Biochemical Engineering Journal 23 (2005) 1–9

Evaluation of porous glass and zeolite as cells carriers for xylitolproduction from sugarcane bagasse hydrolysate

Julio C. Santosa, Solange I. Mussattoa,∗, Giuliano Dragonea, Atılio Convertib, Sılvio S. Silvaa

a Department of Biotechnology, Faculty of Chemical Engineering of Lorena, Rodovia Itajub´a-Lorena, km 74.5, P.O. Box 116, 12600-970 Lorena, SP, Brazilb Department of Chemical and Process Engineering, University of Genova, via Opera Pia 15, I-16145 Genoa, Italy

Received 14 March 2004; received in revised form 21 September 2004; accepted 6 October 2004

Abstract

Adsorbing carriers for immobilisation ofCandida guilliermondiicells to use for xylitol production from sugarcane bagasse hemicellulosehydrolysate were tested. Biomass was immobilised in situ by natural adsorption, i.e. through direct contact between cells for the inoculumand carrier particles at the beginning of fermentations. The carriers employed were: Syrane porous glass beads with 2.53 mm diameter inaverage and pore diameter in the range 60–300�m, supplied by Bioengineering (Wald, Switzerland), and NaX zeolite UOP WE 894 purchasedf tion (a lc ibitedl n top us glassa material.A responsiblef cell size,i e low cella©

K

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oacsdh[o

otheructearchtilisa-secord-r

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rom Plury Quımica S.A. (Diadema, SP, Brazil). At the end of the run with free cells taken as a reference test, xylitol concentraPf )chieved 35.5 g/l, corresponding to a xylose-to-xylitol yield factor (YP/S) of 0.72 g/g and a volumetric productivity (QP) of 0.49 g/l h, while finaell concentration (Xf ) and productivity (Qx) were only 5.32 g/l and 0.048 g/l h, respectively. Both systems with immobilised cells exhower xylitol productions (Pf = 28.8–29.5 g/l,YP/S= 0.52–0.53 g/g,QP = 0.32–0.33 g/l h) and higher cell growth, with particular concerorous glass (Xf = 10.5 g/l,Qx = 0.10 g/l h). Electronic microscopy observations demonstrated that the excellent performance of poros cell support was due to the development of a thick extracellular matrix either within the large pores or on the surface of thiss a consequence, almost 50% of the cells resulted to be adsorbed to the carrier at the end of the run. This growth was also

or a decrease in the fraction of xylose available for xylitol production. Employing zeolite, a material with pore size smaller thanmmobilised cells represented only 30% of the final population and immobilisation was just observed on the carrier surface. Thttachment on this material can be explained by the stress exerted on the outer immobilised cells by the friction among beads.2004 Elsevier B.V. All rights reserved.

eywords:Porous glass; Zeolites; Immobilised cells; Hemicellulose hydrolysate; Fermentation; Xylitol

. Introduction

Lignocellulosic materials represent an abundant source ofrganic compounds, also presenting a large potential for uses raw materials in industrial processes to produce food, fuel,hemicals, enzymes and others[1]. Among these materials,ugarcane bagasse, the major by-product of the sugarcane in-ustry, obtained after crushing the cane to extract the broth,as a world production of about 234 million tonnes per year

2]. Most of bagasse is employed as energetic source in itswn industry, substituting diesel in the heating process of

∗ Corresponding author. Tel.: +55 12 3159 5027; fax: +55 12 3153 3165.E-mail address:solange@debiq.faenquil.br (S.I. Mussatto).

boiler, and to generate electrical energy. Although somecommercial applications of this agro-industrial by-prodhave been evaluated, surpluses still exist and a lot of reswork has been addressed towards alternatives for its ution. Among these, xylitol production from the hemicellulofraction of sugarcane bagasse merits to be stressed. Acing to Pandey et al.[3], this raw material has no utility fosteam and power generation; however, when hydrolyscan provide a useful substrate for microbial cultivationwell as xylitol production.

Xylitol is a naturally occurring five-carbon polyalcohthat can be employed in food, odontological and phaceutical industries[4,5]. The large number of advantageoproperties, such as its sweetening power and anticariog

369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2004.10.001

2 J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9

ity [6,7] justifies the large industrial interest for xylitol. Thispolyalcohol is actually produced from wood hydrolysate bya chemical process that presents some disadvantages, amongwhich the use of high pressure and temperature as well asthe necessity of purifying xylose present in the hydrolysate[8]. These drawbacks encouraged many researchers to lookfor alternative ways to produce xylitol. Nowadays, its mi-crobiological production is very attractive because of severaladvantages, such as the use of mild conditions of pressure andtemperature[9]. In addition, since microorganisms, mainlyyeasts, are able to directly convert xylose into xylitol fromthe hydrolysate, there is no need of xylose purification[10].

Studies about the bioproduction of xylitol have been fo-cused to establish the operational parameters and the processoptions that maximize its yield. One of the leading parametersis the aeration rate: under strong aeration, xylose fermentationis in fact directed towards cell production, whereas yeasts un-der strict anaerobic conditions cannot assimilate xylose. Forthis reason, a suitable control of oxygen input is necessaryfor effective xylitol production[11,12]. Another way to betterthe performance of this bioprocess is the use of immobilised-cell systems. Several advantages have been reported on theuse of immobilised cells, including a high cell concentrationin the fermentation medium and, consequently, an increasein the process efficiency and productivity[13]. Moreover,i bee tions[

im-m f theit ed-bi fectsa anceo isa-t ttle-n ier isn tativeo

2

2

sinaG n-t ed ina gassa perg1 l-y uga-t eh um in

a 6.0 l evaporator at 70± 5◦C to obtain a xylose concentra-tion of about 55 g/l, then detoxified with activated charcoalas described by Alves et al.[18], and finally autoclaved undermanometric pressure of 0.5 atm for 15 min. After this treat-ment the average composition of the hydrolysate was: 55 g/lxylose, 3 g/l glucose, 3 g/l arabinose and 3 g/l acetic acid.

2.2. Microorganism and inoculum

Cells of the yeastC. guilliermondiiFTI 20037 were main-tained at 4◦C on malt extract agar slants. The inoculum wasprepared by cultivation of the yeast in 125 ml Erlenmeyerflasks containing 50 ml of the medium consisting of 30 g/l xy-lose, 3.0 g/l (NH4)2SO4, 0.10 g/l CaCl2·H2O and 10% (v/v)rice bran extract. Cells were cultivated at 200 rpm and 30◦Cfor 24 h, subsequently recovered by centrifugation (2000×g,20 min), washed and resuspended in sterile distilled water.Suitable volumes of this solution were employed as inocu-lum to obtain an initial cell concentration of 1 g/l (dry mass)at the beginning of fermentations. To obtain the rice branextract, an aqueous suspension of rice bran (200 g/l) was au-toclaved during 15 min under 0.5 atm manometric pressure.After cooled, the liquid fraction was separated by centrifu-gation (2200×g, 20 min) to be used for experiments.

2

car-r eteris car-r )d 4 ha 4,p l),w for4 re ast wasp eves.T lisedt s ort sico-c ed inT

2

hy-dC cu-l lisedc (thism xper-i d-s be-g ere

mmobilised-cell systems make it possible for cells toasily recovered for later use in repeated batch opera

14–16].As is well known, good performance of systems using

obilised cells mainly depends on the right selection ommobilisation carrier. Santos et al.[17], investigating xyli-ol production from hemicellulose hydrolysate in a fluidised reactor usingCandida guilliermondiicells immobilised

n porous glass, have recently proposed that inhibiting efnd/or mass transfer limitations could affect the performf such a biosystem, thus requiring experimental optim

ion. In order to point out the actual causes of those boecks, the effectiveness of this macro-porous cell carrow compared with that of zeolite, selected as represenf simply adsorbing support.

. Materials and methods

.1. Sugarcane bagasse hydrolysate

Batches of sugarcane bagasse kindly provided by Uuarany (Olımpia, SP, Brazil) were employed for ferme

ation tests. The hemicellulose hydrolysate was prepar350 l stainless steel reactor loaded with sugarcane band sulphuric acid solution in the ratio of 100 mg acidram of dry matter. The reactor was operated at 121◦C for0 min, employing a solid/liquid ratio of 1/10. After hydrosis, the resulting solid material was removed by centrifion and the hydrolysate was stored at 4◦C. Subsequently, themicellulose hydrolysate was concentrated under vacu

e

.3. Preparation of carriers for cell immobilisation

Porous glass and zeolite were used as immobilisationiers. “Syrane” porous glass beads, with 2.53 mm diamn average and pore diameter in the range 60–300�m, wereupplied by Bioengineering (Wald, Switzerland). Thisier was previously treated with a HNO3 solution (10%, w/vuring 4 h, washed with and kept in distilled water for 2nd dried at 150◦C. Beads of the NaX zeolite UOP WE 89urchased from Plury Quımica S.A. (Diadema, SP, Braziere treated with a 0.5% HCl solution, under agitationh, and went through the same washing/drying procedu

he other support. The granulometric analysis of carrierserformed by passing them through standard Tyler sihe resulting average size distributions were then uti

o calculate either the number of particles per unit mashe external surface area of the beads. The main phyhemical characteristics of both materials are summarizable 1.

.4. Fermentation conditions

To be used as a fermentation medium, detoxifiedrolysate was supplemented with 3.0 g/l (NH4)2SO4, 0.10 g/laCl2·2H2O and 10% (v/v) rice bran extract and ino

ated as aforementioned. For experiments with immobiells, 5 g of carrier (dry mass) were added to each flaskass/volume ratio has already been used in former e

ments[19]). Cells were immobilised in situ by natural aorption through their direct contact with the carrier at theinning of each run. The fermentation conditions used w

J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9 3

Table 1Physico-chemical characteristics of porous glass and zeolite beads utilisedas cell carriers

Carrier Porous glassbeads

Zeolite beads

Commercial namea Syran UOP WE 894Absolute density (g/ml) 1.03 1.91Bulk density (g/ml)a 0.450 0.700Specific number of particles (g−1)b 115.0 67.8Pore diameter (�m) 60–300a (0.3–2)× 10−3c

Porosity (%) 55–60a 57d

Specific surface area (m2/g)a 0.15–0.20External surface area (m2/g)b 0.00231 0.00128

a Information provided by the supplier.b Values estimated by the granulometric analysis.c Information from Moscou[22].d Value estimated from the absolute density assuming for NaX zeolites a

pore volume of 0.3 ml/g according to Breck[35].

the same employed by Alves et al.[18]: 125 ml Erlenmeyerflasks containing 50 ml of the medium were agitated at 30◦Cin an orbital shaker at 200 rpm. The runs, that lasted until atleast 90% xylose was consumed, were monitored by sam-pling every 12 h to follow cell growth, substrate uptake, andproduct formation.

2.5. Analytical methods

The dry mass of cells immobilised in the carrier was deter-mined by drying the samples at 105◦C till constant mass andsubsequently burning all organic substance at 950◦C up tocalcination. The difference between carrier masses measuredat 950 and 105◦C was assumed to represent the dry mass ofimmobilised cells in the sample and was employed to calcu-late the immobilised-cell concentration. The ash content ofcells, determined by similar procedure, was taken into con-sideration for calculations. Adsorbing materials others thancells were previously quantified in blank experiments with-out cells and taken into account when calculating the massof immobilised cells.

Free cells concentration in the medium was determinedat 600 nm using a Beckman spectrophotometer model DU640B (Fullerton, CA), by means of a dry weight versus opticaldensity (OD) calibration curve.

anolc odelL er-ca pre-v d int mnt eda

ng aL s doB en-t anso ford

Inca Energy (Schott Zeiss do Brasil Ltda, Sao Paulo, SP,Brazil).

2.6. Kinetic parameters and yields of fermentations

The xylitol and cell volumetric productivities (QP andQx,g/l h) and the volumetric xylose consumption rate (Qs)werecalculated as the ratios of the concentrations of xylitol (Pf ,g/l), total biomass (Xf , g/l) or xylose consumption (g/l) atthe end of each run to the fermentation time (h). The xyl-itol yield factor (YP/S, g/g) was defined as the ratio ofPfto xylose consumed at the end of each run (g/l). The effi-ciency of xylose-to-xylitol bioconversion (η, dimensionless)was calculated as the ratio ofYP/S(g/g) to the theoretical value(0.917 g/g) of this parameter proposed by Barbosa et al.[20]for yeasts, likeC. guilliermondiiFTI-20037, provided withxylose reductase using NADPH2

+ solely as the cofactor. Thepercentage of immobilised cells was calculated as the ratio ofthe immobilised cells (g/l) toXf , at the end of fermentations.The xylitol specific productivity (qP) was defined as the ratioof QP toXf .

3. Results and discussion

3

e se-l sedi -g pings sso ont nu-t ani-c terizep riori,p isee

lites( ol-u ctb rriers,w thec onet umb -f als ise icm

medb sg , al-m me-t oft and

Glucose, xylose, arabinose, xylitol, acetic acid and ethoncentrations were measured by Shimadzu HPLC, mC-10-AD (Tokyo, Japan), equipped with a Bio-Rad (Hules, CA) Aminex HPX-87H (300 mm× 7.8 mm) columnnd a refractive index RID 6A detector. Samples wereiously filtered through a Sep Pak C18 filter and injectehe chromatograph under the following conditions: coluemperature of 45◦C, 0.01N H2SO4 as the mobile phase ust a flow rate of 0.6 ml/min, injection volume of 20�l.

Micrographs of carrier surfaces were obtained by usiEO1450VP scanning electron microscope (Schott Zeisrasil Ltda, Sao Paulo, SP, Brazil). The superficial elem

al compositions of both carriers were obtained by mef an energy dispersive X-ray spectroscope EDS Ox

.1. Preliminary tests of carrier characterisation

Zeolite UOP WE 480 and Syrane porous glass werected in this work, among the various materials propon the literature as cell carriers[21], with the aim of investiating the performance of typical adsorbing and entrapupports forC. guilliermondii immobilisation. The succef a support for cell immobilisation does in fact depend

he possible occurrence either of diffusion limitations ofrients or products through the biofilm or outer mechal stress. Since these drawbacks, which often characorous or adsorbing materials, cannot be foreseen a preliminary immobilisation tests are necessary to optimvery immobilised-cell system.

Because of the small pore diameter of zeo(0.3–2)× 10−3 �m)[22] with respect to the average cell vme of yeast cells (70�m3) [23], this material should in fae considered as a good representative of adsorbing cahere biofilm can only develop on their outer surface. Onontrary, materials like porous glass, with pore sizes fromo five times the cell size, were shown to allow for maximiofilm development within their pores[24]. The large dif

erence between of the outer structure of these materivident in the micrographs ofFig. 1, obtained by electronicroscopy before cell adhesion.The granulometric analyses of both carriers, perfor

efore their use in fermentations (Fig. 2) show that poroulass is constituted by spheres with quite uniform sizeost 82% of its total mass exhibiting an average dia

er of 2.58 mm. On the other hand, two 44% portionsotal zeolite mass exhibited average diameters of 2.58

4 J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9

Fig. 1. Micrograph of (A) porous glass and (B) zeolite obtained before cell adhesion by scanning electron microscopy (magnification: 2000-fold).

2.17 mm, which reveals a less uniform particle size distribu-tion.

Comparing the physico-chemical properties of these car-riers summarized inTable 1, one can observe that porous

glass is characterised by a specific number of particlesper unit mass (115 g−1) as well as an external surfacearea (0.00231 m2/g) which are nearly twice those of zeo-lite (67.8 g−1 and 0.00128 m2/g, respectively), as a conse-

J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9 5

Fig. 2. Pore size distribution of ( ) porous glass beads and (�) zeolitebeads.

quence of the lower absolute density of the former mate-rial (1.03 g/ml). These characteristics, together with the highporosity (55–60%) and large pore diameter of porous glass(60–300�m), should make the space available for cell coloni-sation of this porous support quite large. As a matter of fact,previous work performed with this material demonstrated thatpore colonisation was quick and effective and that cells keptintact after adsorption[25].

3.2. Tests of cell adhesion and fermentation

The results listed inTable 2 show that total cell con-centration and productivity were, at the end of fermenta-tion with cells immobilised in porous glass, almost twice(Xf = 10.5 g/l andQx = 0.10 g/l h) those observed in the free-cell system, while the growth was comparable in the systemsusing free cells or cells immobilised in zeolites (Xf = 6.65 g/landQx = 0.063 g/l h). Two causes could have positively influ-enced cell growth in porous glass, specifically the increasedoxygen availability to the cells and the porous structure ofthe carrier that favoured cell adhesion and growth within thepores.

As far as the former factor is concerned, solid beads motionin the medium could have made the interfacial area betweenthe liquid and gaseous phases larger, thereby acceleratingo aseda ismt f xy-

lose consumed by this activity could have affected xylitol pro-duction. Similar deviation ofC. guilliermondiimetabolismfrom xylitol formation towards the growth has been previ-ously reported to take place under conditions of strong aer-ation [11,26]. Santos et al.[17], when cultivating this yeastimmobilised in porous glass, have recently observed, in afluidised-bed reactor operating batchwise, an increase in cellconcentration by around 8.4 g/l when the aeration rate wasincreased from 0.031 to 0.093 vvm.

As can be noted inTable 2, the highest xylitol concen-tration (35.5 g/l) was obtained with freeC. guilliermondiicells. Xylitol yield factor (YP/S) and volumetric productivity(QP) were 0.72 g/g and 0.49 g/l h, respectively, correspond-ing to a xylose-to-xylitol bioconversion efficiency of 78.5%.Employing cells immobilised in both carriers, final xylitolconcentration,YP/S andQP were reduced by almost 26, 26and 32% with respect to the free-cell system, respectively.Besides, a slow ethanol formation occurred during all the ex-periments, with particular concern to the fermentation usingzeolite as a carrier (Table 2). These results agree with pre-vious detection of a low ethanol formation with this yeastduring xylitol production from either semi-synthetic media[20] or sugarcane bagasse hydrolysate[19].

Considering that biomass growth requires large oxygenavailability, whereas xylitol and ethanol formations are typi-c pec-t , cellm o thef ownb ss onse-q hep beads uence,i icals bio-c

ousgg ablyt rfaceo ante iesws ongb ally

TR nded o

ob (%)

FPZ

P l conceη produc ;Q

xygen dissolution into the medium. Because of its increvailability, oxygen could have shifted the cell metabolowards biomass formation and the increased fraction o

able 2esults of batch fermentations with cells ofC. guilliermondiieither suspe

Pf (g/l) EtOH (g/l) Xf (g/l) Imm

ree cells 35.5 3.97 5.32 –orous glass immobilised cells 29.5 4.92 10.5 49.6eolite immobilised cells 28.8 5.18 6.65 28.9

f = final xylitol concentration; EtOH = final ethanol concentration;Xf = fina= efficiency of xylose-to-xylitol bioconversion;QP = xylitol volumetric

S = volumetric xylose consumption rate.

al micro-aerobic and anaerobic metabolic activities, resively, these results indicate that, in the free-cell systemetabolism of the yeast was preferentially addressed t

ormation of the desired polyol. On the other hand, as shy the micrographs illustrated inFig. 3, the highly poroutructure of glass beads favoured cell adhesion and cuent formation of biofilm within the pores, while in tresence of zeolite it scarcely developed only on theurface because of the reduced pore size. As a conseqt likely underwent through cell abrasion due to mechantress and/or leading to reductions of both growth andonversion.

With reference to immobilized yeast growth on porlass, Kosaric and Blaszczyk[27] reported that biofilmrown within the porous structure of glass was remark

hicker than that simply adsorbed onto the smoother suf zeolite. It is also evident the formation of an abundxtracellular polymer matrix in which cells and colonere embedded. According to Lazarova and Manem[28],tructure and composition of this matrix varies amiofilms, although polysaccharides and proteins usu

r immobilised in porous glass and zeolite

YP/S (g/g) η (%) QP (g/l h) qP (g/g h) Qx (g/l h) Qs (g/l h)

0.72 78.5 0.49 0.092 0.048 0.690.53 57.8 0.33 0.031 0.10 0.620.52 56.7 0.32 0.048 0.063 0.61

ntration of total cells; Immob = immobilised cells;YP/S= xylitol yield factor;tivity;qP = xylitol specific productivity;Qx = cell volumetric productivity

6 J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9

Fig. 3. Micrograph of (A) porous glass and (B) zeolite obtained after cell adhesion by scanning electron microscopy (magnification: 2000-fold).

predominate, accounting for up to 65% of this materialand 10–15% of total biofilm, respectively. It has beenproposed that extracellular polysaccharide glycocalyx[29]has no other special properties besides its participation in

the cell attachment process[30]. Such a structure appearsto be absent on the zeolite surface, thereby suggesting afundamental role of a macro-porous structure to promote itsformation and consequent cell adhesion to the carrier.

J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9 7

Fig. 4. Spectra of superficial elemental composition of bead surface performed by energy dispersive X-ray spectroscopy after biomass immobilisation: (A)porous glass and (B) zeolite.

As highlighted by Cohen[13], the electrostatic forces arehighly significant mainly in the initial stages of cell adsorp-tion. Since cell surface is usually negatively charged[31] aswell as those of most bedding materials, such as porous glassand zeolites[32], other stronger attractive forces, like thoseexerted by the above adhesive matrix, were necessary to over-come the repulsive barrier between negatively charged par-ticles. Biofilm development on these supports was also con-firmed by the occurrence of a carbon peak in the elementalcomposition of both carriers after cell immobilisation, witha much stronger effect in the case of porous glass (Fig. 4).

A peculiar feature of immobilised-cell systems is the re-lease of free cells in the broth as a result either of natural cellturnover or their release from the support[33]. As a matterof fact, only 50% of total cells resulted to be entrapped inporous glass and less than 30% adsorbed on zeolite beads atthe end of batch immobilisation tests (Table 2). Figs. 5 and 6show that, in the presence of both carriers, cells initially grewin immobilised form, while free cells from their detachmentreached valuable concentrations in the medium only after the

carriers were saturated with biofilm. These figures also showthat zeolite was saturated by a microbial population aroundthree times less than in porous glass.

Using a fluidised-bed reactor and employing commercialxylose as carbon source byC. guilliermondiiimmobilised inporous glass, Silva et al.[34] ascribed the low performanceof the yeast for xylitol production to the mass transfer lim-itation through the biofilm. As suggested by Lazarova andManem[28], very thick biofilms could be in fact not fullyactive because the diffusion of nutrients could become a lim-iting factor beyond a threshold termed “active thickness”. Inthe present work, the substrate transfer to the yeast immobi-lized on porous glass was probably limited, since there wasa reduction in the volumetric xylose consumption rate whencompared with the free-cell system (Table 2). A carbon mate-rial balance of the data suggests that the low specific produc-tivity and reduced final xylitol concentration using this cellcarrier was due to the increased final biomass production.It represents an additional effect to the mass transfer limi-tations. This explanation would not apply to justify the low

8 J.C. Santos et al. / Biochemical Engineering Journal 23 (2005) 1–9

Fig. 5. Behaviours of total cells ofC. guilliermondiiduring the fermentationof sugarcane bagasse hydrolysate. (�) Free-cell system; system with cellsimmobilised in (©) porous glass beads and (�) zeolite beads.

Fig. 6. Behaviours of free and immobilised cells ofC. guilliermondiiduringthe fermentation of sugarcane bagasse hydrolysate. Free cells (solid line)and cells immobilised (dotted line) in (�) porous glass beads and (�) zeolitebeads.

performance of zeolite as a carrier, since the microenviron-ment of cells simply adsorbed on the carrier surface was notso different from that experienced by free cells in the liquidmedium. In this case immobilised cells were directly exposedto the mechanical stress provoked by friction among beads,which was likely responsible for the low xylitol productionobtained with this carrier as well as the scarce cell growthwhen compared with porous glass.

4. Conclusions

Porous glass used to natural immobilisation ofC. guil-liermondii cells behaved as an effective entrapping carrier,being able to reach a final biomass concentration higher than10 g/l. The formation of an adhesive extracellular matrix ap-peared to be a fundamental requisite for biofilm aggregationmainly within its pores. On the contrary, friction among ze-olite beads appeared to exert a negative effect on biomass,

causing a cell stress and consequently lowering the immo-bilisation performance. The results obtained in this work onthe whole suggest that porous glass beads could be employedfor yeast cell entrapment in a continuous process for xylitolproduction from hemicellulose hydrolysates in fluidised-bedreactor.

Acknowledgements

Authors thank the financial support of Brazilian CNPq,CAPES and FAPESP.

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