Thi Loi Dao

Thuan Hoang

Tran Duc

Anh Ly

Jean Christophe Lagier

Sophie Alexandra Baron

Didier Raoult

Philippe Parola

Johan Courjon

Pierre Marty

Hervé Chaudet

Van Thuan Hoang

Sophie Alexandra Baron

Philippe Gautret
email: philippe.gautret@club-internet.fr

Sputum proteomic analysis for distinguishing between pulmonary tuberculosis and non-tuberculosis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): preliminary results

Keywords: MALDI-TOF, proteomic, tuberculosis, respiratory infection, sensitivity, specificity

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

Introduction

In 2018, the World Health Organization reported 10 million new cases of tuberculosis and 1.2 million deaths around the world, making the disease a significant public health concern [START_REF]Global tuberculosis report[END_REF]. The diagnosis of pulmonary tuberculosis is currently based on the detection of M. tuberculosis complex by direct examination with microscopy or by detection of specific DNA sequences by PCR followed by isolation of the mycobacteria by culture.

Sputum contains a wide variety of proteins the composition of which most closely reflects the condition of the lungs. Another advantage is that sputum is easy to sample in a non-invasive way [START_REF] Gray | Sputum proteomics in inflammatory and suppurative respiratory diseases[END_REF]. Numerous studies have analysed the protein profile of sputum to identify protein markers as a new alternative diagnostic method [START_REF] Paone | Blood and sputum biomarkers in COPD and asthma: a review[END_REF][START_REF] Dong | Sputum and salivary protein biomarkers and point-of-care biosensors for the management of COPD[END_REF]. The vast majority of proteomic studies of sputum have been conducted in patients with inflammatory diseases including asthma and chronic obstructive pulmonary disease (COPD) but rarely in patients with lung infections [START_REF] Wheelock | Application of 'omics technologies to biomarker discovery in inflammatory lung diseases[END_REF]. Several authors suggested that specific protein profiles could represent the signature of pulmonary tuberculosis [START_REF] Fu | Proteomic analysis of sputum in patients with active pulmonary tuberculosis[END_REF][START_REF] Bishwal | Sputum Proteomics Reveals a Shift in Vitamin D-binding Protein and Antimicrobial Protein Axis in Tuberculosis Patients[END_REF][START_REF] Mateos | Highresolution quantitative proteomics applied to the study of the specific protein signature in the sputum and saliva of active tuberculosis patients and their infected and uninfected contacts[END_REF].

The objective of this study was to evaluate the feasibility and diagnostic contribution of protein profiling using MALDI-TOF MS when directly applied to sputum from patients with confirmed pulmonary tuberculosis, patients with lower respiratory tract infections other than tuberculosis, and patients with no lower respiratory tract infection in whom tuberculosis had been excluded (controls). We hypothesised that rapid routine and blind MALDI-TOF MS analysis could accurately distinguish these three populations.

Methods

Study population

The population was recruited between January 2019 and June 2020 among hospitalized patients at the IHU, and who were suspected of having pulmonary tuberculosis at the time of admission.

In line with the WHO recommendations for screening for tuberculosis [9], the population included subjects with clinical symptoms suggestive of pulmonary tuberculosis, as well as subjects with an epidemiological context putting them at risk of pulmonary tuberculosis, including people who were in close contact with patients with confirmed tuberculosis, people living with HIV, and people who were immunosuppressed.

Based on the final diagnosis, patients were divided into three groups: (1) the pulmonary tuberculosis group (PTB), in which pulmonary tuberculosis had been confirmed by direct microscopic examination and/or Gene Xpert ® MTB-RIF and/or culture; (2) the lower respiratory tract infections other than tuberculosis (non-TB-LRTI) group, including patients with an LRTI whose tuberculosis tests had all been negative and who received a final diagnosis of pneumonia or exacerbation of COPD with a secondary infection; and (3) non-tuberculosis, non-LRTI (non-TB control) group including patients whose tuberculosis tests were all negative and had excluded any LRTIs. A flow diagram of the study is presented in Supplementary Figure 1. Details of these three groups are shown in Supplementary Table S1.

Clinical samples

Sputum samples were collected as part of the routine diagnostic activity for pulmonary tuberculosis at our clinical microbiology laboratory (IHU). Sputum specimens were collected in accordance with the relevant guidelines and regulations [START_REF] El Khéchine | Diagnosis of pulmonary tuberculosis in a microbiological laboratory[END_REF]. Microscopic detection of acid-fast bacilli (AFB) was performed by laboratory technicians using a commercially available kit featuring Kinyoun staining (RAL Diagnostics, Toulouse, France). Sputum specimens were inoculated onto C-Top COSMO medium (Eurobio SA, Les Ulis, France) after decontamination.

GeneXpert ® MTB/RIF (Cepheid, Sunnyvale, CA, United-States) was used to detect tuberculosis mycobacteria and rifampicin resistance genes in sputum, according to the manufacturer's recommendations.

The remaining sputum material was kept into a dry, sterile container and stored at -80°C until processing for mass spectrometry.

Mass spectrometry

For each sample, a volume of 2.0 μL of sputum was directly deposited on four wells (0.5 μL each well) of a MALDI 96 steel target, then dried at room temperature. Deposits were next coated with 1.0 μL of CHCA matrix solution (α-cyano-4-hydroxycinnamic acid diluted into 500 μL of acetonitrile, 250 μL of HPLC grade water and 250 μL of 10% trifluoroacetic acid and).

The plate was dried at room temperature, then was placed into a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Each sputum sample generated four spectra issued from four deposits. Spectra were recorded within a mass range from 2 to 20 kDa.

Each spectrum was performed in four regions of the same spot and next automatically acquired using the AutoXecute acquisition control in FlexControl software 3.0 (Bruker Daltonics). The BioTyper-RTC v.3.0 software (Bruker Daltonics GmbH) was used to import all mass spectra. The calibration of the mass spectrometer was fully automatic and executed with a commercial solution (BTS: Bacterial Test Standard, Bruker Daltonics GmbH, Germany) and the strategy was completely automated (BioTyper-RTC). The quality criteria of the spectra were based on: (1) -

Bioinformatics analysis

All spectra were analysed using R version 4.0.2 [START_REF] Core | R foundation for Statistical Computing[END_REF][START_REF] Gibb | MALDIquant: a versatile R package for the analysis of mass spectrometry data[END_REF] (Supplementary data). Differences between the two analysed groups were evalued on the discriminant peak identification list using binary predictors. We executed a discriminant analysis between groups using the Binary Discriminant Analysis method [START_REF] Gibb | Differential protein expression and peak selection in mass spectrometry data by binary discriminant analysis[END_REF] for create the list of discriminant peaks for each specimen.

The sensitivity (Se) and specificity (Sp) were calculated by comparing the results of GeneXpert on the same sputum specimens used in the reference method.

Ethics

Mass spectrometry analysis was performed on sputum samples that were collected in the context of routine diagnosis. No additional samples were collected for this study. Clinical data were retrospectively retrieved from medical files and anonymised before analysis. Ethical approval was obtained from the Marseille Institutional Review Board and Ethics Committee (No. 2018-015).

Results

Characteristics of the population

A total of 102 patients were included. Thirty-five cases with confirmed PTB, 36 non-TB LRTI, and 31 non-TB controls were enrolled. Patient details are listed in Table 1 and Supplementary Table S1. PTB and non-TB controls were middle-aged patients, while non-TB LRTI patients were significantly older. The prevalence of clinical symptoms presented by patients varied significantly between groups. In the non-TB control group, 16/31 patients presented clinical symptoms. Most (28/36) patients with non-TB LRTI had acute community-acquired pneumonia.

Differentially expressed peptide peaks

All samples were analysed using MALDI-TOF-MS, and protein profiles were obtained from each sample in the range of 2-20 kDa. Three representative spectra of three groups are shown in Supplementary Figure 2. To distinguish sputum samples collected from PTB patients from the non-TB controls, a total of 86 protein peaks were retrieved from the sputum of 35 PTB patients and 31 non-TB controls. Of these 86 peaks, 11 were significantly different between the two groups (p< 0.05) (Supplementary Table S2).

To distinguish PTB from the non-TB LRTI sputum samples, a total of 92 protein peaks were retrieved from the sputum of 35 PTB patients and 36 non-TB LRTI patients. Of these 92 peaks, eight were significantly different between the two groups (p< 0.05) (Supplementary Table S3).

Protein peaks selected for the decision model of classification

Using the 25 most differentially expressed protein peaks, it was observed that the model distinguished between the PTB patients and the non-TB controls with a sensitivity of 97%, 95% confidence interval (CI) =[85%-100%] and a specificity of 77%, 95%CI=[59%-90%], (Youden index = 0.75) (Table 2 and Supplementary Figure 3 Da, 7348 Da, 3629 Da, 2786 Da and 3671 Da) had higher levels of intensity in the non-TB controls group (Figure 1). A single PTB patient (PTB22) did not clusterise with other PTB patients. This 21-year-old male patient originating from Senegal had a final diagnosis of pulmonary and pleural tuberculosis. This patient was negative for AFB under direct microscopic examination, but positive for M. tuberculosis using GeneXpert ® and sputum culture.

By using the 10 most differentially expressed protein peaks (Figure 2), we found that the model distinguished the PTB patients from the non-TB LRTI patients with a sensitivity of 80% [95%CI=63%-92%] and a specificity of 89% [95%CI=74%-97%], (Youden index= 0.69) (Table 3 and Supplementary Figure 4). Of the top 10 ranking peaks, seven (m/z values: 5687 Da, 5728 Da, 6821 Da, 4242 Da, 6948 Da, 3930 Da, and 5531 Da) had higher levels of intensity in the non-TB LRTI group, and three (m/z values: 4697 Da, 4160 and 4137 Da) had higher levels of intensity in the PTB group (Figure 2).

Evaluation of different diagnostic methods

The performance of the decision model using MALDI-TOF MS, staining methods and culture in comparison to GeneXpert ® is presented in Supplementary Tables 4 and5, along with their sensitivity, specificity, negative predictive value (NPV) values and positive predictive value (PPV). For PTB and non-TB control cases, the sensitivity /specificity was 97% (95%CI=[85%-100%])/77% (95%CI=[59%-90%]) for MALDI-TOF-MS using 25 peaks or when MALDI-TOF-MS and microscopic examination of sputum was combined, 74% (95%CI=[57%-88%])/100% (95%CI=[89%-100%]) for culturing and 74% (95%CI=[57%-88%])/100% (95%CI=[89%-100%]) for microscopic examination of sputum. The PPV was 83% (95%CI=[68%-93%]), 100% (95%CI=[87%-100%]) and 100% (95%CI=[87%-100%]), respectively, and the NPV was 96% (95%CI=[80%-100%]), 78% (95%CI=[62%-89%]) and 78% (95%CI=[62%-89%]), respectively.

For PTB and non-TB LRTI cases, the sensitivity /specificity was 80% (95%CI=[63%-92%])/89% (95%CI=[74%-97%]) for MALDI-TOF MS using 10 peaks, 74% (95%CI=[57%-88%])/100% (95%CI=[90%-100%]) for culturing and 74% (95%CI=[57%-88%])/100% (95%CI=[90%-100%]) for microscopic examination of sputum. The PPV was 88% (95%CI=[71%-96%]), 100% (95%CI=[87%-100%]) and 100% (95%CI=[87%-100%]), respectively, and the NPV was 82% (95%CI=[66%-92%]), 80% (95%CI=[65%-90%]) and 80% (95%CI=[65%-90%]), respectively. When microscopic examination and MALDI-TOF MS were combined, the sensitivity/ specificity was 94% (95%CI=[81%-99%])/89% (95%CI=[74%-97%]) and the PPV /NPV were 89% (95%CI=[75%-97%])/94% (95%CI=[80%-99%]).

By using the same method for classification, we compared the two non-TB groups with 25 most differentially expressed protein peaks. The results are shown in Supplementary Figure 5.

Discussion

Of the 102 patients included in this study (35 with confirmed PTB, 31 non-TB controls, and 36 non-TB LRTI), based on the 25 and 10 most differentially expressed protein peaks, we found that the models distinguished PTB patients from non-TB controls and from non-TB LRTI patients with a sensitivity of 97% and 80% and a specificity of 77% and 89%, respectively. The signal noise ratio threshold 2 was used allowing considering exhaustive peaks and increasing the ability to select peaks to analyze, as previously studied [START_REF] Texier | Insights Into Subspecies Discrimination Potentiality From Bacteria MALDI-TOF Mass Spectra by Using Data Mining and Diversity Studies[END_REF].

In the past, other studies [START_REF] Fu | Proteomic analysis of sputum in patients with active pulmonary tuberculosis[END_REF][START_REF] Bishwal | Sputum Proteomics Reveals a Shift in Vitamin D-binding Protein and Antimicrobial Protein Axis in Tuberculosis Patients[END_REF][START_REF] Mateos | Highresolution quantitative proteomics applied to the study of the specific protein signature in the sputum and saliva of active tuberculosis patients and their infected and uninfected contacts[END_REF] used more resolving methods and could not be applied to a routine diagnostic test. In our work, we did not study for proteomic biomarkers identification. We hypothesized that rapid routine and blinded MALDI-TOF analysis could precisely classify these 10 three groups of patients. We chose a method following as narrowly as possible the standard MALDI-TOF protocol that is used in routine diagnostic.

There are few studies on the use of MALDI-TOF MS directly from biological samples as a diagnostic tool in microbiology. One report showing that direct profiling of small proteins in cerebrospinal fluid (CSF) samples by MALDI-TOF MS may enable the diagnosis of enterovirus (EV) meningitis in comparison to positive and negative CSF samples for EV [START_REF] Torres | Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) proteomic profiling of cerebrospinal fluid in the diagnosis of enteroviral meningitis: a proof-of-principle study[END_REF].

We observed that the NPV of sputum MALDI-TOF MS analysis alone was higher (96%) than that of direct sputum microscopic examination and sputum culture for non-TB controls (78%).

The NPV of sputum MALDI-TOF MS analysis was only 82% for non-TB LRTI patients (compared to 80% for direct sputum microscopic examination and sputum culture). However, when sputum MALDI-TOF MS analysis and direct microscopic examination were combined, the NPV reached 94%. This result suggests that sputum MALDI-TOF MS analysis coupled with microscopic examination could be used as a screening tool for the diagnosis of PTB.

Our study has several limitations, including its small sample size and the fact that it was conducted on frozen samples. The total number of most differentially expressed peaks (10 and 25) may be considered as somewhat high, taking in account the number of patients in each group, but it should be underlined that our method considered both peaks specifically present or absent in any group. Also, we did not identify the specific biomarkers responsible for each peak or group of peaks. There were significant differences of age in the three groups of patients that may have represented a confounding factor. Nevertheless, our study was performed using noninvasive clinical samples from patients suspected of having pulmonary tuberculosis, did not require complex technical expertise and could be conducted by any laboratory personnel. In developing countries, the use of MALDI-TOF MS is limited due to the high price. Yet, when purchased as part of technology platforms with multiple uses in bacteriology, mycology and even entomology, the running costs are much less expensive including the cost of consumables and labour [START_REF] Chabriere | MALDI-TOF MS and point of care are disruptive diagnostic tools in Africa[END_REF][START_REF] Laroche | Medical Entomology: A Reemerging Field of Research to Better Understand Vector-Borne Infectious Diseases[END_REF].

Future studies on fresh sputum samples would be necessary to carry out to assess the optimal conditions of samples for routine diagnostic tests. Studies conducted on a larger sample of patients with confirmed PTB are needed to confirm our results. It would be of interest to include patients with non-tuberculosis Mycobacterium infections as control groups. Finally, there is a need to refine the performance of the method with more than one specimen from a patient at a given time and samples obtained during follow-up in treated patients.

Our study is based on very preliminary results and further investigation must be conducted to confirm the general principle and experimental improvements are needed to strengthen the method. Nevertheless we obtained encouraging first results suggesting that analysis of sputum protein profiles using MALDI-TOF MS directly on samples may enable PTB patients to be distinguished from non-TB patients with or without an LRTI with a good NPV. Because the rapid diagnosis of PTB remains a challenge, the further development of a simple-to-use, rapid and cost-effective tool based on the use of MALDI-TOF MS directly on the sputum would be an attractive screening test, in combination with direct microscopy, in central laboratory settings in future, if results are confirmed.

Table 1: Characteristics of the population in the study with the p-value resulting from the comparison of the three groups Table 2: Sensitivity and specificity using the different number of differentially expressed peptide peaks between the tuberculosis cases and the non-tuberculosis controls Table 3: Sensitivity and specificity using the different number of differentially expressed peptide peaks between the pulmonary tuberculosis cases and the lower respiratory tract infections

  ). Of the top 25 ranking peaks, 15 (m/z values: 4161 Da, 2065 Da, 4137 Da, 6892 Da, 5155 Da, 2128 Da, 2202 Da, 2110 Da, 4355 Da, 6188 Da, 2297 Da, 5227 Da, 5002 Da, 12697 Da and 4283 Da) had higher levels of intensities in the PTB group, and 10 (m/z values: 4901 Da, 2316 Da, 14688 Da, 11769 Da, 11736 Da, 2280

Figure 1 :Figure 2 :

 12 Figure 1: Box plots of the 25 top ranking peaks differentially expressed between the pulmonary tuberculosis and the non-TB control groups. Most of the differentially expressed protein peaks had higher intensity levels in the PTB group than in the non-TB control group

Figure 1 :Figure 2 :

 12 Figure 1: Box plots of the 25 top ranking peaks differentially expressed between the pulmonary tuberculosis and the non-TB control

Table 3 :

 3 Sensitivity and specificity using the different number of differentially expressed peptide peaks between the pulmonary tuberculosis cases and the lower respiratory tract infections

Table 1 :

 1 Characteristics of the population in the study with the p-value resulting from the comparison of the three groups

	Characteristics	Pulmonary tuberculosis case	Non-tuberculosis controls	Non-tuberculosis lower respiratory tract infection	p-value
		N= 35 (%)	N= 31 (%)	N= 36 (%)
	Gender
	Male	30 (85.7)	22 (71.0)	27 (75.0)
					0.33
	Female	5 (14.3)	9 (29.0)	9 (25.0)
	Age, mean (years) ± SD	37.5±15.0	40.1±18.8	59.3±16.8
					<0.000
	Age range	15 -78	15 -85	25 -87
	Chronic respiratory disease	4 (11.4)	7 (22.6)	15 (41.7)	0.01
	Diabetic	1 (2.9)	2 (6.5)	6 (16.7)	0.1
	Cardiovascular disease	4 (11.4)	6 (19.4)	17 (47.2)	0002
	Cancer	0	4 (12.9)	5 (13.9)	0.08
	Immunosuppressed	1 (2.9)	9 (29.0)	9 (25.0)	0.01
	Smoker	19 (54.3)	13 (41.9)	18 (50.0)	0.60
	Alcoholism	12 (34.3)	3 (9.7)	6 (16.7)	0.04
	Clinical symptoms
	General altered state	26 (74.3)	12 (38.7)	19 (52.8)	0.01
	Fever	18 (51.4)	13 (41.9)	29 (80.6)	0.003
	Night sweats	15 (42.9)	6 (19.4)	8 (22.2)	0.06

Table 2 :

 2 Sensitivity and specificity using the different number of differentially expressed peptide peaks between the tuberculosis cases and the non-tuberculosis controls

Acknowledgments

13

We are grateful to Michel Drancourt for critically reviewing the manuscript.

Funding

This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the French National Research Agency under the "Investissements d'avenir" programme, reference ANR-10-IAHU-03, the Région Provence Alpes Côte d'Azur and European ERDF PRIMI funding.

Availability of data and materials

The datasets generated and/or analysed during the current study are available from the corresponding author [P.G.] on reasonable request.

Authors' Contributions

Conceptualization: Philippe Gautret, Didier Raoult, Pierre Marty

Conflict of Interest

The authors declare that they have no conflicts of interest and that they did not receive support by the industry in the last three years.


Nathalie Kouta

Jacqueline Saliba

Nadia Saiyouri

Monitoring of earth concrete damage evolution during drying

Keywords: earth concrete, shrinkage, drying, damage, Acoustic Emission technique

Earth concrete, constituted mainly of raw earth, is a promising ecofriendly material for future constructions. However, it is composed of fine particles like clay which make it very sensitive to drying. The volume change induced by desiccation shrinkage can be responsible of cracking which can have a detrimental effect on the durability and mechanical properties. Thus, in order to design reliable earth concrete structures, considering a coupling between drying shrinkage and damage is important.

The effect of curing conditions on the mechanical properties of earth concrete with different percentages of flax fibers has been studied. The evolution of damage during drying has been monitored continuously using the ultrasound and the acoustic emission (AE) techniques. The mechanical properties of earth concrete decreases for specimens stored at low relative humidity. In addition, the AE results associated with shrinkage and mass loss measurement made it possible to describe qualitatively the drying process.

Introduction

Over the past decade, a large amount of natural resources has been consumed by the construction sector accompanied with the emission of a large amount of CO2 due to the production of cement. Hence, due the increase awareness, many studies are now conducted to find an alternative ecofriendly material. In the case of structures that do not require high mechanical performance, earth concrete can be considered as an acceptable solution in comparison to conventional concrete [START_REF] Kouta | Study of the effect of flax fibers on the fracture behavior of earth concrete by simultaneous application of digital image correlation and acoustic emission[END_REF]. In addition, this latter presents many advantages in terms of hygrothermal and acoustical performance [START_REF] Van Damme | Earth concrete. Stabilization revisited[END_REF].

2 However, the presence of a large quantity of fine particles in earth concrete makes it very sensitive to different types of shrinkage which can induce cracking and affect its strength and durability [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF][START_REF] Tang | Experimental characterization of shrinkage and desiccation cracking in thin clay layer[END_REF][START_REF] Ngo | Design of a soil concrete as a new building material -Effect of clay and hemp proportions[END_REF].

Earth concrete volume change is an unavoidable phenomenon, from very early age to longterm behavior. At early age (before 24 h), earth concrete goes through negative volumetric variations due to water evaporation (plastic shrinkage) or internal reactions of chemical origins (autogenous shrinkage) linked to the hydration of binders. The menisci present at the interface between the waterfilled and empty pores will result in the development of stresses within the liquid phase. These stresses pull earth concrete matrix particles closer together and lead to a volume reduction and strains within the solid framework. The autogenous shrinkage component, being mainly dependent on the composition of earth concrete, develops more rapidly with time than drying shrinkage. Long term drying shrinkage (after 24 h) depends on the age at the beginning of drying and external parameters such as relative humidity (RH) and specimen size [START_REF] Saliba | Influence of shrinkage-reducing admixtures on plastic and long-term shrinkage[END_REF]. The shrinkage of different types of earth construction have been studied in the literature [START_REF] Eid | Drying, cracks and shrinkage evolution of a natural silt intended for a new earth building material. Impact of reinforcement[END_REF][START_REF] Kanema | Shrinkage of earth concrete amended with recycled aggregates and superplasticizer: Impact on mechanical properties and cracks[END_REF]. [START_REF] Walker | Rammed earth: design and construction guidelines[END_REF] studied drying shrinkage for un-stabilized rammed earth walls. Volumetric shrinkage developed in the two direction of the wall with a value that do not exceed 5%. This value depends on clay content, grain size of the soil and drying rate and process that is related to the radius of the pores. Shrinkage of rammed earth is much higher than that of conventional concrete, with a faster evolution in the initial phase, representing 85% of the total shrinkage recorded during the first 28 days [START_REF] Woyciechowski | Shrinkage characteristics of cement stabilized rammed earth[END_REF]. In addition, specimens stored at a RH of 95% have a total shrinkage 20% lower than specimens stored at a RH of 35%. Drying shrinkage for cementstabilized soils is mainly related to the plasticity index of the soil, the clay content [START_REF] Walker | Strength, durability and shrinkage characteristics of cement stabilised soil blocks[END_REF] and the clay minerals present in the soil [START_REF] Reddy | Influence of soil composition on the strength and durability of soil-cement blocks[END_REF]. Increasing clay content increases desiccation shrinkage [START_REF] Walker | Properties of some cement stabilised compressed earth blocks and mortars[END_REF]. If tensile strength is less than stress induced by shrinkage restraint , cracking will occur in earth concrete [START_REF] Saliba | Influence of shrinkage-reducing admixtures on plastic and long-term shrinkage[END_REF].

Micro cracking during shrinkage depends on the type of restraint which can be caused by the structure (externally applied restraint) or the material (internal restraint). Self-restraining mechanisms can be related to moisture/shrinkage gradients that develop at different depths in the specimens and by the presence of aggregates. Those micro cracks increase the permeability of earth concrete and can lead to stress concentrations during loading. Earth concrete structures are therefore vulnerable to inherent stresses or shrinkage-induced cracks that can not only cause structural defects, but also reduce the serviceability, durability and aesthetics of earth concrete [START_REF] Kanema | The influence of soil content on the mechanical properties, drying shrinkage and autogenous shrinkage of earth concrete[END_REF]. Carbonation reactions also occur in the long term due to the penetration of carbon dioxide into earth concrete, leading to the formation of calcite [START_REF] Runigo | Durabilité du limon de Jossigny traité à la chaux et soumis à différentes sollicitations hydriques : comportement hydraulique, microtextural et mécanique[END_REF]. Carbonate crystals have weak cohesion properties and the presence of calcite does not really improve the mechanical performance of earth concrete [START_REF] Diamond | Mechanisms of Soil-Lime Stabilization[END_REF].

Early age shrinkage or plastic shrinkage of earth concrete has been studied in a previous study [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF] and more particularly the effect of flax fibers on cracking sensitivity. In fact, fibers have been widely used for soil reinforcement and to reduce cracking induced by plastic shrinkage in concrete at early age [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF]. Vegetable fibers have the advantage of being light, ecological, with low-cost as they are produced as waste materials, in addition to their good acoustic and hygrothermal properties and compatibility with earth concrete. [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF][START_REF] Kouta | Fracture behavior of flax fibers reinforced earth concrete[END_REF] showed that flax fibers enhanced the ductility of earth concrete by generating a diffused cracking and decreased the horizontal plastic shrinkage. Thus, flax fibers reduce the cracking risk of restrained earth concrete at early age [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF]. The use of natural fibers can be sensitive in the long term due to their natural decomposition and biological attack [START_REF] Sen | Application of Sisal, Bamboo, Coir and Jute Natural Composites in Structural Upgradation[END_REF]. High humidity can promote this kind of activity within the material and accelerate the biodegradation [START_REF] Laborel-Préneron | Plant aggregates and fibers in earth construction materials: A review[END_REF]. Soil stabilization increases the resistance to biological attacks [START_REF] Kymäläinen | Flax and hemp fibres as raw materials for thermal insulations[END_REF]. Shrinkage is also influenced by the fiber's type and content [START_REF] Sangma | Experimental study on shrinkage behaviour of earth walling materials with fibers and stabilizer for cob building[END_REF]. The rate of shrinkage increases with clay and water content but decreases with fiber content [START_REF] Yetgin | The effects of the fiber contents on the mechanic properties of the adobes[END_REF]. The addition of fibers improves also the strength and reduces shrinkage cracking of earth concrete [START_REF] Yetgin | The effects of the fiber contents on the mechanic properties of the adobes[END_REF][START_REF] Bouhicha | Performance of composite soil reinforced with barley straw[END_REF]. The stabilization of shrinkage occurred earlier with adding straw, indicating a rapid drying due to the accelerated evaporation through the air channels formed [START_REF] Bouhicha | Performance of composite soil reinforced with barley straw[END_REF]. The addition of 4% of sisal or coconut fibers reduced tensile shrinkage cracking [START_REF] Ghavami | Behaviour of composite soil reinforced with natural fibres[END_REF].

In this study, long-term shrinkage and the effect of drying on the mechanical properties of earth concrete have been investigated.

In order to monitor damage evolution during shrinkage, the AE technique has been used. This technique has been widely used to monitor damage and crack development during mechanical loading [START_REF] Otsuka | Fracture process zone in concrete tension specimen[END_REF][START_REF] Das | Machine learning based crack mode classification from unlabeled acoustic emission waveform features[END_REF][START_REF] Tsangouri | A fundamental investigation of textile reinforced cementitious composites tensile response by Acoustic Emission[END_REF][START_REF] Saliba | Monitoring of steel-concrete bond with the acoustic emission technique[END_REF][START_REF] Boniface | Evaluation of the acoustic emission 3D localisation accuracy for the mechanical damage monitoring in concrete[END_REF]. Recently, it has been also used to understand some complex phenomena as corrosion [START_REF] Assouli | Detection and identification of concrete cracking during corrosion of reinforced concrete by acoustic emission coupled to the electrochemical techniques[END_REF], creep [START_REF] Saliba | Identification of damage mechanisms in concrete under high level creep by the acoustic emission technique[END_REF], hardening of cement paste and concrete [START_REF] Chotard | Application of the acoustic emission technique to characterise liquid transfer in a porous ceramic during drying[END_REF], drying shrinkage cracking in cement-based materials [START_REF] Hossain | The role of specimen geometry and boundary conditions on stress development and cracking in the restrained ring test[END_REF][START_REF] Assi | Unsupervised and supervised pattern recognition of acoustic emission signals during early hydration of Portland cement paste[END_REF][START_REF] Shiotani | Temporal and spatial development of drying shrinkage cracking in cement-based materials[END_REF][START_REF] Dzaye | Monitoring early-age acoustic emission of cement paste and fly ash paste[END_REF] and drying of ceramics [START_REF] Chotard | Application of the acoustic emission technique to characterise liquid transfer in a porous ceramic during drying[END_REF]. Different mechanisms have been proposed to explain the reasons behind the AE activity during the cement paste hydration as the formation and dissolution of hydrates, the consumption of water and the emptying of capillary pores [START_REF] Chotard | Application of the acoustic emission technique to characterise liquid transfer in a porous ceramic during drying[END_REF]. [START_REF] Lura | Early-age acoustic emission measurements in hydrating cement paste: Evidence for cavitation during solidification due to self-desiccation[END_REF] attributed the AE activity during self-desiccation to cavitation.

A comprehensive study of the damage process during shrinkage of earth concrete is proposed in this paper using the AE technique. First, the materials and the experimental methods are presented.

Then, desiccation shrinkage and its effect on the mechanical properties are analyzed. Finally, the characterization of damage evolution under drying is suggested using the AE method.

Experimental program

Materials

Soil and Hydraulic binders

The soil of earth concrete mixtures is based on an artificial soil composed of 30% of bentonite clay (75% of Smectite, 15% of Illite and 10% of Kaolinite) and 70% of sand [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF]. This artificial soil has been chosen in order to avoid the variability of onsite soil. The artificial soil has been stabilized with 3% of natural hydraulic lime (NHL5/ EN 459-1 [START_REF]EN (European Standards), Lime: Definitions, Specifications and Conformity criteria[END_REF]) and 8% of cement (CEM 1, 52.5 N PM-CP2 / EN197-1 [39]) based on the clay and sand mixture mass. Those percentages have been fixed according to other studies presented in the literature that showed that 2% to 4% of lime are sufficient to stabilize the soil [START_REF] Bell | Lime stabilization of clay minerals and soils[END_REF][START_REF] Khattab | Long-term stability characteristics of a limetreated plastic soil[END_REF], and the optimal percentage of cement to ensure a sufficient strength for earth concrete is 8% [START_REF] Anagnostopoulos | Strength properties of an epoxy resin and cement-stabilized silty clay soil[END_REF]. The pure Portland cement CEM 1 composed mainly of clinker is chosen to prevent the effect of additional components.

Flax fibers

Flax fibers are natural fibers, bio-renewable, recyclable and biodegradable. The fibers were cut at constant length of 12 mm and their diameter is equal to 14.66 µm ±2.95 [START_REF] Page | Influence of different surface treatments on the water absorption capacity of flax fibres : Rheology of fresh reinforced-mortars and mechanical properties in the hardened state[END_REF]. These fibers are rich in cellulose, more than 60% of their constitution, which gives them good tensile strength [START_REF] Page | Multi-physical properties of a structural concrete incorporating short flax fibers[END_REF]. The fibers used in this study were not treated in order to keep them natural and easy to use.

Mixtures

Three mixtures have been tested by varying the percentage of flax fibers: 0, 0.3 and 0.6%. Clay and sand were first mixed during 3 minutes to homogenize of the artificial soil. Then, lime and cement were added to the dry mixture and mixed during 2 minutes. Water and superplasticizer were then added and mixed for 3 minutes. The superplasticizer Tempo 10 has been used due to its efficiency as a water reducer admixture in earth concrete [START_REF] Kanema | Shrinkage of earth concrete amended with recycled aggregates and superplasticizer: Impact on mechanical properties and cracks[END_REF]. Finally, fibers were progressively added and mixed during 3 minutes. Table 1 presents the mixtures components and composition. The quantity of fibers was subtracted from the artificial soil mass to keep an equivalent solid mass. The effective water to binder ratio was kept constant and equal to 0.45. Additional water has been added to take into account water absorbed by flax fibers. Details about the constituents and mixture properties at early age can be found in [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF].

Table 1. Mixtures components and composition.

Methods

Compressive test

Unconfined compressive tests have been conducted using an electromechanical machine with a capacity of 100 kN (Figure 1). A constant loading displacement rate of 0.6 mm/min has been considered. This test has been realized on cubic specimens of 10×10×10 cm 3 at the age of 28 and 180 days. After pouring earth concrete, specimens were covered with a thin sheet of plastic in order to prevent water loss and maintained at a temperature of 20°C during 48 h. Then, specimens were unmolded and stored in two different curing conditions at a RH of 90% and 50% (± 5%) and a temperature of 20 °C.

Components

Ultrasound test

The ultrasound system PUNDIT (Portable Ultrasonic Nondestructive Digital Indicating Tester)

was used to measure the velocity of ultrasound waves. It consists of a pulse generator with two transducers. An electrical pulse is converted into a longitudinal pressure wave with a frequency of 200 kHz by using a transmitter coupled to the surface with grease.

Digital image correlation technique

The compressive tests were monitored with the digital image correlation (DIC) technique. It is a direct and simple method for fracture examination. The usage of this technique implicates an advance surface preparation of earth concrete specimens by using a white and black speckle (white paint at the bottom with small black dots above). The displacement of the tested surface of the specimen is obtained by the analysis of the random speckle at different instances based on the pictures taken during the test.

The photos of the specimen's surface have been captured using a digital camera. The reference images have a resolution of 2452 × 2056 pixels. To improve the luminosity of the photos, two lamps have been used in parallel. Images have been taken at a rate of one image per second during the tests and stored directly. The treatment of the images has been realized using the commercial software Vic 2D with a resolution of 0.05 mm per pixel.

Shrinkage and water loss measurement tests

Shrinkage tests were carried out on prismatic specimens of 7x7x28 cm 3 fitted with metal studs at their ends. Specimens were poured and kept for 24 hours in sealed conditions to avoid drying. After 24 hours, specimens were removed from the molds and shrinkage tests were carried out in an airconditioned room at 20 °C (± 2 °C) and a RH of 50% (± 5%). The measurement of the longitudinal deformation was carried out using LVDT sensors with a precision of 3 µm during several weeks. For total shrinkage, the ends of the specimens were covered with an adhesive aluminum tape to allow identical lateral drying of the four sides of earth concrete. For Autogenous shrinkage, specimens were completely covered with two layers of adhesive aluminum tape to prevent drying. The mass loss was measured simultaneously on specimens with the same dimensions and stored under the same drying conditions.

Acoustic Emission technique

The AE method was used to monitor the damage evolution in earth concrete specimens during shrinkage. The AE system comprised of an eight channel AE Win system, a general-purpose interface bus (2PCI-DISP4) and a PC for data storage analysis. Four piezoelectric transducers with a resonant frequency of 150 KHz were used to convert the mechanical waves to electrical signals (Figure 2). They

Mechanical properties

Figure 3 shows the stress/strain curves for specimens conserved at two different conditions at the age of 28 and 180 days. The strain was calculated as the ratio between the average displacement, obtained using the digital image correlation, and the length of the specimen. In fact, the press displacement does not reflect the real deformation of earth concrete specimens. The deformation calculated from the press displacement is higher than that of DIC due to the deformation of the frame and the crushing of concrete at the contact between the metallic plates and concrete specimen. Different phases can be distinguished. For mixtures stored at 90% of RH, the stress increased linearly in function of the strain almost till the maximal strength showing an important elastic phase. However, for specimens stored at 50% of RH, the elastic phase was observed. The slope of the elastic phase decreases with an important rate as RH decreases while the strain at the peak increases. After the peak, the load decreases quickly for SA0F indicating a brittle behavior of earth concrete conserved at 90% of RH in comparison to that stored at 50% of RH. For specimens with fibers, the post peak region zone was more important indicating an increase of earth concrete ductility due to different softening mechanisms [START_REF] Laborel-Préneron | Plant aggregates and fibers in earth construction materials: A review[END_REF]. Figure 4 shows the maximum compressive stress and the elastic modulus of earth concrete specimens kept at 90% and 50% RH at the age of 28 and 180 days. For the mixtures kept at 90% of RH, the compressive strength decreases slightly with the addition of fibers at the age of 28 days which may be due to the higher porosity [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF]. The compressive strength of earth concrete increased with age specially for specimens with fibers which may be due to time-dependent pozzolanic reactions [START_REF] Bell | Lime stabilization of clay minerals and soils[END_REF]. In fact, the formed hydrates bind the soil particles together and give them a more compact matrix structure.

This limits considerably the particles rearrangement at the interface and increases the effective interfacial contact area. In addition, as natural fibers are characterized by their hydrophilicity, hydrates may cover the surface of fibers due to the presence of water and improve the characteristics of the interfacial bonding and the frictional force between the fibers and the matrix [START_REF] Anggraini | Relationship between the compressive and tensile strengths of lime-treated clay containing coconut fibres[END_REF][START_REF] Muntohar | Engineering properties of silty soil stabilized with lime and rice husk ash and reinforced with waste plastic fiber[END_REF].

For specimens kept at 50% of RH, a high reduction of strength has been observed due to drying with a slight effect of the percentage of fibers. This reduction of strength continues with the age of curing at 180 days. The decrease of the mechanical properties of earth concrete specimens may be due to degradation induced by desiccation shrinkage. This degradation can be observed visually at the surface of the specimens (Figure 5). The photos were taken before the compression test at the age of 180 days for specimens stored at 50% of RH. The photos show the flaking of the surface especially for specimens without fibers in addition to visible cracks. Cracks were detected at the 28 th day for SA0F and spread rapidly over the entire surface of earth concrete, inducing a progressive loss of the surface layer. In fact, micro cracks may develop in earth concrete due to gradient deformation during drying, in addition to the decrease of hydration reactions and the loss of the cohesion between the particles. For the mixtures with fibers, cracks were more visible at the 180 th day, without leading to a significant loss of earth concrete surface. This slower degradation may be associated to a better crack resistance. In fact, the presence of fibers may prevent the propagation of cracks due to the adhesion between the matrix and the fibers and the bridging effect [START_REF] Qiang | Cracking , water permeability and deformation of compacted clay liners improved by straw fi ber[END_REF][START_REF] Barrier | Effects of Fiber Additives on the Desiccation Crack Behavior of the Compacted Akaboku Soil as A Material for Landfill[END_REF]. However, this adhesion may decrease during the drying process due the strain gradient at the interface between the matrix and the fibers which may be responsible of the lower strength at the 180 th day. This may be also due to a different pore size distribution that can modify the capillary pressure and thus the stress gradient that may impact the mechanical and transfer properties of earth concrete.

Digital image correlation monitoring of fracture tests

For a better understanding of the fracturing process, the compressive tests were monitored with the DIC method. Figure 6 presents the 2D strain fields along the X direction for SA0F, SA03F and SA06F stored at 50% and 90% of RH at different loading levels. For specimens kept at 90% of RH, no cracks have been visualized in the elastic phase and macro cracks started to propagate slightly before the peak. For SA0F, a large crack has been observed with an important width. The developed cracks led to the loss of strength and the total rupture of SA0F at 50% of the maximum strength in the post peak region. The rupture type shows clearly a lateral confinement. In fact, due to friction, a triaxial stress state was created within the material in the areas near the loading plates. Vertical cracks appeared mainly near the corners of the lateral surface and were coupled with the horizontal ones due to bending [START_REF] Quagliarini | The influence of natural stabilizers and natural fibres on the mechanical properties of ancient Roman adobe bricks[END_REF]. Consequently, a big part of the specimen fell indicating a brittle fracture of the specimen [START_REF] Bouhicha | Performance of composite soil reinforced with barley straw[END_REF].

However, for specimens with fibers, a diffuse multi cracking has been observed at the peak with small crack openings. This indicates a more ductile behavior due the presence of fibers that limit the crack propagation and opening by bridging the cracks and redistributing the applied stress during the extension [START_REF] Laborel-Préneron | Plant aggregates and fibers in earth construction materials: A review[END_REF][START_REF] Bouhicha | Performance of composite soil reinforced with barley straw[END_REF][START_REF] Quagliarini | The influence of natural stabilizers and natural fibres on the mechanical properties of ancient Roman adobe bricks[END_REF]. Additional cracks have been observed in the post peak region and crack openings increased at the surface with no clear effect of the lateral confinement. This multi cracking is partially responsible of the higher fracture energy and ductility of the material [START_REF] Srikar | A Study on Residual Compression Behavior of Structural Fiber Reinforced Concrete Exposed to Moderate Temperature Using Digital Image Correlation[END_REF].

for SA0F and 80% of the maximum strength for mixtures with fibers. This confirms the degradation of earth concrete specimens and the pre-presence of micro cracks that developed during drying [START_REF] Kanema | Shrinkage of earth concrete amended with recycled aggregates and superplasticizer: Impact on mechanical properties and cracks[END_REF].

Several cracks were observed for SA0F. The crack openings were also more important for SA0F

indicating the effect of fibers in increasing the ductility and preventing the brittle rupture. Figure 7 presents the evolution of a chosen crack width (circled in figure 8) along the height of specimens SA0F and SA06F at 50% and 90% of RH. The crack width increases with the applied load. It started earlier for specimens kept at 50% of RH at 30% of the maximum strength for SA0F and 80% of the maximum strength for SA06F which confirms the pre-cracking of specimens subjected to drying. In addition, the width of the cracks was much larger for specimens kept at 50% of RH than that kept at 90% of RH for the different loading levels. Note that the crack width varied along the crack length due to different stress gradient in addition to the effect of flax fibers distribution and orientation.

The addition of flax fibers slowed down crack openings and increased earth concrete ductility. In fact, flax fibers held the soil matrix together by bridging the cracks, in addition to stress transfer and redistribution in earth concrete [START_REF] Ghavami | Behaviour of composite soil reinforced with natural fibres[END_REF]. The good adhesion between fibers and earth concrete matrix prevented the spread of large crack opening and improved the fracture energy. The rupture occurred due to the debonding of flax fibers from the matrix.

Long term shrinkage

To improve the comprehension of the different mechanisms responsible of the degradation of the mechanical properties of earth concrete due to drying, the evolution of shrinkage in correlation with the mass loss has been studied. The autogenous shrinkage of earth concrete with and without fibers increases slightly in the first 3 days after casting and then stabilized. Desiccation shrinkage occurs in earth concrete due to self-desiccation (internal drying) and mainly due to the loss of water from capillary pores and the gel (external drying).

Total shrinkage is therefore represented as the sum of desiccation and autogenous shrinkage. The reduction of RH in the pore system causes a water-air meniscus that subjects the pore walls to considerable stress and leads to substantial drying shrinkage. The shrinkage rate is slow during the first three days after casting. Then, it increases with an important rate and begins to decrease gradually in function of time to stabilize after 20 days. Desiccation shrinkage is almost 10 times higher than autogenous shrinkage. Note that drying shrinkage values are very important in comparison to ordinary concrete which may be due to a higher capillary pressure and a less rigid solid skeleton. [START_REF] Kanema | The influence of soil content on the mechanical properties, drying shrinkage and autogenous shrinkage of earth concrete[END_REF].

The evolution of desiccation shrinkage in function of the weight loss has been also plotted and shows two phases (Figure 8 (b)). During the first 3 days after casting, even though the weight loss rate is important (about 7%), the generated shrinkage is low. In the second phase, the weight loss and desiccation shrinkage with increased with an important rate proportionally. The slope variation can be explained by the modification of the dimension of the pores that are being emptied and the decrease of the evaporation rate. In fact, during the drying process some of the liquid water cannot reach the surface and evaporates within the pores. The same behavior has been observed for mixtures with and without fibers. From the beginning of the test and till the 6 th day after casting, the weight loss and shrinkage were the same for the two mixtures. However, after this age, the weight loss and the total shrinkage increase slightly for mixtures with fibers. This may be due to the higher water absorption with fibers.

Ultrasound monitoring

Figure 9 shows the comparison between the ultrasonic waves propagation velocity of specimens kept at 50% and 90% of RH in function of the curing age. For specimens kept at 90% of RH, the velocity increases with an important rate during the first days and slightly after the age of 28 days indicating the increase of stiffness [START_REF] Ye | Study on the development of the microstructure in cement-based materials by means of numerical simulation and ultrasonic pulse velocity measurement[END_REF]. For specimens kept at 50% of RH, the wave propagation velocity is smaller. It increases during the first days but the velocity measurement becomes impossible after 8 days for SA03F and 12 days for SA06F. This can be related to water loss and premature damage of specimens due to drying that may cause cracking and a loss of cohesiveness between particles [START_REF] Erdogdu | Yüksek Sıcaklığa Maruz Uçucu Kül İçeren Betonun Ultrases Geçiş Hızı ve Basınç Dayanımının Değerlendirilmesi[END_REF]. For a better understanding of the different phenomena occurring during drying, shrinkage tests have been also monitored using the AE technique.

AE monitoring of drying tests

AE hits provide an indication on the number of burst emissions and wave characteristics, such as the amplitude, duration and absolute energy. The AE hits detected during shrinkage may be generated due to micro cracking or cavitation [START_REF] Lura | Early-age acoustic emission measurements in hydrating cement paste: Evidence for cavitation during solidification due to self-desiccation[END_REF]. In fact, when evaporation occurs at the surface of the pore, the radius of the liquid/vapor interface or meniscus decreases. When the radius of curvature of the meniscus is small, it retreats into the pore and produces a kinetic energy generating the propagation of mechanic waves [START_REF] Dzaye | Monitoring early-age acoustic emission of cement paste and fl y ash paste[END_REF]. Micro cracks can be generated due to moisture gradient that leads to non-uniform drying shrinkage. As the surface of the material exposed to drying shrinks faster, micro cracks are mainly generated at the surface [START_REF] Topolář | Using acoustic emission methods to monitor cement composites during setting and hardening[END_REF]. Shrinkage can be also restrained by the presence of aggregates or fibers inducing micro cracking at the interface with the matrix [START_REF] Bisschop | How to study drying shrinkage microcracking in cement-based materials using optical and scanning electron microscopy?[END_REF].

Figure 10 shows the cumulated number of AE hits in correlation with shrinkage in function of time. For a better analysis, the cumulative number of AE hits has been also plotted in function shrinkage for SA0F and SA06F. Three phases similar to those observed while plotting the relationship between the weight loss and shrinkage have been obtained. During the first phase, few AE hits were detected.

They reached a value of 500 for a shrinkage value equal to 3200 µm/m. This phase is mainly related to the departure of water from the surface and larger porosity. The numbers of AE hits was similar for both mixtures. During the second phase between the 6th and the 11th day after pouring, the number of the cumulative number of AE hits increases with an important rate (from 500 to 4000 signals). This may be related to micro cracks generated by non-uniform desiccation shrinkage at the surfaces exposed to drying. This can be also the reason behind the inability to measure the ultrasonic velocity for SA06F12 after the 11th day (Figure 6). The cumulative number of AE hits was more important for SA0F for the same level of shrinkage indicating a higher damage level. This is confirmed with the compressive tests where the decrease of strength was more important for SA0F. During the third phase, a notable decrease in the acoustic activity was observed indicating the decrease of micro cracks generation in parallel with the decrease of the weight loss and shrinkage stabilization. and reach a value of 85 dB. In fact, during drying, the water present in the pores of the earth concrete evaporates, inducing suction forces. The negative pressure generated in the pores forces the grains to rearrange and move closer to each other. When the tensile stress is equal to the cohesive force of attraction between the particles, cracks will be formed [START_REF] Qiang | Cracking , water permeability and deformation of compacted clay liners improved by straw fi ber[END_REF], [START_REF] Aldaood | Impact of wetting -drying cycles on the microstructure and mechanical properties of lime-stabilized gypseous soils[END_REF]. The primary cracks increase in size and new cracks are formed until they intersect. The external surfaces of the earth concrete specimens are more affected because of the strain gradient. In fact, water loss is faster at the surface than inside the specimen according to the drying kinetics which may explain the spalling observed at the surface.

During the stabilization phase, the generated AE energy and amplitude decrease quickly indicating a lower degradation rate.

The cumulative absolute energy was more important for mixture with fibers. In addition, this energy liberation appeared later. This could be related to a higher toughness with the addition of fibers.

At one hand, micro cracks can initiate at the interface between earth concrete matrix and flax fibers and in the other hand, fibers may be responsible of a different pore size distribution, which can alter drying kinetics, stress redistribution and prevent micro cracks from propagating through the specimen [START_REF] Kouta | Fracture behavior of flax fibers reinforced earth concrete[END_REF][START_REF] Kim | Using acoustic emission to quantify damage in restrained fiberreinforced cement mortars[END_REF].

Evolution of Ib-value

The evolution of the micro cracks dimensions, developed during drying, was carried out by measuring the Ib value. The AE peak amplitude is related to the magnitude of the fractures developed during desiccation shrinkage. To determine the overall fracture quantitatively on basis of peak amplitude, the Ib-value originated from seismology is performed. It is defined as the negative gradient of the seismic magnitude distribution. Statistical values of the amplitude distribution were incorporated to the Ib-value in order to apply it for fracture evaluation in civil engineering materials. According to the cumulative amplitude distribution, the index (Ib-value) changes following the fracture progress [START_REF] Aggelis | Acoustic emission characterization of the fracture process in fibre reinforced concrete[END_REF]. If small-scale fractures are superior to large-scale fractures, Ib-value tend to increase, in the contrary if large-scale fractures are superior to small-scale fractures, Ib-value tends to decrease.

Therefore, Ib-value represents the ratio of weak to strong events [START_REF] Kurz | Stress drop and stress redistribution in concrete quantified over time by the b-value analysis[END_REF]. Micro-cracks generate a large number of weak acoustic emissions with a high Ib-value. Macro-cracks lead to a relatively low Ibvalues since more strong events are generated. The Ib-value was calculated using the peak amplitudes of 100 successive hits, assuming an average amplitude μ and a standard deviation σ of the amplitudes and N the number of hits with an amplitude higher than μα σ or μα σ [START_REF] Shiotani | Evaluation of progressive failure using AE sources and improved b-value on slope model tests[END_REF]. The formula used to calculate the Ib-value is as follows:

= -where α1 and α2 are constants. α1 and α2 values are 0.5 and 1.5 respectively.

Figure 12 shows the evolution of Ib-value during drying in function of time. Ib-value was important at the beginning and decreased after the 5 th day, where damage started to develop with small scale fracturing in the specimen. Ib value attended its minimum stability value after 6 days for SA0F and 8 days for SA06F where the cumulative number of AE hits increased with a high rate. In this phase, micro cracks started to initiate and develop with a larger scale especially near the surface of the specimen where drying is important. Ib value increases again showing stability after 11 days for SA0F and 12 days for SA06F and attend the same value after 14 days. This indicates the presence of two different mechanisms. At the beginning and due to water evaporation, AE hits may be due the retreat of water menisci into the pore. Then, micro cracks initiate and develop with an important rate between the 5 th and 12 th days after concrete casting generating AE signals with higher amplitude and energy.

The third phase indicates a slow propagation of these cracks already developed in the specimen with a lower scale. Even though damage evolution was monitored continuously with the AE technique, however the detailed geometry and pattern of the developed cracks are not shown. Additional techniques as the optical microscopy [START_REF] Shiotani | Temporal and spatial development of drying shrinkage cracking in cement-based materials[END_REF], digital image correlation [START_REF] Lagier | Numerical strategies for prediction of drying cracks in heterogeneous materials : Comparison upon experimental results[END_REF], micro-tomography or others could be used to obtain additional information on micro cracking.

Conclusions

The effect of drying on the mechanical properties of earth concrete has been studied.

  were placed on specimens using silicon grease as a coupling agent. The recorded AE amplitudes range from 0 to 100 dB. The detected signals are amplified with a 40 dB gain differential amplifier. The signal detection threshold is set at a value of about 35 dB to overcome the background noise (RILEM TC212-ACD). The acquisition parameters are set as follows: peak definition time (PDT) = 100 µs, hit definition time (HDT) = 200 µs and hit lock time (HLT) = 400 µs. A high pass filter with a cut-off frequency of 20 KHz, and a low-pass filter with a cut-off frequency of 400 KHz are used to eliminate mechanical and electro-magnetic disturbances. Several signal descriptors were calculated by AEwin system.

Figure 2 .

 2 Figure 2. Prismatic specimen geometry and position of AE transducers

Figure 3 .

 3 Figure 3. Stress-strain curves of earth concrete specimens conserved at 50% and 90% of RH for SA0F, SA03F and SA06F at the age of 28 days (a) and 180 days (b)

Figure 4 .

 4 Figure 4. Maximum strength and Young modulus of SA0F, SA03F and SA06F conserved at 50% and 90% of RH at the age of 28 and 180 days.

Figure 5 .

 5 Figure 5. Photos of SA0F, SA03F and SA06F before the compression test at 50% of RH at the age of 180 days

Figure 6 .

 6 Figure 6. 2D strain fields along X for SA0F, SA03F and SA06F stored at 50% and 90% of RH at different loading levels.

Figure 7 .

 7 Figure 7. Evolution of the crack width for (a) SA0F and (b) SA06F at 50% and 90% of RH

Figure 8

 8 (a) presents the evolution of the total shrinkage, autogenous shrinkage and weight loss of SA0F and SA06F in function of time. Autogenous shrinkage occurs independently of the external water loss and is a result of chemical shrinkage and self-drying shrinkage.

Figure 8 .

 8 Figure 8. (a) Evolution of total shrinkage, autogenous shrinkage and weight loss in function of time and (b) evolution of desiccation shrinkage in function of the weight loss for SA0F and SA06F

Figure 9 .

 9 Figure 9. Evolution of the ultrasonic wave velocity at 50% and 90% of RH in function of time.

Figure 10 .

 10 Figure 10. (a) Cumulative number of AE hits in correlation with shrinkage and (b) cumulative number of AE hits in function of shrinkage for SA0F and SA06F.

Figure 11 .

 11 Figure 11. Cumulative AE energy in correlation with AE amplitude and shrinkage in function of time for SA0F and SA06F

Figure 12 .

 12 Figure 12. Evolution of Ib-value during drying.

  Autogenous and desiccation shrinkage of earth concrete in parallel with the mass loss have been also measured. As a part of the investigation, the ultrasound and the AE techniques were applied to monitor the evolution of damage induced by desiccation shrinkage. AE monitoring allowed a temporal development assessment of damage and the process of drying was characterized by following the AE activity and AE parameters. The results show that the AE technique is able to detect low energy processes during shrinkage as well as damage evolution. The relationship between the AE activity and shrinkage is illustrated and the effect of fibers was investigated. The AE results associated with shrinkage measurements allow to describe qualitatively the drying process and give wealth information on damage evolution. Three different phases were distinguished during drying indicating different mechanisms. Damage increases during the second phase after 6 days of casting indicating the development of self-restraint cracks. This damage may be partly responsible of the strength reduction observed with the compression tests. Additional studies will be carried out thereafter to better understand the effect of flax fibers on shrinkage by the classification of AE signals and conducting addition tests at the microscopic level for a better characterization of the interface between fibers and earth concrete matrix.