Biomussund Bioenrrg~ Vol. 13, Nos I /2.pp. I l-14.

1997
c 1997Published by Elsevier Science Ltd. All rightsreserved
Pergamon
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FERMENTATION OF SUGAR CANE BAGASSE

HEMICELLULOSIC HYDROLYSATE FOR XYLITOL
PRODUCTION: EFFECT OF PH

MARIA G. A. FELIPE*‘, MICHELE ViToLot, ISMAEL M. MANCILHA~ AND SILVA S. SILVA*

*Departamento de Biotecnologia, Faculdade de Engenharia Quimica de Lorena, 12600-000, Lorena,
SBo Paulo, Brazil
TDepartamento de Tecnologia Bioquimico-Farmaceutica, Faculdade de CiCncias Farmaceuticas da
Universidade de SBo Paulo. Sao Paula, Brazil
IDepartamento de Tecnologia de Alimentos. Universidade Federal de Vicosa, Vicosa, Minas Gerais.
Brazil

(Received 5 Noremhrr 1996; raised 3 February 1997; nccepted 25 February 1997)

Abstract-Candida guilliermondii FTI 20037 was grown in sugar cane bagasse hydrolysate supplemented
with (NH&SO, 2.0 g I-‘, CaCI, 0.1 g I-’ and rice bran 20.0 g I-‘, through 45-h batch tests (agitation of
200 min.’ and temperature of 3O’C) with initial pH varying from 2.5 to 7.5. Under pH < 4.5 the
consumption of glucose, xylose and arabinose as well as the production of xylitol and cells were inhibited.
Nevertheless, at pH values >- 5.5 the yeast produced xylitol with a yield of 0.75 g g-’ and productivity
of 0.57 g I-’ h-‘. Moreover, the yeast was also capable of metabolizing the acetic acid, which is always
present in media made from hydrolysates of plant material. The inhibition of xylose/xylitol bioconversion
could be related to the effects of low pH and undissociated acetic acid concentration over 5.0 g I-‘. (~‘1
1997 Published by Elsevier Science Ltd

Keywords-Xylitol; sugar cane bagasse; hemicellulosic hydrosylate; pH; acetic acid; Candida guilliwmondii

1. INTRODUCTION and eucalyptus.7 However, these hydrolysates

contain furfural, hydroxymethylfurfuraP and
Sugar cane bagasse is a residue having a
acetic acid’ as by-products, which are toxic to
hemicellulosic fraction rich in fermentable
the yeast. Acetic acid occurs in high concen-
carbohydrates. As bagasse hydrolysate contains
trations in most hydrolysates. Acetic acid
significant amounts of xylose, it is a suitable
concentrations higher than 3.0 g 1-l inhibits the
culture medium for microbial production of
ability of C. guilliermondii to convert xylose into
xylitol.’ 3 This product is a sweetener compar-
xylitol.’ The non-ionised acetic acid, which is
able to sucrose, but less cariogenic and a useful
found in the medium at pH ~7.0, probably
component of dietetic foods.4 The industrial
acts as an inhibitor of the yeast metabolism.“.”
production of xylitol is based on the chemical
It has been reported that at pH 4.5 the
reduction of pure xylose attained from lignocel-
xylose/xylitol conversion in synthetic medium
lulosic hydrolysates. However, this process is
occurred at a high yield.” However, no
expensive since the yield and quality of the
bioconversion occurred when the yeast was
xylitol depends on the purity of the pentose.5
grown in sugar cane bagasse hydrolysate
An alternative to chemical reduction is the
containing acetic acid concentrations greater
direct bioconversion of xylose to xylitol, which
than 5.0 g ll’ and pH ~4.5.”
can be achieved using any of a number of
This papers reports experiments in which the
microorganisms, such as those of the genus
effect of the initial pH on the xylose/xylitol
Candidu. It has previously been reported that
conversion was investigated, using sugar cane
Candida guilliermondii FTI 20037 can produce
bagasse hydrolysate as the main constituent of
xylitol from the xylose present in hemicellulosic
the culture medium. Batch fermentations were
hydrolysates of sugar cane bagasse,2,’rice straw”
carried out using clarified, supplemented and
‘Author to whom correspondence should be addressed concentrated hydrolysate, with initial pH
Fax: 0055 12 5533165; e-mail: feqlps@eu.ansp.br). varying from 2.5 to 7.5.
II
12 M. G. A. FELIPE et al.

2. MATERIALS AND METHODS 2.3. Growth conditions

2.1. Microorganism and inoculum preparation
A stock culture of Candida guilliermondii FTI
20037 maintained on an agar malt extract
(Merck) slant was transferred to a 125 ml
Erlenmeyer flask containing 50 ml liquid
medium (xylose 30.0 g l-‘, glucose 7.0 g l-‘,
ammonium sulphate 2.0 g l-‘, calcium
chloride 0.1 g 1, rice bran 20.0 g ll’) and
incubated with shaking at 200
brations min’ at 30°C for 24 h. The cells
were then recovered by centrifugation
2000 x g for 15 min and resuspended in
sterile distilled water to reach a final
concentration of 1.0 g 1-l (0.92 x IO6
cells ml-‘).
2.2. Preparation of the bagasse hydrolysate
Sugar cane bagasse from Usina Nova
America (Assis, SP, Brazil) was impregnated
with H,SO, (100 mg g-’ dry matter) and then
heated to 140°C for 20 min. After that, the
liquid fraction was concentrated by heating
at 58°C under vacuum. The hydrolysate
had the following composition (pH = 0.56):
glucose 8.32 g 1-l; xylose 48.0 g 1-l; arabinose
6.36 g 1-l; furfural 0.09 g 1-l; hydroxymethyl-
furfural 0.08 g 1-l; acetic acid 5.72 g 1-l.
The hydrolysate was the treated as follows:
the pH was raised to 10.0 by addition of
solid CaO and then decreased to 5.5 with
H,SO,. Then, the pH of various samples
was further adjusted with H,SO, to 2.5, 3.5,
4.5 and 5.5 and with NaOH to 6.5 and 7.5.
These samples were autoclaved at 115°C
0.5 atm for 15 min, then centrifuged at
1500 x g for 15 min to remove the precipitate
formed.
The hydrolysate was supplemented with the
same nutrients as used in the inoculum
preparation, xylose and glucose being omitted.
Fermentations were carried out in 125 ml
Erlenmeyer flasks containing 50 ml of the
culture medium, under constant agitation as
previously at 30°C for 45 h.
2.4. Analytical methods
vi-
Glucose, xylose, arabinose, xylitol, acetic
at acid, furfural and hydroxymethylfurfural were
determined using HPLC (HP 1082 B) as
previously described by Felipe et al. (1996b).
The cell number was. determined directly by
counting in a Neubauer chamber (area = l/
400 mm*; height = 0.100 mm).
3. RESULTS AND DISCUSSION
The effect of the initial pH on the xylose/xyl-
itol conversion by Candida guilliermondii FTI
20037 grown in sugar cane bagasse hydrolysate
is summarised in Table 1. At pH values lower
than 4.5, the consumption of glucose, xylose,
arabinose and the production of xylitol were
strongly inhibited. Conversely, at pH 2 4.5 the
sugars, except arabinose, were consumed and
xylitol was produced. At pH 4.5 most of the
sugar had been consumed after 29 h of
fermentation. Similar results were found with
this yeast grown on rice straw hydrolysate
containing an acetic acid concentration of
1.82 g l-‘.6 Moreover, C. guilliermondii FTI
20037 grown in synthetic medium without acetic
acid, metabolised xylose even at pH = 2.5.12
This behaviour, which was also observed for
Pachysolen tannophilus,‘4 Candida shehatae,15,16

Table 1. Yield (Y) and productivity (P) of xylitol, acetic acid (A) and cell (X) concentration in the fermentation of sugar
cane bagasse hydrolysate by C. guiNiermondii FTI 20037 carried out at different pH after 45 h of incubation

PH
2.5”,b 3.5 4.5 5.5 6.5 7.5
Glucose 8.718.9 8.9/8.9 8.9/0.0 s.s/o.o 7.710.0 8.2jO.O
Xylose 40.0/41.0 42.0142.5 42.5123.0 40.0/5.4 37.0/0.0 38.0/0.0
Arabinose 6.8/7.0 8.717.8 7.817.8 7.716.0 6.815.7 7.515.8
Xylitol 0.0 0.0 14.2 25.9 25.3 23.8
Y (g g-‘) 0.0 0.0 0.78 0.75 0.65 0.60
P (g I-’ h) 0.0 0.0 0.38 0.57 0.54 0.51
X (cells ml-’ 108) nd nd 0.37 1.15 0.72 0.96
A (g I-‘) 4.814.2 4.514.0 4.4/1.6 4.3/0.0 4.ojo.o 4.4/0.0
PH 2.6 3.7 5.5 6.5 6.8 6.9
“Initial concentration.
bFinal concentration.
nd. not detected.
 Fermentation of sugar cane 13

Candida boidini” and Pichia stipitis,” could be the end of the fermentation (Table 1). A similar
due to the high amount of undissociated acetic behaviour was also observed for Candida blankii
acid molecules in the medium under low pH.lY and Candida utilis”.” grown in a synthetic
The acetic acid would freely diffuse across the medium, whose composition resembled that of
cell membrane and once in the cytoplasm would bagasse hydrolysate, as well as for P. stipitis
ionise, thereby affecting the intracellular pH. cultured in wood hydrolysate.”
As a consequence, the energy and anabolici Among the yeast strains, a comparison of the
catabolic pathways (xylose uptake and con- pH effect on cell metabolism with the acetic acid
sumption included) can be uncoupled, resulting inhibitory effect on biomass and xylitol pro-
in a reduction of cell growth.” duction is difficult, owing to the interference of
In the present study a reduction in cell other factors, such as aeration4 and tempera-
concentration and morphological alterations ture.15 However, the effects of low pH and acetic
were also observed through a microscopic acid concentration over 5.0 g ll’ on the
analysis (data not shown). Disturbance of the xylose/xylitol conversion are a matter of
transmembrane active transport due either to concern, since hydrolysates of plant origin are
pH alteration or competition between acetic always contaminated by varying amounts of
acid and sugar molecules for the active sites, acetic acid. Thus, the hydrolysates must be
could also contribute to the effects seen. cleaned up using ionic resins and active
Xylitol production is also influenced by the charcoal’” or calcium oxide plus sulphuric acid.’
pH of the culture medium. At pH lower than 4.5 The latter treatment, which is the less costly of
the yeast did not produce xylitol as a result of the alternatives, was successfully employed for
reduced xylose consumption. The highest values minimising the inhibition effects on the fermen-
of xylitol production were observed at pH 5.5, tative activity. In short, bioconversion of xylose
6.5 and 7.5, which corresponded to 25.9, 24.3 into xylitol could be improved through the
and 22.8 g 1-I, respectively. It must be pointed fermentation of CaO/HSO,-treated sugar cane
out that in the absence of acetic acid, high bagasse hydrolysate at pH 5.0 and acetic acid
xylitol productivity occurs at pH values of 4.0 concentration below 5.0 g 1-l.
and 5.0.” This was also observed for C.
.~hehatae,‘5.‘h Schizosaccharomyces pomhae” and A~knol~lrcf~emmts~The authors acknowledge financial
C. hoidini.” assistance from CNPq/Brazil, and Maria Eunice M. Coelho
fo1 the helpful revision of this paper
In the present work the highest xylitol yield
(0.91 g g-‘) occurred after 22 h of fermentation
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