food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

A model of furfural-inhibited growth and xylitol

production by Candida magnoliae TISTR 5663

Siwaporn Wannawilai a,b , Yusuf Chisti c , Sarote Sirisansaneeyakul a,b,

a Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
b Center for Advanced Studies in Tropical Natural Resources (CASTNAR), National Research University-Kasetsart
University (NRU-KU), Kasetsart University, Bangkok 10900, Thailand
c School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: Furfural, an inhibitor of yeast growth, occurs in xylose-containing lignocellulosic

Received 21 December 2016 hydrolysates that are potential substrates for the fermentative production of the natural
Received in revised form 9 June 2017 sweetener xylitol from xylose. Effects of furfural on growth and xylitol production by the
Accepted 4 July 2017 yeast Candida magnoliae TISTR 5663 are reported. Aerobic as well as oxygen limited con-
Available online 12 July 2017 ditions were used to assess the effects of furfural. A mathematical model was developed
to predict the behavior of this fermentation. The model was validated using independent
Keywords: experimental data. The model and its kinetic parameters are reported. Furfural was found
Xylitol to be a competitive inhibitor of growth. Nevertheless, the presence of a certain amount of
Xylose furfural (0.3 g L1 ) in the production medium actually improved the productivity of xylitol
Furfural and its yield from xylose under suitable oxygen limited conditions.
Fermentation model 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Candida magnoliae

1. Introduction of energy and carbon for growth. If the intracellular conversion

Xylitol (C5 H12 O5 ) is a commercially used natural sweetener.
Its consumption does not raise the blood sugar level and,
therefore, it is suitable for diabetics. Bacteria found in the
mouth do not metabolize xylitol to acids that promote tooth
decay. Xylitol is produced commercially by catalytic hydro-
genation of xylose, a sugar recovered typically from acid
hydrolysates of birchwood or corncobs. In principle, microbial
fermentation processes can be used to convert xylose to xyli-
tol (de Albuquerque et al., 2014; Sirisansaneeyakul et al., 2013;
Wannawilai and Sirisansaneeyakul, 2015).
Xylose metabolizing yeasts take up xylose from the cul-
ture medium and convert it to xylitol through the action of
the enzyme xylose reductase. This process requires the coen-
zyme NADPH (Sirisansaneeyakul et al., 1995; Tochampa et al.,
2005). The xylitol formed is reduced to xylulose by the NAD+ -
dependent xylitol dehydrogenase (Sirisansaneeyakul et al.,
1995; Tochampa et al., 2005). The cell uses xylulose as a source
of xylitol to xylulose is somehow decreased, the excess xylitol
is excreted.
Xylitol excretion can be enhanced for example by decreas-
ing the supply of the necessary cofactor NAD+ required to
convert it to xylulose. NAD+ is generated via the respiratory
chain (Parajo et al., 1998; Vsquez et al., 2006) and any slowing
of respiration would reduce its regeneration. Oxygen limita-
tion can be used to slow respiration and the regeneration of
NAD+ . This would cause a redox imbalance between xylose
reductase and xylitol dehydrogenase to make more xylitol
available for excretion (Sirisansaneeyakul et al., 1995). Thus,
an oxygen limited environment is optimal for production of
xylitol from xylose (Sirisansaneeyakul et al., 2013; Tochampa
et al., 2005). Oxygen limitation of course reduces the produc-
tion of biomass, the biocatalyst needed for xylitol production.
Therefore, an optimal production process requires two distinct
sequential phases: an initial phase that promotes the growth
of the biomass and a subsequent oxygen-limited phase that
promotes the production of xylitol.

Corresponding author at: Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
E-mail address: sarote.s@ku.ac.th (S. Sirisansaneeyakul).
http://dx.doi.org/10.1016/j.fbp.2017.07.002
0960-3085/ 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
130 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140

ated during the hydrolysis reactions. Typically, xylose for use

Nomenclature in fermentation processes is puried from the above men-
tioned hydrolysates to remove the inhibitory compounds but
A Parameter dened by Eq. (2) this adds to the cost of procuring xylose. The expense of a
a Toxic severity subsequent fermentation can be substantially reduced if the
B Parameter dened by Eq. (3) xylose-containing hydrolysate can be used directly without a
Ccal Calculated value of a variable from the model purication step.
(g L1 ) Furfural inhibits fermentation processes in multiple ways
Cexp Experimentally measured value of a variable (Almeida et al., 2007; Liu et al., 2009; Modig et al., 2002; Sanchez
(g L1 ) and Bautista, 1988), although many microorganisms are able
Cexp Average of all the experimentally measured val- to convert it to less toxic products (Matos et al., 2016; Wang
ues of a variable (g L1 ) et al., 2017). Under aerobic conditions, some yeasts overcome
I Furfural concentration (g L1 ) furfural toxicity by oxidatively converting it to furoic acid
I0 Initial concentration of furfural (g L1 ) using the coenzyme NAD+ (Horvth et al., 2003). This pro-
I50 Furfural concentration to reduce specic cess can actually improve xylitol production by diverting NAD+
growth rate to 50% (g L1 ) away NAD+ -mediated conversion of xylitol to xylulose so that
Im Critical furfural concentration (g L1 ) excess xylitol is available to be excreted. In view of the impor-
kL a Volumetric gasliquid oxygen transfer coef- tance of this effect (Wannawilai et al., 2017), the present study
cient (h1 ) focussed on the effects of furfural on yeast growth and xylitol
KF Saturation constant for furfural (g L1 ) production under aerobic as well as oxygen limited conditions.
KI Inhibition constant (g L1 ) Production of xylitol from xylose has been previously math-
KO Dimensionless oxygen coefcient ematically modeled (Sirisansaneeyakul et al., 1995; Tochampa
KS Saturation constant for substrate (g L1 ) et al., 2005; Wannawilai et al., 2015), but only for the cases
mS Biomass maintenance coefcient (g g1 h1 ) of substrates free of any inhibitory compounds. In practice,
n Agitation speed of shaker platform (min1 ) xylose for conversion to xylitol is obtained via acid hydrolysis
P Xylitol concentration (g L1 ) of wood (e.g. birchwood) and such hydrolysates are inevitably
qI Specic rate of furfural consumption (g g1 h1 ) contaminated with furfural. Furfural is a well-known inhibitor
qmax
I Maximum specic rate of furfural consumption of microbial fermentations, including fermentation of xylose
(g g1 h1 ) to xylitol (Wannawilai et al., 2017). While inhibition of xylitol
qP Specic rate of xylitol production (g g1 h1 ) fermentation by furfural has been demonstrated (Wannawilai
qS Specic rate of xylose consumption (g g1 h1 ) et al., 2017), it has not been mathematically modelled. Fermen-
QI Volumetric rate of furfural consumption tation models applicable to furfural-contaminated substrates
(g L1 h1 ) are of practical importance in commercial processes. Using a
QP Volumetric rate of xylitol production (g L1 h1 ) xylose-containing wood hydrolysate as a fermentation sub-
QS Volumetric rate of xylose consumption strate is far less expensive than using xylose puried from
(g L1 h1 ) such a hydrolysate. Models accounting for inhibitory effects
QX Volumetric rate of biomass production allow the behavior of a fermentation to be predicted for vari-
(g L1 h1 ) ous levels of contamination of the substrate with furfural.
R2 Determination coefcient The yeast Candida magnoliae TISTR 5663 was used as it
S Substrate concentration (either glu- has been shown to effectively convert pure xylose to xyl-
cose + xylose, or xylose only) (g L1 ) itol (Sirisansaneeyakul et al., 2013). Effects of furfural on
t Time (h) growth and xylitol production kinetics were modeled. Model-
V Flask working volume as percent of total ask predicted optimal conditions for xylitol production in the
volume (%) presence of furfural were validated using experimental data
X Biomass concentration (g L1 ) not used in developing the model.
YP/xyl Xylitol yield from xylose (g g1 )
YX/S Biomass yield from substrate (either glu-
cose + xylose, or xylose only) (g g1 ) 2. Materials and methods

Greek letters 2.1. Microorganism and culture media

Growth-associated production coefcient
(g g1 ) Pure cultures of the yeast C. magnoliae TISTR 5663 were
Non-growth-associated production coefcient maintained on agar slants. The agar medium contained the
(g g1 h1 ) following components (g L1 ) in deionized water: malt extract
 Specic growth rate (h1 ) 3, yeast extract 3, peptone 5, glucose 10 and agar 15. A fresh
I Specic growth rate in the presence of the slant was incubated at 30 C for 24 h and then stored at 4 C.
inhibitor (h1 ) The slants were refreshed monthly.
max Maximum specic growth rate (h1 ) The growth medium contained the following (g L1 )
in deionized water: glucose 10, xylose 5, KH2 PO4 18.75,
(NH4 )2 HPO4 6, MgSO4 7H2 O 1.13, CaCl2 0.1, myo-inositol
Xylose is commercially produced via dilute acid hydrolysis 36.5 103 , calcium pantothenate 18.2 103 , thiamine-HCl
of birchwood, corncobs and other lignocellulosic substrates. 3.66 103 , pyridoxal-HCl 0.9 103 , biotin 1.8 105 , FeCl3
In addition to xylose, the acid hydrolysate contains other 9.1 103 , MnSO4 H2 O 6.4 103 , ZnSO4 7H2 O 5.46 103
sugars and inhibitory compounds (mainly furfural) gener- and CuSO4 5H2 O 1.46 103 (Sirisansaneeyakul et al., 1995;
 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140
Tochampa et al., 2005; Wannawilai and Sirisansaneeyakul,
2015). The initial pH of the medium was adjusted to pH 4.0
with 3 M HCl (Sirisansaneeyakul et al., 2013). All media were
sterilized by autoclaving (121 C, 15 min).
2.2. Preparation of inoculum
Two loopfuls of cells from a slant were inoculated into 25 mL
of the growth medium in a 250-mL Erlenmeyer ask. The cul-
ture was grown aerobically by incubating the ask on a rotary
shaker (250 min1 , 30 C, 24 h). Subsequently, all the culture
broth was transferred to a 500-mL Erlenmeyer ask containing
225 mL of the growth medium. The inoculum was incubated
for 24 h under the above specied conditions. This culture was
used to seed all experiments.
2.3. Shake ask cultures
2.3.1. Effects of furfural on biomass production phase
The experiments were carried out in 500-mL Erlenmeyer
asks. Twenty ve milliliters of inoculum was added to 225 mL
of the growth medium. This medium contained 10 g L1 glu-
cose and 5 g L1 xylose as carbon sources. The initial pH was
4.0. The initial furfural concentration (0, 0.164, 0.462, 0.977,
1.723 and 2.615 g L1 ) in different asks varied. All asks were
held on a rotary shaker (250 min1 ) at 30 C until the glucose
and xylose were exhausted. Samples were taken every 4 h to
measure the concentrations of the biomass, furfural, xylose
and xylitol.
2.3.2. The effect of furfural on xylitol production phase
Our previously reported data on effects of furfural on produc-
tion of xylitol (Wannawilai et al., 2017) was used in modeling
and verication of the fermentation kinetics in the present
work. In brief, the experiments used 500-mL Erlenmeyer asks
with an initial working volume of 250 mL. Growth medium
containing glucose (10 g L1 ) and xylose (5 g L1 ) but no furfural
was used in an initial biomass production phase. The initial
pH was 4.0. All asks were held on a rotary shaker (250 min1 )
at 30 C until the glucose and xylose were exhausted. At this
point, xylose (30 g L1 ) and (NH4 )2 HPO4 (6 g L1 ) were added.
Furfural was added to different asks at various initial con-
centrations (0, 0.1, 0.2, 0.3, 0.5, 0.75, 1.0 and 2.0 g L1 ). The
initial working volume was now 300 mL. The pH was 7.0. The
agitation speed was reduced to 150 min1 and the incuba-
tion temperature was 30 C. All experiments were in duplicate.
Samples were taken periodically to measure the concentra-
tions of the biomass, furfural, xylose and xylitol.
2.3.3. Effects of agitation speed on xylitol production
Cultures were started as explained above (Section 2.3.2) for
the biomass production phase. Subsequently, in the xylitol
production phase (see above), different asks were agitated at
110 min1 and 250 min1 . At the start of the xylitol production
phase, furfural was added to all asks to obtain a concen-
tration of 0.3 g L1 . The initial pH was 7.0 and the incubation
temperature was 30 C. All experiments were in duplicate. The
samples were taken periodically for the above specied mea-
surements.
2.4. Analytical methods
The samples of the culture broth were centrifuged (1700 g,
10 min) to separate the cells and the supernatant. The cells
131
were washed twice with distilled water and dried at 105 C for
24 h to measure the dry cell weight (DCW). The supernatant
was used to measure xylose, glucose, xylitol and furfural by
HPLC (Knauer, Berlin, Germany). A VertiSepTM SUGAR CMP
HPLC column (7.8 mm 300 mm, 9 m; Vertical Chromatog-
raphy Co., Ltd., Thailand) was used for measuring xylose,
glucose and xylitol. The mobile phase was deionized water
at a ow rate of 0.4 mL min1 . The column was held at 80 C.
A refractive index detector was used. The injection volume
was 20 L. Furfural concentration was measured using an
Eurospher 100-5 C18 column (250 4.6 mm; Knauer, Berlin,
Germany). The mobile phase was 5% (v/v) acetonitrile at a
ow rate of 1.0 mL min1 . The injection volume was 20 L.
The column was kept at 25 C. A UV detector measuring at
a wavelength of 254 nm was used. The fermentation kinetic
parameters were calculated according to Sirisansaneeyakul
et al. (2013).
The volumetric oxygen transfer coefcient (kL a) for a
500 mL nonbafed Erlenmeyer ask was estimated at 30 C
using the following equations:
kL a = A ln V + B (1)
where V (%) was the percentage of the total ask volume lled
with the liquid and the parameters A and B depended on the
shaking speed (n, min1 ) as follows:
A = 7.0 104 n2 0.4879n 32.622 (2)
B = 2.1 103 n2 + 1.836n 123.82 (3)
Eq. (1) is similar to that recommended by Schiefelbein et al.
(2013).
2.5. Kinetic model for yeast growth and xylitol
production
The effects of furfural were modeled by treating it as a growth
inhibitor in view of its known effects (Almeida et al., 2007;
Liu et al., 2009; Modig et al., 2002; Wannawilai et al., 2017).
In the absence of the inhibitor, the growth was assumed to
follow Monod type kinetics in which the specic growth rate
depended on the concentration of the substrate (S) that could
be glucose, xylose or a combination of the two. Thus, the
biomass concentration (X) at time t depended on the specic
growth rate with the inhibitor present (I ), as follows:
dX
= I X (4)
dt
and the specic growth rate I depended on the concentration
of the substrate (S) and the inhibitor (I), as follows:
max S
 I
a
I = 1 (5)
KS + S Im
In the above equation, max is the maximum specic growth
rate, KS is the saturation constant, Im is the threshold con-
centration of furfural at which the growth is fully suppressed
(i.e. I becomes zero) and a is the toxic severity coefcient,
a measure of the inhibitory power. For a = 1, an increasing
concentration of furfural reduces the specic growth rate lin-
early. If a < 1, the specic growth rate declines hyperbolically
as the concentration of furfural increases. For a > 1, the specic
growth rate shows a rapid parabolic decline as the concentra-
132 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140

tion of furfural increases. Therefore, a high a-value represents where and are the growth-associated and
a higher toxicity compared to a lower value (Georgieva et al., nongrowth-associated coefcients for product formation,
2007; Lee et al., 2000; Levenspiel, 1980; Wannawilai et al., 2015). respectively.
To determine the type of inhibition that may be occurring In the cell, furfural is converted to less toxic compounds
(e.g. competitive inhibition, uncompetitive inhibition, non- such as furfuryl alcohol (Wang et al., 2017) and furoic acid
competitive inhibition), the various inhibition models (Cer (Horvth et al., 2003; Kundiyana et al., 2009; Liu et al., 2009;
et al., 2009) were tested. These were as follows: Palmqvist et al., 1999). This conversion is assumed to follow
the Michaelis-Menten kinetics; thus,
max S
I =   for competitive inhibition (6)
KS 1 + I
+S dI qmax I
KI = I X (13)
dt KF + I
maxI S
1+ K In the above equation qmax
I is the maximum specic consump-
I
I = for uncompetitive inhibition (7) tion rate of furfural and KF is the corresponding saturation
 KS
 +S
1+ KI constant.
I
The measured furfural concentration was used to calcu-
maxI S late the volumetric (QI ) and specic (qI ) consumption rates of
1+ K furfural as follows:
I
I = for noncompetitive inhibition (8)
KS + S
I2 I1
QI = (14)
In the above equations, KI is an inhibition constant. Depend- t2 t1
ing of the value of KS , furfural may behave as a competitive
QI X2
inhibitor, an uncompetitive inhibitor or a noncompetitive qI = ln (15)
X2 X1 X1
inhibitor (Cer et al., 2009). If  is the specic growth rate in the
absence of the inhibitor (i.e. I = 0), then for competitive inhibi- where I1 and I2 are the measured concentrations of furfural at
tion at I = I50 ,  = 2 I (Cheng and Prusoff, 1973) and, therefore, the beginning (time = t1 ) of fermentation and at the instance
Eq. (6) reduced to the following: (time = t2 ) of total consumption of furfural, respectively; X2 is
the biomass concentration at time t2 ; and X1 is the biomass
I50 concentration at time t1 . Eq. (13) was used to estimate the
KI = S
(9)
KS +1 kinetic parameters qmax and KF .
I

In Eq. (9), I50 is the inhibitor concentration required to reduce 2.6. Estimation of model parameters and statistical
specic growth rate to 50% of the -value in the absence of analyses
the inhibitor. For the case of competitive inhibition, if S = KS ,
KI = 0.5I50 . On the other hand, if S >> KS , the KI << I50 and if The experimental data were used to estimate the kinetic
S << KS , the KI
= I50 . parameters (i.e. max , KS , YX/S , mS , and ) of the
For the case of uncompetitive inhibition at I = I50 , Eq. (7) above referenced models. The parameter values were
modies to the following: selected to produce the best t between the model equa-
tions and the experimental data. Berkeley MadonnaTM
I50
KI = (10) (http://www.berkeleymadonna.com/) software was used for
KS
S +1 the tting. The value of coefcient of determination (R2 )
(Kumar, 2007; Lang et al., 2009) was used to decide on the good-
If S = KS , then KI = 0.5I50 . On the other hand, if S >> KS , then ness of t between the model and the data. R2 was calculated
KI
= I50 and if S << KS , then KI << I50 . Similarly, for noncompet- as follows:
itive inhibition at I = I50 , KI = I50 when S = KS , or S >> KS or S << KS
(Cer et al., 2009; Cheng and Prusoff, 1973).

2
(Ccal Cexp )
The substrate (whether single or mixed) uptaken by the R2 =
2
(16)
2
(Ccal Cexp ) + (Ccal Cexp )
yeast is metabolized for growth, cell maintenance and xylitol
production. Therefore, the substrate consumption rate can be
where Ccal is the value of a variable calculated using the model
modeled as follows:
and Cexp is the corresponding experimentally measured value.
   dX    
dS 1 1 dP Cexp is the average of all the experimentally measured values
= + mS X+ (11) of the relevant variable.
dt YX/S dt YP/xyl dt

where YX/S is the biomass yield on substrate, mS is the biomass
maintenance coefcient, YP/xyl is the xylitol yield on xylose
( = 0.912 g g1 ; Barbosa et al., 1988), and P is the concentration
of xylitol (the product) at time t.
Xylitol production from xylose is generally described as
a mixed growth-associated product formation. Therefore,
the production kinetics of xylitol can be modeled using
Luedeking-Piret equation, as follows:
dP
= (+)X
dt
3. Results and discussion
3.1. Effect of furfural in biomass and xylitol
production phases
Furfural inhibited the yeast growth. It reduced the specic
growth rate (Fig. 1A) and the nal biomass concentration
(Fig. 1B). Furfural extended the duration of the lag phase
(Fig. 1B). The specic growth rate was reduced with increasing
furfural concentration in a concentration-dependent manner
(12)
(Fig. 1A). The length of the lag phase increased nonlin-
 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140 133

Fig. 1 Initial furfural concentration (I0 ) in Erlenmeyer asks affects the specic growth rate (I ) (A), the nal biomass
concentration (grey bars) (B) and the length of the lag phase (B). The nal biomass concentration was the corrected
concentration at 28 h in batch culture. The length of the lag phase (lled squares, B) was calculated by tting the batch
growth data using the DMFit software (http://browser.combase.cc/DMFit.aspx).

early with an increasing initial concentration of furfural once
the concentration exceeded 0.164 g L1 (Fig. 1B). Although
the lag phase was not inuenced by furfural concentrations
of <0.164 g L1 , both the specic growth rate and the nal
biomass concentration were negatively impacted relative to
controls (no furfural) also by low concentrations (Fig. 1). For
example, the nal biomass concentration was reduced to
4.94 g L1 , or 21% relative with control (no furfural) in the
presence of furfural at an initial concentration of 0.164 g L1
(Fig. 1B). Growth was not completely inhibited by furfural up
to 2.615 g L1 (Fig. 1A). Yeasts are known to reduce the adverse
impact of furfural by metabolizing it to less toxic products
such as furoic acid or furfuryl alcohol (Horvth et al., 2003;
Liu et al., 2009; Wang et al., 2017). Therefore, as furfural was
progressively detoxied the growth rate increased.
In view of the growth inhibitory effects of furfural (Fig. 1),
the biomass production phase of the culture should ideally
be completely free of furfural. Once a substantial amount of
biomass has been generated rapidly in a furfural-free oper-
ation, the conversion of xylose to xylitol may be benecially
carried out in the presence of a certain amount of furfural
in a separate phase of culture as shown later in this work.
Although the presence of furfural in the second stage still
inhibited growth (data not shown), this was actually use-
ful as it allowed more of the xylitol formed from xylose to
be excreted into the culture medium instead being further
metabolized to xylulose for production of biomass. An ini-
tial furfural concentration of approximately 0.3 g L1 in the
xylitol production phase has been previously reported to
improve production of xylitol in C. magnoliae (Wannawilai
et al., 2017).
3.2. Kinetic modeling of biomass and xylitol
production
In modeling biomass growth, the specic growth rate (I ) was
taken to depend on the concentration of the inhibitor I (i.e.
furfural). In view of the linear dependence of  on I (Fig. 1A),
the value of a (Eq. (5)) was taken to be unity. A critical thresh-
old concentration of furfural (Im ) was dened as the value that
just completely inhibited growth (I = 0). The measured values
of , i.e. the specic growth rate in the absence of furfural, and
I (i.e. the specic growth rate with furfural present at an ini-
tial concentration I) were used to calculate the ratio I /. This
ratio was plotted against the initial concentration of furfural as
shown in Fig. 2. The resulting straight lines were extended to
the x-axis (i.e. to I / = 0) to read the threshold inhibitory con-
centrations Im for the biomass growth phase and the xylitol
production phase (Fig. 2).
In the biomass growth phase with mixed glucose and
xylose as substrates, the Im -value was 10.183 g L1 (Fig. 2). For
the xylitol production phase with xylose as the sole carbon
substrate, the Im value was 7.148 g L1 (Fig. 2). This was con-
sistent with values reported for other yeasts. For example,
the critical concentration of furfural for completely inhibit-
ing growth of eight strains of the yeast Candida spp. has been
reported to be 8 g L1 (Lazarova et al., 1990). Similarly, for
the yeast Saccharomyces cerevisiae, a furfural concentration of
53 mM ( = 5.1 g L1 ) reduced the specic growth rate to nil as
reported by Palmqvist et al. (1999). Based on the Im -values,
furfural was more toxic to C. magnoliae TISTR 5663 cells dur-
ing the xylitol production phase which operated under oxygen
limiting conditions with xylose as the sole substrate. For any
134 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140
Fig. 2 Plots of I / versus initial furfural concentration for
evaluating the critical furfural concentrations (Im ) for the
biomass production phase (lled circles) and the xylitol
production phase (open circles).
Fig. 3 Biomass specic rate of furfural consumption (qI )
versus initial concentration of furfural (I). Filled symbols are
for the biomass production phase (qmax I = 0.447 g g1 h1 ;
KF = 0.706 g L1 ; R2 = 0.9318). Open symbols are for the
xylitol production phase (qmax I = 0.254 g g1 h1 ;
1 2
KF = 1.169 g L ; R = 0.9794).
growth to occur during the xylitol production phase with
xylose as the only substrate, some of the xylitol formed must
be converted to xylulose via the action of NAD+ -dependent
xylitol dehydrogenase. In the presence of furfural, some of the
NAD+ is used in its metabolism to furoic acid (Horvth et al.,
2003), a less toxic metabolite compared to furfural. Therefore,
metabolism of furfural competes with the metabolism of xyl-
itol for the essential resource of NAD+ . The presence of xylitol
and, therefore, xylose, competitively inhibits the metabolism
of furfural (Matos et al., 2016).
These results were veried experimentally as shown in
Fig. 3. In Fig. 3, the specic consumption rate of furfural (qI )
is plotted against its concentration. A MichaelisMenten type
relationship is observed both during the biomass production
phase (glucose and xylose used as mixed substrates) and the
xylitol production phase (xylose used as the sole carbon sub-
strate). From the plots shown, during the biomass production
phase, the maximum specic consumption rate of furfural
(qmax
I ) was 0.4465 g g1 h1 and the saturation constant (KF )
was 0.7055 g L1 (Fig. 3). During the xylitol production phase,
the qmax
I value was 0.2536 g g1 h1 and KF was 1.169 g L1
(Fig. 3). Thus, under oxygen limiting conditions and with
xylose as the only substrate, qmax I was only 57% of its
value in the biomass production phase. Thus, furfural was
barely detoxied, or consumed, during the xylitol production
phase.
The fermentation models and the optimal estimated val-
ues of the relevant kinetic parameters are summarized in
Table 1. In the highly aerobic biomass production phase (agi-
tation speed of 250 min1 ; glucose = 10 g L1 ; xylose = 5 g L1 )
with the two cosubstrates supplied, a high value of the maxi-
mum specic growth rate (max = 0.148 h1 ; Table 1) occurred.
The saturation constant for the mixed substrate was high
(KS = 2.319 g L1 ; Table 1). The substrates were used mainly
to produce biomass and, therefore, the biomass yield on
substrate was high (YX/S = 0.327 g g1 ; Table 1). During rapid
growth, relatively little substrate is consumed for mainte-
nance metabolism and, therefore, the best t value of the
maintenance coefcient was nil (mS = 0 g g1 h1 ; Table 1).
Glucose was metabolized more readily compared to xylose
and a certain low amount of xylitol was produced in the
late exponential growth phase. As the operation was highly
aerobic, production of xylitol was not favored. Thus, the
value of the growth-associated ( = 0.0097 g g1 ; Table 1) and
nongrowth-associated ( = 0.0027 g g1 h1 ; Table 1) produc-
tion coefcients were fairly low.
In the xylitol production phase, the oxygen supply was
limited (agitation speed = 150 min1 ) and xylose was the sole
carbon source. During this phase barely any biomass was
produced and the inhibitory effect of furfural was strong.
The maximum specic growth rate (max = 0.0086 h1 ; Table 1)
was a mere 6% of its value in the biomass growth phase
and the biomass yield on substrate (YX/S = 0.228 g g1 ; Table 1)
was 70% of the yield during the growth phase. The main-
tenance coefcient was high (mS = 0.0014 g g1 h1 ; Table 1).
The oxygen-limited environment supported xylitol produc-
tion. Although the yield of xylitol on xylose (YP/xyl = 0.912 g g1 ;
Table 1) was no different than in the biomass production
phase, the rate of production was much higher. Thus, the value
of the parameter was more than 600-fold greater (Table 1)
compared to the biomass production phase.
The model predictions and the measured data are com-
pared in Fig. 4 for the biomass production phase and Fig. 5 for
the xylitol production phase. The comparisons are for batch
fermentations carried out at various initial concentrations of
furfural including control. Both during biomass production
and xylitol production, the predictions generally agreed well
with the measurements (Figs. 4AE, 5) of the key variables of
the biomass concentration (X), the substrate concentration
(S), the xylitol concentration (P) and furfural concentration
(I). With few exceptions (discussed later), the determination
coefcient (R2 ) values were generally high: 0.77790.9975 for
the biomass concentrations; 0.90190.9994 for substrate con-
centrations; 0.90350.9912 for furfural concentrations; and
0.78320.9990 for xylitol concentrations. The exceptions were
the data in Fig. 4F at an initial furfural concentration of
2.615 g L1 . This was the highest concentration of furfural
tested in the biomass growth phase. In this case the model
predictions did follow the broad trends of the data but the
determination coefcient values for several of the key mea-
surements were low (R2 0.66; Fig. 4F). Therefore, the model is
satisfactory for furfural concentrations of at least up to 2 g L1 .
To understand the nature of growth inhibition by furfural,
the values of the inhibition constant (KI ) were calculated for
 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140 135

Table 1 Kinetic parametersa of the fermentation process.

Model equation Parameter Biomass growth phaseb Xylitol production phasec

max S
 I
a
Cell growth dX
dt
= KS +S 1 Im X max (h1 ) 0.148 0.0086
KS (g L1 ) 2.319 0.430
Im (g L1 ) 10.183 7.148
a 1 1
      
Xylose consumption dS
dt
= 1
YX/S
dX
dt
+ mS X+ 1
YP/xyl
dP
dt
mS (g g1 h1 ) 0 0.0014

YX/S (g g1 ) 0.327 0.228

YP/xyl (g g1 ) 0.912 0.912

Xylitol production dP
dt
= ( + ) X (g g1 ) 0.0097 5.956
(g g1 h1 ) 0.0027 0.0018
qmax I
Furfural reduction dI
dt
= KF +I X
I qmax
I
(g g1 h1 ) 0.4465 0.2536
KF (g L1 ) 0.7055 1.169

a
Kinetic parameters were estimated by tting the model equations to the experimental data for the biomass growth and xylitol production
phases with and without furfural in shake ask cultures.
b
In biomass production phase glucose (10 g L1 ) and xylose (5 g L1 ) were used together in 500-mL Erlenmeyer asks with an initial working
volume of 250 mL of growth medium (initial pH = 4.0; temperature = 30 C; agitation speed = 250 min1 ).
c
Xylitol production phase occurred under oxygen-limited conditions with 30 g L1 xylose as the only carbon substrate in 500-mL Erlenmeyer
asks (initial working volume = 300 mL of growth medium; initial pH = 7.0; temperature = 30 C; agitation speed = 150 min1 ).

Table 2 The KI values estimated using the various inhibition models during the biomass production phase in shake
asks.
Inhibition model KI (g L1 ) R2 valuesa

X S P I

Competitive inhibition dX
dt
= maxI S X 0.253 0.8771 0.0408 0.9301 0.0308 0.8146 0.0228 0.9497 0.0275
KS 1+ +S
KI
max S

1+ I
dX KI
Uncompetitive inhibition = X 1.534 0.8770 0.0408 0.9301 0.0309 0.8145 0.0228 0.9494 0.0277
dt
 KS

+S
1+ I
KI
 max S

1+ I
dX KI
Noncompetitive inhibition dt
= KS +S X 1.788 0.8770 0.0408 0.9301 0.0309 0.8146 0.0228 0.9494 0.0277

X = biomass concentration; S = cosubstrate (glucose and xylose) concentration; P = xylitol concentration; and I = furfural concentration.
a
R2 values are shown as average standard derivation for the model ttings for initial furfural concentrations of 0.164, 0.462, 0.977 and
1.723 g L1 .

Table 3 The KI values estimated using the various inhibition models during the xylitol production phase in shake
asks.
Inhibition model KI (g L1 ) R2 valuesa

X S P I

Competitive inhibition dX
dt
= 
max S
X 0.220 0.9874 0.0145 0.9970 0.0022 0.9909 0.0044 0.9272 0.0124
KS 1+ I +S
KI

max S

1+ I
dX KI
Uncompetitive inhibition = X 0.807 0.9899 0.0107 0.9983 0.0011 0.9944 0.0028 0.9265 0.0130
dt
 KS
I
+S 
1+
KI
max S

1+ I
dX KI
Noncompetitive inhibition dt
= KS +S X 0.820 0.9899 0.0107 0.9983 0.0011 0.9944 0.0028 0.9265 0.0130

X = biomass concentration; S = concentration of xylose; P = xylitol concentration; and I = furfural concentration.

a
R2 values are shown as average standard derivation for the model ttings for initial furfural concentrations of 0.1, 0.2, 0.3. 0.5, 0.75, 1.0 and
2.0 g L1 .

three different kinds of inhibition kinetics as summarized in
Table 2 for the biomass growth phase and Table 3 for the xyl-
itol production phase. Irrespective of the inhibition model,
the KI value for the biomass production phase (Table 2) was
higher compared to the KI value estimated using the same
model in the xylitol production phase (Table 3). This indicated
that furfural was much more toxic to yeast during the xylitol
production phase compared to the biomass production phase.
During the biomass production phase when the sub-
strate concentration (S = 15 g L1 ) was higher than the KS
value ( = 2.319 g L1 ), furfural acted as a competitive inhibitor
as the KI -value ( = 0.253 g L1 ) was less than the I50 -value
136 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140

Fig. 4 Comparison of experimental (symbols) and model predicted (lines) fermentation proles for the biomass production
phase with the following initial concentrations of furfural (g L1 ): (A) 0; (B) 0.164; (C) 0.462; (D) 0.977; (E) 1.723; and (F) 2.615.
All fermentations used aerobic conditions at an agitation speed of 250 min1 (X, biomass concentration; S, concentrations of
cosubstrates (glucose and xylose); P, concentration of xylitol; and I, concentration of furfural).

( = 5.092 g L1 ). Similarly, during the early stages of the
xylitol production phase, furfural likely acted as a compet-
itive inhibitor of growth because the xylose concentration
( = 30 g L1 ) was higher than the KS value ( = 0.430 g L1 )
and, therefore, the KI -value ( = 0.220 g L1 ) was less than I50
( = 3.574 g L1 ).
3.3. Effects of agitation speed on xylitol production in
shake ask cultures
Xylitol production in shake asks was compared at agitation
speeds of 110 and 250 min1 and with the data obtained earlier
at 150 min1 . Xylitol production was carried out using 500-mL
Erlenmeyer asks with an initial working volume of 300 mL.
The initial xylose concentration was 30 g L1 and furfural was
added at an initial concentration of 0.3 g L1 . The initial pH
was 7.0 and the incubation temperature was 30 C.
The production kinetic parameters for various agitation
speeds are summarized in Table 4. A high agitation speed of
250 min1 (i.e. an initial kL a 17.22 h1 ; Eq. (1)(3)) resulted
in the highest yield (YP/xyl = 0.536 g g1 ) and productivity
(QP = 0.340 g L1 h1 ) of xylitol as a consequence of a relatively
improved supply of oxygen. Although too high a concentra-
tion of dissolved oxygen is not wanted for xylitol production,
a certain amount of oxygen is necessary. Oxygen transfer
is improved by increasing the kL a value. At the two lower
agitation speeds the kL a values were low (kL a 1.24 h1 at
110 min1 and 2.74 h1 at 150 min1 ).
 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140 137

Fig. 5 Comparison of experimental (symbols) and model predicted (lines) fermentation proles for the xylitol production
phase with the following initial concentrations of furfural (g L1 ): (A) 0; (B) 0.1; (C) 0.2; (D) 0.3; (E) 0.5; (F) 0.75; (G) 1.0; and (H)
2.0. All fermentations used oxygen limited conditions at an agitation speed of 150 min1 (X, biomass concentration; S,
concentration of xylose; P, concentration of xylitol; and I, concentration of furfural).

The effect of dissolved oxygen on the culture could be correlate with the agitation speed (n) of the shaker platform
empirically modeled by modifying Eq. (5) as follows: (Fig. 7), as follows:

max S I
a
I = 1 KO (17)
KS + S Im KO = 8.4 103 n 0.2363 (18)

where the empirical coefcient KO was used as a tting param-

eter. As shown in Fig. 6, the measured data and the model
predictions for xylitol concentration (initial furfural concen-
tration = 0.3 g L1 ; oxygen limiting conditions with Erlenmeyer
ask agitation speeds of 110, 150 and 250 min1 ) agreed
closely. The best t values of the parameter KO were found to
The correlation coefcient for the above equation was 0.999.
The range of agitation speeds spanned by the above equation
corresponded to an approximate range of the initial kL a values
of 1.2417.22 h1 in 500 mL Erlenmeyer asks with an initial
working volume of 300 mL.
138 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140

Fig. 6 Comparison of experimental (symbols) and model predicted (lines) fermentation proles for the xylitol production
phase at the following combinations of agitation speeds (n) and KO values: (A) n = 110 min1 , KO = 0.705; (B) n = 150 min1 ,
KO = 1.0; and (C) n = 250 min1 , KO = 1.871. All experiments were done in 500 mL Erlenmeyer asks with furfural added at an
initial concentration of 0.3 g L1 . The initial working volume was 300 mL (X, Biomass concentration; S, concentration of
xylose; P, concentration of xylitol; and I, concentration of furfural).

3.4. Model validation
The model was validated against experimental data not previ-
ously used in model development. For this, batch experiments
were carried out in 500 mL Erlenmeyer asks, as above, but at
an agitation speed of 290 min1 . This agitation speed corre-
sponded to an estimated initial kL a value of 27 h1 that had
been previously reported to be optimal for xylitol production
with a different yeast (Candida guilliermondii FTI 20037; Silva
et al., 1997). The initial working volume was 300 mL. The initial
concentration of furfural was 0.3 g L1 . The model equations
(Eqs. (4), (11)(13) and (17)) with the earlier established kinetic
parameters shown in Table 1 were used to predict the concen-
trations of biomass, xylose, xylitol and furfural during xylitol
production. The measured data and the model predictions
are compared in Fig. 8. The predicted biomass concentrations
 food and bioproducts processing 1 0 5 ( 2 0 1 7 ) 129140 139

Table 4 Kinetic parameters of xylitol production at

Biomass (X)
various shaking speeds.a 30 Xylose (S) 0.6
Xylitol (P)
Kinetic parameter Shaking speed (min1 )b Furfural (I)
25 0.5

110 150 250 20 0.4

Volumetric rates (g L1 h1 )
15 0.3
QX 0.003 0.001 0.029 0.003 0.046 0.009
QS 0.301 0.053 0.416 0.010 0.635 0.007 10 0.2
QP 0.132 0.046 0.198 0.007 0.340 0.031
5 0.1
Specic rates
 (h1 ) 0.001 0.000 0.007 0.001 0.012 0.002 0 0.0
qS (g g1 h1 ) 0.085 0.002 0.106 0.004 0.164 0.002 0 4 8 12 16 20 24 28 32 36 40
qP (g g1 h1 ) 0.036 0.006 0.050 0.002 0.088 0.010

Yields (g g1 )
YX/S 0.010 0.002 0.069 0.005 0.073 0.014
Fig. 8 The model validation. Comparison of the measured
YP/xyl 0.430 0.076 0.476 0.007 0.536 0.054 data (symbols) with the model predictions (lines) for xylitol
production phase. The agitation speed was 290 min1 . The
a
The experiments were carried out in 500-mL Erlenmeyer asks KO ( = 2.2) used for verication had been calculated with Eq.
using xylose (30 g L1 ) as sole substrate and furfural (0.3 g L1 ). The
(18) (Fig. 7). The data shown are average values of two
initial working volume was 300 mL. The temperature was 30 C
experiments done in parallel in 500 mL Erlenmeyer asks
and the initial pH was 7.0.
b
The estimated initial kL a values (see Eqs. (1)(3)) were as follows: with an initial furfural concentration of 0.3 g L1 and an
1.24 h1 at agitation speed of 110 min1 ; 2.74 h1 at agitation speed initial working volume of 300 mL (X, biomass concentration
of 150 min1 ; and 17.22 h1 at agitation speed of 250 min1 . (open circles); S, concentration of xylose (open triangles
up); P, concentration of xylitol (open squares); and I,
concentration of furfural (lled triangles down)).

published (Mohamad et al., 2016; Tochampa et al., 2005), none

consider the effects of furfural or relate to C. magnoliae.

4. Conclusion

Furfural was a competitive inhibitor of growth of the yeast C.

magnoliae TISTR 5663 both during the highly aerobic biomass
generation phase and the oxygen-limited xylitol production
phase. A mathematical model was shown to predict well the
biomass growth proles, the substrate consumption proles,
the xylitol production proles and furfural consumption pro-
les in both phases.

Fig. 7 Plot of KO versus agitation speed. The regression

Acknowledgements
coefcient for the line shown was 0.9988.

The authors would like to thank the Higher Education

after the rst 8-h were a little low, but otherwise the predic-
tions agreed well with the measured data (Fig. 8). In summary,
the model was satisfactorily validated against independent
data.
For the data in Fig. 8, the volumetric and specic
rates of xylose consumption were 0.862 0.008 g L1 h1
and 0.199 0.012 g g1 h1 , respectively. The volumet-
ric and the specic rates of biomass production were
0.099 0.010 g L1 h1 and 0.0228 0.003 h1 , respectively.
The biomass yield on xylose was 0.114 0.011 g g1 . All
these values were substantially higher compared to the data
obtained at a lower agitation speed of 250 min1 . A sufciently
high supply of oxygen assured a sufciency of NAD+ as this
coenzyme is produced via the oxygen-dependent respiratory
pathway. A good supply of NAD+ of course meant a chan-
neling of more of the xylitol to xylulose and on to biomass
production. Therefore, the biomass specic production rate of
the excreted xylitol ( = 0.086 0.006 g g1 h1 ) and the xylitol
yield on xylose ( = 0.433 0.002 g g1 ) were both reduced (Fig. 8)
compared to the shake ask cultures agitated at 250 min1 .
Although other models of xylitol fermentation have been
Research Promotion and National Research University Project
of Thailand, Ofce of the Higher Education Commission, and
the Kasetsart University Research and Development Institute
(KURDI), Bangkok, Thailand, for nancial support and the
Department of Biotechnology, Kasetsart University, for facil-
itating this research.
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