Weissland Thierry

Cozette Maryne
email: maryne.cozette@u-picardie.fr

Leprêtre Pierre-Marie

Maryne Cozette

Angular ranges as a reliable isokinetic method to assess shoulder rotator muscles

Keywords: data accuracy, muscle balance, strength, sport, rehabilitation

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

INTRODUCTION

The balance between internal (IR) and external (ER) rotator muscles has an important role in mobility and in the stability of glenohumeral joint [START_REF] Noffal | Isokinetic eccentric-to-concentric strength ratios of the shoulder rotator muscles in throwers and nonthrowers[END_REF] . The imbalance between IR and ER could be responsible for a faulty centering of the humeral head [START_REF] Howell | Normal and abnormal mechanics of the glenohumeral joint in the horizontal plane[END_REF] . Tanaka et al. (2012) [START_REF] Tanaka | Evaluation of dislocation position in patients with recurrent anterior shoulder dislocation[END_REF] and Clément et al.

(2017) [START_REF] Clément | Three-dimensional analysis of the locked position in patients with recurrent shoulder instability[END_REF] showed that the range of instability was in the middle of the range of motion (ROM) rather than in maximum external rotation. The muscle strength balance is classically researched by peak torque (PT) ratios of antagonist and agonist muscles in isokinetic standardized conditions [START_REF] Edouard | L'épaule du lanceur : Quel équilibre musculaire pour une meilleure performance et la prévention des blessures?[END_REF] . The PT values are defined as the highest data point of the curve for torque output in the ROM for each muscle group. However, the angular position at which PT is reached is not, therefore, taken into account. It has been observed that agonist and antagonist muscles did not attain their respective PT at the same angle [START_REF] Croisier | Exploration isocinétique : analyse des courbes[END_REF][START_REF] Sonza | Analysis of the isokinetic torque curves in shoulder movement[END_REF] . The torque analysis of rotator muscles generally shows that PT values are reached at angles near to the limit of the ROM [START_REF] Sonza | Analysis of the isokinetic torque curves in shoulder movement[END_REF] . However, Brown et al. (1995) [START_REF] Brown | Isokinetic load range during shoulder rotation exercise in elite male junior tennis players[END_REF] showed that imposed velocity is instable precisly at this angles near to the limit of the ROM. These observations may explain the relative unreliability of the physiological (antagonist-PT in concentric / agonist-PT in concentric) and functional (antagonist-PT in eccentric / agonist-PT in concentric) ratios of shoulder rotator muscles found by Edouard et al.

(2011, 2013) [START_REF] Edouard | Reliability of isokinetic assessment of shoulder-rotator strength: a systematic review of the effect of position[END_REF][START_REF] Edouard | Reliability of shoulder rotators isokinetic strength imbalance measured using the Biodex dynamometer[END_REF] . Finally, the isokinetic PT method to calculate ratios did not appear to be adapted to the shoulder muscle balance assessment according to Tanaka et al. (2012) [START_REF] Tanaka | Evaluation of dislocation position in patients with recurrent anterior shoulder dislocation[END_REF] and Clément et al. (2017) [START_REF] Clément | Three-dimensional analysis of the locked position in patients with recurrent shoulder instability[END_REF] . Indeed, the muscle balance must be assessed by angular sectors, in particularly in range of instability that corresponds to the middle of ROM.

Together, these observations questioned the interest of physiological and functional ratios based on PT values in the dynamic exploration of shoulder muscles balance. From a dynamic perspective, it is therefore important to determine the muscular potential in its entirety. To that end, we here suggest an alternative and reliable method based on angular analysis for isokinetic assessment of shoulder rotator muscles strength balance.

METHODS

Twenty-two male non-overhead trained collegiate athletes (23.0±2.7 years, 180.6±5.7 cm, 74.6±6.5 kg), voluntarily participated in the study, including ten subjects (22.8±2.0 years, 180.8±6.8 cm, 74.8±6.6 kg) who had also taken part in a reliability session. Each subject was familiarized with the experimental procedures before the study. All were free of upper extremity musculoskeletal injuries and did not present any contraindication to upper limb isokinetic testing. Before the experiments, the subjects were informed of the risks and stresses associated with the protocol and gave their written informed consent. The study was performed in accordance with the Ethical Standards in Sport and Exercise Research 11 and approved by the national ethics committee of France (CERSTAPS 2015/07/07).

The muscular shoulder parameters of each subject were measured in seated position, the shoulder in scapular plane [START_REF] Forthomme | Isokinetic assessment of the shoulder rotators : a study of optimal position[END_REF] on an isokinetic dynamometer (Cybex Norm ® , division of Lumex, Inc., Ronkonkoma, New York, USA) in an air-conditioned room (Temperature: ~20.3°C, Relative humidity: ~35.0% and Atmospheric pressure: ~762mmHg). The seat and handlebar heights were set for each subject according to the Cybex isolated joint testing manual (Cybex/ Isolated-Joint testing and Exercise. Ronkohoma, NY, USA: Cybex, 1983). After 10 minutes of standardized warm-up on an arm ergometer (Cybex metabolic systems MET300 ® , Ronkonkoma, New York, USA), and 3 minutes at rest, each subject performed two blocks in a random order ; one block for the dominant shoulder (D) and another block for the non-dominant shoulder (ND). Each block consisted of 1 series of 3 repetitions at 60°.s -1 in concentric contraction, 2 minutes rest, and 1 series of 3 repetitions at 60°.s -1 in eccentric contraction. One repetition consisted in internal rotation (ranging from 0 to 110°) followed by external rotation (ranging from 110 to 0°). The range of motion was 110°. The subjects of the test-retest reliability study were tested with the same protocol at one-week interval. To minimize physical activity effects, the subjects were asked to avoid high-intensity exercise training one week before and during the study.

The torque (T) and peak torque (PT) values were measured for internal (IR) and external (ER) rotators of dominant (D) and non-dominant (ND) shoulder in concentric (CON) and eccentric (ECC) modalities without gravitational correction. All data were extracted and recorded with an acquisition unit (PowerLab 16/35 PL3516, ADInstruments ® , Bella Vista, NSW 2153, Australia). The physiological ratios (ER/IR) measured exclusively in concentric modality, and the functional ratios, i.e the torque value of ER measured in eccentric modality related to the torque value of IR measured in concentric modality (ERecc/IRcon) and conversely (IRecc/ERcon), were calculated. We distinguished the ratios calculated with the averaged torque by the angular range of 10° as T-ratios (TER/IR ; TERecc/IRcon ; TIRecc/ERcon) and the ratios calculated with the peak torque as PT-ratios (PTER/IR ; PTERecc/IRcon ; PTIRecc/ERcon).

The isokinetic dynamometer software transfers the best PT value in a series of n repetitions, i.e for three repetitions, only the repetition associated with the highest PT was selected for IR and ER. To use the same method, we retained only the repetitions associated with PT of IR and ER.

Data was acquired at a frequency of 100Hz at the angular velocity of 60.s -1 . The Torque data was averaged by angular range of 10°. To simplify the angular ranges reporting we used integers (i.e 10-19.9° was reported as 10-20°). Two extreme ranges (i.e 0-10° and 100-110°) were systematically excluded from analysis because they correspond to the delay time to acceleration and deceleration phases, so they are not really at constant velocity. Data recorded on these angular ranges are presented for illustrative purpose but our analysis focused on the angular ranges from 10° to 100°. For sake of clarify, data were rounded to tenths.

Descriptive statistics are expressed as means and standard deviation. The normal distribution and homogeneity of variance were verified for each parameter (Shapiro-Wilk test). T-test was used for comparing paired samples. We used ANOVA with repeated measures followed by post-hoc analysis with Bonferroni t-test to compare the means for each angular range of 10°.

The coefficient of variation (CV) and the intraclass correlation coefficient (ICC) were calculated to test the absolute and relative reliability [START_REF] Atkinson | Statistical methods for assessing measurement error (reliability) in variables relevant to sports medicine[END_REF] . The tests were performed using the full version of SPSS (IBM ® SPSS ® Statistics, Version 20.0.0, Chicago, USA). A significance level of P<0.05 was selected for all tests.

RESULTS

The PT and T values are reported in Table 1. Three observations were made on the reliability indexes of the averaged torque by angular range (Fig. 1). Firstly, this approach exhibited better reliability indexes of T-ratios compared to PT-ratios. Secondly, the reliability was better in CON compared to ECC modality. Thirdly, the reliability of IR was better than ER. ICC and CV were calculated for all ratios (Fig. 1). For both shoulders, functional and physiological PT-ratios presented variable and unsatisfactory ICC (from 0.14 to 0.75) and CV (from 7.0 to 28.5%) compared to T-ratios. Lower ICC and CV were found at the onset and the end of ROM. Hence, for the majority of the angular ranges, the reliability indexes of T-ratios were better than PT-ratios (Fig. 1). For the TER/IR physiological ratio, the ICC scores were 0.33-0.85 (CV : 11.7-14.8%) and 0.14-0.84 (CV : 7.1-11.0%) for D and ND shoulders compared to 0.21 (CV : 12.7%) and 0.15 (CV : 16.8%) respectively for the PTER/IR ratio. For the functional ratios, TERecc/IRcon ICC values were 0.18-0.79 (CV : 11.5-20.1%) and 0.03-0.79 (CV : 7.4-14.9%) and ICC of TIRecc/ERcon were 0.08-0.88 (CV : 12.2-32.2%) and 0.04-0.27 (CV : 19.0-36.3%) for D and ND shoulders, respectively.

IR reached higher values of averaged torque between 20 to 40°, while averaged torque of ER muscles reached their maximum between 60 to 90° (Fig. 2). All ratio values exhibited significant changes with the angular range (P<0.05, Table 1). The maximal TER/IR and TERecc/IRcon ratios occurred between 60 to 100°. In contrast, the higher values of TIRecc/ERcon ratio were found at angular ranges of 20-40°.

DISCUSSION

The main purpose of this study was to propose an alternative method to explore shoulder rotator muscles balance that was representative to the functional reality and reliable. Our main results showed significant differences between the PT-ratio and the T-ratio for the three calculated ratios (ER/IR ; ERecc/IRcon ; IRecc/ERcon), and significant differences between angular ranges for T-ratios. Moreover, reliability was higher for the 10° steps angular approach compared to ratios calculation based on the PT.

The physiological TER/IR ratio was lower in external rotation and tended to increase in internal rotation. Indeed, the potential difference beetween agonist and antagonist muscles was less important in internal rotation. This is explained by the length/tension relationship which animates the rotator muscles: internal rotators were less effective when in slightly shortened position, and external rotators were more efficient in slightly stretched position. Cibulka et al.

(2014) [START_REF] Cibulka | The influence of muscle length on one-joint shoulder internal and external rotator muscle strength[END_REF] showed that IR and ER muscles were weakest when placed in a position of shortest muscle length, i.e. at end-range of internal rotation for IR and at end-range of external rotation for ER. For functional ratios, our results showed a better ability of the antagonists to counteract the concentric action of the agonists at the end of the dynamic movement, i.e. at the end of the internal rotation for the TERecc/IRcon and at the end of the external rotation for the TIRecc/ERcon ratio. This eccentric control was more effective at the end of the dynamic movements of the throwing: the cocking and the acceleration phases which are respectively associated with PTIRecc/ERcon and PTERecc/IRcon ratios [START_REF] Vogelpohl | Isokinetic rotator cuff functional ratios and the development 332 of shoulder injury in collegiate baseball pitchers[END_REF] . [START_REF] Ruas | Specific joint angle assessment of the shoulder rotators[END_REF] studied the evolution of physiological and functional ratios of the shoulder rotator muscles using the specific angular torque (AST) method. The AST method consists of measuring the agonist and antagonist muscle torque and associated ratios at specific joint angles [START_REF] Cozette | Isokinetic strength ratios: conventional methods, current limits and perspectives[END_REF] . They showed significant differences of the AST-ratio values between angles, and between the PTERecc/IRcon ratio and the ASTERecc/IRcon ratio. However there was no significant difference between the PTER/IR ratio and the ASTER/IR ratio. The AST method allows to have more information during the ROM, but corresponds to one data point at one angle. This method applied to shoulder assessement has not been studied for reliability.

Ruas et al. (2014)

The most widely used ratios in the literature (ER/IR and ERecc/IRcon) were more reliable by using angular range method than PT method (Fig. 1). This is explained by smoothing since the torques were averaged by angular ranges of 10°. Thus, at an acquisition frequency of 100 Hz and the angular velocity of 60°.s -1 , the data is averaged with 20 points per angular range. This makes the averaged torque by angular range more constant and less variable than a raw and isolated value (PT). Moreover, the reliability of the T-ratios was better in the middle of the ROM, when the angular velocity was stabilised, i.e apart from the extreme ranges of the ROM which are non-isokinetic (phases of acceleration and deceleration) [START_REF] Brown | Isokinetic load range during shoulder rotation exercise in elite male junior tennis players[END_REF] . The reliability indexes could be improved by changing the test position. Hence, Forthomme et al. (2011) [START_REF] Forthomme | Isokinetic assessment of the shoulder rotators : a study of optimal position[END_REF] showed better absolute reliability in supine position.

The results of our study support the proposed methodology, but there are certain aspects that deserve discussion. The amplitude of angular ranges was set at 10° for two reasons. First, it is the best compromise between precision and practicality. Indeed, the objective being to provide a tool to the clinician, most of the measurements carried out have an accuracy to within 10°.

Second, it allowed comparison of our results with the majority of previous sectorial studies [START_REF] Cozette | Isokinetic strength ratios: conventional methods, current limits and perspectives[END_REF] .

The seated position with shoulder in scapular plane was chosen because it is more physiological, safe, comfortable and allows the development of optimal torque 9 . However, it would have been interesting to compare the two most used positions [START_REF] Edouard | Reliability of isokinetic assessment of shoulder-rotator strength: a systematic review of the effect of position[END_REF][START_REF] Forthomme | Isokinetic assessment of the shoulder rotators : a study of optimal position[END_REF] to assess their effect on the T-ratios reliability: the seated position with the shoulder in scapular plane vs. the supine position with shoulder in frontal plane. Finally, the absence of women in the sample represents a limit. Indeed Forthomme et al. ( 2013) [START_REF] Forthomme | Shoulder pain among highlevel volleyball players and preseason features[END_REF] showed significantly different PT-ratio values between women and men.

In summary, isokinetic parameters evolved significantly during the ROM and the torque ratios were different of the ratios calculated with the peak torque. The physiological and functional ratios had a satisfactory reliability on the isokinetic angular ranges unlike the ratios usually used and associated to the peak torque. Thus, in view of these elements, the angular range method is a reliable method to determine dynamic stability during the ROM. Table 1 : Means (± SD) of Peak Torque (PT) and Averaged Torque par angular range of 10° (in N.m) during concentric (CON) and eccentric (ECC) at 60°.s-1 of internal (IR) and external (ER) shoulder rotation for dominant (D) and non-dominant shoulder (ND), associated physiological (ER/IR) and functional ratios (ERecc/IRcon ; IRecc/ERcon). Non-isokinetic ranges highlighted in grey.

Captions

Figure 1 .

 1 Figure 1. Relative (intraclass correlation coefficient ; ICC) and absolute (coefficient of variation ; CV) reliability indexes of averaged torque by angular range of 10° and peak torque (PT) for internal rotators (IR) in concentric (CON) and eccentric (ECC) modality (A), external rotators (ER) in concentric (CON) and eccentric (ECC) modality (B) and physiological (ER/IR) and functional (ERecc/IRcon ; IRecc/ERcon) ratios (C) at 60°.s -1 for dominant shoulder. Nonisokinetic ranges are highlighted in grey. $ CV ≤ 15%.

Figure 2 .

 2 Figure 2. Evolution of averaged torque per angular range of 10° (in N.m) and peak torque (PT) production sector for internal rotators (IR) in concentric (CON) and eccentric (ECC) modality (A), external rotators (ER) in concentric (CON) and eccentric (ECC) modality (B) and associated angular range ratios (C,D) with PT-ratios values at 60°.s -1 for dominant shoulder.Non-isokinetic ranges are highlighted in grey. * Significant difference (P<0.05) between averaged torque per angular range of 10° and PT-ratios.

ACKNOWLEDGMENTS

The authors thank professor Charles Vincent for commenting an early draft of the manuscript.

Conflicts of interest: None

Funding: Ministry of national education and research of France

Ethical Approval: National sport sciences ethics committee


Tilia Dahou

Françoise Defoort

Mejdi Jeguirim
email: mejdi.jeguirim@uha.fr

Capucine Dupont

Towards understanding the role of K during biomass steam gasification

Keywords: steam gasification, reaction kinetics, catalysis, potassium, silica, rice husk

Highlights

• Rice husk steam gasification was conducted with K 2 CO 3 added without direct contact • A two-compartment thermobalance crucible was used to assess the gas phase influence

• The K-catalysis mechanisms involve the gas phase, most likely with formed KOH(g)

• Both pyrolysis and steam gasification steps are catalyzed by K

• The K-catalysis competes with the reaction of K with SiO 2 inherent to biomass

Abstract

Steam gasification is known to be catalyzed by K. However, the exact mechanisms are not identified to this day. In the present study, the catalytic influence of K on biomass steam

Introduction

To develop bioenergy production, agricultural residues represent an interesting source as they have an important availability and do not decrease the surface of land available for food production. However, agricultural residues can be problematic during the gasification process due to high inorganic contents compared to woods. Their main inorganic elements are generally Si, K and Ca [START_REF] Vassilev | An overview of the chemical composition of biomass[END_REF]. Such elements can be detrimental to the gasification process by inducing slag formation and/or agglomeration of the gasifier bed [START_REF] Bartels | Agglomeration in fluidized beds at high temperatures: Mechanisms, detection and prevention[END_REF][START_REF] Sikarwar | An overview of advances in biomass gasification[END_REF]. Moreover, they can have a strong effect on the reaction kinetics which may influence the biomass residence time inside the reactor and therefore the gasifier design. In this context, the residence time must be adapted either to reach the highest conversion possible or to purposely have a remaining char that can be burnt in order to bring the required heat to the process, depending on the gasifier technology.

Alkali and alkaline earth metals (AAEMs) tend to have a catalytic effect that enhances biomass gasification kinetics. More specifically, alkali metals, and in particular K which is more present in biomass than Na [START_REF] Vassilev | An overview of the chemical composition of biomass[END_REF], are reported to be more active than alkaline earth metals. The catalytic activity of AAEMs was reported to be, in decreasing order, K, Na, Ca and Mg [START_REF] Huang | Effects of metal catalysts on CO2 gasification reactivity of biomass char[END_REF][START_REF] Yip | Effect of Alkali and Alkaline Earth Metallic Species on Biochar Reactivity and Syngas Compositions during Steam Gasification[END_REF][START_REF] Zhang | Proposal of a semi-empirical kinetic model to reconcile with gasification reactivity profiles of biomass chars[END_REF]. This ranking has been determined for materials with various ash contents.

Catalytic gasification uses this effect to improve the gasification process. Catalysts usually studied are compounds of AAEMs as well as some heavy metal compounds such as Fecompounds and Ni-compounds [START_REF] Arnold | Catalysts for gasification: a review[END_REF][START_REF] Nzihou | A review of catalysts for the gasification of biomass char, with some reference to coal[END_REF]. In the case of K-catalysis, added compounds are mostly K 2 CO 3 and KOH which are compounds naturally found in biomass [START_REF] Arnold | Catalysts for gasification: a review[END_REF]. KNO 3 , KCl or K 2 SO 4 have also been studied [START_REF] Bouraoui | CO2 gasification of woody biomass chars: The influence of K and Si on char reactivity[END_REF][START_REF] Kramb | The effects of calcium and potassium on CO2 gasification of birch wood in a fluidized bed[END_REF][START_REF] Lahijani | CO2 gasification reactivity of biomass char: Catalytic influence of alkali, alkaline earth and transition metal salts[END_REF]. Authors have found the following relative activity: KOH ~ K 2 CO 3 ~ KNO 3 > K 2 SO 4 > KCl [START_REF] Kramb | Modeling of the catalytic effects of potassium and calcium on spruce wood gasification in CO2[END_REF][START_REF] Hüttinger | Influence of the catalyst precursor anion in catalysis of water vapour gasification of carbon by potassium: 1. Activation of the catalyst precursors[END_REF][START_REF] Hüttinger | The influence of the catalyst precursor anion in catalysis of water vapour gasification of carbon by potassium: 2. Catalytic activity as influenced by activation and deactivation reactions[END_REF]. They have also demonstrated that under steam the intermediate species KOH was formed from all salts [START_REF] Hüttinger | The influence of the catalyst precursor anion in catalysis of water vapour gasification of carbon by potassium: 2. Catalytic activity as influenced by activation and deactivation reactions[END_REF].

Several methods have been used in the literature to highlight the catalytic effects of these inorganic elements. Studies have used raw biomass or biomass char [START_REF] Degroot | Gasification of agricultural residues (biomass): influence of inorganic constituents[END_REF][START_REF] Dupont | Kinetic modelling of steam gasification of various woody biomass chars: Influence of inorganic elements[END_REF][START_REF] Dupont | How inorganic elements of biomass influence char steam gasification kinetics[END_REF][START_REF] González-Vázquez | Unconventional biomass fuels for steam gasification: Kinetic analysis and effect of ash composition on reactivity[END_REF][START_REF] Hognon | Comparison of steam gasification reactivity of algal and lignocellulosic biomass: Influence of inorganic elements[END_REF][START_REF] Kannan | Gasification of biomass chars in carbon dioxide: dependence of gasification rate on the indigenous metal content[END_REF][START_REF] Link | Reactivity of the Biomass Chars Originating from Reed, Douglas Fir, and Pine[END_REF][START_REF] López-González | Gasification of lignocellulosic biomass char obtained from pyrolysis: Kinetic and evolved gas analyses[END_REF][START_REF] Romero Millán | Steam gasification behavior of tropical agrowaste: A new modeling approach based on the inorganic composition[END_REF][START_REF] Strandberg | Effects of Pyrolysis Conditions and Ash Formation on Gasification Rates of Biomass Char[END_REF], biomass or char washed with deionized water and/or with acid to remove the inherent inorganic matter [START_REF] Yip | Effect of Alkali and Alkaline Earth Metallic Species on Biochar Reactivity and Syngas Compositions during Steam Gasification[END_REF][START_REF] Kramb | The effects of calcium and potassium on CO2 gasification of birch wood in a fluidized bed[END_REF][START_REF] Kannan | Gasification of biomass chars in carbon dioxide: dependence of gasification rate on the indigenous metal content[END_REF][START_REF] Feng | Steam Gasification of Sawdust Biochar Influenced by Chemical Speciation of Alkali and Alkaline Earth Metallic Species[END_REF][START_REF] Jiang | Formation, fates and roles of catalytic precursors generated from the K2CO3-carbon interactions in the K2CO3-catalyzed CO2 gasification of coal char[END_REF][START_REF] Lv | Experimental study on the optimization of parameters during biomass pyrolysis and char gasification for hydrogen-rich gas[END_REF][START_REF] Zahara | CO2 Gasification of Sugar Cane Bagasse: Quantitative Understanding of Kinetics and Catalytic Roles of Inherent Metallic Species[END_REF], or biomass or char with added inorganic compounds [START_REF] Zhang | Proposal of a semi-empirical kinetic model to reconcile with gasification reactivity profiles of biomass chars[END_REF][START_REF] Bouraoui | CO2 gasification of woody biomass chars: The influence of K and Si on char reactivity[END_REF][START_REF] Kramb | The effects of calcium and potassium on CO2 gasification of birch wood in a fluidized bed[END_REF][START_REF] Lahijani | CO2 gasification reactivity of biomass char: Catalytic influence of alkali, alkaline earth and transition metal salts[END_REF][START_REF] Kramb | Modeling of the catalytic effects of potassium and calcium on spruce wood gasification in CO2[END_REF][START_REF] Kannan | Gasification of biomass chars in carbon dioxide: dependence of gasification rate on the indigenous metal content[END_REF][START_REF] Jiang | Formation, fates and roles of catalytic precursors generated from the K2CO3-carbon interactions in the K2CO3-catalyzed CO2 gasification of coal char[END_REF][START_REF] Lv | Experimental study on the optimization of parameters during biomass pyrolysis and char gasification for hydrogen-rich gas[END_REF][START_REF] Arnold | Interaction of Potassium and Calcium in the Catalytic Gasification of Biosolids and Switchgrass[END_REF][START_REF] Bach-Oller | Effect of potassium impregnation on the emission of tar and soot from biomass gasification[END_REF][START_REF] Degroot | Kinetics of gasification of Douglas Fir and Cottonwood chars by carbon dioxide[END_REF][START_REF] Feng | Catalytic mechanism of ionexchanging alkali and alkaline earth metallic species on biochar reactivity during CO2/H2O gasification[END_REF][START_REF] Kirtania | Kinetic study of catalytic gasification of wood char impregnated with different alkali salts[END_REF][START_REF] Meijer | Kinetics of the alkali-carbonate catalysed gasification of carbon: 3. H2O gasification[END_REF][START_REF] Mudge | Catalyzed steam gasification of biomass[END_REF][START_REF] Perander | Catalytic effect of Ca and K on CO2 gasification of spruce wood char[END_REF][START_REF] Elliott | Alkali catalysis in biomass gasification[END_REF]. These latter ones were prepared through solid mixing [START_REF] Jiang | Formation, fates and roles of catalytic precursors generated from the K2CO3-carbon interactions in the K2CO3-catalyzed CO2 gasification of coal char[END_REF][START_REF] Arnold | Interaction of Potassium and Calcium in the Catalytic Gasification of Biosolids and Switchgrass[END_REF][START_REF] Bach-Oller | Effect of potassium impregnation on the emission of tar and soot from biomass gasification[END_REF][START_REF] Mudge | Catalyzed steam gasification of biomass[END_REF][START_REF] Elliott | Alkali catalysis in biomass gasification[END_REF] or impregnated with a solution containing the inorganic compound [START_REF] Zhang | Proposal of a semi-empirical kinetic model to reconcile with gasification reactivity profiles of biomass chars[END_REF][START_REF] Bouraoui | CO2 gasification of woody biomass chars: The influence of K and Si on char reactivity[END_REF][START_REF] Kramb | The effects of calcium and potassium on CO2 gasification of birch wood in a fluidized bed[END_REF][START_REF] Lahijani | CO2 gasification reactivity of biomass char: Catalytic influence of alkali, alkaline earth and transition metal salts[END_REF][START_REF] Kramb | Modeling of the catalytic effects of potassium and calcium on spruce wood gasification in CO2[END_REF][START_REF] Lv | Experimental study on the optimization of parameters during biomass pyrolysis and char gasification for hydrogen-rich gas[END_REF][START_REF] Bach-Oller | Effect of potassium impregnation on the emission of tar and soot from biomass gasification[END_REF][START_REF] Degroot | Kinetics of gasification of Douglas Fir and Cottonwood chars by carbon dioxide[END_REF][START_REF] Feng | Catalytic mechanism of ionexchanging alkali and alkaline earth metallic species on biochar reactivity during CO2/H2O gasification[END_REF][START_REF] Kirtania | Kinetic study of catalytic gasification of wood char impregnated with different alkali salts[END_REF][START_REF] Meijer | Kinetics of the alkali-carbonate catalysed gasification of carbon: 3. H2O gasification[END_REF][START_REF] Mudge | Catalyzed steam gasification of biomass[END_REF][START_REF] Perander | Catalytic effect of Ca and K on CO2 gasification of spruce wood char[END_REF][START_REF] Elliott | Alkali catalysis in biomass gasification[END_REF]. Impregnation has been the major addition method used in literature since it is simple to carry out and allows a close contact between the inorganics and the biomass.

Few authors have compared the effects of various addition methods. Elliott et al. [START_REF] Elliott | Alkali catalysis in biomass gasification[END_REF] and Bach-Oller et al. [START_REF] Bach-Oller | Effect of potassium impregnation on the emission of tar and soot from biomass gasification[END_REF] have observed a slightly stronger catalytic effect of K 2 CO 3 in the case of impregnation compared to dry mixing. Authors have supposed an activity through the gas phase and attributed the slight decrease in activity with dry mixing to a lower inorganic compound volatilization due to mass transfer limitations. However, to our knowledge, no available investigations have been conducted to identify the gas phase effect of K-catalysts.

In addition, several studies in the literature have examined the catalytic CO2 gasification of biomass species having a low inorganic content [9-12, 25, 31, 33, 35-37].

In this context, the present work aims to contribute to the understanding of the K influence on biomass steam gasification in the case of a Si-rich biomass, in order to better understand the K behavior, its effect through the gas phase and its potential interaction with the inherent SiO 2 of the biomass.

Materials and Methods

Materials

The selected biomass for this study was rice husks (RH) from Camargue, France, a Si-rich biomass. Its behavior was compared to sunflower seed shells (SSS) from Bordeaux, France, a Si-poor and K-rich biomass, and beech wood (BW) from France, Si-poor and more generally ash-poor biomass. The samples were ground below 200 µm in a Pulverisette 14 rotor mill (FRITSCH, Idar-Oberstein, Germany). The ash content and inorganic element composition of the samples were measured according to solid fuel standards NF EN 14775 [START_REF]Solid biofuels -Determination of ash content (EN 14775)[END_REF] and NF EN ISO 16967 [START_REF] Ca | International Organization for Standardization. Solid biofuels -Determination of major elements -Al[END_REF], respectively. The properties of each biomass are summarized in Table 1. A model K-compound was selected for the study: potassium carbonate (K 2 CO 3 ). It was selected since it can be found naturally in biomass [START_REF] Vassilev | An overview of the organic and inorganic phase composition of biomass[END_REF]. Merck K 2 CO 3 EMSURE® ACS, ISO, Reag. Ph. Eur, ≥ 99.0 % was used. The K 2 CO 3 powder was ground from around 500 µm to <100 µm with a mortar and pestle, in a N 2 atmosphere to prevent its hydration. It was then kept in a vacuum desiccator.

Table 1. Composition of rice husks and beech wood (in dry basis

Experimental installation and procedure

The steam gasification behavior of the samples was investigated through thermogravimetric analysis (TGA). Experiments were carried out at atmospheric pressure using a Setsys thermobalance (SETARAM, Caluire, France) coupled with a Wetsys steam generator described elsewhere [START_REF] Dupont | Kinetic modelling of steam gasification of various woody biomass chars: Influence of inorganic elements[END_REF].

A home-made crucible was designed for this present investigation. It is a crucible consisting of a cylindrical platinum crucible of 2 mm height and 10 mm diameter with a partition of the same height along its diameter made up of platinum with 5% gold (Figure 1). The feed gas is injected from below the crucible. Experiments were conducted in three different configurations: biomass or inorganic compound alone; mixtures of biomass and inorganic powders; biomass and inorganic compound without direct contact in the crucible.

Inorganic concentrations were expressed as a percentage of the total sample:

% = + (1)
With the mass of inorganic added and the mass of biomass.

Mixtures of biomass and inorganic powders were prepared with approximately 140 mg of biomass and the necessary mass of inorganic compound to obtain the desired concentration.

The two powders were added to a plastic flask with a few glass beads of 3 mm diameter.

Homogeneous mixing was obtained by shaking the flask by hand for around one minute.

The TGA sample mass was determined through preliminary experiments on pure biomass to be in the chemical regime in this configuration.

Approximately 15 mg of sample were heated at 24 °C.min -1 until 450 °C under 0.05 L.min -1 N 2 and kept for 60 min at this temperature. They were then heated at 24 °C.min -1 until 800 °C and kept at this temperature until the end of the experiment. Samples were swept by N 2 after the final temperature was reached to ensure pyrolysis completion and mass stability (12 min). Gas was then switched to a mixture of 20 vol% H 2 O in N 2 with the same gas flow of 0.05 L.min -1 . This marked the beginning of the gasification step. The experiment was completed when the mass was stabilized, i.e. when gasification was finished and only ashes remained.

Experiments were conducted at least in duplicates to verify repeatability. Only one of the repetitions is plotted.

Results were plotted as the normalized mass:

, ( 2
)
where and are the mass of initial raw biomass and the mass measured at the time t, respectively.

Gasification solid conversion was defined from the mass loss measured as a function of time during TGA by using the following expression:

= , (3)
where and are the mass of char before gasification (at the time of steam injection)

and the mass measured at the end of gasification (remaining ash), respectively.

The gasification rate was then defined as the variation of conversion versus time:

= . (4)
An average reactivity between two values of conversion " and # was also defined:

$ % &$ ' = ( 1 * + ' * + % $ ' $ % . (5)
There is no standardized definition for the reactivity [START_REF] Barrio | Steam Gasification of Wood Char and the Effect of Hydrogen Inhibition on the Chemical Kinetics[END_REF]. In this study, the average reactivity was calculated between 1 and 80% conversion as in several investigations in literature [START_REF] Dupont | Kinetic modelling of steam gasification of various woody biomass chars: Influence of inorganic elements[END_REF][START_REF] Hognon | Comparison of steam gasification reactivity of algal and lignocellulosic biomass: Influence of inorganic elements[END_REF][START_REF] Barrio | Steam Gasification of Wood Char and the Effect of Hydrogen Inhibition on the Chemical Kinetics[END_REF]. This choice allowed to obtain a trend that was not affected by the particular phenomena that can occur at high conversion values.

Additionally, the derivative thermogravimetric (DTG) curve was obtained with the following equation:

,-. = /" &" /" &" . (6)
where &" , and /" are consecutive time steps, &" is the mass measured at time &" and /" is the mass measured at time /" .

To allow comparison between the different samples, results from pyrolysis and gasification of samples with added K 2 CO 3 (mixtures and without contact) were corrected by subtracting the equivalent mass profiles of pure K 2 CO 3 to the measured mass profiles. An example is given in Figure 2. The black curve is the mass as directly measured in TGA. The orange curve is the pure K 2 CO 3 profile that is subtracted. Finally, the blue curve is the equivalent char mass with the K 2 CO 3 subtracted. It is this last mass that is used in further data treatments and interpretations. and corresponding pure K 2 CO 3 mass.

Characterization techniques

Optical microscopy

Optical microscopy was used to observe the sample morphology before and after reaction.

An Olympus SZX12 microscope was used with a ToupTek Photonics ToupCam camera and the associated ToupView software.

Scanning electron microscopy coupled to energy dispersive X-ray spectroscopy

Scanning electron microscopy coupled to energy dispersive X-ray spectroscopy (SEM-EDX)

was used to study the morphology and the surface elemental composition of the TGA residues. A Philips XL30 microscope was used with a 15 kV electron beam. Back-scattered electrons (BSE) detection was used. It creates a contrast related to atomic number. Samples were placed on graphite tape and graphitized before analysis.

Thermodynamic equilibrium simulation method

Calculations at thermodynamic equilibrium were performed to simulate the behavior of K 2 CO 3 under steam at 800 °C.

They were performed by minimization of the Gibbs free energy of the total system with the FactSage 7.2 software and the databases FTsalt and FactPS [START_REF] Bale | FactSage thermochemical software and databases[END_REF][START_REF] Hack | A novel thermodynamic database for slag systems and refractory materials[END_REF].

The steam gasification procedure applied to pure K 2 CO 3 was simulated at thermodynamic equilibrium. The initial data are listed in Table 2. K 2 CO 3 initial mass was slightly different from the experimental initial mass put in the TGA since calculations dealt with the gasification step and there was a slight mass loss before gasification (Figure 4).

The gas flow is constant, therefore the effect of time is simulated by the effect of the initial gas mass [START_REF] Defoort | Thermodynamical analysis of the fission product release in the ORNL VI-3 and VI-5 tests[END_REF].

Results and Discussion

Pure biomass steam gasification

The results obtained during the pyrolysis and steam gasification of the pure biomass samples are plotted in Figure 3. (average reactivity r 1-80% = 1.4%.min -1 ) while gasification of sunflower seed shells require less than 20 minutes (average reactivity r 1-80% = 30.3%.min -1 ). Rice husks gasification rate is continuously decreasing whereas sunflower seed shells has a constant and then decreasing conversion rate. Beech wood has an intermediate behavior with a gasification time of around 40 minutes (average reactivity r 1-80% = 4.9%.min -1 ) and a gasification rate that is constant and then increases. Another work showed that these significant differences are mainly due to the inorganic composition of the biomass species, and not to the char physical properties [START_REF] Dahou | Contribution to the understanding of the role of inorganic elements in biomass steam gasification[END_REF].

Pure K 2 CO 3 thermogravimetric analysis (TGA)

The pyrolysis and gasification procedure was applied to various mass samples of K 2 CO 3 .

Figure 4 shows the mass evolution versus time. A very low volatilization of K 2 CO 3 is observed during the pyrolysis step. On the contrary, the sample is completely volatilized when steam is injected. This behavior is in accordance with literature [START_REF] Arvelakis | Simultaneous Thermal Analysis (STA) on Ash from High-Alkali Biomass[END_REF][START_REF] Knudsen | Transformation and Release to the Gas Phase of Cl, K, and S during Combustion of Annual Biomass[END_REF][START_REF] Zhao | Effect of Steam and SiO2 on the Release and Transformation of K2CO3 and KCl during Biomass Thermal Conversion[END_REF]. The volatilization time depends on the initial mass varying from 40 min to 2h for samples between 0.68 and 3.20 mg.

In order to explain this behavior, a thermodynamic equilibrium simulation with pure K 2 CO 3 was conducted. Under thermodynamic equilibrium conditions, in the case where K 2 CO 3 is in an inert atmosphere, it is slightly volatilized as K(g) and CO 2 (g). On the opposite, in the case where K 2 CO 3 is in a steam atmosphere, it is largely volatilized as KOH(g). These volatilized species can be explained by the following reactions [START_REF] Sergeev | Vaporization behavior of Na2CO3 and K2CO3[END_REF][START_REF] Wood | The Mechanism of the Catalytic Gasification of Coal Char: A Critical Review[END_REF]:

0 # 12 3 4 = 2 0 + 12 # + 0.5 2 # (7) 0 + 9 # 2 = 029 + 0.5 9 # (8)
The simulated mass evolution of K 2 CO 3 during the gasification step is plotted in dotted lines in Figure 4. The simulation predicts that the whole amount of K 2 CO 3 volatilizes in 2 to 20 minutes in thermodynamic equilibrium conditions. It shows mass transfer limitations during K 2 CO 3 volatilization. They could be due to the configuration of the crucible in the carrier gas and/or to the K 2 CO 3 particle size.

The mass evolution of K 2 CO 3 as a function of time and initial mass was modeled through a second-degree polynomial of general equation as shown in Figure 4. The model is presented in Supplementary Material. Here, the objective was only to fit experimental results in the TGA configuration to be able to extrapolate the results to other initial masses in the studied range. It has not been attempted in the present work to give a signification to these coefficients as in the literature [START_REF] Knudsen | Transformation and Release to the Gas Phase of Cl, K, and S during Combustion of Annual Biomass[END_REF]. For example, Knudsen et al. [START_REF] Knudsen | Transformation and Release to the Gas Phase of Cl, K, and S during Combustion of Annual Biomass[END_REF] have modelled the evaporation of pure K-compounds based on mass transfer for a carrier gas sweeping the crucible parallel to its surface, on the contrary to the TGA configuration where the gas is perpendicular. Authors have assumed K transport was controlled by diffusion through an external gas film. They have used mass transfer correlations to obtain the coefficients.

Interactions between materials

Pyrolysis and gasification of rice husks-slow-gasifying biomass-were carried out with K 2 CO 3 added in two configurations: mixed and without contact in a divided crucible. Both configurations had approximately the same proportion of added K 2 CO 3 , i.e. 7 wt%. This proportion corresponds to 4 wt% of K in the initial mixture (Table 1). Such a high content (see natural contents < 1 wt% of K in raw biomass, in Table 1) was selected to clearly observe the phenomena. Results were compared to rice husks and sunflower seed shells Above 450 °C, the curves indicate that K 2 CO 3 addition has an effect on both pyrolysis and gasification steps. This effect is discussed in the following sections.

Influence of K 2 CO 3 addition on the pyrolysis reaction

Though the present study focuses on the gasification reaction, an effect of K addition already appeared during the pyrolysis step at high temperature, before steam injection. Since the char formed during pyrolysis is the starting material of the gasification reaction, it is important to also investigate these phenomena. Above 450 °C, a second DTG peak is observed around 82 min and 500 °C for all samples with and without added K 2 CO 3 . It corresponds to the typical mass loss observed when heating the samples between 450 and 800 °C, as seen with pure biomass, and is due to a residual char devolatilization.

High temperature pyrolysis phenomena observation

However, when K 2 CO 3 is added to rice husks (mixed and without contact), a third DTG peak is observed close to the beginning of the 800 °C holding time. An additional experiment where heating is conducted up until 1000 °C instead of 800 °C shows that the peak is maximum around 860 °C (see Supplementary materials). The effect is stronger when K 2 CO 3 is mixed with rice husks. Nevertheless, it appears even if rice husks are not in contact with the added K 2 CO 3 which means it is related to the gas phase and probably attributed to K volatilization. The stronger effect in the case of the mixture can then be explained by a higher concentration of K-compound in the gas layer around the biomass in this case.

Third DTG peak investigation during pyrolysis step

In order to determine the origin of this third DTG peak, the same experiment was conducted on rice husks with K 2 CO 3 added without contact but it was stopped after the pyrolysis step at 800 °C, i.e. before steam injection. Optical microscope observations of the crucible before and after the experiment are presented in Figure 7. Rice husk chars have no visual particularity at this scale. On the K 2 CO 3 side, grains which were initially white are covered with a black layer after pyrolysis until 800 °C and material under each grain suggests a partial fusion. The black layer most likely is carbon deposited on top of the inorganic residue. These observations are in agreement with literature where higher char yields have been observed with K impregnation due to condensation of tars [START_REF] Blasi | Effects of Potassium Hydroxide Impregnation on Wood Pyrolysis[END_REF][START_REF] Zaror | Secondary char formation in the catalytic pyrolysis of biomass[END_REF][START_REF] Zhao | Effect of potassium inorganic and organic salts on the pyrolysis kinetics of cigarette paper[END_REF]. However, a competition between this char formation and tar cracking have been noted [START_REF] Zaror | Secondary char formation in the catalytic pyrolysis of biomass[END_REF]. Regarding the residue under the black layer, it cannot be pure K 2 CO 3 (whose melting point is 901°C [START_REF] Bale | FactSage thermochemical software and databases[END_REF] i.e. above the pyrolysis temperature) but a material with a lower melting point. Since liquid formation is associated to a higher volatilization, it is in agreement with the fact that the third DTG peak also occurs before the pure K 2 CO 3 melting point. These results

indicate that a reaction occurs on the K 2 CO 3 side when it is in presence of biomass, even without contact.

It is known that pyrolysis gases (CO2, H2O, CO, H2) and inorganic gases (H2S, NH3, HCl, KCl, etc.) are released during biomass devolatilization [START_REF] Björkman | Release of Chlorine from Biomass at Pyrolysis and Gasification Conditions[END_REF][START_REF] Bridgwater | Pyrolysis of Biomass[END_REF]. Therefore, the liquid observed at 800 °C could be due to the reaction of K 2 CO 3 with one of these gases.

The residue at 800 °C on the K 2 CO 3 side was analyzed by SEM-EDX and only K, C and O were detected. H cannot be detected by this methods.

Therefore K 2 CO 3 most likely reacts with H 2 O(g) originating from biomass devolatilization. The residue is then a mixture of K 2 CO 3 and KOH where a core of K 2 CO 3 is at equilibrium with a liquid phase (solidified at ambient temperature).

The K 2 CO 3 -KOH phase diagram (see Supplementary Material) predicts that, at 800 °C, solid K 2 CO 3 is in equilibrium with a liquid for compositions up to 23.5mol% KOH. This is a reasonable composition that could have been obtained from the reaction with H 2 O(g) from the biomass devolatilization. It also predicts that a liquid phase can appear at 450 °C, the temperature of the first plateau of the TGA procedure. This behavior has been confirmed by an additional experiment where a higher mass loss of K 2 CO 3 was observed during high temperature pyrolysis after it had been in contact with steam during the 450 °C plateau than when it had only been in a N2 atmosphere (see Supplementary Material).

Therefore, from these results, the third DTG peak can be attributed to the devolatilization of the carbon matrix catalyzed by a K-compound, probably, KOH(g), released to the gas phase from the K 2 CO 3 side of the crucible.

In literature, authors do not usually conduct pyrolysis at such high temperatures. Indeed, as previously stated, the main mass loss occurs below 450 °C so studies are focused on that temperature range. However, studies in the carbon activation literature [START_REF] Marsh | CHAPTER 6 -Activation Processes (Chemical)[END_REF] are in accordance with the results from the present work where pyrolysis at high temperature in the presence of a K-compound induces an additional mass loss, i.e. third DTG peak. However, in the case of activated carbons the K-compound is added through impregnation of an aqueous solution, or occasionally through dry mixing (Amoco process) [START_REF] Marsh | CHAPTER 6 -Activation Processes (Chemical)[END_REF], whereas in the present study the effects are observed including when K 2 CO 3 is not in contact with the biomass.

For the first time it is here demonstrated that the effect occurs through the gas phase. The main explanation given in literature regarding the mechanism of action involves the intercalation of K followed by its explosive removal to form the pores [START_REF] Marsh | CHAPTER 6 -Activation Processes (Chemical)[END_REF].

Influence of K 2 CO 3 addition on the gasification reaction

Gasification phenomena observation

Figure 8 shows the gasification solid conversion and conversion rate of rice husks with and without added K 2 CO 3 compared to that of sunflower seed shells. It is clearly seen that, K 2 CO 3 has an effect on the gasification step. As stated in the Materials and Methods section (see 2.2), the results were corrected by subtracting the equivalent mass profiles of pure K 2 CO 3 to the measured mass profiles of the total sample. Therefore, if the rice husks and K 2 CO 3 behaviors were independent, their mass profiles would have been additive and the presented equivalent mass profile of rice husks would have been similar to rice husks alone. Yet, the conversion profile of the sample of rice husks and K 2 CO 3 mixed goes above 100%. These high values, which are not physically possible, occur around the time where the profile of pure K 2 CO 3 is subtracted. It means that it is an effect of the mathematical treatment of the data, i.e. of the K 2 CO 3 profile subtraction, and not a measured effect. In addition, both samples with added K 2 CO 3 , mixed and without contact, have a sudden slope change approximately 50 min after steam injection, which corresponds to the completion of the pure K 2 CO 3 volatilization that is subtracted to the results. Moreover, both curves are above the curve of rice husks alone. This last difference compared to the curve of rice husks alone is significant even in the case where K 2 CO 3 is added without contact. Indeed, husks alone that are all grouped compared to curves with added K 2 CO 3 . All the above observations show that there is an interaction between the biomass and the inorganic sample both when K 2 CO 3 is mixed and without contact. The behaviors of the samples cannot be assessed through the simple addition of the behaviors of the pure rice husks and K 2 CO 3 .

This means that the equivalent rice husks profiles presented do not represent the real mass loss on the rice husks side. Therefore, quantitative assumptions cannot be drawn from these data but trends still can be analyzed.

When comparing the addition mode of K 2 CO 3 , it can be seen that mixing induces a stronger catalytic effect during gasification. However, even if it is weak, the effect of K 2 CO 3 without contact with rice husks is present. This demonstrates that it acts, at least partially, through the gas phase. The stronger effect in the case of the mixture can then be explained by a higher concentration of K-compound in the gas layer around the biomass in this case.

Influence of the pyrolysis step on gasification

An additional experiment was designed in order to check if a reaction during pyrolysis influences the behavior during gasification. Pyrolysis of rice husks with added K 2 CO 3 without contact was carried out and the resulting char was gasified alone, after the residue on the During this experiment, only char was gasified, therefore no subtraction of a K 2 CO 3 profile was required. This explains that the curve does not have the sudden slope change like the previous ones. It is seen directly that the gasification rate of rice husks is higher than for pure RH alone while K 2 CO 3 is not present anymore during gasification. Its average reactivity between 1 and 80% conversion increased from 1.4 to 2.2 %.min -1 . To our knowledge, this result has never been demonstrated in the literature. It indicates that the interaction between K 2 CO 3 and char during the pyrolysis step catalyzes a subsequent gasification. Moreover, this effect occurs without contact between the biomass and the inorganic compound, therefore through the gas phase during pyrolysis step by the volatilization of K compounds and their condensation on the biomass char.

Ash characterization

Effects of K 2 CO 3 addition on ash yield and ashes appearance

From the normalized mass profiles presented in Figure 5, it can be observed that when K 2 CO 3 is added to rice husks, mixed or without contact, the mass stabilizes at a higher ash yield (19.7 and 15.2wt% respectively) than rice husks alone (13.6wt%) (Table 3). This higher ash yield suggests that at least a part of the added K 2 CO 3 is incorporated to the biomass char and therefore to the biomass ash at the end of the gasification step. The increase is observed including in the case of addition without contact. Therefore, this proves that K 2 CO 3 is incorporated after its volatilization during the pyrolysis step. As a result, the Kspecies in the ash can be in another form than directly K 2 CO 3 , such as K 2 O. The higher ash yield value corresponds to the theoretical ash yield calculated if all the added K 2 CO 3 is incorporated to the ashes, either directly as K 2 CO 3 (19.6wt%) or as K 2 O (17.4wt%).

In order to investigate this supposed reaction of a K-species with the rice husk ashes, the ashes from the previous experiments were characterized through optical microscope and SEM-EDX. Again with SEM, the typical rice husk ashes and the glassy agglomerates can be distinguished. The EDX analysis confirms that the typical rice husks ashes contain mainly SiO 2 . In the glassy agglomerates, the detected elements are K, Si and O. This supports the idea of K-silicates formation.

This demonstrates that, in addition to influencing the pyrolysis and gasification of the carbonaceous matrix, the added K 2 CO 3 also reacts with the inorganic elements inherent to the rice husks. This behavior is supported by literature observations of the reaction between K-compounds and SiO 2 to form K-silicates [START_REF] Kannan | Gasification of biomass chars in carbon dioxide: dependence of gasification rate on the indigenous metal content[END_REF][START_REF] Link | Reactivity of the Biomass Chars Originating from Reed, Douglas Fir, and Pine[END_REF][START_REF] Gupta | CO2 gasification of char from lignocellulosic garden waste: Experimental and kinetic study[END_REF]]. However, our study suggests that the reaction involves the gas phase, which has not been proved in literature.

This leads to a limitation of the catalysis of the gasification by K 2 CO 3 due to its reaction on SiO 2 inherent to the rice husks.

Competing interactions of K with carbon and silica

To demonstrate these competing interactions, the same experimental procedure was applied to beech wood with K 2 CO 3 added without contact. Beech wood had a very low SiO 2 content so it was expected that K 2 CO 3 would have induced a stronger catalysis than for rice husks.

Synthesis of the several effects of K along biomass pyrolysis and steam gasification

During the present investigation, we have observed that the addition of K during the pyrolysis and gasification of rice husks has significant effects at several levels. It influences the kinetics of both pyrolysis and gasification steps and it interacts with the inorganic compounds inherent to the biomass.

It is important to note that all these effects occur whether K 2 CO 3 is in contact or not with the biomass. This behavior highlights the fact that K 2 CO 3 mechanisms of action are through the gas phase, with the formation of KOH(g). This is permitted by the fact that experiments use a low gas flow that enables the mass transfers and reactions to occur at the proximity of the crucible. Conducting experiments with a high gas flow would then be expected to reduce or suppress the catalytic effect which would confirm this result. Effects observed in the case of mixtures are stronger than without contact which can be explained by a higher concentration of K-compound in the gas layer around the biomass in this case.

The effects of K 2 CO 3 on biomass pyrolysis and gasification demonstrated in the present study are summarized in Figure 13. During the pyrolysis, steam is produced and reacts with K 2 CO 3 to form a mixture of K 2 CO 3 and KOH where a core of K 2 CO 3 is at equilibrium with a liquid phase. When the pyrolysis reaches a high temperature, KOH(g) is volatilized from this mixture and catalyzes an additional devolatilization of the char. Then, when steam is added, the mixture completely volatilizes as KOH(g). This formed KOH(g) catalyzes the gasification and reacts with the biomass inherent SiO2 to form K-silicates. It is demonstrated that the interaction of K 2 CO 3 with the biomass during the pyrolysis is decisive for the subsequent gasification. Indeed, when the two materials are in presence only during pyrolysis but not during gasification, catalysis is still observed.

However, the catalytic effect remains weaker than what has been observed in literature in the case of impregnation or occasionally mixing [START_REF] Feng | Catalytic mechanism of ionexchanging alkali and alkaline earth metallic species on biochar reactivity during CO2/H2O gasification[END_REF][START_REF] Mudge | Catalyzed steam gasification of biomass[END_REF][START_REF] Sueyasu | Simultaneous Steam Reforming of Tar and Steam Gasification of Char from the Pyrolysis of Potassium-Loaded Woody Biomass[END_REF]. This is attributed to the fact that the present work intends to catalyze rice husks that have a high content in Si, while most literature studies work with materials with a low ash content [START_REF] Huang | Effects of metal catalysts on CO2 gasification reactivity of biomass char[END_REF][START_REF] Zhang | Proposal of a semi-empirical kinetic model to reconcile with gasification reactivity profiles of biomass chars[END_REF][START_REF] Bouraoui | CO2 gasification of woody biomass chars: The influence of K and Si on char reactivity[END_REF][START_REF] Kramb | The effects of calcium and potassium on CO2 gasification of birch wood in a fluidized bed[END_REF][START_REF] Lahijani | CO2 gasification reactivity of biomass char: Catalytic influence of alkali, alkaline earth and transition metal salts[END_REF][START_REF] Kramb | Modeling of the catalytic effects of potassium and calcium on spruce wood gasification in CO2[END_REF][START_REF] Kirtania | Kinetic study of catalytic gasification of wood char impregnated with different alkali salts[END_REF][START_REF] Meijer | Kinetics of the alkali-carbonate catalysed gasification of carbon: 3. H2O gasification[END_REF][START_REF] Perander | Catalytic effect of Ca and K on CO2 gasification of spruce wood char[END_REF]. Hence, we have confirmed this fact by experiments on beech wood that has a very low Si content. Along with the ash characterization during the TGA experiments, these results highlight a competition between on the one hand the influence of K 2 CO 3 on the biomass pyrolysis and char gasification kinetics and on the other hand its interaction with SiO 2 inherent to the rice husks.

Conclusions

The present work contributes to the understanding of the K role in biomass steam gasification. It has demonstrated the competition between SiO 2 and the carbonaceous matrix to react with a K-catalyst. On the one hand, the K-compound catalyzes the high temperature pyrolysis and the steam gasification of the carbonaceous matrix of biomass. On the other hand, its decomposition product reacts with the SiO 2 inherent to biomass. In both cases, effects are observed including when the K-compound is not in direct contact with the biomass which demonstrates that the mechanisms of action involve the gas phase.

During the pyrolysis at high temperature, an additional mass loss occurs in presence of K 2 CO 3 . This observation is in accordance with the carbon chemical activation literature. It has been demonstrated in the present work that it is related to an additional volatilization of the carbon matrix in relation with the formation of KOH(g). The latter is formed from the reaction of K 2 CO 3 with H 2 O(g) released from the devolatilization of the carbon matrix.

Regarding the gasification step itself, K 2 CO 3 increases the reaction rate both with and without contact with the biomass. It has been demonstrated in the present study that the interaction of K 2 CO 3 with the biomass during the pyrolysis step is decisive for the ulterior gasification.

Figure 1 .

 1 Figure 1. Divided crucible (here containing RH and K 2 CO 3 ).

Figure 2 .

 2 Figure 2. Measured mass, equivalent char mass (as used in further interpretations)

Figure 3 .

 3 Figure 3. TGA of rice husks (RH), sunflower seed shells (SSS) and beech wood (BW) presented as a. normalized mass versus time and b. gasification solid conversion rate versus gasification solid conversion.

Figure 4 .

 4 Figure 4. Mass evolution versus time of various initial masses of K 2 CO 3 alone during the pyrolysis and gasification procedure. Full line: measurement. Dotted line: thermodynamic equilibrium simulation. Dashed line: mathematical model fitting experimental data.

  alone. The evolution with time of the normalized mass of the samples-including ashes-and the DTG curves are presented in Figure 5. As stated in the Materials and Methods section (section 2.2), the measured mass profiles were corrected by subtracting the equivalent mass profiles of pure inorganic compound.

Figure 5 .

 5 Figure 5. a. TGA and b. DTG of RH with and without added K 2 CO 3 and of SSS.

Figure 6

 6 Figure 6 is a zoom of the high temperature pyrolysis from Figure 5. It shows the evolution of the normalized mass and DTG of the samples during the 30 minutes preceding steam injection, i.e. during heating from 450 to 800 °C.

Figure 6 .

 6 Figure 6. a. TGA and b. DTG of RH with and without added K 2 CO 3 and of SSS. Zoom on the high temperature pyrolysis. Full line: alone. Dotted line: mixed. Dashed line: without contact. Oscillations observed in b. are due to the experimental set-up and are considered as not significant.

Figure 7 .

 7 Figure 7. RH with K 2 CO 3 added without contact a. before and b. after pyrolysis until 800 °C in the TGA, i.e. procedure stopped before steam injection.

Figure 8 .

 8 Figure 8. Gasification at 800°C of RH with and without added K 2 CO 3 and of SSS. a. Solid conversion as a function of time (for RH alone, three replicates are plotted which explains the apparent large line). b. Solid conversion rate as a function of the conversion.

Figure 8 .

 8 a shows three replicates of the experiments on rice b. a.

K 2

 2 CO 3 side was removed. Results are shown in Figure 9.

Figure 9 .

 9 Figure 9. Gasification solid conversion as a function of time of RH char alone produced in presence of K 2 CO 3 without contact, compared to pure RH and SSS alone.

Figure 11 .

 11 Figure 11. SEM observation of ashes from pyrolysis and gasification of RH a. alone and b. with K 2 CO 3 added without contact (same aspect mixed).

Figure 13 .

 13 Figure 13. Scheme of the effects of K 2 CO 3 on biomass pyrolysis and gasification, in the case of K 2 CO 3 added without contact.

  ).

	S	wt%	0.1	0.09	0.1	0.058
	Cl	wt%	0.1	0.09	0.1	0.005
	Si	mg.kg -1	63955	59518	194	115
	K	mg.kg -1	5822	44632	9729	910
	Ca	mg.kg -1	1797	1672	4489	2516
	Mg	mg.kg -1	659	613	1838	475
	P	mg.kg -1	981	913	896	75
	Na	mg.kg -1	413	384	9	3.6
	Al	mg.kg -1	228	212	150	12
	Fe	mg.kg -1	192	179	1099	34
	Main inorganic elements	-	Si -K -Ca Si -K -Ca K -Ca -Mg Ca -K -Mg
	Property	Unit	Rice husks (RH)	RH + 7wt% K 2 CO 3	Sunflower seed shells (SSS)	Beech wood (BW)
	Ash at 550 °C	wt%	14.6	20.3**	3.3	0.6
	C	wt%	41.6	39.0	50.2	45.9
	H	wt%	5.2	4.7	6.5	6.2
	O*	wt%	45.1	35.6	40.6	47.1
	N	wt%	0.5	0.50	0.7	0.3

*Total oxygen calculated by difference with all other elements. **Theoretical, calculated as: %K 2 CO 3 + (100 -%K 2 CO 3 ) × Ash of rice husks

Table 2 . Initial data for the thermodynamic equilibrium calculations.

 2

	Temperature	800 °C
		K 2 CO 3
		0.54 mg
	Initial solid	0.70 mg 1.23 mg
		1.73 mg
		2.99 mg
		0 to 1.5 g N 2
	Initial gas	+ 0 to 0.241 g H 2 O
		(0.5 L.min -1 , 0 to 30 min)

Table 3 . Gasification ash yield measured in TGA and associated theoretical yields.

 3

	Rice husks	Rice husks + K 2 CO 3 without contact	Rice husks + K 2 CO 3 mixed

Acknowledgements

The authors would like to thank Sébastien THIERY for his help with TGA experiments.

Funding

This research was funded by the Agence de l'Environnement et de la Maîtrise de l'Energie.

Pictures from microscope observation of ashes from rice husks alone and mixed with K 2 CO 3 are presented Figure 10. Ashes in the presence of K 2 CO 3 show some black glassy agglomerates on top of the white ashes (Figure 10.b) that are absent from rice husks alone (Figure 10.a). It suggests the formation at the process temperature of a liquid that solidified at ambient temperature. The SiO 2 -K 2 O phase diagram (see Supplementary Material) confirms that liquid can be present at the process temperature of 800 °C. The SiO 2 /(K 2 O+SiO 2 ) ratio for a mixture of rice husks and 7wt% of K 2 CO 3 is of 0.79 (from rice husks composition in Table 1). However, local concentrations could vary largely to reach lower or higher ratios. The change in appearance in the presence of K 2 CO 3 -including without contact-confirms that a reaction occurs between the added K 2 CO 3 or one of its decomposition products and the inorganic compounds naturally contained in biomass-most likely SiO 2 which represents 94 wt% of the inorganic content of rice husks.

The same ashes were characterized through SEM-EDX, as presented in

Glassy agglomerate

The normalized mass evolution and the gasification conversion profiles are shown in Figure 12 for beech wood alone and with K 2 CO 3 without contact during pyrolysis and gasification. Regarding gasification (Figure 12.b) when K 2 CO 3 is added without contact, beech wood reactivity greatly increases (average reactivity increasing from r 1-80% = 4.9 to 42.4%min -1 ). It reaches sunflower seed shells level (r 1-80% = 30.3%.min -1 ). The catalysis is very strong compared to what is observed in the case of rice husks (average reactivity increasing from r 1- 80% = 1.4 to 2.2 %.min -1 ). As stated previously, beech has very low ash and Si contents while rice husks is a Si-rich biomass. Therefore it confirms that the competing reaction of K 2 CO 3 decomposition product on the inherent SiO 2 prevents it to catalyze the gasification. b. a.


Fabrizio Di Pinto

Cheikh Sokhna

P.-E Fournier

Didier Raoult

Florence Fenollar
email: florence.fenollar@univ-amu.fr.

Ibrahima Sory

Traore

Issa Isaac Ngom

Cheikh Ibrahima Lo

Pierre-Edouard Fournier

Detailed description of Senegalia massiliensis strain SIT17T, a bacterium isolated from the human gut

Keywords: Senegalia massiliensis, culturomics, taxonogenomics, human gut, Bacteria

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

Introduction

Recently, the culturomics concept developed in our laboratory allowed to change the paradigm of the human gut microbiota [START_REF] Lagier | Microbial culturomics: paradigm shift in the human gut microbiome study[END_REF]. Indeed, by this method, more than 50% of the microorganisms present in the human gut microbiota are known. In order to improve culture and bacterial identification, culturomics is associated with a new process named taxonogenomics to provide exhaustive information and to better characterize bacterial species [START_REF] Seng | Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry[END_REF][START_REF] Fournier | New Microbes New Infections promotes modern prokaryotic taxonomy: a new section "TaxonoGenomics: new genomes of microorganisms in humans[END_REF]. Combining phenotypic characteristics and genomic analysis and comparison, this polyphasic approach exceeds the limits of conventional methods long used for the description of new species [START_REF] Ramasamy | A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species[END_REF][START_REF] Vandamme | Polyphasic taxonomy, a consensus approach to bacterial systematics[END_REF].

Here, we present the classification and features of Senegalia massiliensis strain Marseille-P2130 T , including a description of the complete genome sequencing and annotation.

Isolation and growth conditions

Strain Marseille-P2130 T was first isolated in 2015 from the stool of a healthy 13month-old Senegalese boy [START_REF] Traore | Senegalia massiliensis," a new bacterium isolated from the human gastrointestinal tract[END_REF]. The sample was collected in Senegal and was then frozen at -80°C. Subsequently, it was transported in dry ice to Marseille, where the bacterial culture was started. The initial growth of bacterial cells was obtained on Columbia agar with 5% sheep's blood after 2 days of anaerobic incubation at 37°C. The identification of strain Marseille-P2130 T using Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry (MALDI-TOF MS) was unsuccessful. The process was performed on a Microflex LT spectrometer (Bruker, Daltonics, Bremen, Germany) as previously described [START_REF] Sankar | The human gut microbiome, a taxonomic conundrum[END_REF]. The spectra obtained were imported and analyzed using the Biotyper 3.0 software against the Bruker database permanently improved with the local MEPHI database (Figure 1).

Strain identification and phylogenetic analysis

In order to identify the strain Marseille-P2130 T , the 16S rRNA gene was amplified using the fD1 and rP2 primer pair (Eurogentec, Angers, France) and sequenced using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and 3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously reported [START_REF] Stackebrandt | Taxonomic parameters revisited : tarnished gold standards[END_REF]. The 16S rRNA nucleotide sequences were assembled and corrected using CodonCode Aligner software (http://www.codoncode.com). The polymerase chain reaction-amplified genes coding for 16S rRNA of Senegalia massiliensis yielded 92.74% similarity level with Brassicibacter thermophilus strain Cel2f (GenBank accession no: NR137216) [START_REF] Wang | Brassicibacter thermophilus sp. nov., a thermophilic bacterium isolated from coastal sediment[END_REF], the phylogenetically closest species with standing in nomenclature (Figure 2). This value was lower than the 95%, the recommended threshold for delineating a new bacterial genus based on 16S rRNA gene sequence without DNA-DNA hybridization [START_REF] Meier-Kolthoff | Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age[END_REF]. Classification and general features are summarized in Table 1.

Phenotypic and biochemical characteristics

Colonies of the strain Marseille-P2130 T were grey and translucent with a size of 0.5-1 mm on Columbia agar with 5% sheep's blood. Growth was observed from 28 to 45°C, with an optimal growth at 37°C, and colonies were obtained after 48 hours of culture. Bacterial cells were Gram-positive, rod-shaped and motile, but non spore forming (Figure 3A). Observed under electronic microscopy, the cells presented a mean diameter of 0.4 μm and a mean length of 3.2 μm (Figure 3B). Bacterium had a catalase positive but no oxidase negative activities. Senegalia massiliensis is able to grow in an environment with a pH ranging from 6 to 8.5, with an optimal value of 7. Strain Marseille-P2130 T is an anaerobic bacterium that can grow in a microaerophilic atmosphere. On the other hand, no growth was observed under aerobic conditions. The biochemical and phenotypic features of strain Marseille-P2130 T were compared to other close representative strains in the Clostridiaceae family (Table 2). Using API ZYM strips (bioMérieux), positive reactions were observed for esterase, esterase lipase, alkaline phosphatase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-galactosidase. However, we noted that the enzymatic activities for lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, αgalactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase (FAME) analysis of the strain Marseille-P2130 T was carried out by operating gas chromatography/mass spectrometry (GC/MS) as previously described [START_REF] Sasser | Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME)[END_REF][START_REF] Dione | Genome sequence and description of Anaerosalibacter massiliensis sp[END_REF]. The result showed that Hexadecanoic acid (32.6%), 9-Octadecenoic acid (21.6%) and 13-methyltetradecanoic acid (11.9%), are the most abundant fatty. Other saturated and unsaturated fatty acids are also found (Table 3).

Genomic properties and comparison

The genome of strain Marseille-P2130 is 2,866,883 bp long with 27.39 mol% G+C content and contains 2,933 coding genes (Figure 4). It is composed of [START_REF] Sasser | Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME)[END_REF] 5).

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Senegalia massiliensis gen. nov. sp. nov., that contains the strain Marseille-P2130 T . Thus, the combination of culturomics and taxono-genomics would contribute to a better knowledge of the associated human microorganisms and could help to better understand physiological functioning in health and disease.

Description of Senegalia gen. nov.

Senegalia (Se.ne.ga.lia. L. gen. n. Senegalia) is the Latin name of Senegal, where the stool specimen was collected. Cells are Gram-positive, non spore-forming, motile and aeroanaerobic bacilli. The type species is Senegalia massiliensis sp. nov.

Description of Senegalia massiliensis sp. nov.

Senegalia massiliensis gen. nov., sp. nov. (mas.si.li.en'sis. L. fem. adj., from massiliensis, of Massilia), the Latin name of Marseille where the strain was first isolated. It is classified as a member of the family Clostridiaceae within the phylum Firmicutes. The strain Marseille-P2130 T designed the type strain of Senegalia massiliensis gen. nov., sp. nov., and was deposited in CSUR (CSURP2130) and DSMZ (DSM 103071) collections. It is Gram positive bacilli, motile, catalase positive, oxidase negative and non-spore forming. Strain Marseille-P2130 T was first isolated from the stool of a healthy 13-month-old Senegalese boy. Its genome is 2,866,883 bp long with 27.39 mol% G+C content and possesses 2933 coding genes. The genome and 16S rRNA sequences of the strain Marseille-P2130 T are both deposited in GenBank under accession numbers UZAQ00000000 and LN881608, respectively.

  and α-fucosidase, were negative. Using API 50 CH, positives reactions were observed for glycerol, D-ribose, L-xylose, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, inositol, D-mannitol, D-sorbitol, methyl α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, D-lactose, D-sucrose, D-trehalose, Dmelezitose, D-raffinose, D-turanose, D-xylose, D-fucose, L-fucose, D-arabitol, potassium gluconate and starch. However, there was no metabolism for the following carbohydrates: erythritol, L-arabinose, D-adonitol, methyl β-D-xylopyranoside, methyl α-Dmannopyranoside, D-arabinose, inulin and glycogen. Cellular fatty acid methyl esters

Figure 1 .

 1 Figure 1. Reference mass spectrum from Senegalia massiliensis strain Marseille-P2130 T .

Figure 2 .

 2 Figure 2. Phylogenetic tree highlighting the position of Senegalia massiliensis strain

Figure 3 .

 3 Figure 3. The morphology of bacterial cells of strain Marseille-P2130:

Figure 4 .

 4 Figure 4. Graphical circular map of the chromosome. From outside to the center: Contigs

Figure 5 .

 5 Figure 5. Heatmap generated with OrthoANI values calculated using the OAT software

Table 4 .

 4 contigs. By comparing it with others related species, its genome (2.87 Mbp) is smaller than Alkaliphilus oremlandii strain OhILAsm, Proteiniborus ethanoligenes strain DSM 21650, Clostridiisalibacter paucivorans strain DSM 22131, Alkaliphilus transvaalensis strain ATCC 700919, Paramaledivibacter caminithermalis strain DSM 15212, Alkaliphilus peptidifermentans strain DSM 18978 and Alkaliphilus metalliredigens strain L21-TH-D2 (3.12, 3.16, 3.24, 4.02, 4.05, 4.45 and 4.93 Mbp, respectively), but larger than the genome of Caldisalinibacter kiritimatiensis (2.79 Mbp). The G+C content of strain Marseille-P2130 T (27.39 mol%) is smaller than A. oremlandii, P. ethanoligenes, C. paucivorans, A. transvaalensis, P. caminithermalis, A. peptidifermentans, A. metalliredigens and C. kiritimatiensis (36.3, 32.6, 31.4, 34, 30.5, 34.1, 36.8 and 30.1 mol%, respectively). The gene Results from pairwise genome comparison obtained from analysis of the digital DNA-DNA hybridization (dDDH) using GGDC software are shown in Table 5. OrthoANI values among the closely related species ranged from 64.37%, between A. metalliredigens and C. paucivorans, to 70.05 % between C. kiritimatiensis and S. massiliensis. When S. massiliensis was compared to these closely species, values ranged from 65.93% with A.

	content of strain Marseille-P2130 T (2,933 genes) is larger than A. oremlandii (n=2,898), C.
	kiritimatiensis (n=2,557) and P. ethanoligenes (n=2,846), but smaller than C. paucivorans
	(n=3,014), A. peptidifermentans (n=4,072), A. metalliredigens (n=4,641), A. transvaalensis
	(n=3,640) and P. caminithermalis (n=3,543). Distribution of functional classes of predicted
	genes according to the clusters of orthologous groups (COGs) was reported in

metalliredigens to 70.05 % with C. kiritimatiensis (Figure

Table 1 .

 1 Classification and general features of Senegalia massiliensis strain Marseille-P2130.

Table 2 .

 2 Differential characteristics of Senegalia massiliensis strain Marseille-P2130 (data from this study) compared to other close bacteria

	281
	Properties	Senegalia massiliensis	Clostridiisalibacter paucivorans	Alkaliphilus oremlandii	Alkaliphilus transvaalensis	Proteiniborus ethanoligenes	Sporosalibacterium faouarense
	Cell diameter (µm)	0.3-0.5	0.5	0.5	0.4-0.7	0.5-0.6	0.5
	Oxygen requirement	Aero-anaerobic	Anaerobic	Anaerobic	Anaerobic	Anaerobic	Anaerobic
	Shape	bacilli	bacilli	bacilli	bacilli	bacilli	bacilli
	Gram stain	+	+	+	+	+	+
	Motility	+	-	+	+	-	+
	Indole	-	-	NA	+	-	NA
	Production of
	Alkaline phosphatase	+	NA	NA	NA	NA	NA
	Catalase	+	NA	NA	NA	NA	NA
	Oxidase	-	NA	NA	NA	NA	NA
	Nitrate reductase	-	NA	-	+	+	NA
	Urease	-	NA	NA	NA	NA	NA
	ß-galactosidase	+	NA	NA	NA	NA	NA
	N-acetyl glucosamine	-	NA	NA	NA	NA	NA

Table 5 .

 5 Pairwise comparison of Senegalia massiliensis strain Marseille-P2130 with other species using GGDC formula 2 (DDH estimates based on identities 291 / HSP length)*. 292 *The confidence intervals indicate the inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical 293 test data sets (which are always limited in size) These results are in accordance with the 16S rRNA and phylogenomic analyses as well as the GGDC results 294

		Senegalia	Alkaliphilus	Alkaliphilus	Alkaliphilus	Alkaliphilus	Proteiniborus	Clostridiisalibacter	Paramaledivibacter	Caldisalinibacter
		massiliensis	metalliredigens	oremlandii	transvaalensis	peptidifermentans	ethanoligenes	paucivorans	caminithermalis	kiritimatiensis
	Caldisalinibacter	19.7±2.4	37.2±5	34±4.9	17.5±4.4	18±4.5	18.9±4.6	16.9±4.4	23.6±4.7	100%
	kiritimatiensis
	Paramaledivibacter	15.2±4.3	28.4±4.9	26.4±4.8 16±4.3	19.5±4.6	19±4.5	23.6±4.7	100%
	caminithermalis
	Clostridiisalibacter	17.5±4.5	25±4.8	16.7±4.4 31±4.9	17.8±5.4	18.6±5.5	100%
	paucivorans
	Proteiniborus	17.3±4.1	19.9±4.6	19.8±4.6 18.7±4.6	16.2±4.3	100%
	ethanoligenes
	Alkaliphilus	17.2±4.4	25.5±5.2	24.8±4.8 19.9±4.7	100%
	peptidifermentans
	Alkaliphilus	27.4±4.8	23.5±4.8	22.2±4.7 100%
	transvaalensis
	Alkaliphilus	29.9±4.9	26.8±4.9	100%
	oremlandii
	Alkaliphilus	33.9±4.9	100%
	metalliredigens
	Senegalia	100%
	massiliensis

Acknowledgements

The authors thank Catherine Robert for sequencing the genome and Aurelia Caputo for submitting the genomic sequence to GenBank.

Funding sources

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

Conflict of interest

The authors declare no conflict of interest.

Ethics and consent

The child's parents provided a signed informed consent and the study was approved by the ethics committee of the Institut Fédératif de Recherche IFR48 under number 09-022.