Susanna Heikkinen
email: susanna.l.heikkinen@helsinki.fi

Les Lie Jacquemin

Antoine Rouilly

Caroline Sablayrolles

Maija Tenkanen

Pierre-Yves Pontalier

Comparison of two wheat bran extracts in the sheet extrusion process

Wheat bran extract Sheet extrusion Material properties

ln this study the applicability oftwo differently purified crude wheat bran extracts for sheet manufactur ing was evaluated. The sheet extrusion was chosen for material production because it is an industrially feasible large-scale process. The wheat bran extracts had different starch ( 45% and 11%) and protein (18% and 38%) contents but similar arabinoxylan (16% and 17%) and lignin (11 % and 12%) contents. Glycerol, sorbitol, and their blends were used as external polyol plasticizers to enhance the continuous formation of the sheet during the process. With the chosen extrusion parameters, cohesive 100-mm-wide sheets were successfully produced with single screw extruder. The mechanical and thermal properties of the sheets were evaluated, as well as their water sensitivity. Sorbitol plasticization made the sheets stronger and stiffer and significantly lowered the watervapor permeability(WVP)and watervapor sorption (WVS) of the sheets as compared to glycerol plasticization. The sheets rich in starch had a higher tensile strength and elongation at break and a lower WVP and WVS than sheets with low starch content and high prote in content. The strongest and stiffest sheet with the lowest WVP was the sorbitol-plasticized sheet with high starch and low protein content.

Introduction

Wheat (Tritium spp.) is widely cultivated in the world, and its processing produces large amounts of by-products, such as bran and straw, which are a substantial reservoir of biopolymers. Wheat bran contains up to 30% arabinoxylan (AX). approximately 20% starch, and 12-20% proteins [START_REF] Bataillon | Extraction and purification of arabinoxylan from destarched wheat bran in a pilot scale[END_REF][START_REF] Hollmann | Pilot-scale isolation of glucuronoarabinoxylans from wheat bran[END_REF]Ebringerova, 2006;[START_REF] Xie | Isolation and characterization of wheat bran starch[END_REF][START_REF] Jacquemin | Comparison of different twin-screw extraction conditions for the production of arabinoxylans[END_REF]. Ali of these biopolymers form films individually and could be utilized more efficiently than they are currently being used, for example, in biodegradable packaging and coat ings. Biopolymer films are usually produced in a laboratory scale via solution casting method onto Petri/Teflon dishes. Depending on their structural features, such as molecular weight, they may require an external plasticizer to enhance cohesive film formation. ln addition, with external plasticization film properties, such as the flexibility of the film can be adjusted. Cereal-based AX has been used in various film studies, either with or without external plastihttp://dx.doi.org/10.1016/j.indcrop2016.06.020 cization. For example, oat spelt AX films were successfully formed when plasticized with glycerol, sorbitol, or their blends [START_REF] Heikkinen | Long-term physical stability of plasticized hemicellulose films[END_REF][START_REF] Mikkonen | Films from oat spelt arabinoxylan plasticized with glycerol and sorbitol[END_REF], whereas barley husk AX formed cohesive but brittle films without plasticization (Hoije et al., 2005). Glycerol and sorbitol are often used polyol plasticizers in starch and protein films (Mc.Hugh and Krochta 1994;[START_REF] Talja | Effect of amylose content on physical and mechanical properties of potato-starch-based edible films[END_REF]. Although casting is a widely used technique on a laboratory scale, it has hardly ever been scaled up and is difficult to carry out con tinuously. Unlike casting, extrusion is a continuous process used to make films/sheets from biopolymers. Film extrusion of polysaccha rides has been reported; corn cob arabinoglucuronoxylan, starch, pectin, and sugar beet pulp have been successfully utilized in sheet extrusion [START_REF] Fishman | Extrusion of pectin/starch blends plasticized with glycerol[END_REF][START_REF] Fishman | Extrusion of pectin and glycerol with various combinations of orange albedo and starch[END_REF][START_REF] Rouilly | Thermo-mechanical processing of sugar beet pulp. III. Study of extruded films improvement with various plasticizers and cross-linkers[END_REF][START_REF] Bahcegul | Extrusion of xylans extracted from corn cobs into biodegradable polymeric materials[END_REF][START_REF] Hietala | Bionanocomposites of thermoplastic starch and cellulose nanofibers manufactured using twin screw extrusion[END_REF][START_REF] Akkus | Post-extrusion heat-treatment as a facile method to enhance the mechanical properties of extruded xylan-based polymeric materials[END_REF]. ln addition, sunflower protein isolate and soy protein films have been pro duced by extrusion [START_REF] Zhang | Mechanical and thermal properties of extruded soy protein sheets[END_REF][START_REF] Rouilly | Film extrusion of sunflower protein isolate[END_REF]. Crude biopolymer fractions can be a feasible choice in film production and even enhance the film's material properties. A study with wood hydrolysate demonstrated that high-level purification is not always required; instead, crude hydrolysates may work as well as the upgraded fractions [START_REF] Saadatmand | Turning hardwood dissolving pulp polysaccharide residual material into barrier packaging[END_REF].

The extraction of wheat bran and straw mixture using twin screw extrusion has been reported in a recent work (Jacquemin et al., 2015). Here, we present the further processing of two of these extracts via single screw extrusion in an attempt to obtain continuous 100-mm-wide sheets. The composition of the extracts varied in terms of their starch and protein content, whereas the arabinoxylan and lignin content remained similar. The effects of both the extract composition and the external plasticizer on the mechanical and thermal properties, as well as water sensitivity, were evaluated.

Materials and methods

A twin screw extrusion fractionation process was used in the production of the two wheat bran extracts, one with high starch content (WBE-S) and another with high protein content and low starch content (WBE-P) [START_REF] Jacquemin | Comparison of different twin-screw extraction conditions for the production of arabinoxylans[END_REF]. Prior to the extraction, bran for WBE-S was de-starched by washing the bran with 40 • C water, and bran for WBE-P was de-starched enzymatically using ␣-amylase (BAN 480L, Novozyme, Denmark). The main components and monosaccharide compositions of the extracts are shown in Table 1 [START_REF] Jacquemin | Comparison of different twin-screw extraction conditions for the production of arabinoxylans[END_REF].

Prior to sheet extrusion, the dried extracts were mixed with external plasticizers, water (30%, w/w of dry extract) and polyol (30%, w/w of dry extract), in a Perrier 32.00 mixer (Montrouge, France) to enhance cohesive sheet production in the extrusion process. Glycerol, sorbitol, and their blends (2:1, 1:1, and 1:2) were the polyols used in this study. Due to limited availability of WBE-S isolate, only 1:1 blend was tested. The mixture was stored in hermetically sealed bags at 4 • C for 12 h prior extrusion. The sheets were extruded using a Polylab Haake system (Karlsruhe, Germany) with a single-screw mechanism. The diameter of the screw was 18 mm, the screw speed was 100 rpm, and the compression rate was 1.8. The barrel temperature was adjusted as 50 • C/80 • C/100 • C/110 • C (die). The system was fitted with a 100-mm-wide sheet die with a 250 m slit thickness. After sheet extrusion, the samples were stored at 25 • C RH 60% prior to measurements, except the samples for the water vapor sorption study, which were stored at 23 • C RH 0% (P 2 O 5 ). The thickness of the sheets was measured at five points by using a digital micrometer (Mitutoyo, Japan) with an accuracy of 0.001 mm, and the average value was calculated.

The tensile properties of the sheets were determined with a H5KT (JFC, France) universal testing machine fitted with a 100 N load cell. At least ten specimens (75 mm long and 5 mm wide, according to ISO 527-2, type 1BA) were tested for each kind of film. The grip separation and the crosshead speed were set to 55 mm and 1 mm/s, respectively.

Dynamic mechanical analysis (DMA) experiments were performed using a Tritec 2000 DMA instrument (Triton Technology Ltd., UK) in single-cantilever bending mode. From the sheet samples, pieces of about 30 mm in length and 5 mm in width were cut, after which they were placed in a 'pocket' designed for biomaterials to be investigated by DMA. The temperature was ramped from -100 to 100 • C at 2 • C/min, with an applied frequency of 1 and 10 Hz and amplitude of 0.05 mm. The tan ␦ was plotted as a function of temperature in order to determine the thermal relaxations, of which the ␣-relaxation was taken as a glass transition.

In water vapor permeability measurements, sheet specimens were clamped on 6.4-cm-radius aluminum cups containing 7 g of CaCl 2 (RH 0%) as a desiccant. The cups were then placed in a climate room with a controlled atmosphere (25 • C and RH 60%) and weighed once a day for 7-10 days. The water vapor transfer rate (WVTR) and water vapor permeability (WVP) were calculated as described earlier [START_REF] Heikkinen | Specific enzymatic tailoring of wheat arabinoxylan reveals the role of substitution on xylan film properties[END_REF]. Three parallel samples were tested for each sheet type.

A DVS intrinsic sorption microbalance (Surface Measurement Systems, Alperton, UK) was used for the water vapor sorption study, as described previously [START_REF] Heikkinen | Specific enzymatic tailoring of wheat arabinoxylan reveals the role of substitution on xylan film properties[END_REF]. The measurements were carried out at a humidity range of 0-90% at 25 • C. Two parallel samples were tested.

Results and discussion

With both studied extracts (WBE-S and WBE-P) plasticized with polyol, 100-mm-wide continuous sheets were successfully produced by single-screw extrusion (Fig. 1). The thickness of the sheets varied, with the average values being between 280 m to 655 m. The thickness was larger than the slit of the extrusion die, which was caused by the expansion of the material at the extruder output. In pre-tests, slit thicknesses of 100, 250, and 400 m were tested, and the most regular and continuous sheets were obtained with a slit thickness of 250 m, regardless of the screw rotation speed used (75, 100, and 125 rpm). For traditional thermoplastics, low thicknesses are achieved by using post-extrusion treatments, such as blowing or calendaring, which are rarely possible with natural polymers due to their poor melt strengths in the extrusion conditions. Blowing has been successfully used with corn starch blends and milk caseinates [START_REF] Belyamani | Production and characterization of sodium caseinate edible films made by blown-film extrusion[END_REF][START_REF] López | Acetylated and native corn starch blend films produced by blown extrusion[END_REF]. The effect of cutting direction on the mechanical properties of the sample specimens was tested with glycerol-and sorbitol-plasticized sheets. The specimens cut in the direction of the extrusion flow had somewhat better mechanical properties than the specimens cut in the perpendicular direction because of the chain alignment at the die exit; therefore, the extrusion flow direction was chosen for the analysis of all the samples. The tensile strengths of the sheets varied from 2.4 MPa to 20.7 MPa, depending on the plasticizer and extract type used (Table 2). Additionally, stiffness and elongation, as well as water vapor permeability (WVP), were affected by the type of polyol plasticizer used. The same effect was seen in both sheet types (Table 2). With both extracts tensile strength and Young's modulus increased and elongation at break decreased when sorbitol was used as a plasticizer instead of glycerol. When polyol blends were used, the same effect was seen with increasing sorbitol content in the blend, especially in the WBE-S sheets, but to a lesser extent in the WBE-P sheets. The effect of polyol type on the film properties has been seen earlier for example with plasticized oat spelt AX films prepared via casting [START_REF] Heikkinen | Long-term physical stability of plasticized hemicellulose films[END_REF]. Starch content clearly increased the strength and the stiffness of the sorbitol-plasticized sheets and decreased the WVP. In the glycerol-plasticized sheets, the effect was similar, and furthermore, elongation at break increased.

The results indicate that the thermoplasticization of starch that occurs during extrusion improved the material properties. The sorbitol-plasticized sheet from WBE-S clearly had the highest tensile strength (20.7 MPa) in this study, whereas the elongation at break was similar to that seen in the WBE-P sheets plasticized with glycerol, sorbitol, or their blends (Table 2). The strongest sheet in this study had a higher tensile strength than sheets prepared from glycerol-plasticized soy protein (15.6 MPa, at RH 50% and RT), glycerol-plasticized sunflower protein isolate (6.7 MPa, at RH 60% and 25 • C), and sorbitol-plasticized potato starch (8.8 MPa, at RH 53% and RT), but it had a clearly lower elongation at break (2.2% vs. 8.8%, 46%, and 23%) [START_REF] Zhang | Mechanical and thermal properties of extruded soy protein sheets[END_REF][START_REF] Rouilly | Film extrusion of sunflower protein isolate[END_REF][START_REF] Hietala | Bionanocomposites of thermoplastic starch and cellulose nanofibers manufactured using twin screw extrusion[END_REF]. In contrast, corn cob AGX sheet strips produced without external polyol plasticizer were stronger and elongated more (76 MPa and 35%, at RH 50% and 23 • C) as compared to the sheets in this study [START_REF] Bahcegul | Extrusion of xylans extracted from corn cobs into biodegradable polymeric materials[END_REF].

Water vapor sorption (WVS) was higher in the glycerolplasticized sheets than in the sorbitol-plasticized sheets for both extract types. Starch removal increased the WVS as the glycerolplasticized sheets (WBE-P) showed lower WVS as compared to the starch-containing sheets (WBE-S) (Fig. 1). When sorbitol was used as a plasticizer, no similar effect was observed.

The glass transitions (T g ) of the glycerol-and sorbitol-plasticized sheets were analyzed using DMA (Table 2.). In all the measurements, two separate tan ␦ peaks were obtained indicating two T g values (Fig. 3). WBE-S data is shown in Fig. 2 (WBE-P showed similar behavior, results not shown). It is plausible that there are separate plasticizer-rich (T g1 ) and polymer-rich (T g2 ) phases. The protein/starch content did not have an impact on T g , as the values were dependent on the type of polyol rather than the extract content (Table 2). Two relaxation peaks have been observed in polysaccharide films when the phase separation of the polyol plasticizer in the film matrix occurred [START_REF] Mathew | Plasticized waxy maize starch: effect of polyols and relative humidity on material properties[END_REF][START_REF] Mikkonen | Effect of polysaccharide structure on mechanical and thermal properties of galactomannan-based films[END_REF]. In this study, the extruded sheets were a mixture of arabinoxylan, lignin, protein, starch, and polyol plasticizer, and it is possible that polyol was not evenly distributed in the sheet, causing two relaxation peaks in the DMA measurements. Glycerol-plasticized extracts were in rubbery state at RT whereas sorbitol-plasticized extracts were in glassy state, explaining the highest strength and moduli, as well as the lowest WVP of the sorbitol-plasticized samples.

Conclusions

This study demonstrated that crude biomass extracts, such as WBEs, have potential as raw materials for sheet extrusion. The WBEs formed continuous 100-mm-wide sheets when polyol and water were added as external plasticizers. Water sensitivity and mechanical properties of the extruded sheets were affected both by the chosen polyol and the extract composition, i.e. starch content. Sorbitol plasticized WBE-S sheets had the lowest WVP and WVS. The relatively high starch content in the extract made the sorbitol plasticized WBE-S sheets stronger and stiffer compared to sheets less starch and more protein (WBE-P). The effect was similar in the glycerol-plasticized WBE-S sheets; furthermore, elongation at break increased. This result highlights the role of starch in WBEs and potential of less refined extracts in sheet preparation.

Fig. 1 .

 1 Fig. 1. Sheet production from plasticized wheat bran extract via extrusion. Width of the die is 100 mm and slit thickness 250 m.

Fig. 2 .

 2 Fig. 2. Water vapor sorption of the plasticized sheets produced from wheat bran extracts (WBE) at RT. WBE-S contained higher amounts of starch and lower amounts of protein than WBE-P.

Fig. 3 .

 3 Fig. 3. Variation of tan ␦ for WBE-S extruded sheets plasticized with glycerol and sorbitol.

Table 1

 1 Main components and monosaccharide composition of the wheat bran extracts shown as a percentage of the dry matter[START_REF] Jacquemin | Comparison of different twin-screw extraction conditions for the production of arabinoxylans[END_REF].

		WBE-S	WBE-P
	Protein	17.9	38.2
	Lignin	11.0	11.9
	Total carbohydrates:	61.8	29.8
	Arabinose	5.1	5.7
	Xylose	11.0	11.5
	Mannose	0.4	0.2
	Galactose	0.8	1.0
	Glucose	44.5 a	11.4
	Ara/Xyl	0.46	0.50
	a Origin of glucose mainly starch.

Table 2

 2 Effect of plasticizer and extract type on the mechanical properties and water vapor permeability of extruded sheets.

	Plasticizer	Tensile strength (MPa)	Elongation at break (%)	Young's modulus (MPa)	WVP (g mm/m 2 d kPa)	Tg1 ( • C)	Tg2 ( • C)
	WBE-S
	Gly 30%	7.3 ± 0.5	7.4 ± 2.8	222 ± 25	28.8 ± 0.4	-59.9	+9.1
	Gly 15% Sor 15%	7.8 ± 1.9	4.9 ± 1.9	168 ± 38	10.5 ± 0.1
	Sor 30%	20.7 ± 2.6	2.2 ± 0.5	736 ± 59	3.2 ± 0.1	-21.2	+33.5
	WBE-P
	Gly 30%	2.4 ± 0.6	3.1 ± 1.3	84 ± 18	34.8 ± 0.4	-56.9	+3.6
	Gly 20% Sor 10%	3.2 ± 0.7	2.3 ± 0.5	149 ± 3	19.9 ± 0.4
	Gly 15% Sor 15%	2.9 ± 0.9	2.2 ± 0.6	138 ± 54	21.7 ± 0.6
	Gly 10% Sor 20%	3.3 ± 1.0	2.9 ± 0.8	137 ± 51	15.3 ± 0.3
	Sor 30%	5.3 ± 1.3	1.9 ± 0.6	297 ± 32	8.7 ± 0.1	-20.7	+33.3

Acknowledgments

We thank Docent Kirsi Mikkonen for the valuable discussions and comments. The Academy of Finland (contract no 132150), the French National Research Agency (ANR) (ANR-09-CP2D-16), and the COST Action FP1003 are thanked for their financial support.


Les Lie Jacquemin
email: leslie.jacquemin@ensiacet.fr

Assad Mogni

Rawan Zeitoun

Cécile Guinot

Caroline Sablayrolles

Luc Saulnier

Pierre-Yves Pontalier
email: pierreyves.pontalier@ensiacet.fr

Comparison of different twin-screw extraction conditions for the production of arabinoxylans

Keywords: Arabinoxylans Destarching Alkali extraction Twin-screw extrusion Ultrafiltration Resin chromatography EV, evaporation, P, precipitation, FD, freeze drying, UF, ultrafiltration, CHR, ion exchange chromatography, AXa, rabinoxylans, GXg, lucuronoxylans, GAX, glucoronoarabinoxylans, TMP, transmembrane pressure, MWCO, molecular weight eut-off

The aim of this article is to compare two different sets of optimal conditions for twin-screw extraction of xylans and define their influence on the purification steps, combining ultrafiltration and industrial chromatography. Iwo xylan extracts were obtained by twin-screw extrusion of straw and bran. Condition 1 used a high straw{bran ratio (equal to 6) and high sodium hydroxide content, and condition 2 used a lower straw{bran ratio (equal to 2) and low sodium hydroxide content.

Introduction

Wheat bran and wheat straw are sources of cell-wall polysaccharides such as cellulose. hemicelluloses. and lignin. Ara binoxylans are the most abundant hemicellulose polymers in wheat bran. They mainly consist of a chain of o-xylopyranosyl with �-(1-4) links, substituted on position 2 and/or 3 with a-L arabinofuranosyl residues [START_REF] Spiridon | Hemicelluloses: Major sources, properties and applications[END_REF]. Hemicelluloses from bran have the potential to be incorporated into a wide vari ety of applications, such as thickeners, emulsifiers. stabilizers and binders in the food, pharmaceutical and cosmetics industries. but film-forming ability is one oftheir most attractive properties Oain, Sjostedt, & Classer, 2000;[START_REF] Spiridon | Hemicelluloses: Major sources, properties and applications[END_REF]. Therefore, much work has been undertaken investigating their possible fractiona tion.

A large range of procedures has been proposed for the extrac tion and purification of arabinoxylans. One of the most widespread consists of stirred extraction, with sodium hydroxide [START_REF] Brillouet | Isolation, purification and characterization of a complex heteroxylan from industrial wheat bran[END_REF]Bataillon, Mathaly, Numes Cardinali. & Duchironet. 1998;[START_REF] Maes | Alkaline hydrogen peroxide extraction of wheat bran non-starch polysaccharides[END_REF]. but many others such as barium hydroxide (Bergmans, Beldman, Cruppen, & Voragenet, 1996), or dimethyl sulfoxide [START_REF] Lawther | Influence of alkaline pre-treatments on the cell wall components of wheat straw[END_REF] can be used. Alkaline conditions disrupt the cell walls by hydrolyzing ester linkages between the hemicelluloses and the other cell-wall components thus dissolving these hemicelluloses, but also lignin. The liberated hemicelluloses can then be recovered in aqueous media [START_REF] Fang | Comparative study of hemicelluloses from wheat straw by alkali and hydrogen peroxide extractions[END_REF] with an extraction yield of50% after 2 h in 1% NaOH solution [START_REF] Sun | Physico-chemical and structural characterization of hemicelluloses from wheat straw by alkaline peroxide extraction[END_REF]. Higher recovery yields can be obtained but with longer residence time [START_REF] Chanliaud | Alkaline extraction and characterisation of heteroxylans from maize bran[END_REF]. However, other extraction technologies have been studied to improve recovery efficiency, such as steam explosion (Classer & Wright. 1998;[START_REF] Montané | Fractionation of wheat straw by steam-explosion pretreatment and alkali delignification. cellulose pulp and byproducts from hemicellulose and lignin[END_REF] or chemical extraction assisted by ultrasonication [START_REF] Ebringerovà | Effect of ultrasound on the extractibility of corn bran hemicelluloses[END_REF][START_REF] Hromàdkovà | Ultrasonic extraction of plant materials-Investigation of hemicellulose release from buckwheat hulls[END_REF][START_REF] Sun | Fractional and physico-chemical characterization of hemicelluloses from ultrasonic irradiated sugarcane bagasse[END_REF]. Combined with the use ofalkaline agents in order to accelerate solubilization of the arabinoxylans instead of the lignin and cellulose, steam explosion is an efficient tool for monomeric sugar recovery [START_REF] Sun | Characteristics of degraded hemicellulosic polymers obtained from steam exploded wheat straw[END_REF][START_REF] Krawczyk | Isolation of hemicelluloses from barley husks?[END_REF]. However, it results in hydrolytic depolymerization of hemicelluloses, which is attractive for alcoholic fermentation or xylose transformation [START_REF] Puls | Industrially isolated hemicelluloses[END_REF], but not for isolation of hetero-polysaccharides [START_REF] Glasser | Isolation options for non-cellulosic heteropolysaccharides (HetPS)[END_REF].

All the treatments lead to the production of a complex extract that requires different purification steps, and alcoholic precipitation is the most useful and efficient procedure on a laboratory scale. Other purification treatments have been studied, such as microfiltration [START_REF] Bataillon | Extraction and purification of arabinoxylan from destarched wheat bran in a pilot scale[END_REF] and ultrafiltration [START_REF] Jorda | Biopolymer purification by ultrafiltration[END_REF] in order to reduce the cost of the purification step. However, the production of solid arabinoxylans (in powder form) from an aqueous solution or suspension requires either a freeze-drying or a spray-drying step [START_REF] Glasser | Isolation options for non-cellulosic heteropolysaccharides (HetPS)[END_REF].

Ultrafiltration, a well-established separation process in industry, was used to separate polysaccharides from co-extracted molecules, small oligosaccharides, monosaccharides and salts, in order to concentrate the solution and reduce the volume of alcohol used for precipitation [START_REF] Schlesinger | Evaluation of alkali resistant nanofiltration membranes for the separation of hemicellulose from concentrated alkaline process liquors[END_REF][START_REF] Montané | Fractionation of wheat straw by steam-explosion pretreatment and alkali delignification. cellulose pulp and byproducts from hemicellulose and lignin[END_REF][START_REF] Nabarlatz | Purification of xylo-oligosaccharides from almond shells by ultrafiltration[END_REF][START_REF] Persson | Isolation of hemicelluloses by ultrafiltration of thermo-mechanical pulp process water-Influence of operating conditions[END_REF]. This is a very attractive process because it results in the partial demineralization and removal of small molecules [START_REF] Glasser | Isolation options for non-cellulosic heteropolysaccharides (HetPS)[END_REF]. Hemicelluloses are purified because large molecules are retained and low molecular mass solutes pass through the membrane. However, in some cases, coloration of the extract remains too dark after the ultrafiltration treatment, producing a dark-brown arabinoxylan powder, therefore anionexchange chromatography can be combined with ultrafiltration to improve purification prior to powder recovery [START_REF] Glasser | Isolation options for non-cellulosic heteropolysaccharides (HetPS)[END_REF][START_REF] Andersson | Comparison of diafiltration and size-exclusion chromatography to recover hemicelluloses from process water from thermomechanical pulping of spruce[END_REF].

Nonetheless, even if only a few studies have dealt with pilot scale extraction of hemicelluloses (Annison, Choct, & Cheethamet, 1992;[START_REF] Bataillon | Extraction and purification of arabinoxylan from destarched wheat bran in a pilot scale[END_REF][START_REF] Zeitoun | Twin-screw extrusion for hemicellulose recovery: Influence on extract purity and purification performance[END_REF][START_REF] Montané | Fractionation of wheat straw by steam-explosion pretreatment and alkali delignification. cellulose pulp and byproducts from hemicellulose and lignin[END_REF], it needs to be developed on an industrial scale [START_REF] Puls | Industrially isolated hemicelluloses[END_REF]. As twinscrew extrusion is a continuous process, which could be scaled up to an industrial scale, it was investigated for straw/bran co-extraction using sodium hydroxide. This technique combines chemical, thermal and mechanical actions to remove the dissolved molecules from the plant tissue by continuous squeezing in a screw press [START_REF] Zeitoun | Twin-screw extrusion for hemicellulose recovery: Influence on extract purity and purification performance[END_REF], and it has proved to be an efficient method for alkaline extraction of hemicelluloses from wheat bran [START_REF] Maréchal | Twin screw extrusion and ultrafiltration for xylan production from wheat straw and bran[END_REF]. It gives lower extraction yields and selectivity than batch extraction but has the advantage of being continuous and requiring smaller quantities of reagents and water [START_REF] Montané | Fractionation of wheat straw by steam-explosion pretreatment and alkali delignification. cellulose pulp and byproducts from hemicellulose and lignin[END_REF][START_REF] Maréchal | Twin screw extrusion and ultrafiltration for xylan production from wheat straw and bran[END_REF]. The extrusion process has been studied on a laboratory scale, using an experimental design, covering the influence of the straw/bran ratio, the bran/sodium hydroxide ratio and screw rotation speed. The results showed that two optimal extraction conditions exist, the first with a high straw/bran ratio (equal to 6) and high sodium hydroxide content, and the second with a low straw/bran ratio (equal to 2) and low sodium hydroxide content [START_REF] Maréchal | Twin screw extrusion and ultrafiltration for xylan production from wheat straw and bran[END_REF], but the comparison was only made using the recovery yield with alcoholic precipitation.

Twin-screw fractionation leads to the production of very complex solutions containing the biopolymers, but also their hydrolyzed products and co-extracted molecules (proteins, lignin, and inorganic salts). Hence, it is important to develop efficient purification steps regarding efficiency, cost and environmental impact [START_REF] Jorda | Biopolymer purification by ultrafiltration[END_REF]. For example, precipitation of the hemicellulose alkaline extract using acids and alcohols to obtain a certain degree of purification, remains expensive because it requires several volumes of alcohol for one volume of solution. Thus, work is needed to devise more cost-effective separation methods.

In this study, extraction has been performed on a larger scale (a few hundred kilos), using the first set of optimal extraction conditions above, in order to evaluate the performance of the purification process, including ultrafiltration and anion exchange chromatography. Thus, the aim of the present work is to compare both optimal extraction conditions on a larger scale and define their influence on the efficiency of the refining procedure.

Materials and methods

General description of the process

Two sets of large-scale optimal extrusion conditions were evaluated in order to produce a large volume of extract. Each extract was refined using three refining pathways (Fig. 1) in order to study the influence of the extraction conditions on purification performance. The three pathways were:

• fractionation of straw and bran and direct extract precipitation:

("C + P + FD"); • extract precipitation after concentration by ultrafiltration:

("C + UF + P + FD"); and • as above but with an ion exchange chromatography purification step: ("C + UF + CHR + P + FD").

In addition, because the destarching step was derived from a laboratory scale protocol, it also had to be adapted, and so three different procedures have been tested, as shown below. For each procedure, the bran was then dried at 45 • C for 24 h. All wheat bran and straw was from the same origin (Arteris, France).

Twin screw extraction

Experiments were conducted using a co-penetrating and corotating twin-screw extruder (Clextral BC45) (Fig. 2).

The extruder was composed of a 1.4 m-long barrel containing seven dedicated profile modules for alkaline treatment of plant matter. Modules C, D, E and G were heated using induction belts (5 kW). There were two reverse-pitch screw elements with grooved peripheral slots in the screw for leakage flow, and a series of 1 cmlong neutral pitch bilobal elements splayed at 90 degrees to each other, were used to knead the plant matter. The 10 cm-long filter element mounted on the last section, was perforated with conical holes (1 mm inlet, 2 mm outlet) and was used to optimize solid/liquid separation [START_REF] Zeitoun | Twin-screw extrusion for hemicellulose recovery: Influence on extract purity and purification performance[END_REF]. One hour before extrusion, wheat bran and sodium hydroxide were blended at room temperature, giving a liquid/solid ratio of 7, adjusted to 10 just before the extrusion. The mixture was injected into the extruder in section B, using an exocentric-screw pump (Nemo, 2NE40A). Straw was also introduced in section A using a screw feed, and mixed with the alkaline dough in the first zone of the barrel through the neutral pitch element and the reverse-pitch screw element successively. Wash water was injected downstream of this zone (E) by piston pump (Clextral, DKM K20-2-P32), and the mixture was carried through the second reverse pitch element located just downstream of the filtration module. Screw rotation speed and barrel temperature were monitored from a control panel. The extract was collected and kept in a cold room before further processing, while the refined cellulosic fibres were collected at the barrel outlet.

Optimal conditions for extrusion concerning hemicelluloses yields and purity, have been defined in previous studies [START_REF] Maréchal | Twin screw extrusion and ultrafiltration for xylan production from wheat straw and bran[END_REF] and are summed up in Table 1.

Liquid-solid separation

Clarification of the extract from suspended matter that could hinder the subsequent concentration step, was performed using a continuous centrifuge separator (Alpha-Laval, CLARA20 LAPX) equipped with 400 m spaced discs. The extract was introduced continuously at a flow rate of 100 L/h via an impeller pump (Schneider, Reform B-FU). Clarification was at 11,130 × g, and separated sludge was drawn off every 4 min for trials 1 and 3 and every 5 min for trial 2.

Purification process

Ultrafiltration

The ultrafiltration (UF) membrane used had a molecular weight cut-off (MWCO) of 30 kDa and a surface area of 1.15 m 2 . The 52 cm long, 5.4 cm external diameter module was composed of 1300, 0.5 mm-diameter polyethersulfone hollow fibres, (GE Healthcare, UFP-30-C9A). This membrane was selected in the light of previous results [START_REF] Zeitoun | Twin-screw extrusion for hemicellulose recovery: Influence on extract purity and purification performance[END_REF] as discussed in Section 3. The filtration apparatus used a 36 L capacity feed reservoir, a rheostat controlled bilobal-pump, a filtration cartridge and a pressure gauge allowing manual membrane pressure adjustment. Transmembrane pressure (TMP), the average of the inlet and outlet membrane module pressures, was measured using manometers.

In order to determine the influence of transmembrane pressure and circulation flow rate on the permeate flux and define optimal operating conditions for extract concentration, ultrafiltration was first carried out by recycling both permeate and retentate flux in the feed tank, to maintain a constant concentration.

The extract was then concentrated by extracting the permeate flux, while still recycling the retentate flux in the feed tank. The permeate flow rate was measured gravimetrically using an electronic balance. The volume reduction factor (VRF) was evaluated by taking the extract density as equivalent to that of water.

Membrane cleaning used the following procedure:

-demineralized water rinse for 30 min; -cleaning with 1 N sodium hydroxide solution for 1 h at 40 • C; and -demineralized water rinsing until pH 7 obtained.

The concentration of the whole extract was made with the same membrane, in 20 L batches, and there were 5 batches for each trial. A new membrane was used for each of these trials.

Anion-exchange chromatography

Chromatography was performed with a strong anion-exchange resin (Rohm and Haas, Amberlite IRA958-Cl) on 12.5 L batches. 2 L of resin was stirred with the extract for 1 h and separation between extract and resin performed by Büchner-filtration. Before use, the resin was degassed for 24 h in 6 L demineralized water, and after use it was reconditioned for 30 min using deionized water, and regenerated with 5 L of 2% sodium hydroxide solution.

Powder recovery

Alcoholic precipitation

Hemicelluloses were recovered at different stages of the process by precipitating the alkaline extracts with acid and alcohol. Acetic acid was added to the extract up to pH 5.5, followed by 3 volumes of ethanol for 1 volume of solution. To improve precipitation, the solution was stored at 4 • C overnight, and aggregated hemicelluloses were then recovered, by wringing out the simple filtration cloth, and drying to obtain powders.

Freeze-drying

Hemicellulosic precipitates were freeze-dried in all cases (Cryo Rivoire, PILOT27). Samples were placed in aluminium trays and cooled to-40 • C. Decreasing the pressure to -300 mbar caused sublimation of moisture content which then froze in a cold-trap. Finally, the temperature was gradually increased to 20 • C and the dried samples recovered.

Analytical

Starch in bran quantification before and after destarching was made with a total starch assay kit using thermostable ␣-amylase and amyloglucosidase (Megazyme, Ireland). Dry matter was gravimetrically determined at 105 • C over a period of 24 h, and ash content was determined by thermogravimetric analysis after incineration at 550 • C for 5 h. An estimation of the three cell-wall components contained in the solid fraction (cellulose, hemicelluloses, and lignins), was made using the ADF-NDF method of Van Soest andWine (1967, 1968). Lignin composition in powders was measured using the Tapi norm T222 om 88 for acid-insoluble lignin. Matter treated with sulfuric acid was filtered on Wattman preweighed glass-fibre filters, washed with deionized water, and dried at 105 • C overnight before being weighed. Proteins were measured on a Kjeldahl automated device. The specific factor (5.7) used for the conversion of nitrogen content to protein content, uses the average molecular weight of amino acids in the bran endosperm compared to the molar mass of nitrogen [START_REF] Sosulski | Amino acid composition and nitrogen-toprotein conversion factors for animal and plant foods[END_REF]. Individual neutral sugars were analyzed by gas-liquid chromatography (GLC) after hydrolysis (2 h in 1 M H 2 SO 4 at 100 • C), and conversion of individual sugars into aldidol acetates [START_REF] Englyst | Improved method for measurement of dietary fiber as non-starch polysaccharides in plant foods[END_REF]. A prehydrolysis step in concentrated sulfuric acid (72% w/v) was carried out for 30 min at 25 • C on the fresh bran and straw. All determinations were carried out in triplicate.

Results and discussion

Raw material

For the first two trials, starch was removed by water washing. For trial 1, filtration was done from the top of the tank by pumping the starch milk through an external loop, while for trial 2, water was removed by filtration through the bottom of the tank. The bran used for trial 3 was destarched using enzymes. The starch composition and amount of destarching for each batch of bran are shown in Table 2. The decrease in level of destarching from 70.7% to 46.5% between trial 1 and 2 indicates that during filtration through the bottom of the tank, the bran cake has retained a part of the starch molecules in suspension in the wash water. Moreover, the high level of destarching observed in trial 3 shows that enzymatic hydrolysis is much more efficient, leading to a starch content of 2.5% of the dry matter weight.

Analysis of the main fractions of destarched bran allows characterization of at least 90% of the dry matter.

Bran 1 is slightly different from bran 2 and 3, especially for mineral content, which is lower (around 3% for bran 1 and around 5% for bran 2 and 3) and proteins (around 14% for bran 1 and around 20% for bran 2 and 3). This is due to the fact that bran 1 came from a different batch of the same production. Cellulose and lignin contents obtained using Van Soest's characterization are equivalent, whereas a significant difference between bran 2 and bran 3 can be seen for hemicelluloses content. However, this fraction seems to be overvalued for bran 3 compared to the gas chromatography analysis, making the total carbohydrate obtained by gas chromatography appear to be lower than the hemicellulose content evaluated gravimetrically. Therefore, as bran contains a low proportion of cellulose and because Van Soest's method is more accurate for raw and fibrous materials, only the gas chromatography analysis results will be used for the rest of this article.

Starch has been analyzed using a total starch assay procedure with megazyne on bran, before and after the destarching treatment.

In Table 2, it appears that the destarching yield is better under condition 1 than condition 2, and even though the two brans were not from the same batch and did not contain the same amount of starch initially, the amount of starch remaining in the bran is very similar (10%). Nevertheless, it can be assumed that in the second case, starch has been solubilized and then entrapped in the bran during the bottom filtration. Thus its recovery was higher during extrusion, and the X/G ratio confirms this hypothesis since it is far higher in condition 1 (0.8) than in condition 2 (0.2). The X/G ratio can thus be used as an indicator of starch contamination in the extract. Further analyses (data from another partner not shown) on the final powder also confirm this hypothesis.

Extraction

Composition of refined straw fibres

It has been shown in previous studies that straw is mainly added to improve liquid-solid separation, and so the source of extracted molecules is mainly the bran, pre-soaked in soda 1 h before extrusion, [START_REF] Zeitoun | Twin-screw extrusion for hemicellulose recovery: Influence on extract purity and purification performance[END_REF][START_REF] Jacquemin | Evaluation of the technical and environmental performances of extraction and purification processes of arabinoxylans from wheat straw and bran[END_REF]. However, after extraction, slight differences can be observed between the composition of the straw and the refined straw fibres produced during extrusion (Table 3). These can be explained by the thermo-mechanical and chemical treatments applied simultaneously to the straw and bran, which lead to the extraction of a few compounds from straw (hemicelluloses) whereas some bran compounds remain trapped in the refined straw fibres (proteins).

Conditions used for trial 1 involved a high straw/bran ratio, a high sodium hydroxide concentration and a low liquid/solid ratio, whereas those for trials 2 and 3 had a lower straw/bran ratio, a lower sodium hydroxide concentration and a higher liquid/solid ratio. All the trials were performed with low mechanical action (slow screw rotation speed, 150 rpm). For trial 1, the high straw/bran ratio combined with a low liquid/solid ratio increases the mechanical action on the raw material, despite the slow screw rotation speed. This results in better liquid-solid separation at the outlet of the extruder and gives a higher dry matter content of refined straw fibres (34.5% versus 21.5%). The use of a lower straw/bran ratio combined with a lower sodium hydroxide concentration for trials 2 and 3, means that more bran compounds remain entrapped in the refined straw fibres implying higher proteins and hemicelluloses in these. The less effective liquid-solid separation also means that the refined straw fibres have a higher moisture and a higher mineral content. Thus, despite using less sodium hydroxide for extraction in trials 2 and 3, a higher mineral content is found in the residue, compared to trial 1.

Extracts and filtration cake compositions

Table 4 shows the composition of extracts obtained just after extrusion, and the extract and centrifugation cake obtained after centrifugation.

Two extraction conditions were evaluated, the first with a low liquid/solid ratio and a high straw/bran ratio and the second, with a higher liquid/solid ratio and a lower straw/bran ratio favouring chemical action, and thus higher solubilization of the hemicelluloses. There is only a slight difference in the appearance of the crude extracts, but this difference increases after centrifugation which removes the non-solubilized molecules but not the solubilized ones. Mechanical action dominates compared to chemical action when extraction is carried out under extraction condition 1, and part of the extracted xylan is in the form of bran particles. This explains why there is a greater reduction in dry matter after the centrifugation step for condition 1.

The dry matter concentration of the crude extract obtained in trial 1 reached 3.1% versus 2.6% for trial 2 and 3.2% for trial 3. The lower value obtained in trial 2 compared to trial 3 may be the result of the less effective destarching step. However, under alkaline conditions at ambient temperature, starch can form a gel that could hinder the water washing of the molecules released at the a "Crude extract" is the extract obtained at the outlet of the extruder. b "Centrifuged extract" is the extract obtained after centrifugation. c "Cake" is the matter in suspension removed from the crude extract during centrifugation. d Refers to X + A introduced by bran and straw. extrusion outlet [START_REF] Ring | Some studies on starch gelation[END_REF][START_REF] Morris | Starch gelation and retrogradation[END_REF]. The combined use of a low straw/bran ratio and low sodium hydroxide concentration for trials 2 and 3, gives greater protein and carbohydrate fractions recovered in the extract, and a lower proportion of ash compared to trial 1. After centrifugation, the reduction in dry matter is 22.6% for trial 1 whereas it is only 15.6% for trial 3. This difference can be explained by the higher mechanical and chemical action applied to the plant matter during trial 1, due to the high straw content and sodium hydroxide concentration. Hence, a greater amount of suspended matter is recovered in this extract 1, and removed during centrifugation, whereas for trials 2 and 3 extracted molecules are mainly in solution. For all the trials, the proportion of proteins and ash in the extract has increased, and this is because a major part of them are solubilized and cannot be evacuated with the suspended matter in the cake. However, cake also contains some ash and proteins from the part of the extract evacuated with it during flushing. The variations in the proportion of carbohydrates in the extract are not significant, implying that part of them are probably in a non-solubilized form and evacuated with the cake.

The Xylose + Arabinose (X + A) extraction yield can be calculated by using only the X + A coming from bran, or all the X + A introduced, from both bran and straw. Taking all the X + A introduced, there is almost no difference between the conditions tested. Assuming that carbohydrates only come from bran, condition 1 gives the best results, with an extraction yield of 50% while this is only 22% for condition 2. For both conditions 1 and 2 the centrifugation recovery yield is only 50% of the X + A and this result indicates that the mechanical action of the extruder is producing particles containing xylans, which are not dissolved and so are recovered in the centrifugation cake.

The centrifugation cakes show differences in dry matter content (9.8% for trial 1, 14.7% for trial 2 and 9.2% for trial 3), that can be explained by a slight difference in flushing frequency used (every 4 min for trials 1 and 3 and every 5 min for trial 2). During centrifugation, the flush interval should be short enough to be able to push the matter out of the pipes, but not so short as to cause large losses in the extract. Optimization of this frequency is one of the main opportunities for improving the process.

Powder compositions

A fraction of each centrifuged extract was treated using alcoholic precipitation, and hemicellulosic powders were recovered after freeze-drying, used because it was assumed it would allow 100% recovery yield of the precipitated molecules. The composition of all these fractions was evaluated and the results are shown in Table 5.

The dry matter yields of the powder recovery steps (precipitation + freeze drying) after centrifugation are around 38.5% for all powders, which indicates that not all the solubilized compounds are precipitated and confirms that precipitation also has a purifying action [START_REF] Jacquemin | Evaluation of the technical and environmental performances of extraction and purification processes of arabinoxylans from wheat straw and bran[END_REF]. The ash content is higher for the powder produced during trial 1 (as well as for its corresponding extract; Table 4), once again because of the larger amount of sodium hydroxide used here. The carbohydrate content, reached 58.4% in trial 2. This is a very high value, mainly due to the high glucose content (42.1%). And the origin of this glucose is starch, because the content from the powder produced using better-destarched bran under the same conditions (trial 3), is only 11.7%. Therefore, the total carbohydrate content of the powder from trial 3 is only 31.7% whereas powder from trial 1 reached 48.4% (but still with a substantial fraction of glucose, due to the less efficient destarching procedure). The X/G ratio can therefore be used as an indicator of starch contamination in the extract. Since it is low in extract 2, it can be supposed that this contains a larger amount of starch than extract 1 with a higher X/G ratio. Further analysis (data from another partner not shown) of the final powder also confirms this hypothesis.

The proportion of proteins in powders is consistent with previous results. Nonetheless, because less bran was used in trial 1, less protein was recovered in the extract and thus their content in the powders is lower (12.9%). There are two reasons for the smaller proportion of proteins in trial 2 compared with trial 1 (respectively, 37.0% and 18.0%). The first is that the gel formed by starch during extrusion had probably hindered their extraction. The second is that because the proportion of carbohydrate is higher, the relative proportion of proteins is lower.

The content in arabinoxylans (X + A) of trial 1 powder is 24.2% while it is only 14.9% and 18.8% for those from trials 2 and 3, respectively. This can be explained by the presence of more glucose for trial 2 and more proteins for trial 3. The X/A ratio is slightly higher for trial 1 (2.8) which confirms that a part of the arabinoxylans from the straw (that have an X/A ratio around 9; [START_REF] Jacquemin | Evaluation of the technical and environmental performances of extraction and purification processes of arabinoxylans from wheat straw and bran[END_REF] are also extracted due to the higher mechanical action applied during this experiment.

Concentration and purification

Ultrafiltration

Alcoholic precipitation is the most widely used technique for recovery of hemicelluloses contained in alkaline extracts on a laboratory scale. It is a selective technique, which separates large molecules (proteins, polysaccharides) from small molecules (salts, monosaccharides). However, it requires the addition of at least 3 volumes of ethanol per volume of extract, meaning that it is expensive and has been singled out as the main restriction to the development of hemicellulose production on an industrial scale. Thus, the combination of ultrafiltration and anion-exchange chromatography has been investigated in order to define how it could be introduced into the process to replace precipitation [START_REF] Jacquemin | Evaluation of the technical and environmental performances of extraction and purification processes of arabinoxylans from wheat straw and bran[END_REF]. Optimal conditions for filtration, defined in a previous work, showed that the best results were obtained with a 30 kDa membrane. The polarization layer that appears during ultrafiltration, changes the filtering properties of the membrane and leads to a high rejection rate of the hemicelluloses. [START_REF] Zeitoun | Twin-screw extrusion for hemicellulose recovery: Influence on extract purity and purification performance[END_REF]. Concentration of the different extracts was performed with a transmembrane pressure of 2.3 bar and a circulation flow rate of 0.23 m/s (210 kg/h). During ultrafiltration of the trial 2 extract, it was impossible to apply the same filtration conditions as for the other extracts, because the permeate flux was too low. Therefore filtration was performed with a higher transmembrane pressure. Despite this increase, the permeate flux for trial 2 extract remained below those obtained with the two other conditions (Fig. 3). For trials 1 and 3, the flux decreases from 13 to 9 kg/h/m 2 while for trial 2, it decreases from 7 to 5 kg/h/m 2 . This difference can be explained by the gelling properties of the starch contained in the extract, which increased its viscosity, assuming that the high value of carbohydrate in the extract of trial 2 (Table 4) is linked to high starch content.

After ultrafiltration, a part of the extract was precipitated in order to analyse the effect of the sequences of operations on the powder composition. Results are presented in Table 5.

For powder content, comparing data from Table 5 shows a slight decrease in carbohydrate while proteins increase. The amount of arabinoxylans (X + A) and proteins recovered from 100 kg of extract after centrifugation is 0.16 kg and 0.20 kg, respectively, for trial 2. This 100 kg of extract produced 50 kg of retentate, leading to the production of 0.16 kg of arabinoxylans and 0.23 kg of proteins. It can be concluded from this result that ultrafiltration of extract from trial 2 implies a very low loss in protein and arabinoxylans. The results are very similar for trial 3 but the recovery yields are lower than for trial 1.

It has been shown in previous studies that ultrafiltration is an efficient tool for polymer concentration and for the elimination of ash and small molecules [START_REF] Jacquemin | Evaluation of the technical and environmental performances of extraction and purification processes of arabinoxylans from wheat straw and bran[END_REF]. However, the demineralization effect is not visible in these results because alcoholic precipitation also eliminates ash. One interesting result is the yield in dry matter of the precipitation step after ultrafiltration. This increased from 38.0% to 53.1% for trial 1, from 38.9% to 61.9% for trial 2 and from 38.6% to 87.3% for trial 3. Two reasons can be put forward to explain this result. The first is that purification by ultrafiltration removes molecules that hinder the precipitation. The second is that higher solute concentrations increase the efficiency of the precipitation step. Which could explain why for trial 3, more proteins are recovered by precipitation of retentate than by precipitation of the extract before ultrafiltration.

It can be assumed that the decrease in carbohydrates after ultrafiltration is mainly due to elimination of small molecules and monosaccharides, while the polymers are retained.

Ion exchange chromatography

Anion exchange chromatography has been evaluated for the removal of the coloured molecules, lignin and proteins, in order to obtain purer hemicelluloses. This step obviously decreases the lignin content (from 24.7% to 23.3% for trial 1, from 18.3% to 11.0% for trial 2 and from 22.6% to 11.9% for trial 3), leading to a change in carbohydrate composition. In fact, compared with powders from extracts that have not been percolated on resin, powders here show an increase in the proportion of carbohydrates while that of proteins remains stable or slightly lower (Table 5). Although this step is efficient for lignin removal, the result indicates that the resin also retains a fraction of proteins, maybe due to aromatic amino acids. The X/A ratio also decreases in all cases proving that some arabinoxylans are similarly retained on the resin beads. The ash content remains stable meaning that the mechanism responsible for the purification does not involve ion exchange from resin. Furthermore, the global composition of the powder produced does not change, trial 2 still contains a large part of glucose (44.5%) whereas a Calculation refers to (bran + straw), expressed as the dry weight of recovered matter in the powder/weight of this matter in (bran + straw). b (X + A) is the sum of xylose and arabinose. c Content of (X + A) in the powder (wt% dry matter).

glucose content is lower for trial 3 (11.4%), and the protein content for trial 3 is still very high.

Recovery yields

Production yield of each fraction produced under the three conditions has been evaluated for dry matter, organic matter, xylose + arabinose and protein (Table 6).

For all process routes, and for all trials, the yields in (X + A) are below 10%, which is partly explained by the brevity of the residence time in the extruder. The small differences observed between the different process pathways for the same trial, can be explained by the fact that a precipitation step is always involved, which masks the effect of ultrafiltration and chromatography. Nevertheless, it can be seen that ultrafiltration is efficient at concentrating all polymers, including protein and lignin, and that chromatography seems to be an effective procedure to purify the resulting extract (Trial 3) even if it still requires optimization.

Hence, trial 3 is much more efficient (twice) than trial 2, because remaining starch created a gel during extrusion that entrapped a part of the released molecules. Trial 1 gives lower recovery yields than trial 2 because the calculation includes the mass derived from straw while in fact only a small part of the molecules in the extract come from this, as shown by the X/A difference between trial 1 extracts and the others. However, if the calculations were made using only bran-derived (X + A), the (X + A) yields would increase above 20%.

Conclusions

Two different large scale feed conditions (considered optimal for small scale xylan recovery; [START_REF] Maréchal | Twin screw extrusion and ultrafiltration for xylan production from wheat straw and bran[END_REF], have been investigated for the extraction of arabinoxylans by straw-bran co-extrusion in a twin-screw extruder. These large-scale results confirm those found on a smaller scale, with very similar arabinoxylan recovery yields of between 9% and 10% taking into account all the arabinoxylan introduced (from both bran and straw). For the xylans coming only from bran, condition 1 gives the best result with an extraction yield of 28% after centrifugation compared to 12% with condition 2. The centrifugation step had a carbohydrate recovery yield of approximately 50% under all the conditions tested, which is because of the large amount of small particles generated during the extrusion.

The 30 kDa ultrafiltration membrane is adapted to hemicellulose concentration, and to solution demineralization, but it also concentrated large molecules such as proteins. As the loss of arabinoxylans remains low, it should be possible to wash the extract by diafiltration to ensure complete demineralization. The results also indicate that pre-extraction starch removal is necessary, because starch reduces extraction efficiency and leads to fouling of the ultrafiltration membrane. Chromatography is efficient for the elimination of coloured molecules, but it also retains proteins, mainly aromatic compounds. Thus, the combination of ultrafiltration and chromatography enables partial purification of the extract with a final arabinoxylan purity ranging from 16% to 26%. The carbohydrate content of the final powder may exceed 50%, and the other main components are protein and lignin.

This large-scale study almost defines the industrial performance of a continuous process including extrusion, ultrafiltration and industrial chromatography. On a large scale, extraction must be made with a large straw/bran ratio, and the extract can be efficiently concentrated by ultrafiltration instead of evaporation. Nevertheless, under these conditions, extraction of the proteins is more efficient than for polysaccharides, with protein recovery yield ranging from 10% to 30%. Twin-screw extrusion is an efficient tool for the extraction of the main soluble fractions of plant matter and allows polysaccharides, proteins and lignin to be washed from the cellulose fibres. Nonetheless, the separation of these fractions remains difficult because a part of them is extracted in non-soluble particle forms, subsequently lost during the centrifugation step. Production of a refined hemicellulose powder would require an extra step to separate the large molecules (proteins and lignins) and xylans. However, the purity required for the final powder depends on its future application. For biofuel production, requirements are as yet undefined, but for material applications (data not shown) starch, protein and lignin bring properties that could be interesting, with no further purification required.

Fig. 1 .

 1 Fig. 1. Schematic representation of the processes. C-centrifugation; UF-ultrafiltration; CHR-chromatography; P-precipitation; FD-freeze drying.

Fig. 2 .

 2 Fig. 2. Twin-screw extruder configurations for the alkaline treatment of plant matter.

Fig. 3 .

 3 Fig. 3. Evolution of the permeate flux during concentration of the extract by a volume reduction factor of 2. All trials were conducted at a tangential flow rate of 0.23 m/s (210 kg/h) at a temperature of 20 • C.

Table 1

 1 Extrusion conditions.

		Conditions	Conditions used for
		used for trial 1	trials 2 and 3
	Bran/NaOH	2	7
	Straw/bran	6.2	2
	Liquid/solid	5.9	9.4
	Screw rotation speed (rpm)	150	150
	Wash water flow (kg/h)	92	92
	Induction belt temperature ( • C)	50	50
	2.2. Extraction process
	2.2.1. Raw material pre-treatments
	Wheat straw was crushed before extrusion with a hammer-mill
	(Electra BC-P) fitted with a 6 mm-screen. However, because it con-
	tains around 20% starch, which could hinder subsequent extraction
	and purification steps, the bran had to be destarched.
	Starch was removed using two different procedures:
	-Three hot water extractions by mixing 15 kg of dry bran with
	150 L of water at 40 • C for 15 min. The liquid/solid separation was
	made either by aspiration of the liquid from the top (trial 1) or
	by bottom filtration (trial 2).
	-Enzymatic hydrolysis using BAN 480L (Novozyme, Denmark), 1 h
	stirring at 60 • C followed by water washing for 15 min at 40 • C by
	top filtration (trial 3).

Table 2

 2 Detailed composition of destarched bran.

	Bran, trial 1	Bran, trial 2	Bran, trial 3

a X/A is the ratio between xylose and arabinose contents, X/G is the ratio between xylose and glucose contents.

Table 3

 3 Comparison between the refined straw fibres and the initial straw.

		Straw	Refined straw fibres 1	Refined straw fibres 2	Refined straw fibres 3
	Dry matter (%)	91.5	34.5	21.5	21.4
	Ash (wt% dry matter)	8.1	3.3	8.3	8.5
	Organic matter (wt% dry matter)	91.9	96.7	91.7	91.5
	Protein (wt% dry matter)	2.1	2.2	3.7	6.2
	Cellulose (wt% dry matter)	39	29.8	37.8	33.8
	Hemicelluloses (wt% dry matter)	32	27.6	24.4	36.1
	Lignin (wt% dry matter)	9	19.8	7.8	6.9

Table 4

 4 Composition of extracts after extrusion and after centrifugation.

		Trial 1			Trial 2			Trial 3
		Crude	Centrifuged	Cake c	Crude	Centrifuged	Cake c	Crude	Centrifuged	Cake c
		extract a	extract b		extract a	extract b		extract a	extract b
	Dry matter (%)	3.1	2.4	9.8	2.6	2.1	14.7	3.2	2.7	9.2
	Ash (wt% dry matter)	34.6	42.9	16.4	20.3	22.9	13.6	20.9	23.8	13.7
	Organic matter (wt% dry matter)	65.4	57.1	83.6	79.7	77.1	86.4	79.1	76.2	86.3
	Protein (wt% dry matter)	8.5	10.3	3.9	15.4	18.7	6.4	22.9	27.5	12.1
	Carbohydrate (wt% dry matter)	22.7	20.0	n.d.	31.2	32.9	n.d.	n.d.	n.d.	n.d.
	X + A recovery yield d (%)	12.9	9.9	n.d.	12.4	9.8	n.d.	n.d	n.d.	n.d.

n.d. not determined.

Table 5

 5 Composition of powders obtained after alcoholic precipitation and freeze-drying of the centrifuged extracts (C + P + FD), of the centrifuged and ultrafiltered extracts (C + UF + P + FD) and of the extracts percolated through the chromatographic resin (C + UF + CH + P + FD). X/A is the ratio between xylose and arabinose contents, X/G is the ratio between xylose and glucose contents.

		C + P + FD			C + UF + P + FD			C + UF + CH + P + FD
		Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3
	Weight (kg of powder/100 kg of extract) a	1.2	1.1	1.1	1.7	2.5	3.6	1.8	1.7	2.3
	Dry matter (%)	94.1	97.2	96.5	95.5	97.4	87.2	95.4	95.6	94.9
	Ash (wt% dry matter)	9.4	3.5	5.9	5.5	3.6	6.3	5.2	4.3	7.8
	Organic matter (wt% dry matter)	90.6	96.5	94.1	94.5	96.4	93.7	94.8	95.7	92.2
	Protein (wt% dry matter)	12.9	18.8	37.0	15.6	18.8	39.3	13.5	17.9	38.2
	Lignin (wt% dry matter)	-	-	-	24.7	18.3	22.6	23.3	11.0	11.9
	Total carbohydrates (wt% dry matter)	48.4	58.4	31.7	46.7	57	26.6	50.3	61.8	29.8
	Arabinose	6.4	4.8	6.3	6.6	4.4	4.9	7.1	5.1	5.7
	Xylose	17.8	10.1	12.5	17.6	9.9	10.4	18.8	11.0	11.5
	Mannose	0.3	0.4	0.2	0.3	0.4	0.0	0.3	0.4	0.2
	Galactose	1.1	0.8	1.0	1.2	0.7	0.9	1.2	0.8	1.0
	Glucose	22.5	42.1	11.7	20.7	41.8	10.4	22.9	44.5	11.4
	X/A b	2.8	2.1	2.0	2.7	2.2	2.1	2.6	2.2	2.0
	X/G b	0.8	0.2	1.1	0.8	0.2	1.0	0.8	0.2	1.0
	Dry matter yield of precipitation step (%)	38.0	38.9	38.6	53.1	61.9	87.3	56.5	57.6	53.4
	a Mass of powder recovered from 100 kg of extract.

b

Table 6

 6 Purification yields for the main fractions obtained during the three trials.

		C + P + FD			C + UF + P + FD			C + UF + CH + P + FD
		Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3
	Dry matter yield (%) a	4.7	6.2	8.2	4.9	5	11.7	4.3	4.6	7.6
	Organic matter yield (%) a	4.6	5.0	8.2	5.0	5.4	11.7	4.4	4.7	7.6
	(X + A) yield (%) a,b	4.5	3.3	5.8	4.3	2.7	6.9	4.4	2.7	5.1
	Protein yield (%) a	14.8	12.5	29.7	19.1	10.5	45	14.3	10	28.8
	(X + A) purity (wt% dry matter) c	24.2	14.9	18.8	24.2	14.3	15.3	25.9	16.1	17.2

Acknowledgment

We gratefully acknowledged the financial support allocated to the VALOWHEAT project by the French National Research Agency (ANR) (ANR-09-CP2D-16).


A X Sengissen

Alexis Giauque

Gabriel Staffelbach

M Porta

W Krebs

P Kaufmann

Thierry Poinsot

Large Eddy Simulation of piloting effects on turbulent swirling flames

Keywords: Combustion instabilities, Partially premixed, Swirled, Large Eddy Simulations

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Introduction

Modern heavy duty gas turbines usually operate in lean premixed regimes to satisfy emissions regulations and can be very sensitive to combustion instabilities [1][2][START_REF] Smith | 21st Joint Propulsion Conference[END_REF]. In most cases, flame stabilisation is provided by swirl injectors. A key zone of the chamber controlling instabilities is the burner outlet section where swirl is very intense and must provide flame stabilisation. In these regions, the natural unstable modes of swirling flows (Precessing Vortex Cores or PVCs [START_REF] Billant | [END_REF][START_REF] Gupta | Swirl flows[END_REF][START_REF] Hall | [END_REF][7][8]) can interact with stabilisation and lift-off phenomena [9][10][11][12] to produce undesired oscillations.

A method to encourage robust stabilisation is to use small pilot flames in these regions, usually by adding pure fuel injection. This leads to increased N OX lev-els and therefore a compromise between stabilisation and pollution levels must be sought. Furthermore, stabilisation is a difficult task because its basic mechanisms in a piloted swirled zone are not well understood. Proof of the importance of fuel injection are observed in active control examples in which a small modulation of flow rate in the fuel lines feeding the pilot flame can be sufficient to alter the stability of the combustor [13][14][15].

Large Eddy Simulation (LES) is becoming a standard tool to study the dynamics of turbulent flames [16][START_REF] Peters | Turbulent combustion[END_REF][START_REF] Poinsot | Theoretical and numerical combustion[END_REF]. The objective of this paper is to use LES (section 2) to compare two different cases of piloting in a gas turbine burner. Either 2 or 6 percent of the total methane used in the burner is injected additionally through pilot fuel lines. The burner is described in section 3. The 6% pilot fuel case leads to a robust and stabilized flame while the 2% case induces a small lift-off zone of the flame where a PVC can develop and lead to flame oscillations (section 4).

Because of the complexity of the burner, no detailed measurements are available. However, observations in the full scale atmospheric test rig confirm LES predictions: the 2% leads to a more unstable flame than the 6% case.

Numerical approach used in Large Eddy Simulations

A fully compressible explicit code is used to solve the multi-species Navier-Stokes equations on hybrid grids [8,[START_REF] Moureau | [END_REF]20]. Subgrid stresses are described by the classical Smagorinsky model [21]. A two-step chemical scheme is fitted for lean regimes on the GRI-Mech V3 reference [20]. The objective of the fit procedure is that the two-step mechanism and the GRI mechanism must produce the same flame speeds and maximum temperatures for laminar premixed onedimensional flames for equivalence ratios ranging between φ = 0.4 and φ = 1.2.

The flame / turbulence interaction is modeled by the Dynamic Thickened Flame (DTF) model [START_REF] Légier | Summer Program 2000[END_REF] which accounts for both mixing and combustion, and is crucial in partially premixed flames.

The explicit Lax-Wendroff numerical scheme uses second-order spatial accuracy and second-order time accuracy. The boundary condition treatment is based on a multi-species extension [START_REF] Moureau | [END_REF] of the NSCBC method [START_REF] Poinsot | [END_REF], for which the acoustic impedance is controlled to minimise the unwanted reflections [24]. The adiabatic walls are handled using a logarithmic lawof-the-wall formulation which is known to perform well with the classical Smagorinsky model [25]. Typical runs are performed on a grid composed of 1.4 million tetrahedra on parallel architectures. Multiple validations of this LES tool are available for nonreacting [26,27] and reacting flows [8,20,28,29].

Target configuration

The test geometry is an axisymmetric combustion chamber (Fig. 1-a), with a 3MW full scale burner inlet (Fig. 1-b). This burner is composed of two coaxial swirlers:

• The premix passage swirler contains 24 vanes.

Methane is injected through 10 small holes on each vane, ensuring efficient mixing and delivering approximately 90% of the total mass flow rate. In the LES, this flow is assumed to be fully premixed.

• The computational domain includes all pilot passage vanes as well as the pilot fuel tubes, but not the premix passage vanes. Appropriate profiles of velocity and species are imposed to mimic the inlet experimental data [20] downstream of the premix passage vanes (Fig. 2).

The two operating points simulated in this study (2% and 6%) only differ by the fuel mass flow rates in the pilot fuel inlets:

• In the pilot passage stream, the fuel delivered by pilot injection leads to a global equivalence ratio of φ = 0.36 (case 6%) and φ = 0.12 (case 2%), of which the latter is outside the flammability limits. However, the very heterogeneous mixture may allow combustion to develop locally in rich pockets or in diffusion flamelets, depending on the mixing efficiency.

• In the premix passage stream, the imposed profiles in both cases correspond to a perfectly premixed flow with an equivalence ratio of φ = 0.53.

Results and discussion

In swirling flows, the general mechanism leading to flame stabilisation is well known [8,20]: a central core of hot gases is maintained along the burner axis by the strong recirculation zone induced by swirl. This section shows how this classical stabilisation mechanism is affected by the pilot flames. In the 6% pilot fuel case, the flame is clearly anchored on the central hub of the pilot passage, and the temperature fluctuations remain small (Fig. 3-b): burnt gases are found along the axis from x = 0 to x = 2R. Flame lift-off appears in the 2% case (Fig. 3-a). The gases between x = 0 and x = 2R are cold. Hot pockets begin to appear after x = 2R but they are very intermittent, as demonstrated by the very large values of the RMS temperature (error bars on Fig. 3-a). A clearer understanding of the differences between the two cases can be gained by plotting isosurfaces of temperature and stoichiometric equivalence ratio (Fig. 4). For the 6% case, the hot zone (T = 2/3 • T ref ) is directly connected to the pilot passage hub (Fig. 4-b). For the 2% case, the flame is stabilised on a 'finger' of burnt gases which is rotating around the x-axis (Fig. 4-a), thereby inducing the a) Cut at : large RMS fluctuations of temperature seen in Fig. 3a.

0 R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 1
T/Tref b) 0 R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 1
The axial velocity fields (Fig. 5) also present significant differences. For the 6% case, a very large zone with small velocities (mean as well as RMS) develops between x = 0 and x = 3R (Fig. 5-b). This zone contains the hot gases (Fig. 3-b) which provide stabilisation. The 2% case (Fig. 5-a) is characterised by a more intense recirculation (see for example cuts at x = 2R or x = 3R) and a much higher level of RMS velocities. This zone (between x = 0 and x = 3R) contains cold gases (Fig. 3-a) which experience intense fluctuations. Even when the temperature increases (downstream of x = 3R), the velocity RMS values (Fig. 5-a) remain much higher for the 2% than for the 6% case, confirming that the 2% flame is not only lifted but also more hydrodynamically unstable.

Instantaneous combustion regimes can be visualised by scatter plots of reaction rate versus local mixture fraction Z (Fig. 6). In both cases, most reacting points are located very close to the global mixture fraction of the combustor Zmean, but in the 6% case, combustion also takes place at richer regimes, even slightly above stoichiometric (Z st ), yielding higher maximum heat release. These points correspond to the roughly stoichiometric mixture issuing from the four pilot fuel jets after it has mixed with the premix Typical instantaneous fields of equivalence ratio are displayed on Fig. 7. While the 6% case remains roughly axisymmetric and stoichiometric near the pilot passage hub, the 2% case in this zone has an asymmetric pattern below the flammability limit (φ < 0.4), which rotates around the x-axis.

The near stoichiometric zone of Fig. 7-b for the 6% case is the source of the robust stabilisation of this regime: this allows the flame to propagate back to the burner and anchor to the hub. On the other hand, for the 2% case (Fig. 7-a), mixing between the pilot fuel and the pilot passage air is too fast and leads to a mixture at the pilot passage mouth which is too lean for flame propagation. Figure 7 also shows a zone within which the flow is reversed. This central recirculation zone is delimited by the white isoline that the 6% case exhibits a smaller zone of reversed flow (as expected from the mean velocity profiles of Fig. 5) than the 2% case. Obviously, having reversed flow is not a sufficient criterion for stabilisation: having robust burning pilot flames is more important (as for the 6% case). For the 2% case, the absence of combustion in this zone leads to a lean cold region in which even reversed flow can not anchor the flame.

U = 0. Note a) 0 R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 -6 -4
U/Uref b) 0 R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 3 2 1 0 -1 -6 -4
The existence of such a lean and cold zone leads to the formation of a PVC [START_REF] Billant | [END_REF][START_REF] Gupta | Swirl flows[END_REF][START_REF] Hall | [END_REF][7][8]. This PVC only occurs in the 2% case and precesses at 408Hz. The drastic change of velocity field near the pilot passage mouth for this case presented on Fig. 5 is one of the factors which most probably facilitate its development.

A specific feature of the 2% case is the correlation between the lean jet of methane and cold air issuing from the pilot passage and the low pressure zone due to the PVC structure. Figure 8 displays fields of pressure, temperature and local equivalence ratio (reconstructed through the mixture fraction) for both pilot fuel cases in a transverse plane at x = 3R.

The low pressure regions are a good indicator of the PVC presence (Fig. 8-a), and are well correlated with the cold (Fig. 8-b) and lean (Fig. 8-c) regions for the 2% case. The PVC appears to capture some of the lean cold gases produced by the pilot passage and prevents their mixing with the surrounding products. This observation is consistent with detailed mixing studies of jet / vortex interaction which show that mixing can be strongly decreased within vortex structures [30].

The flame then features a cold non-reacting "finger-like" rotating structure protruding within the stabilisation zone (illustrated by Fig. 4-a and sketched on Fig. 9). This is clearly not favorable either for flame stabilisation nor for thermoacoustic stability: RMS pressure levels for the 2% case can be as high as 6000 Pa (170 dB) on the axis and the noise is radiated to 2000 Pa (160 dB) at the wall while they do not exceed 500 Pa for the 6% case. For the 6% case, the situation is very different: a PVC is not observed (Fig. 8-a'), less cold gas reaches the plane at x = 3R (Fig. 8-b') and lean gases are not found around the axis (Fig. 8-c').

The mechanism leading to the PVC formation in the 2% case is purely due to hydrodynamic and combustion effects but not to acoustic coupling. A basic proof of the absence of acoustic coupling can be assessed by comparing the acoustic eigenfrequencies of the combustion chamber with the precessing frequency of the PVC (408 Hz). The fundamental transverse eigenmode is 575 Hz, and the non reflecting inlet/outlet treatment [24] is built to damp all the longitudinal modes.

Conclusions

This study presents Large Eddy Simulations (LES) of piloting effects in a full-scale gas turbine burner. By computing explicitely all details of the pilot passage zone where pure pilot methane is injected upstream of the vanes, LES provides new insights on the key mechanisms that control flame stability. When enough methane is injected in the pilot zone (in the 6% case), a roughly stoichiometric zone is formed at the burner mouth, allowing flame propagation within this zone and preventing the formation of a Precessing Vortex Core (PVC). On the other hand, when the flow rate of pilot fuel is too small (in the 2% case), the mixture issuing from the pilot passage is too lean, preventing flame stabilisation and leading to the formation of a PVC containing lean cold gases which diminishes the effect of piloting even more. Obviously, between the 2% and 6% piloting cases, a bifurcation takes place in the basic flow structure. The significant effects of this bifurcation captured by LES coincide with observations on stability limits in the full scale test rig.

Fig. 1 :

 1 Fig. 1: a) Global view of the test geometry and b) zoom on the burner (pilot and premix passages) inner parts.

Fig. 2 :

 2 Fig. 2: Cross section of the test geometry and location of the planes used in Fig. 3, 5 and 8.

Fig. 3 :

 3 Fig. 3: Profiles of mean temperature (thick line), RMS of temperature (error bars) and T = T ref reference line (dashed line) for a) 2% case and b) 6% case.

Fig. 4 :

 4 Fig. 4: Instantaneous isosurface of temperature (T = 2/3 • T ref , colored by axial velocity) and isosurface of equivalence ratio (φ = 1, colored in blue) for a) 2% case and b) 6% case. color figure in print

Fig. 5 :Fig. 6 :

 56 Fig. 5: Profiles of mean velocity (thick line), RMS of velocity (error bars) and U = 0 reference line (dashed line) for a) 2% case and b) 6% case.

Fig. 7 :

 7 Fig. 7: Instantaneous field of equivalence ratio on central plane for a) 2% case and b) 6% case. Flammability zone (φ = 0.4): black line. Recirculation zone (U = 0): white line.

Fig. 8 :Fig. 9 :

 89 Fig. 8: Instantaneous fields of a) pressure, b) dimensionless temperature and c) equivalence ratio on transverse plane (see Fig. 2) for 2% case (left) and 6% case (right).

Acknowledgments

This work was carried out with the support of Siemens PG in the framework of EC project DESIRE.

Most numerical simulations have been conducted on the computers of CINES, the French national computing center, on a SGI origin 3800.