Xylitol Production from Wheat Straw Hydrolysate Using

Stirred Tank Reactor

Larissa Canilha, Elisângela J. Cândido, João Batista de Almeida e Silva

Department of Biotechnology, Faculty of Chemical Engineering of Lorena, Rod. Itajubá- Lorena, Km 74.5,
Lorena- S.P., 12608-970 Phone: +(12)5533422; Fax: +(12)5533133
Correspondence to: larissa@debiq.faenquil.br

ABSTRACT

Wheat straw hemicellulosic hydrolysate was fermented in a stirred tank reactor (STR) in
order to verify the behavior of the yeast Candida guilliermondii FTI 20037 for xylitol
production. Fermentation was carried out in a 5.0 L STR, using an inoculum concentration of
0.5 g/L. The reactor was operated at an agitation speed of 300 rpm, at 30 °C, aeration of
0.6 vvm for 60 hours. The results showed that the xylose consumption by the yeast was high,
and that glucose, arabinose and acetic acid assimilated in small concentrations do not
interfere with the bioconversion of xylose into xylitol. The bioconversion yield was 0.89 g
xylitol /g xylose and the conversion efficiency, 97%.

Index Entries: wheat straw hydrolysate, Candida guilliermondii, fermentation, xylitol

production.

INTRODUCTION
Lignocellulosic materials are composed of mainly cellulose, hemicellulose and lignin.
Hemicellulose is composed of linear and branched heteropolymers of L-arabinose, D-
galactose, D-glucose, D-mannose and D-xylose. The composition of hemicellulose vary
according to the plant species, such as wheat straw (32%), barley straw (32%), rice straw
(25%), corn cobs (37%), sugarcane (22%) and eucalyptus wood (15-22%) (Sun, 1996; Vital
and Della Lúcia, 1986). The hemicellulose fraction can be hydrolyzed and used for
fermentation of xylose, with a view to producing xylitol (Canettieri et al., 2001).
Xylitol (C5H12O5) is a sugar alcohol with sweetening power superior to that of sorbitol and
mannitol, and comparable to that of sucrose (Makinen, 1992; Gales and Nguyen, 2000). It is
anticariogenic, prevents the formation of acids that attack the tooth enamel (Makinen, 1992;
Gales and Nguyen, 2000; Shen et al., 2001) and can be used as a sugar substitute in dietetic
food for diabetics (Parajó et al., 1998). The literature reports that this poliol can also be used
in the treatment of osteoporosis, since it considerably improves the biomechanical properties
of the bones and prevents reduction both in their density and in their contents of minerals,
calcium and phosphorus (Mattila et al., 2002). Moreover, xylitol inhibits the growth of the
bacterial species Streptococcus pneumoniae and Haenophilus influenzae, which cause acute
medium otitis, so it could be employed, instead of antibiotics, to combat this disease
(Erramoupse and Heyneman, 2000; Tapiainen et al., 2002). Owing to all these characteristics,
xylitol is a feedstock of particular interest to the food, odontological and pharmaceutical
industries.
The biotechnological process for bioconversion of xylose to xylitol is attractive because it
does not require a pure xylose solution as is the case when xylitol is produced by the chemical
pathway. (Heikkilä et al., 1992; Nigam and Singh, 1995). Yeasts are considered to be the best
xylitol producers, especially those belonging to the genus Candida (Winkelhausen and
Kuzmanova, 1998), for example, Candida guilliermondii (Barbosa et al., 1988; Carvalho et
al.,2002). During the yeast metabolism, D-xylose is initially transported across the cell
membrane by the diffusion transport system (Hahn-Hägerdal et al., 1994; Winkelhausen and
Kuzmanova, 1998) and then reduced to xylitol by either NADH or NADPH with xylose
reductase. In C. guilliermondii cells, D-xylose is reduced by NADPH (Sene et al., 2001).
Xylitol is formed and secreted from the cell and also oxidized to D-xylulose by NAD or
NADP with xylitol dehydrogenase. D-xylulose is phosphorylated and enters the pentose-
phosphate pathway, which generates the NADPH cofactors, used in the first step of the xylose
metabolism (Hahn-Hägerdal et al., 1994; Nigam and Singh, 1995; Winkelhausen and
Kuzmanova, 1998).
The objective the present study was to evaluate the bioconversion of xylose to xylitol by
C. guilliermondii FTI20037 in the wheat straw hydrolysate.

MATERIALS AND METHODS

Microorganism
The experimenter was carried out with C. guilliermondii FTI20037 described by Barbosa et
al. (1988). The yeast was maintained on malt-extract agar slants at 4 °C.
Preparation of wheat straw hydrolysate
Wheat straw was hydrolyzed in a 30 L reactor at 144.5 °C for 30 min with 2.51% acid
concentration and 1:17.5 solid-liquid ratio. The hydrolysate was concentrated under vacuum
at 70 °C to increase xylose concentration. As a result, all the hydrolysate components had
their concentrations increased about fourfold: glucose increased from 2.79 to 8.36 g/L, xylose
from 10.65 to 39.57 g/L, arabinose from 1.78 to 6.41 g/L, acetic acid from 0.68 to 1.60 g/L,
furfural from 0.34 to 0.05 g/L and hydroxymethylfurfural from 0.03 to 0.12 g/L. The
hydrolysate was then treated in order to reduce the concentrations of toxic compounds. The
initial pH was raised to 7.0 with calcium oxide and decreased to pH 5.5 with phosphoric acid.
The hydrolysate was mixed with 10% activated charcoal, agitated (200 rpm, 30 °C, 1 h) then
filtered. The filtrate was autoclaved at 111 °C for 15 min.
Preparation of microorganism and inoculum cultivation
Cells of C. guilliermondii FTI20037 were inoculated into synthetic medium containing
(g/L) 30.0 D-xylose, 7.0 glucose, 2.0 ammonium sulfate, 0.1 calcium chloride and 20.0 rice
bran extract. The cells were incubated for 24 hours on a rotary shaker (30 °C, 200 rpm),
collected by a 15-minute centrifugation at 2000 x g and resuspended in sterile distilled water.
Xylose and glucose solutions and rice bran extract were autoclaved at 111 °C for 15 min and
ammonium sulfate and calcium chloride solutions were sterilized at 121 °C for 20 min. The
rice bran suspension was then aseptically centrifuged at 2000 x g for 20 min.
Medium and fermentation conditions
The fermentation medium consisted in concentrated and treated hydrolysate supplemented
with 1.0 g/L ammonium sulfate and 5.0 g/L rice bran extract. The medium was introduced
into a 5.0 L bioreactor and fermented for 60 hours at 30°C. The agitation and aeration rates
were the same as those used by Felipe et al. in 1997 (300 rpm and 0.6 vvm) and the inoculum
concentration was 0.5 g/L.
Analytical methods
The concentrations of sugars, xylitol and acetic acid were determined by HPLC in a Biorad
Aminex HPX-87H column (300 x 7.8mm) at 45 °C using 0.01N sulfuric acid solution as the
eluent, flow rate of 0.6 mL/min and 20µL sample volume. Concentrations of furfural and
hydroxymethylfurfural (HMF) were also determined by HPLC using Resolve 5µ Spherical
C18 (3.8x300 mm) column at 25 °C, solution of acetonitrile/water (1:8) as the eluent and
20µL sample volume. The cell number was determined by direct counting in a Neubauer
chamber (area =1400mm2; height = 0.100mm).
The xylitol yield factor is represented by Equation 1:
(1)
∆p Pf − Pi
ΥP / S = =
∆S Sf − Si
in which and correspond to the initial and final xylose concentrations and and
Si Sf Pi Pf

correspond to the initial and final xylitol concentrations.

The volumetric productivity was calculated by Equation 2:
Qp = (2)
∆p Pf − Pi
=
∆t tf − ti
in which and correspond to the initial and final xylitol concentrations and and
Pi Pf ti tf
correspond to the initial and final fermentation times.
The conversion efficiency is the ratio between the calculated YP/S and the theoretical
conversion factor of 0.917 g/g calculated by Barbosa et al., 1988.

RESULTS AND DISCUSSION

Table 1 shows the concentrations of glucose, xylose, acetic acid, arabinose and xylitol, as
well as cell concentration and xylose consumption during fermentation. The concentrations of
xylose, glucose and arabinose, as well as the xylitol production by C. guilliermondii
FTI20037 are in Figure 1.

Table 1 – Concentrations of glucose, xylose, acetic acid, arabinose, xylitol and cells, and
xylose consumption rates during the fermentation of wheat straw hydrolysate by
Candida guilliermondii yeast.

Time Glucose Xylose Xylose Acetic Arabinose Xylitol Cells

(h) (g/L) (g/L) consumptio acid (g/L) (g/L) (cells/mLx108)
n (%) (g/L)
0 6.927 30.93 0.000 1.268 5.380 - 0.21
6 2.255 27.72 10.38 1.301 6.423 1.841 1.50
12 0.133 27.50 11.09 0.927 5.434 2.387 2.38
18 0.294 23.27 24.77 0.972 6.271 6.724 2.43
24 0.187 16.63 46.23 0.855 5.583 10.97 3.06
36 0.201 11.32 63.40 0.815 5.427 16.18 2.72
42 0.408 3.886 75.43 0.747 4.988 18.46 3.60
48 0.407 1.447 92.83 0.605 3.995 24.04 3.20
54 0.358 0.367 98.81 0.480 2.701 27.86 3.63
60 0.347 0.157 99.49 0.446 1.778 24.54 3.34
66 0.295 0.107 99.65 0.446 1.368 25.22 4.00
72 0.103 0.103 99.67 0.366 0.642 24.33 4.90
78 0.104 0.106 99.66 0.144 0.218 24.00 3.26

As can be seen, in the first twelve hours of fermentation glucose was rapidly consumed by
the yeast, and the xylose consumption was consequently affected. After this period, xylose
consumption rate remained low until the 18th hour of fermentation, and then increased
significantly, reaching more than 90% after 48 hours. Starting from the 54th hour, the xylose
concentration remained almost stable, indicating the end of the fermentation process. Studies
employing C. guilliermondii (Alves et al.,1998) and C. shehatae (Jeffries, 1988) report that
the arabinose assimilation by the yeast was small when xylose (carbon source) was present in
the hydrolysate. Likewise, in our experiment, the slow arabinose consumption detected during
the first 48 hours of fermentation was possibly due to an inhibiting action of xylose. The
amount of xylitol produced by C. guilliermondii FTI 20037 from wheat straw hemicellulosic
hydrolysate was proportional to the consumption of xylose and arabinose, stabilizing after 48
hours. All the acetic acid concentrations were much lower than 3.0 g/L, which is the
minimum amount considered as inhibitory (Felipe et al., 1997).

FIGURE 1 – Xylose, glucose and arabinose concentrations and xylitol production by Candida
guilliermondii FTI20037 in wheat straw hydrolysate.

Roberto (1997) used rice straw hydrolysate and observed that xylitol production is
associated with cell growth. It is also known from the literature that bioreactors provide more
oxygenation for the medium, favoring cell growth. Therefore, in our work, a small cell
concentration was used as the inoculum (0.5 g/L), on the assumption that it would result in an
increased oxygen availability in the medium and consequently in a higher cell growth rate,
which actually occurred.
The fermentative parameters of the bioconversion of xylose into xylitol concerning our
work and the work of other authors are in Table 2. The maximum yield of bioconversion
attained by us with wheat straw hydrolysate (0.89g/g) was very close to the maximum
theoretical yield (0.917 g/g) proposed by Barbosa et al. (1988) and higher than the yields
provided by hydrolysates from rice straw (0.72 and 0.65 g/g) (Mussato and Roberto, 2001;
Silva and Roberto, 2001), sugar cane bagasse (0.82 and 0.79 g/g) (Felipe et al., 1997; Alves
et al., 1998) and eucalyptus chips (0.84 and 0.20 g/g) (Parajó et al., 1997; Canettieri et al.,
2001). With regard to productivity, the value obtained in our work (0.50 g/L.h) was similar to
those reported for rice straw (0.61 and 0.54 g/L.h) (Mussato and Roberto, 2001; Silva and
Roberto, 2001), cane bagasse (0.57 and 0.47 g/L.h) (Felipe et al., 1997; Alves et al., 1998)
and eucalyptus wood (0.53 g/L.h) (Parajó et al., 1997) and higher than the value found by
Canettieri et al., 2001 (0.10 g/L.h).

Table 2 – Fermentative parameters of the bioconversion of xylose into xylitol by different

yeasts using different raw materials

Microorganism Raw Fermentative parameters References

material
Yield Efficiency Productivity
(g/g) (%) (g/L.s)
C. Wheat straw 0.89 97 0.50 Our work
guilliermondii
FTI 20037
C. Rice straw 0.72 79 0.61 Mussato and
guilliermondii Roberto, 2001
FTI 20037
C. Rice straw 0.65 71 0.54 Silva and Roberto,
guilliermondii 2001
FTI 20037
C. Sugarcane 0.79 86 0.47 Alves et al., 1998
guilliermondii bagasse
FTI 20037
 C. Sugarcane 0.75 82 0.57 Felipe et al., 1997
guilliermondii bagasse
FTI 20037
C. Eucalyptus 0.20 22 0,10 Canettieri et al., 2001
guilliermondii wood
FTI 20037
D. hansenii Eucalyptus 0.84 92 0.53 Parajó et al., 1997
Y-7426 wood

CONCLUSIONS

The maximum xylitol yield obtained in this study (0.89 g xylitol /g xylose), which
corresponded to a bioconversion efficiency of 97%, was very close to the maximum
theoretical value proposed by Barbosa et al. (1988) and higher than the values obtained with
rice straw, sugar cane bagasse and eucalyptus wood hydrolysates. This result evidences the
potentiality of wheat straw as a raw material for xylitol production by Candida guilliermondii
FTI20037.

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