscillator, and assuming only two components (the substrate s and the coating c), the cantilever loss angle is written:

φ = E lost c + E lost s 2π E = φ c 2π E c + φ s 2π E s 2π E = φ c D + φ s (1 -D) (5)
In the previous expression the relation between energy loss and φ is applied twice, for the substrate and the coating and, finally, the dilution factor D has been defined as E c /E. E c and E s are the energy stored in the coating and in the substrate respectively, E = E c + E s .

In order to calculate the dilution factor it is assumed that the oscillator is at its maximum displacement; therefore the total energy is equivalent to the maximum potential energy that, for an unidimensional elastic beam, is:

E = V max = Y I L 0 ∂ 2 w ∂z 2 2 dz ( 6
)
where Y is the Young's modulus, I the cross moment of inertia calculated with respect to the neutral line (Y I is called also the beam rigidity), w is the transversal displacement of the neutral line with respect to its position at rest and z is the coordinate along the beam of length L. When equation ( 6) applies to the coating and to the substrate, only the rigidity makes the difference between the two energies because the mode shape w(z) is the same for both. Hence, the dilution factor becomes:

D = V c V = Y c I c Y c I c + Y s I c s (7)
In the previous expression it appears I c s which is the substrate cross moment of inertia when the coating is present. In facts, the uncoated-substrate cross-moment of inertia I u s is different from the previous one because the position of the neutral line may change when the coating is applied.

Using the dimensions reported in Section III we have calculated the beam rigidities for the various components in the two measured sampled, assuming 70 GPa, 140 GPa and 169 GPa the Young's modulus of coated silica, coated tantala and silicon along the direction < 110 >, respectively. The expression of the rigidities with all the parameters is rather complicated and it is not of any particular use to have it written here. Therefore we report only the results of the dilution factor calculations: D calc Si = 0.41 and D calc Ta = 0.54. On measuring the frequency shift of the resonant modes before and after the coating deposition one can have a way to measure directly the dilution factor. In facts the resonant angular frequencies ω n are related to the potential energy V n of each mode n through the equivalence V n = K n where K n is the kinetic energy. In an explicit form the previous equation reads:

V n = ω 2 n µ L 0 [w n (z)] 2 dz ( 8
)
where µ is the linear mass density defined as the integration of density ρ over the beam cross section. The previous equation is fulfilled independently of a multiplicative factor (the amplitude of the mode) of w n (z). The energy equation ( 8) written for the uncoated and the coated beams follows:

V u s,n = (ω u n ) 2 µ s L 0 [w n (z)] 2 dz (9) V c s,n + V c c,n = (ω c n ) 2 (µ s + µ c ) L 0 [w n (z)] 2 dz (10)
Dividing term by term the previous equations and remembering the definition of potential energy (6) and dilution factor (7) a simple relation between dilution factor and frequency shifts is obtained:

I u s I c s [1 -D] = f u f c 2 µ s µ s + µ c (11)
On cantilevers and wafers substrates that are bent by the action of the stress in the coating the previous relation does not apply. The reason of that is still unclear and it is being investigated by several research groups. Our samples have the coating deposited on both sides and the cantilever is almost straight (the tip angle is only 0.5 • with respect to the cantilever base). In the previous expression only one quantity (that needs to be calculated, being impossible to measure it) still depends on the Young's modulus of coating: I c s . However for the cantilevers and coatings used in this study, its difference with I u s is relatively small, of the order of one part in 10 4 , so the ratio I u s /I c s is from now on considered equal to 1. With this assumption, equation (11) shows the link of the dilution factor to quantities that are easily measurable and that are not related to the knowledge of the elastic constants of coating.

Contrary to previous works [START_REF] Berry | Vibratind Reed Internal Friction Apparatus for Films and Foils[END_REF] where the authors gave a linearized version of the equation (11) valid for coatings much thinner as compared to the substrates, in our experiment the measurement of the dilution factor or of the coating elastic constant by frequency shift seems to be reliable, probably due to the small difference between coating and substrate thickness. Taking the dimensions of samples in Section III and the density worked out by mass measurements on silicon wafers, one can calculate the dilution factors from the measured frequency shift as reported in the table III. The measured and calculated dilution factor for coated silica are equal within the experimental uncertainties whereas for tantala the difference is significant and consistent with other measurements of dilution factors of coated tantala on silicon wafers (not yet published) done by the authors. The difference can be explained by an actual tantala Young's modulus of 118 GPa rather than 140 GPa as assumed in the dilution factor calculation.

Once the dilution factor is known, equation ( 5) is used to work out the loss angle of the The characterizing parameter used here is the loss angle φ that allows the calculation of thermal noise in any system where the same coating is deposited. The loss angle of annealed 500 • C tantala and silica are independent of frequency whereas as-deposited silica shows a slight dependence on frequency as f -0.025 . The measurements of coating loss done with direct measurement of thermal noise were compared with measurements on some 3" diameter silicon wafers using the resonant method. The wafers were coated using the same coater and the same coating parameters as for the micro cantilevers. The dilution factors were both calculated and measured. The results of these measurements are shown in table V.

The loss angle measurements made by thermal noise method are in a fairly good agreement with the values obtained by the resonant method. In particular, the values obtained for tantala are in agreement within the experimental uncertainties. For as-coated silica the comparison shows a significant disagreement that might be explained by a large variability on the optical and mechanical parameters that has been observed by the authors on the ascoated samples. The annealing process, beside lowering both optical and mechanical losses, contributes to stabilize their values.

A comparison can be made also with results 26 , 27 of coating deposited on silicon substrates and measured by the resonant method obtained by other authors. Their results for the loss of tantala annealed at different temperatures are in the range 2.7 × 10 -4 to 5 × 10 -4 .

The direct measurement of thermal noise done on micro cantilever as compared to the resonant method applied on larger substrates offer a smaller statistical uncertainty on the measurement of the loss angle and the possibility to cover continuously more than 3 decades of frequency. At the moment the major uncertainty comes from the systematic error related to the poor knowledge of the uncoated substrate noise. A lower residual gas pressure and greater statistics will lower this uncertainty.

FIG. 2 .

 2 FIG. 2. Electron microscope image of a cantilever without coating and later used in the measurements. The length of the cantilever protruding from the silicon block is about 505 µm. The photo should show the irregularity of the width and of the slanted faces due to the etching process. In the inset: position of the laser beams relative to the cantilever.

  FIG. 3. Electron microscope image of the sample Si A : side view at the middle of the cantilever.Measurement of the total coating thickness t (sum of the coatings deposited on the narrow and wide sides) and total cantilever thickness t + h are shown. t and h are respectively 12% and 2% higher than the values that come from the parameters reported in TableI. At right: sketch of the coated-cantilever cross section that helps to understand the electron microscope image.

21 FIG. 6 .

 216 FIG. 6. PSD of sample Ta A coated with tantala obtained by subtraction of the averaged PSD of measured signals S meas and averaged PSD of background noise S bg . The fit of the resonance with equation (4) is still valid at low frequencies, demonstrating the frequency independence of φ. Peaks around 10 kHz and high noise level below 5 Hz are residual environmental noise.

φ 4 Frequency 6 FIG. 7 .

 467 FIG. 7. Internal damping φ as a function of frequency for the three measured samples. The result of the reconstruction process through Kramers-Kronig's relations φ KK is in very good agreement

  as-coated annealed at 500 • C Tantala with D 11.4 ± 0.2 4.90 ± 0.25 with D calc 8.2 ± 0.7 3.8 ± 0.4 Silica with D calc 3.9 ± 0.4 -Direct measurement of thermal noise on micro cantilevers coated with tantala and silica have been performed in the frequency range from 10 Hz to 20 kHz. The measurement were used to characterize the noise properties of the two amorphous materials deposited by IBS.

TABLE I .

 I Coating parameters for the two measured samples.

	Si	SiO 2	424 ± 4	541 ± 5 2.52 ± 0.02 70
	Si A	SiO 2	432 ± 4	552 ± 5 2.47 ± 0.02 70
	Ta A	Ta 2 O 5 378 ± 4	482 ± 5 7.39 ± 0.02 140

Sample Coating Thickness Thickness

ρ E material on a (nm) on b (nm) (g/cm 3 ) (GPa)

  Table I are corrected by this effect from the nominal values coming from the deposition process. The Young's moduli reported in this table are compatible with values measured by nanoindentation 23 . Two cantilevers have been coated in total.

  are listed the

		10 -21	exp. PSD: Smeas
			PSD fit: S fit
		10 -22	exp. background noise: S bg
	PSD S d (m 2 /Hz)	10 -25 10 -24 10 -23	PSD S d (m 2 /Hz)	10 -24 10 -23 10 -21 10 -22	f 1
		10 -26
		10 -27
		10 0	10 1	10 2	10 3	10 4
			Frequency f (Hz)

FIG. 4. Averaged PSD of measured deflection for 140 data sets for a silica coated cantilever measured in vacuum (sample Si). The curve is compared to the fitting function (4) and the to background noise, measured on a rigid mirror. Inset: zoom on a 100 Hz frequency interval around resonance.

TABLE II .

 II Fitting parameters 24 . The uncertainty corresponds to the statistical error on the fits of more than 120 spectra (only 20 for uncoated samples).

		10 -20
		10 -21	Sample Si: Smeas -S bg
	PSD difference (m 2 /Hz)	10 -28 10 -27 10 -26 10 -25 10 -24 10 -23 10 -22	Structural damping model: S fit Sample Si A : Smeas -S bg
		10 -29
		10 0	10 1	10 2	10 3	10 4
				Frequency f (Hz)
		Sample		f 0 ( Hz)	φ 0 /10 -4
		Uncoated Si & Si A 16501 ± 2	0.3 ± 0.3
		Sample Si		17732.2 ± 0.1 2.59 ± 0.04
		Sample Si A		17835.2 ± 0.1 0.43 ± 0.02
		Uncoated Ta A		16110 ± 1	0.3 ± 0.3
		Sample Ta A		14771.6 ± 0.1 2.28 ± 0.03

FIG. 5. PSD of samples Si and Si A coated with silica obtained by subtraction of the averaged PSD of measured signals S meas and averaged PSD of background noise S bg (PSDs of figure

TABLE III .

 III The measured vs. calculated dilution factors.Sample Ta A 16110 ± 1 14771.6 ± 0.1 171 ± 9 200 ± 5 0.45 ± 0.02 0.54 ± 0.02 coating from the loss angles of the substrate φ s and of the whole oscillator φ. With a noise measurement on an uncoated cantilever we have estimated the substrate loss φ s = (0.3 ± 0.3) × 10 -4 and then the resulting coating losses for the two types of coatings are listed in TableIV. The values in brackets are the systematic errors related to the estimation

	Sample	f u [Hz]	f c [Hz]	µ s × 10 8 µ c × 10 8	D	D calc
				[g/cm] [g/cm]
	Sample Si 16501 ± 2 17732.2 ± 0.1 174 ± 9 76 ± 2 0.40 ± 0.02 0.40 ± 0.02
	Sample Si A 16501 ± 2 17835.2 ± 0.1 174 ± 9 76 ± 2 0.41 ± 0.02 0.41 ± 0.02

TABLE IV .

 IV Loss angle of coatings deposited on Silicon micro cantilevers Coating φ × 10 4 φ s × 10 4 D φ c × 10 4

	SiO 2 as-coated 2.59 ± 0.04 0.3 ± 0.3 0.40 ± 0.02 6.0 ± 0.3 (±0.5)
	SiO 2 annealed 0.43 ± 0.02 0.3 ± 0.3 0.41 ± 0.02 0.62 ± 0.05 (±0.43)
	Ta 2 O 5 annealed 2.28 ± 0.03 0.3 ± 0.3 0.45 ± 0.02 4.7 ± 0.2 (±0.4)

of the substrate losses. The maximum systematic error correspond to a substrate loss angle equal to zero.

TABLE V .

 V Coating loss angles measured with the resonant method on Silicon wafers Coating φ c × 10 4 φ c × 10 4

ACKNOWLEDGMENT

The authors would like to thank the Engineer Armel Descamps of the Lyon Nanotechnology Intitute (INL) for the electron microscope analysis. The authors are grateful to the LABEX Lyon Institute of Origins (ANR-10-LABX-0066) of the Université de Lyon for its financial support within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) of the French government operated by the National Research Agency (ANR). This work has been also partially supported by the Fédération de Physique Ampére. Finally, T.J. Li acknowledges the financial support from the Chinese Scholar Council (CSC).


Thuy Doan

Dung My Jusselme

Jean-Christophe Lata

Bo Van Nguyen

Pascal Jouquet
email: pascal.jouquet@ird.fr

The earthworm species Metaphire posthuma modulates the effect of organic amendments (compost vs. vermicompost from buffalo manure) on soil microbial properties. A laboratory experiment

Keywords: Biolog analysis, DGGE, Enzymatic activities, Metaphire posthuma, organic fertilization, Tropical soil

The aim of this study was to determine the influence of compost and vermicompost produced from buffalo manure on soil bacterial diversity and activity in the presence and absence of the endogeic earthworm Metaphire posthuma. This experiment was carried out for 15 months with a maize-tomato-maize cycle under greenhouse conditions in Northern Vietnam. It showed a positive influence of compost and vermicompost on soil microbial properties, with higher cultivable bacteria, higher bacterial and catabolic diversity (Shannon diversity 'H' and Richness 'S') indices and higher enzymatic activities than control soils which only received mineral fertilizers.

Differences also occurred between compost and vermicompost with lower activity and diversity in the soil amended with vermicompost, probably because of its higher molecular stability. The presence of M. posthuma led to divergent dynamics of bacterial community in soils amended with compost and vermicompost. Earthworms negatively influenced soil microbial properties in composted soil (lower Average Well Color Development 'AWCD'), probably because of competition between bacteria and earthworms for organic resources and/or because of the consumption of microbes by earthworms. Conversely, the presence of earthworms increased bacterial diversity and activity with higher AWCD, and H and S indices for the vermicompost treatment, probably as a result of a stimulation of microorganisms that allow the degradation of stable organic matter and its further consumption by earthworms. In conclusion, this study clearly confirmed the different impacts of compost and vermicompost on bacterial activity and diversity and highlighted the importance considering the interaction of these organic substrates with local endogeic earthworms.

Introduction

Increasing soil organic matter (SOM) stocks and stability by addition of organic amendment offers a good way to substantially improve soil quality and therefore agricultural sustainability [START_REF] Diacono | Long-term effects of organic amendments on soil fertility[END_REF][START_REF] Gomiero | Energy and environmental issues in organic and conventional agriculture[END_REF][START_REF] Moeskops | The impact of exogenous organic matter on SOM contents and microbial soil quality[END_REF]. Most of the studies aimed at comparing the influence of animal waste amendment on soil quality have been done using swine, poultry, cow or horse manure as original substrates [START_REF] Arancon | Vermicompost tea production and plant growth impacts[END_REF][START_REF] Pramanik | Changes in Organic -C, N, P and K and Enzyme Activities in Vermicompost of Biodegradable Organic Wastes under Liming and Microbial Inoculants[END_REF]. However, comparatively less research had been focused on buffalo manure, although it is often the main source of organic amendment for many farmers in Asia, especially in Vietnam [START_REF] Yajima | High latrine coverage is not reducing the prevalence of soil transmitted helminthiasis in Hoa Binh province, Vietnam[END_REF] where it is usually applied on the fields several times per year as compost to improve soil fertility [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF][START_REF] Ngo | Transformation of buffalo manure by composting or vermicomposting to rehabilitate degraded tropical soils[END_REF][START_REF] Ngo | The effect of earthworms on carbon storage and soil organic matter composition in tropical soil amended with compost and vermicompost[END_REF].

Understanding microbial and biochemical processes in soils are important for the management of farming systems [START_REF] Sebastiana | Chemical and biochemical properties in a silty loam soil under conventional and organic management[END_REF]. The utilization of organic amendment usually leads to important modifications of both soil microbiological and biochemical properties [START_REF] Hueso | Severe drought conditions modify the microbial community structure, size and activity in amended and unamended soils[END_REF][START_REF] Macci | Almond tree and organic fertilization for soil quality improvement in southern Italy[END_REF]. Several studies have indeed pointed out the higher microbial biomass and diversity in soils amended with compost [START_REF] Bulluck | Organic and synthetic fertility amendments influence soil microbial, physical and chemical properties on organic and conventional farms[END_REF]. Microbial respiration, enzymatic activities such as dehydrogenase and phosphatase activities, and N mineralization rates are also usually enhanced after the amendment of organic substrates [START_REF] Gil-Sotres | Different approaches to evaluating soil quality using biochemical properties[END_REF][START_REF] Liu | The earthworm Aporrectodea caliginosa stimulates abundance and activity of phenoxyalkanoic acid herbicide degraders[END_REF][START_REF] Marinari | Influence of organic and mineral fertilisers on soil biological and physical properties[END_REF]. Soil macrofauna has also been considered as an important component of soil quality [START_REF] Ruiz | IBQS: A synthetic index of soil quality based on soil macro-invertebrate communities[END_REF]. Amongst soil organisms, earthworms are considered as key soil engineers due to their influences on soil structural properties [START_REF] Jouquet | Soil invertebrates as ecosystem engineers: intended and accidental effects on soil and feedback loops[END_REF][START_REF] Lavelle | Soil function in a changing world: the role of invertebrate ecosystem engineers[END_REF]. As a consequence, earthworms regulate microbial populations with the elimination of some microorganisms and the proliferation of others [START_REF] Byzov | Fate of soil bacteria and fungi in the gut of earthworms[END_REF][START_REF] Drake | As the worm turns: the earthworm gut as a transient habitat for soil microbial biomes[END_REF][START_REF] Marhan | Phospholipid fatty acid profiles and xylanase activity in particle size fractions of forest soil and casts of Lumbricus terrestris L. (Oligochaeta, Lumbricidae)[END_REF][START_REF] Mummey | Endogeic earthworms differentially influence bacterial communities associated with different soil aggregate size fractions[END_REF]. Earthworms have also been used for the management of organic wastes and the production of high-quality compost called 'vermicompost' [START_REF] Edwards | The influence of vermicomposts on plant growth and pest incidence[END_REF]. Vermicompost is especially interesting as an organic fertilizer because of its high contents of plant available nutrients (N, P, K) [START_REF] Edwards | The use of earthworms in the breakdown of organic wastes to produce vermicomposts and animal feed protein[END_REF][START_REF] Edwards | The influence of vermicomposts on plant growth and pest incidence[END_REF][START_REF] Jouquet | Does compost and vermicompost improve macronutrient retention and plant growth in degraded tropical soils?[END_REF]. The formation of vermicompost, which involves the activity of epigeic earthworms in controlled environments, appears to be a useful indirect avenue for improving physico-chemical properties of soils [START_REF] Edwards | The influence of vermicomposts on plant growth and pest incidence[END_REF][START_REF] Gonzalez | Influence of organic amendments on soil quality potential indicators in an urban horticultural system[END_REF]. Several studies also pointed out the specific structure and higher activity of bacterial communities in vermicompost [START_REF] Aira | Optimizing vermicomposting of animal wastes: Effects of rate of manure application on carbon loss and microbial stabilization[END_REF][START_REF] Pramanik | Changes in Organic -C, N, P and K and Enzyme Activities in Vermicompost of Biodegradable Organic Wastes under Liming and Microbial Inoculants[END_REF][START_REF] Sen | Do earthworms affect dynamics of functional response and genetic structure of microbial community in a lab-scale composting system?[END_REF][START_REF] Yakushev | The effect of earthworms on the physiological state of the microbial community at vermicomposting[END_REF][START_REF] Yakushev | Estimation of the effects of earthworms and initial substrates on the bacterial community in vermicomposts[END_REF][START_REF] Yasir | Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity[END_REF].

Recent studies have compared the influence of compost and vermicompost from buffalo manure and purely chemical fertilizers on tropical soil quality and plant growth [START_REF] Amossé | The flows of nitrogen, bacteria and viruses from the soil to water compartments are influenced by earthworm activity and organic fertilization (compost vs. vermicompost)[END_REF][START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF][START_REF] Jouquet | The rehabilitation of tropical soils using compost and vermicompost is affected by the presence of endogeic earthworms[END_REF][START_REF] Jouquet | Does compost and vermicompost improve macronutrient retention and plant growth in degraded tropical soils?[END_REF][START_REF] Ngo | Transformation of buffalo manure by composting or vermicomposting to rehabilitate degraded tropical soils[END_REF][START_REF] Ngo | The effect of earthworms on carbon storage and soil organic matter composition in tropical soil amended with compost and vermicompost[END_REF]. These studies suggest that the application of composted and vermicomposted buffalo manure reduces significantly the amount of chemical fertilizers needed, as well as improving soil quality and reducing off-site effects such as nutrient leaching and water pollution. Recently, Amossé et al. [START_REF] Amossé | The flows of nitrogen, bacteria and viruses from the soil to water compartments are influenced by earthworm activity and organic fertilization (compost vs. vermicompost)[END_REF] and Doan et al. [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF] also showed that endogeic earthworms are more active when compost is used as fertilizer in comparison with vermicompost with a better development of earthworm abundance and biomass and cast production but variable effects on plant growth.

Therefore, the aim of this study was to determine the influence of the interaction between organic fertilization (compost vs. vermicompost produced from buffalo manure) and endogeic earthworm activity on soil microbial diversity, activity and some associated biochemical properties. We used soil samples from the experiment carried out by Doan et al. [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF] to test our hypotheses: (1) that organic fertilization should increase microbial activities and diversity but that this effect should be more pronounced when vermicompost was used, and (2) that these effects should be increased in the presence of endogeic earthworms.

Materials and Methods

Soil properties and organic amendments

The soil was sampled in the 0-10 cm layer of a fallow in the red river delta (Dong Ngac commune, Ha Noi, Vietnam, 21°5'28"N, 105°47'2"E), air-dried and sieved at 2 mm to discard stones and litter residues. This soil is described as Eutric Fluvial [START_REF] Fao | Soil map of the world revised legend[END_REF][START_REF] Haruyama | Problem Soils of the Paddy Fields in the Eastern Coastal Plain of the Red River Delta, VietNam[END_REF] with low organic carbon (OC = 9.7 mg g -1 ) and nutrient content (total nitrogen = 0.9 mg g -1 ; total phosphorus, P 2 O 5 = 1.8 mg g -1 ; available phosphorous = 0.46 mg g -1 ; and total potassium, K 2 O = 1.1 mg g -1 ). Soil was alkaline (pH H2O = 8.0, pH KCl = 7.7) and mainly sandy (61.0% sand, 28.7% silt and 10.3% clay, mainly kaolinite). Compost and vermicompost were produced in a farm in Dong Cao village from domestic buffalo manure after 3 months of maturation in two different and separated units, as reported by Jouquet et al. [START_REF] Jouquet | The rehabilitation of tropical soils using compost and vermicompost is affected by the presence of endogeic earthworms[END_REF]. The procedure adopted was based on local farmer's knowledge.

Briefly, buffalo manure was placed in 500 L bags and covered by a lid to prevent evapotranspiration and moisture addition due to rainfall, thereby conserving compost humidity and preventing anaerobic conditions due to rainwater addition. Every 1-2 weeks the compost was mixed thereby increasing aeration and favouring aerobic conditions. Vermicomposting was carried out in a similar way, the only difference being the addition of earthworms. The compost was significantly more alkaline and with higher C but lower N contents than the vermicompost substrate (pH H2O = 8.5 (SE: 0.2) and 7.5 (SE: 0.1), OC = 178 (SE: 3) and 164 mg g -1 (SE: 8), and N = 14 (SE: 1) and 16 mg g -1 (SE: 1) respectively for compost and vermicompost). Concentration in P and K were similar between compost and vermicompost with 14 (SE: 2) and 11 mg g -1 (SE: 1), respectively for total P and K. More information on the chemical properties of these substrates and those of the buffalo manure that was used to produce compost and vermicompost can be found in Ngo et al. [START_REF] Ngo | The effect of earthworms on carbon storage and soil organic matter composition in tropical soil amended with compost and vermicompost[END_REF]. The earthworm species used to produce vermicompost was Eisenia andrei.

Greenhouse experiment

The experiment was carried out during 15 months in a greenhouse at the Soil and Fertilizer Research Institute (SFRI), in Hanoi, Vietnam. The air humidity was always high, between 75 and 100% and the average daily temperature varied from 15 to 25 °C during the year. The experiment was set up in 10 L baked clay pots filled with 5 kg of soil mixed thoroughly with compost, vermicompost, or the equivalent amount of inorganic nutrients, applied as mineral fertilizers (n = 3 replicates). These amendments were repeated after each vegetation period, in total 3 times during the experiment. The amount of compost and vermicompost was calculated to be equivalent to applying 20 t ha -1 (dry weight) of organic substrate (90.5 g pot -1 ). Mineral fertilizers were urea (CH 4 N 2 O, %N = 46.3%), potash (K 2 O, %K = 60%) and phosphate (P 2 O 5 , %P = 16%).

The equivalent amount of N, P and K found in compost and vermicompost was applied in the control soil as mineral fertilizers. When necessary, additional nutrients were also applied to the compost and vermicompost substrates (more information on the experiment is available in Doan et al. [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF]). This protocol allowed us to compare the influence of substrates which had exactly the same N, P and K content. Thereafter we planted common agricultural crops: maize (June to September 2008, variety LNS 222), tomato (November 2008 to April 2009, variety HT14), and maize again (June to September 2009, variety LNS 222). For the three dates, only one plant was grown per pot. The influence of the treatments on plant growth and yield, and SOM quality can be found in Doan et al. [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF] and Ngo et al. [START_REF] Ngo | The effect of earthworms on carbon storage and soil organic matter composition in tropical soil amended with compost and vermicompost[END_REF], respectively. Briefly, this study showed that plant yield was always higher for the mineral treatment and that the beneficial positive effect of vermicompost decreased over time. Overall plant yields were always the highest for the mineral and lowest for compost treatments. Although the application of vermicompost led to a similar yield as the control treatment for the first maize planting, its beneficial influence decreased during the experiment until its effect was similar to that of the compost treatment.

The influence of earthworms on soil microbial properties was investigated by adding two adults of the endogeic Metaphire posthuma per pot (n = 3 replicates) before each cultivation (before the 2 maize and tomato plantings), then reaching a density close to the one observed in the field. This species was found in the garden of the SFRI institute. Metaphire posthuma is a medium size endogeic geophagous earthworm ~10 cm in length and 5 mm in diameter on average at the adult stage which produces approximately five to 10 fold its own weight in casts per day [START_REF] Bottinelli | How earthworms accelerate soil porosity under watering events[END_REF][START_REF] Jouquet | Does compost and vermicompost improve macronutrient retention and plant growth in degraded tropical soils?[END_REF][START_REF] Jouquet | What are the limits of the drilosphere? An incubation experiment using Metaphire posthuma[END_REF][START_REF] Jouquet | Does the influence of earthworms on water infiltration, nitrogen leaching and soil respiration depend on the initial soil bulk density? A mesocosm experiment with the endogeic species Metaphire posthuma[END_REF]. At the end of the experiment, the number of earthworms reached 0, 1.2 (±0.2) and 3.5 (±0.8), in the mineral, vermicompost and control treatments respectively [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF]. Earthworms only had an impact on plant growth and yield in the compost treatment. The effect of earthworms was initially null (i.e. for the first maize crop), positive (i.e. for tomato planting) and finally negative (i.e. for the second maize planting).

In total, 18 pots were set up (3 fertilization treatments x 2 earthworm treatments x 3 replicates). Soil samples were collected at the end of the experiment.

Soil bacterial diversity

Total DNA samples were extracted from 1 g of dry soil from three randomly selected soil cores for each plot. The DNA extracts were purified using a Spin Column S-400 HR (Amersham) and quantified with a spectrophotometer at 260 and 280 nm (NanoDrop® ND-1000 UV-Vis Spectrophotometer). Amplifications were attempted using universal primers 338F/518R [START_REF] Lane | Nucleic acid techniques in bacterial systematics[END_REF][START_REF] Muyzer | Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA[END_REF] with a GC-clamp [START_REF] Sheffield | A simple and efficient method for 632 attachment of a 40base pair, GC rich sequence to PCR amplified DNA[END_REF] attached to the 5' extremity to the forward primer for DGGE optimization [START_REF] Muyzer | Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA[END_REF]. Taq polymerase Ready-To-Go (Amersham Pharmacia, USA) was added to 0.25 µM of each primer and 50 ng of template DNA. Amplifications were performed in 25 µl reaction mixtures with a thermo cycler (Eppendorf). PCR cycles were: initial denaturation 95°C for 5 min, and 30 cycles of denaturation 94°C for 1 min, annealing 65°C for 1 min and elongation 72°C for 3 min, and a final extension step at 72°C for 10 min. After amplification, PCR products were applied to 8% (w/v) polyacrylamide gels, except that denaturing gradient was made from 40 to 70% (100% denaturant contains 7M urea and 40% formamide). The electrophoresis was run for 16 h at 60°C and 75 V in a DcodeTM Universal Mutation Detection System (Biorad, USA) containing 1 x TAE buffer. The gels were after stained with ethidium bromide for 15 min, washed with MilliQ water and visualized by UV transillumination with the GelDoc 2000 system (BioRad, Richmond, California, USA). Scanned images of the DGGE gels were analyzed with TotalLab Quant software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK) and the density and mobility of the bands (OTU: Operation Taxonomy Units) were calculated.

Sole carbon source utilization profile

Bacterial cells were extracted from soil for inoculation in Ecoplates TM (AES Laboratory France). The Ecoplates TM contain 3 replicates of 31 different environmentally relevant carbon sources and one control well per replicate [START_REF] Choi | Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities[END_REF]. A volume of 150 µl supernatant was inoculated in each well, and the plates were incubated in darkness on a shaker (150 rpm) at 25°C for 7 days. The color development was measured as absorbance at 595 nm using a Microplate spectrophotometer (EL800-PC, Biotek Instruments, Winooski, USA) every 24h for one week. All kinetic curves suggested that after 72h incubation time, the wells with the most active microbial communities reached the asymptote of color development. Therefore, this point was considered as the optimal incubation time for further statistical analyses. Data were collected using Microlog 4.01 software. They were normalized by average well color development (AWCD), as described by Garland and Mills [START_REF] Garland | Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level-sole carbonsource-utilization[END_REF]: AWCD = ∑ (C-R)/n, where C is color production within each well, R is the value of the no-substrate control well of each plate, and n is the number of substrates (n = 31). The sole carbon source utilization profiles were determined on three replicate samples of each soil.

Enzyme activity

Enzyme activities were used as a broad-spectrum indicator of soil biological activity [START_REF] Miambi | Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas[END_REF]. Enzyme activity measurements were made on samples with 75% of their water holding capacity and incubated at 25°C for 48 h to reactivate the microflora. These assays were carried out in triplicate to measure activities. All enzyme activities were determined at optimum conditions of pH, temperature and substrate concentration in order to get an assessment of their maximum potential activity in soil. N-acetylglucosamidase, β-Glucosidase, amylase and xylanase activities were determined according to Mora et al. [START_REF] Miambi | Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas[END_REF]. Urease activity was determined according to Tabatabai [START_REF] Tabatabai | Soil Enzymes, in: Methods of Soil Analysis[END_REF] and alkaline and acid phosphatase activities were measured following Kandeler et al. [START_REF] Kandeler | Tillage changes microbial biomass and enzyme activities in particle-size fractions of a Haplic Chernozem[END_REF]. These enzymes were chosen based on their importance in the nutrient cycles of C (β-glucosidase, amylase and xylanase), N (urease and N-acetyl-glucosamidase) and P (alkaline and acid phosphatase).

Statistical analyses

Data from DGGE analysis and catabolic potential using Biolog EcoPlate™ were used to evaluate the structural and functional diversity of bacterial community as suggested by Eicherner et al. [START_REF] Eicherner | Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shock in the activated sludge microbial community[END_REF] and Zak et al. [START_REF] Zak | Functional diversity of Microbial communities: a quantitative approach[END_REF], respectively for the DGGE and Biolog analyses. The Shannon indices (H) were obtained as follows: H = -∑ P i ln (P i ), where P i = n i / N, and n i corresponded to the height of a DGGE band peak, or OTU (Operation Taxonomy Unit), and N the sum of all the peak heights. For the Biolog analysis, P i corresponded to the ratio of the consumption of a particular substrate to the sums of consumption of all substrates after 72 h of incubation. Richness indices (S) were either the number of OTUs detected from each lane or the number of wells with a corrected absorbance greater than 0.25 in Biolog EcoPlate TM .

Results were analysed by analysis of variance (two-way ANOVA) with treatments (i.e., type of fertilizer and presence or absence of earthworms) as independent variables. Comparisons between means were tested with LSD test. A Principal Component Analysis (PCA) was carried out to describe enzyme activity analyses. All statistical calculations were carried out using R [START_REF] Pramanik | Changes in Organic -C, N, P and K and Enzyme Activities in Vermicompost of Biodegradable Organic Wastes under Liming and Microbial Inoculants[END_REF]. Differences among treatments were declared significant at the <0.05 probability level.

Results

Genetic structure of bacterial community

A dendrogram of similarity showed a similarity of 64% between the initial soil substrate and those sampled after 15 months of cultivation (Figure 1). The percentage of similarity was also very high between organically and chemically fertilized soils (77% of similarity) on one hand and composted and vermicomposted soils on the other hand (85% of similarity). Earthworms had no effect on the structure of bacterial communities in soils amended with mineral fertilization (95% of similarity) and their effects were low in soils amended with vermicompost and compost (90% and 85% of similarity, respectively).

The influence of the different treatments on the genetic diversity of bacterial populations is shown in Table 1a.

Functional diversity by Biolog analysis and enzyme activity

Microbial metabolic activity was assessed through the measurement of AWCD in Biolog EcoPlate TM (Figure 2), and through substrate H and S indices (Table1b). The amendment of compost and vermicompost led to increase AWCD, H and S with higher values for composted soils when compared to vermicomposted and mineral fertilized soils with and without earthworms. The inoculation of earthworms did not influence these indices for the mineral treatment. Conversely, an interaction between organic fertilization and earthworm presence was observed for the compost and vermicompost treatments. Earthworms increased AWCD, H and S indices for vermicompost treatment but the opposite effect was observed for the compost treatment.

A principal component analysis was carried out from the enzyme metabolic properties of soils (Figure 3, details are shown in Table 2). Treatments were mainly separated along the first axis which explained 68.3% of the total variability. Chemical fertilization led to lowest enzymatic activities, with no influence of earthworms. The amendment of vermicompost led to higher xylanase, amylase and β-Glucosidase activities and lower N-acetyl-glucosamidase and alkaline phosphatase activities than for the compost treatment. The presence of earthworms entailed a reduction in xylanase, βglucosidase and alkaline phosphatase for the vermicompost treatment. Conversely, the presence of earthworms had negative effect on N-acetyl-glucosamidase, alkaline and acid phosphatase, and urease activities for the compost treatment.

Discussion

Effect of organic amendments on soil microbial properties

Maintenance of SOM is important for the long-term productivity of agroecosystems.

The amendment of organic substrates, such as crop residues and compost, aims to counteract the progressive loss of OM in agricultural soils [START_REF] Marinari | Influence of organic and mineral fertilisers on soil biological and physical properties[END_REF][START_REF] Tejada | Application of a green manure and green manure composted with beet vinasse on soil restoration: effects on soil properties[END_REF]. As a consequence, soil microbiological properties are usually improved after the amendment of OM [START_REF] Kl Bailey | Suppressing soil-borne diseases with residue management and organic amendments[END_REF][START_REF] García-Ruiz | Suitability of enzyme activities for the monitoring of soil quality improvement in organic agricultural systems[END_REF]67,[START_REF] Stark | Influence of organic and mineral amendments on soil microbial properties and processes[END_REF]. Our study confirmed this general trend and showed the positive influence of buffalo manure amendment, either in form of compost or vermicompost, on soil microbial properties. Indeed, our study showed that soils amended with compost and vermicompost were characterized by higher bacterial and catabolic diversity (H and S) indices and higher enzymatic activities than control soils which only received mineral fertilizers.

Organic amendments are sources of available energy for soil microorganisms [START_REF] Gomez | Soil bacterial functional diversity as influenced by organic amendment application[END_REF]. However, the quality of the organic amendments is of major importance in the regulation of microbiological properties. Several studies related the quality and stability of composted and vermicomposted organic substrates to their effects on microbiological properties [START_REF] Aira | Eisenia fetida (Oligochaeta: Lumbricidae) modifies the structure and physiological capabilities of microbial communities improving carbon mineralization during vermicomposting of pig manure[END_REF][START_REF] Atiyeh | Pig manure vermicomposts as a component of a horticultural bedding plant medium: effects on physicochemical properties and plant growth[END_REF][START_REF] Sen | Do earthworms affect dynamics of functional response and genetic structure of microbial community in a lab-scale composting system?[END_REF][START_REF] Yakushev | Estimation of the effects of earthworms and initial substrates on the bacterial community in vermicomposts[END_REF]. These studies indicate that the bacterial community structure in vermicompost considerably differs from that in analogous composts. Vermicomposts are usually more stable than composts, with higher availability of nutrients and improved microbiological properties [START_REF] Anastasi | Isolation and identification of fungal communities in compost and vermicompost[END_REF][START_REF] Pramanik | Changes in Organic -C, N, P and K and Enzyme Activities in Vermicompost of Biodegradable Organic Wastes under Liming and Microbial Inoculants[END_REF][START_REF] Vivas | Assessing the Impact of Composting and Vermicomposting on Bacterial Community Size and Structure, and Microbial Functional Diversity of an Olive-Mill Waste[END_REF][START_REF] Yakushev | The effect of earthworms on the physiological state of the microbial community at vermicomposting[END_REF]. In our study, vermicompost was characterized by lower C and higher N content in comparison with the compost substrate (see Material and Method). This suggests that vermicomposting led to higher microbial activity and higher rate of OM decomposition than composting. After 15 months of experiment, soils amended with vermicompost were characterized lower metabolic activity (Figure 3) and lower genetic and functional diversity and richness (Table 1). Therefore, these results do not confirm the general assumption that vermicompost enhances microbial and enzymatic activities in soils [START_REF] Pramanik | Changes in Organic -C, N, P and K and Enzyme Activities in Vermicompost of Biodegradable Organic Wastes under Liming and Microbial Inoculants[END_REF][START_REF] Yakushev | The effect of earthworms on the physiological state of the microbial community at vermicomposting[END_REF]. They are however in line with those of Aira and Dominguez [START_REF] Aira | Microbial and nutrient stabilization of two animal manures after the transit through the gut of the earthworm Eisenia fetida (Savigny, 1826)[END_REF] who found higher microbial biomass but lower microbial activity in vermicompost in comparison with compost. As suggested by Ngo et al. [START_REF] Ngo | Transformation of buffalo manure by composting or vermicomposting to rehabilitate degraded tropical soils[END_REF], the epigeic E. andrei accelerates the decomposition of the buffalo manure during vermicomposting probably through an enhancement of the microbial activity. The end-product is a more decomposed and stabilized organic substrate with lower forms of C and more N available to microorganisms. As a consequence, microbial populations were more active and diversified in the soil amended with compost, as shown by higher AWCD, and H and S indices which reflects the oxidative capacity of soil microorganisms, but also H and S indices analyzed by DGGE which represent the number and diversity of bacterial community.

Earthworms modulate the effect of organic fertilizers on soil microbial properties

Despite the high diversity of tropical earthworms, only a small number of species have been intensively studied in relation to soil processes and ecosystem functioning. The fast turnover of OM in tropical environments and the low amount of litter and fresh OM debris in tropical agro-ecosystems lead to a predominance of endogeic earthworm species [START_REF] Lavelle | Soil Ecology[END_REF]. As endogeic earthworms are known to affect soil physical, chemical and microbial properties [START_REF] Lavelle | Soil Ecology[END_REF], we hypothesized that endogeic earthworm activity can modulate the effect of organic fertilizers on soil microbial properties. Whether the positive influence of compost on soil microbial properties is maintained in the presence of endogeic earthworms remains however unexplored. As shown with the dendrogram of similarity, the bacterial community structure was mainly determined by the fertilization treatment (mineral, compost vs. vermicompost amendment), since its effect exceeded the effect of earthworms. From the same experiment, Doan et al. [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF] showed that M. posthuma development was the highest in presence of compost, low in presence of vermicompost while earthworms did not survive in the mineral treatment. These results can easily be explained by the higher availability of C and N in compost than in vermicompost, and by the absence of easily available C resources in the mineral treatment [START_REF] Ngo | The effect of earthworms on carbon storage and soil organic matter composition in tropical soil amended with compost and vermicompost[END_REF]. These results also confirm the study of the Bisht et al. [START_REF] Bisht | Reproductive potential of the earthworm Metaphire posthuma (Oligochaeta) in different food substrate[END_REF] who showed that M. posthuma develops very well in presence of cow dung and they suggest that this species is belonging to the polyhumic endogeic functional group [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF]. Consequently, M. posthuma did not influence soil microbiological properties in the mineral treatment but it led to significant changes in compost and vermicompost treatments. Interestingly, its effects in compost treatment were very different, if not the opposite, from those observed for the vermicompost treatment. Indeed, although the diversity of bacteria was not influenced by M. posthuma in the compost treatments, significant increases in CFU and H and S diversity indices were observed for vermicompost treatment. Earthworms also differently influenced catabolic activities with higher AWCD and H and S diversity indices for vermicompost and lower AWCD and H diversity index in the compost treatment. Although increased enzyme activities are usually observed in presence of earthworms [START_REF] Sen | Do earthworms affect dynamics of functional response and genetic structure of microbial community in a lab-scale composting system?[END_REF], our study showed decreasing soil enzyme activities for both compost and vermicompost treatments. However, all the enzyme activities were not similarly affected, therefore reflecting an interaction between the nature of the organic amendment and M. posthuma. Despite a high degree of DGGE band similarity (OTU) between treatments, changes in the activity of enzymes of C, N and P cycles reflect the activity of specific microbial populations [START_REF] Sen | Do earthworms affect dynamics of functional response and genetic structure of microbial community in a lab-scale composting system?[END_REF] and support the hypothesis that M. posthuma led to divergent dynamics of bacterial communities in soils amended with compost and vermicompost. These findings could partially be explained by the nature of the organic substrates. The negative effect of earthworms on soil microbial properties in composted soil could result from a competition between bacteria and earthworms for organic resources and/or from the consumption of microbes by earthworms [START_REF] Tiunov | Carbon availability controls the growth of detritivores (Lumbricidae) and their effect on nitrogen mineralization[END_REF]. Conversely, the higher metabolic activity of microbes in vermicomposted soil suggests that earthworm activity led to a stimulation of microbes in the presence of vermicompost. Litter and OM decomposition rates are often stimulated in experiments comparing soils with and without earthworms [START_REF] Brown | Effects of earthworms on plant production in the tropics[END_REF]. It is therefore likely that earthworms stimulate microbial activity through the fragmentation and alteration of vermicompost in their gut and/or through the production of more easily degradable organic substances (i.e., the mucus) leading to priming effect processes [START_REF] Bernard | Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil[END_REF]. These results can be seen in line with the niche construction, or feedback loop hypothesis applied to soil engineers [START_REF] Jouquet | Soil invertebrates as ecosystem engineers: intended and accidental effects on soil and feedback loops[END_REF]. The stimulation of bacterial activity in vermicomposted soil led to increased palatability of the OM from highly polymerised organic substances to more simple molecules and/or microbial biomass which can afterwards be consumed by earthworms [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. This hypothesis is supported by the fact that earthworm development was difficult at the beginning of the experiment in vermicomposted soil, probably because of the low palatability of vermicompost for earthworms, but increased over time [START_REF] Doan | Interaction between compost, vermicompost and earthworm activity. A one year greenhouse experiment[END_REF].

The presence of endogeic earthworms in soil is more a generality than an exception. This study therefore highlights the importance of the quality of organic resources applied as amendments and the importance of endogeic earthworms in evaluating the relevance of compost and vermicompost in the field.

[67] M. Zaman, K.C. Cameron, H.J. Di, K. Inubushi, Changes in mineral N, microbial and enzyme activities in different soil depths after applications of dairy shed effluent and chemical fertilizer. Nutr. Cycl. Agroecosyst. 63 (2002) 275-290.

  Composted soils were characterized by highest H and S values and mineral soils by lowest values. The utilization of vermicompost led to intermediate results. Earthworms had no influence on the bacterial community structure, except for the vermicompost treatment where they increased H and S indices in soil.

  which were best predicted by the variation in clusters, are identified as OTUs (Operation Taxonomy Units).

Figure 1 .

 1 Figure 1. Dendrogram of similarity derived from DGGE analyses of 16S rRNA

Figure 2 .

 2 Figure 2. Average well color development (AWCD) calculated from Biolog EcoPlate TM absorbance data after 72 h of incubation (n = 3) in the soil

Table 1 .

 1 Shannon (H) and Richness (S) diversity indices calculated from (a) OTUs * of DGGE gel and (b) individual substrate consumption in Biolog EcoPlate TM in the initial soil (T 0 ) and in soil after 15 months of cultivation from a greenhouse experiment. Treatments are mineral, compost and vermicompost amendments incubated with (EW+) and without (EW-) earthworms. Standard errors are given in parenthesis. Values with the same letter are not different at P = 0.05 (LSD test, n=3).

	Soil amendment
	(a) Structural diversity

c EW+ 2.18 (0.2)

Table 2 .

 2 Enzyme activities in soil (U g -1 ) before the experiment (initial soil T 0 ) and after 15 months of cultivation with (EW+) or without (EW-) earthworms. Standard errors are given in parenthesis. For each variable, values with the same letter are not different at P = 0.05 (LSD test, n=3). nd: not detected.

		N-acetyl
	Soil amendment	-glucosamidase β-Glucosidase alkaline phosphatase acid phosphatase	Urease	Amylase	Xylanase
	Initial soil To	0.73 (0.04)	0.68 (0.04)	13.81 (0.56)	4.42 (0.15)	0.19 (0.04)	nd	nd
	Mineral
	EW-	0.12 (0.03) c	0.83 (0.06) d	17.95 (0.82) e	6.95 (0.24) c	1.00 (0.19) c	nd	nd
	EW+	0.11 (0.02) c	1.04 (0.21) d	17.45 (0.62) e	7.87 (0.5) c	0.80 (0.07) c	nd	nd
	Compost
	EW-	2.13 (0.16) a	2.46 (0.08) c	42.41 (2.72) a	14.68 (1.44) a	2.11 (0.14) a 2.51 (0.18) b	nd
	EW+	1.83 (0.16) b	2.43 (0.08) c	33.07 (2.72) c	10.39 (1.44) b	1.74 (0.14) b 2.4 (0.18) b	nd
	Vermicompost
	EW-	1.86 (0.07) b	3.16 (0.77) a	34.61 (1.89) b	13.55 (0.71) a	2.12 (0.08) a 5.32 (0.70) a 0.50 (0.10) a
	EW+	1.82 (0.05) b	2.82 (0.67) b	30.06 (0.42) d	13.49 (1.03) a	2.19 (0.02) a 4.87 (0.61) a 0.33 (0.00) b

Acknowledgment

This project was financially supported by CNRS (SystéMO project) and IRD (unit research UMR-211-BIOEMCO) French institutes. We acknowledge with gratitude Dr.

Tran Duc Toan and the SFRI (Soils and Fertilizers Research Institute) Vietnamese institute for providing access to the greenhouse and laboratories and to Emma Rochelle-Newalle, Corinne Rouland-Lefevre and Didier Orange for valuable discussions.