Biochemical Engineering Journal 25 (2005) 25–31

Xylitol production from sugarcane bagasse hydrolysateMetabolic behaviour ofCandida guilliermondiicells entrapped

in Ca-alginate

W. Carvalhoa, J.C. Santosa, L. Canilhaa, S.S. Silvaa, P. Peregob, A. Convertib,∗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 7 February 2004; received in revised form 13 February 2005; accepted 25 March 2005

Abstract

Sugarcane bagasse hydrolysate was used for batch xylitol production in stirred tank reactor withCandida guilliermondiicells entrappedin Ca-alginate beads. Experiments were carried out using five-fold concentrated hydrolysate, agitation speed of 300 rpm, air flowrate of1 ,ri at xylose wass le and cellg intenance.T them©

K

1

ocfwbt

‘Nnhd

to

cellct tove

luesostEi-litol

uc-tion,tion

esti-se hy-

1d

.3 l min−1, initial cell concentration of 1.4 gDM l−1, and starting pH 6.0. Xylitol production reached 47.5 g l−1 within 120 h of fermentationesulting in a bioconversion yield of 0.81 g g−1 and a productivity of 0.40 g l−1 h−1. The metabolic behaviour ofC. guilliermondiiwas thennvestigated through material balances using the concentrations of consumed substrates and formed products and assuming thimultaneously assimilated by the yeast for anaerobic and semi-aerobic xylitol productions, complete oxidation by the TCA cycrowth. Data collected at different times were finally used to estimate the overall ATP requirements for biomass growth and mahe energy expenditure increased from 2.1 to 6.6 molATP C mol−1

DM throughout the fermentation, highlighting a progressive difficulty oficrobial system in facing the energy needs of its semi-aerobic metabolism.2005 Elsevier B.V. All rights reserved.

eywords:Xylitol; Immobilised cells; Aeration; Bioconversion; Kinetics; Material and bioenergetic balances

. Introduction

Lignocellulosics are an abundant and inexpensive sourcef carbohydrates that can be used to produce high-valuehemicals[1]. Xylitol is an alternative sweetener used inood, odontological, and pharmaceutical preparations[2–6],hose present chemical production is very expensive[7]. Theiotechnological process could lower the production costs by

he direct use of hydrolysates under moderate temperature

Abbreviations:Ac, acetate; ATP, adenosine triphosphate; EtOH, ethanol;H2’, reducing equivalents; NAD+, nicotinamide adenin dinucleotide;ADP+, nicotinamide adenin dinucleotide phosphate; NADPH, reducedicotinamide adenin dinucleotide phosphate; SBHH, sugarcane bagasseemicellulose hydrolysate; TCA, tricarboxylic acids; XDH, xylitol dehy-rogenase; XR, xylose reductase∗ Corresponding author. Tel.: +39 010 353 2593; fax: +39 010 353 2586.E-mail address:converti@unige.it (A. Converti).

and pressure conditions[8], and would allow the productbe classified as natural.

Immobilised cell reactors allow the use of increasedconcentrations, resulting in better productivity with respesuspended cell reactors[9]. However, very few studies habeen published on their use for xylitol production[10–14],and cell concentrations attained did not reach high valikely due to growth limitations. Oxygen supply is the mimportant parameter influencing xylitol bioproduction.ther excess or lack of this nutrient can in fact hinder xyformation in pentose-fermenting yeasts[8,15–18]. Thus, theperformance of immobilised cell systems for xylitol prodtion is expected to depend on a careful control of oxygenawhich is influenced by the aeration rate, the immobilisacarrier, and the bioreactor[13,14].

In this study, the production of xylitol byCandida guil-liermondii cells entrapped in Ca-alginate beads was invgated, on concentrated sugarcane bagasse hemicellulo

369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2005.03.006

26 W. Carvalho et al. / Biochemical Engineering Journal 25 (2005) 25–31

Nomenclature

ai,j stoichiometric coefficients of semi-aerobic metabolism ofC. guilliermondii(C molj C mol−1

DM or molj mol−1j′ )

P xylitol concentration (g l−1)P/O moles of ATP produced per 0.5 molO2 con-

sumed (molATP mol−1O )

q◦ATP specific rate of ATP generation linked

to the electron transport system(molATP C mol−1

DM h−1)q

f,GluATP specific rate of ATP generated by glucose fer-

mentation to ethanol (molATP C mol−1DM h−1)

qr,PenATP specific rate of ATP generation linked to

pentose consumption by the TCA cycle(molATP C mol−1

DM h−1)qi,y specific variation rates (C moly C mol−1

DM h−1

or moly mol−1y′ h−1)

qO2 specific oxygen uptake rate(molO2 C mol−1

DM h−1)QP xylitol productivity (g l−1 h−1)S xylose concentration (g l−1)XR total cell concentration in the reactor (gDM l−1)XRI immobilised cell concentration in the reactor

(gDM l−1)XRM free cell concentration in the reactor (gDM l−1)YP/S xylitol yield based on xylose consumption

(gP gS−1)(1/YATP) +mATP/µ overall ATP requirements

for biomass growth and maintenance(molATP C mol−1

DM)

SubscriptsDM dry matterF final valuei generic metabolic activityj, j′, y, y′ generic metabolitesO oxygen0 starting value

drolysate (SBHH), in a stirred tank reactor (STR). The batchculture was selected not only because of its simplicity but alsowith a view for future continuous application. For this pur-pose, the system was tested under optimum oxygen supplyconditions, previously selected using screening design and re-sponse surface methodology[19]. Fermentations were evalu-ated in terms of substrates consumption, products formation,and growth of free and immobilised cells. The average dataresulting from duplicate experiments were then used to per-form a kinetic study and material balance of xylose simulta-neously consumed by the yeast through assumed metabolicroutes. Bioenergetic balances[20] were employed to estimate

the specific rates of ATP generated by each metabolic activityand the ATP requirements for biomass growth and mainte-nance. A similar approach has recently been applied to thestudy of batch xylitol production from SBHH byC. guil-liermondii cells immobilised on porous glass spheres[14]as well as from corncob hydrolysate by suspended cells ofDebaryomyces hansenii[21].

2. Materials and methods

2.1. Preparation and treatment of the sugarcanebagasse hemicellulose hydrolysate

Sugarcane bagasse was hydrolysed with sulphuric acid ina 250-l reactor as previously described[1]. The hydrolysatewas then concentrated five-fold at 70◦C under vacuum. To re-duce the amounts of inhibitors, the concentrated hydrolysatewas detoxified, as proposed by Alves et al.[22], by raisingthe pH to 7.0 with calcium oxide and decreasing it to 5.5 withphosphoric acid. Active charcoal (2.5%) was added to the hy-drolysate, which was then mixed at 200 rpm and 30◦C for 1 h.The precipitates resulting from pH adjustment and addition ofactive charcoal were removed by vacuum filtration after eachone of these procedures. After this treatment, the concentra-tions of the main inhibitors in the hydrolysate were 3.9 g l−1

af

2i

al.[l sksc( andn a lo-c lswc dr ppedi sus-p lgineS previ-o nsor pro-dr alticp rides

2

s l-

cetic acid, 0.01 g l−1 furfural, 0.01 g l−1 hydroxymethylfur-ural, and 1.8 g l−1 lignin (acid soluble Klason lignin).

.2. Microorganism, inoculum cultivation, and cellmmobilisation

C. guilliermondiiFTI 20037, described by Barbosa et23], was maintained on agar malt extract slants at 4◦C. Aoopful of cells was transferred into 125-ml Erlenmeyer flaontaining 50 ml of a medium consisting of (g l−1): xylose30), ammonium sulphate (3.0), calcium chloride (0.1),atural rice bran extract (20). Rice bran was supplied byal rice-processing mill (Canas, Sao Paulo, Brasil). The celere incubated for 24 h in a rotatory shaker (200 rpm, 30◦C),ollected by centrifugation (2000×g, 15 min), washed anesuspended in sterile distilled water, and then entran Ca-alginate beads. An adequate volume of the cellension was added to a solution of Na-alginate (Satia1100, Degussa Texturant Systems, Baupte, France)usly heated to 111◦C for 15 min. The final concentratiof Na-alginate and cells were 20 and 6 g l−1 (dry-weight),espectively. Cell–gel beads (2.75 mm in diameter) wereuced by dripping this mixture into a 11 g l−1 calcium chlo-ide solution, using a 19-G needle (1.5 in.) and a peristump. The beads were maintained in the calcium chloolution for 24 h and washed with sterile distilled water.

.3. Medium and fermentation conditions

The treated hydrolysate was heated to 111◦C for 15 min,upplemented with (g l−1) ammonium sulphate (3.0), ca

W. Carvalho et al. / Biochemical Engineering Journal 25 (2005) 25–31 27

cium chloride (0.1), and rice bran extract (20). The pH wasadjusted to 6.0 with NaOH 2.0 M to be used as the fermen-tation medium. Next, 1.3 l of the medium was introducedinto a 2.4-l stirred tank reactor[13] containing 200 g of Ca-alginate beads with the immobilised cells. The concentrationof immobilised cells in the reactor at the beginning of fer-mentation was 1.4 g l−1. The reactor was equipped with sen-sors for the measurements of dissolved oxygen concentration,pH, and temperature. Sparging the reactor with air streamsat a flowrate of 1.3 l min−1 and using an agitation speed of300 rpm ensured the desired level of semi-aerobiosis[19].The reactor was operated at 30◦C for 144 h.

2.4. Analytical methods

Glucose, xylose, arabinose, xylitol, ethanol, arabitol, andacetic acid concentrations were determined by high perfor-mance liquid chromatography[14].

Free and immobilised cell concentrations were determinedby absorbance at 600 nm and correlated with the cell dry-weight through a calibration curve. Aliquots of the cultiva-tion broth taken during the runs were centrifuged (2000×g,15 min), and the cells were resuspended in water for the deter-mination of the free cell concentration. Known masses (ac-curacy within 0.01 g) of Ca-alginate beads from the sames olvedi era on oft den-s ther

s oft ium

(XM), immobilised cells in the Ca-alginate beads (XI ), andtotal cells in the reactor (XR) [13].

2.5. Kinetic parameters

The experimental data of initial and final concentrationsof products and substrates in a given period of time wereused to calculate their respective average volumetric vari-ation rates. These were then divided by the average cellconcentration in the same period to give the correspondingspecific rates, which were finally expressed as g g−1

DM h−1 orC mol C mol−1

DM h−1, according to circumstances.Xylitol yield (YP/S) was defined as the ratio of final xyli-

tol concentration (PF) to xylose consumption during the run(S0 −SF), while xylitol productivity (QP) was calculated asthe ratio ofPF to the fermentation time. Variations of theconcentrations of the same compounds within a given timeinterval were used for the metabolic study.

2.6. Material and bioenergetic balances

Material balances have been previously described forC.guilliermondiicells immobilised on porous glass and grownon SBHH [14]. These balances were based on the aver-age yeast composition (CH1.79O0.50N0.20) reported by Roels[m theb mingyN Dd on-st ouslyd

TM ads an

1

2+a2,10‘

3

4

5

6H1.79O

7

activityeducer

amples were dried with absorbent paper and then dissn a potassium citrate solution (20 g l−1) under agitation. Thesulting suspensions were centrifuged (2000×g, 15 min),nd the cells resuspended in water for the determinati

he immobilised cell concentration. Ca-alginate beadsity (1.0 g ml−1) was assumed to keep constant duringuns.

Cell growth determination was based on the variationhe concentrations of free cells in the fermentation med

able 1etabolic model forC. guilliermondiicells entrapped in Ca-alginate be

. Glucose fermentation (f, Glu)a

a1,2CH2O→a1,7CH3O0.5 +a1,8CO2 +a1,9ATP

. Anaerobic pentose-to-pentitol conversion (an, Pen)b

a2,1CH2O +a2,5H2O→a2,6CH3O0.5 +a2,7CH3O0.5 +a2,8CO2 +a2,9ATP

. Semi-aerobic pentose-to-pentitol conversion (sa, Pen)a3,1CH2O +a3,5H2O→a3,6CH3O0.5 +a3,8CO2 +a3,9ATP +a3,10‘H2’

. Aerobic pentose oxidation by the TCA cycle (r, Pen)a4,1CH2O +a4,5H2O→a4,8CO2 +a4,9ATP +a4,10‘H2’

. Aerobic acetate oxidation by the TCA cycle (r, Ac)a5,3CH2O +a5,5H2O→a5,8CO2 +a5,10‘H2’

. Biomass growth on pentoses (b, Pen)a6,1CH2O +a6,4NH3 +a6,5H2O→a6,8CO2 +a6,9ATP +a6,10‘H2’ + a6,11C

. Reducing power regeneration by the electron transport system (o)a7,10‘H2’ + a7,12O2 +a7,5H2O→a7,9ATP

a Subscripts in stoichiometric coefficientsai,j refer to (i) each metabolicb ‘H2’ stands for reducing equivalents in the form of any metabolic r

24], the models proposed by Barbosa et al.[23] for the fer-entation of xylose by different metabolic pathways, andasic knowledge about the metabolism of pentose-consueasts. In agreement with previous studies[25–27], onlyADPH-dependent xylose reductase (XR) and NA+-ependent xylitol dehydrogenase (XDH) activities were cidered forC. guilliermondiiFTI 20037.Table 1illustrateshe metabolic activities assumed to take place simultaneuring the semi-aerobic metabolism of this yeast.

d cultivated on SBHH

H2’

0.50N0.20

or to (j) each metabolite involved..

28 W. Carvalho et al. / Biochemical Engineering Journal 25 (2005) 25–31

The main bioenergetic parameters as well as the stoichio-metric coefficients ofTable 1were estimated by the exper-imental data of the specific rates of variations of the con-centrations of the main substrates (xylose, glucose, arabi-nose, and acetic acid) and products (xylitol, ethanol, ara-bitol, biomass, and carbon dioxide), as previously described[14].

3. Results

After concentration and detoxification, sugarcane bagassehydrolysate was used for xylitol production byC. guillier-mondiicells entrapped in Ca-alginate.Fig. 1depicts the pro-files of sugars and acetic acid consumptions as well as ofxylitol production during the bioconversion. The best fer-mentation parameters were achieved after 120 h of fermen-tation: xylitol production, xylose-to-xylitol bioconversionyield, and xylitol productivity reached 47.5 g l−1, 0.81 g g−1,and 0.40 g l−1 h−1, respectively. Xylitol yield then decreaseddown to 0.73 g g−1, owing to likely product consumption bythe yeast cells after complete xylose depletion. Such a be-haviour at low xylose levels, already observed by other au-thors[17], suggests that the fermentation should be stoppedbefore the complete consumption of the main substrate. Withr s ex-h anda ieldsd

dt tion.A ells,s stantt cellsa run,l lk,w

F d( e-t

Fig. 2. Profiles of immobilised cell (XRI) (�), free cell (XRM) (�), andtotal cell (XR) (�) concentrations in the reactor during the xylose-to-xylitolbioconversion.

In spite of this, the bioconversion yield achieved in thepresent study (0.81 g g−1) was only little lower than the max-imum theoretical value (0.917 g g−1) proposed forC.guillier-mondiiFTI 20037[23]. Even higher yields could be expectedfrom a repeated-batch culture, because of the progressive celladaptation to the toxic compounds present in the hydrolysate[28]. On the other hand, xylitol productivity of the batch cul-ture (0.40 g l−1 h−1) was lower than that reported for freeC.guilliermondiicells[8,28–31]cultivated according to differ-ent modes of operation, probably due to mass transfer limi-tations within the gel matrix[13].

4. Discussion and conclusions

Application of material and energy balances to the resultsof previous tests, performed on more dilute hydrolysate, andusingC. guilliermondii cells immobilised on porous glassbeads, demonstrated that a rigorous regulation of the aerationrate is crucial to achieve satisfactory xylitol yields[14]. Themetabolism of this yeast was shown to head, under optimumsemi-aerobic conditions, for xylitol production, whereas fur-ther increase in the aeration rate caused both the fractionof immobilised cells and the xylitol yield to remarkably de-crease.

rgeticb angeso ea g top ceo ef-fi ing aP oi-c e endoc lite’s

egard to the other carbon sources, while glucose waausted within the first 12 h of fermentation, arabinosecetic acid were uptaken slowly and their consumption yid not exceed 60% at the end of the run.

As can be seen inFig. 2, the immobilised cells showehe highest growth rate at the beginning of the fermentafter 12 h, the Ca-alginate beads were apparently full of cince their concentration in the beads was almost conhroughout the rest of the experiment. The suspendedccounted for almost 60% of total cells at the end of the

ikely due to the absence of diffusion limitations in the buhich highlights their abundant growth as well.

ig. 1. Profiles of glucose (�), xylose (�), arabinose (©), and acetic aci♦) consumptions, as well as of xylitol (�) production, during the xyloso-xylitol bioconversion.

The same approach based on material and bioenealances is used here to investigate the metabolic chccurring inC. guilliermondii immobilised in Ca-alginatnd cultivated under semi-aerobic conditions. Accordinrevious results[14] and supposing a negligible influenf the immobilisation on the oxidative phosphorylationciency, bioenergetic balances were performed assum/Oratio of 1.37 molATP C mol−1

O . The average values of sthiometric coefficients estimated by these balances at thf fermentation are listed inTable 2, whileTable 3shows theorresponding specific rates of variation of each metabo

W. Carvalho et al. / Biochemical Engineering Journal 25 (2005) 25–31 29

Table 2Stoichiometric coefficients,ai,j (C-molj C mol−1

DM or molj mol−1j′ ), of the material balances used to study the metabolism of entrapped cells ofC. guilliermondii

cultivated on SBHH

ja Metabolites ib

1 2 3 4 5 6 7f, Glu an, Pen sa, Pen r, Pen r, Ac b, Pen o

1 Pentoses 0 1.000 1.000 1.000 0 1.095 02 Glucose 1.000 0 0 0 0 0 03 Ac 0 0 0 0 1.000 0 04 NH3 0 0 0 0 0 0.200 05 H2O 0 0.100 0.100 1.000 1.000 −0.405 −4.1556 Pentitols 0 0.800 0.900 0 0 0 07 EtOH 0.667 0.067 0 0 0 0 08 CO2 0.333 0.133 0.100 1.000 1.000 0.095 09 ATP 0.333 0.027 −0.020 0.550 0 −6.588c 5.700d

10 ‘H2’ 0 0.400 0.020 2.000 2.000 0.095 4.15511 Biomass 0 0 0 0 0 1.000 012 O2 0 0 0 0 0 0 2.077

a Metabolites’ numbering is the same as inTable 1.b For metabolic activity numbering and symbols, seeTable 1.c a6,9 =−[(1/YATP) +mATP/µ].d a7,9 = 2a7,12P/O = (4.155 molO mol−1

DM) × (1.37 molATP mol−1O ).

Table 3Specific rates,qi,y (C mmoly C mol−1

DM h−1 or mmoly mol−1y′ h−1), of variations of metabolites’ (y) concentrations in the different activities (i) constituting the

metabolism ofC. guilliermondiicells cultivated on SBHH

ya Metabolites ib

1 2 3 4 5 6 7f, Glu an, Pen sa, Pen r, Pen r, Ac b, Pen o

1 Pentoses 0 15.4 66.6 14.3 0 9.82 06 Pentitols 0 12.3 59.9 0 0 0 07 EtOH 4.61 1.02 0 0 0 0 09 ATP 2.31 0.410 −1.33 7.87 0 −59.1 49.8

10 ‘H2’ 0 0.614 1.33 28.6 4.90 0.852 −36.3a Metabolites’ numbering is the same as inTable 1.b For metabolic activity numbering and symbols, seeTable 1.

concentration. Percentage errors between theoretical and ex-perimental carbon material balances varied from−4.3 to3.6%, whereas those referring to the reduction degree bal-ances were negligible, thereby demonstrating the validity ofthe aforementioned metabolic assumptions.

Fig. 3shows that about 15% of xylose was consumed forthe anaerobic production of xylitol during the run. Such ametabolic pathway was mainly active during the first 12 h,which suggests that the fast growth rate of immobilised cellsobserved during this period (Fig. 2) could have led to quickoxygen consumption and formation of anaerobic zones insidethe Ca-alginate beads. The occurrence of anaerobic condi-tions inside the support, favouring the anaerobic productionsof xylitol, arabitol, and ethanol, would be consistent with theresults ofFig. 4that depicts the behaviours of the specific ratesof ATP production associated with each one of the assumedmetabolic pathways. The highest values of these parametersobserved at the start of the run would be due not only to typi-cal aerobic activities, such as the reducing power regenerationby the electron transport system (q◦

ATP) and pentose oxidationby the TCA cycle (qr,Pen

ATP ), but even to the anaerobic fermen-

Fig. 3. Influence of the fermentation time on the fractions of xylose pro-gressively consumed byC. guilliermondiicells for (�) anaerobic xylose-to-xylitol conversion, ( ) semi-aerobic xylose-to-xylitol conversion, ()biomass growth from xylose, and (�) aerobic xylose oxidation by the TCAcycle.

30 W. Carvalho et al. / Biochemical Engineering Journal 25 (2005) 25–31

Fig. 4. Influence of the fermentation time on the specific rates of ATP gen-eration by the (�) electron transport system, (�) glucose fermentation, (♦)semi-aerobic penitols production, (×) anaerobic pentitols production, and() pentoses consumptions by the TCA cycle.

tation of glucose (qf,GluATP ). This trend is consistent with both

the initial occurrence of anaerobiosis within the support andits substitution by semi-aerobiosis after about 24 h, likely dueto stop of biomass growth within the support. These resultsconfirm the need of oxygen supply optimisation.

Since the cell must consume, under steady-state condi-tions, any excess production of energy in the form of ATPeither for growth or maintenance, the very highqATP valuesestimated during the first fermentation phases were also con-sistent with the rapid cell growth observed during the sameperiod. All the relevant activities in terms of ATP generationwere related to the consumption of substrates other than xy-lose (Fig. 4), thereby stressing their fundamental energeticrole already highlighted for xylitol production from such acomplex medium[12]. As can be seen inFig. 3, under ad-equate aeration conditions, 73% of xylose consumed within108 h of fermentation was in fact utilized for xylitol semi-aerobic production, almost 14% for xylitol anaerobic forma-

F wtha

tion, 9% for biomass growth, and only 4% by the aerobicoxidation through the TCA cycle.

The ATP requirements for growth and maintenance pro-gressively increased throughout the fermentation, whichmeans that either activity became more and more expensivefor the cell (Fig. 5). At the start of the run, this parame-ter reached a value (2.1 molATP C mol−1

DM) that is comparableto those of micro-aerobic fermentations in complex media(2.0–2.6 molATP C mol−1

DM) [21] and is appreciably lower thanthose observed forC. guilliermondiiimmobilised on porousglass (3.4 molATP C mol−1

DM) [14]. On the other hand, at theend of fermentation, these energy requirements increased upto 6.6 molATP C mol−1

DM, practically reaching those of typicalanaerobic fermentation systems (5.5–7.0 molATP C mol−1

DM)[20].

Acknowledgements

The authors gratefully acknowledge the financial supportof FAPESP and CNPq (Brazil) and thank Ms. M.E.M. Coelhofor revising this manuscript.

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te

n of

helling563.with

[ m4)

[

[ av--chnol.

[ ro-rent

ig. 5. Time behaviour of the yield of ATP consumption for biomass grond maintenance.

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