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Industrial Crops and Products 56 (2014) 160165

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Pilot scale simultaneous saccharication and fermentation at very

high gravity of cassava our for ethanol production
Chinh-Nghia Nguyen, Thanh-Mai Le, Son Chu-Ky
Department of Food Technology, School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet, Hai Ba Trung,
Hanoi 10000, Viet Nam

a r t i c l e

i n f o

Article history:
Received 30 August 2013
Received in revised form 5 February 2014
Accepted 8 February 2014
Simultaneous saccharication and
fermentation (SSF)
Very high gravity (VHG)
Cassava our

a b s t r a c t
We developed a simultaneous saccharication and fermentation (SSF) process of cassava our at very
high gravity (VHG). Cassava our (CF) was dissolved in water to reach 315.4 g/l dry matter, and then the
mixture was liqueed at 80 C for 90 min by using alpha-amylase (3532 AAU/kg CF) and beta-glucanase
(2812 U/kg CF). SSF of liqueed mash of cassava was performed at 30 C with the simultaneous addition of
two glucoamylases (Distillase ASP at 540 GAU/kg CF and Amigase Mega L at 0.035% w/w), active dry yeast
(1.5 107 cells/l), urea (12 mM) and KH2 PO4 (4 mM). Under these conditions, the SSF process nished
after 72 h. The ethanol content achieved 17.2% v/v corresponding to 86.1% of the theoretical ethanol yield
at lab scale and decreased to 16.5% v/v corresponding to 83.6% of the theoretical ethanol yield at pilot
scale. Therefore, the SSF of cassava our under VHG condition could have a great potential for the ethanol
industry in Vietnam and South East Asia.
2014 Elsevier B.V. All rights reserved.

1. Introduction
According to the increasing price of oil, bio-ethanol is known
as an ideal candidate to replace the role of fossil fuel. Thus, the
research on this renewable source becomes growingly important
for humans, especially in terms of improving the productivity, the
efciency and decreasing production cost. In Vietnam and in South
East Asia, cassava is considered an attractive raw material for bioethanol production thanks to the following advantages: (i) the ease
of plantation in various soil types and climate conditions; (ii) a
very low input and investment for planting; (iii) all year round
availability of feedstock in the form of fresh roots and dry chips;
(iv) a high starch-containing raw materials and a lower proportion
of bers (Sriroth et al., 2007). Indeed, the Vietnamese Ministry of
Industry and Trade declared that bio-fuel production will achieve
1.8 million tons in 2025, which accounts for 5% of countrys demand
(Ministry of Industry and Trade, 2007b). Moreover, the government
also adapted the policy to improve the beverage ethanol industry
in Vietnam. By the development strategy of beverage ethanol production in Vietnam (Ministry of Industry and Trade, 2007a), ethanol
industry will produce 188 million liters ethanol for food industry
in 2025. Overall, the beverage and bio-ethanol industry has a great
potential in Vietnam in the future.

Corresponding author. Tel.: +84 4 3868 0119; fax: +84 4 3868 2470.
E-mail addresses:, (S. Chu-Ky).
0926-6690/ 2014 Elsevier B.V. All rights reserved.

Besides the conventional process of ethanol production, simultaneous saccharication and fermentation (SSF) process has been
widely used in the world, but only recently introduced to Vietnam
in order to augment ethanol yield and shorten time production.
Indeed, after liquefaction by alpha-amylase, glucoamylase is added
to the slurry, concomitantly with yeasts, and the SSF is conducted
in a single reactor. The presence of yeast along with enzymes minimizes the sugar accumulation in the bioreactor. Moreover, since
the sugar produced during starch or cellulosic breakdown slows
down alpha-amylase action, higher yields and concentrations of
ethanol are possible using SSF (Das Neves, 2006; Klasson et al.,
2013; Molaverdi et al., 2013; Scordia et al., 2013; Wang et al., 2013;
Yingling et al., 2011a,b). The SSF process has been successfully carried out on different substrates such as ax shive (Klasson et al.,
2013), sweet sorghum stalk (Molaverdi et al., 2013), giant reed
(Scordia et al., 2013), sweet sorghum bagasse (Wang et al., 2013),
potato tubers (Srichuwong et al., 2009) and cassava (Chu-Ky et al.,
2009; Yingling et al., 2011b). Therefore, it is of interest to improve
the efciency of the SSF process in the ethanol industry in Vietnam.
Very high gravity (VHG) technology has been introduced to
increase the volumetric productivity and the cost effectiveness
of the SSF process. In VHG technology, mash preparation contains at minimum of 270 g/l dry matter (Bayrock and Ingledew,
2001). This technology has a great deal of advantages in ethanol
production: (i) increasing plant capacity and reduction in capital
costs; (ii) increasing plant efciency; (iii) reducing risk of contaminating bacteria (Thomas et al., 1996; Yingling et al., 2011a,b).

C.-N. Nguyen et al. / Industrial Crops and Products 56 (2014) 160165


Table 1
Characterization of the enzyme products used in this work.

Enzymes products


Optimal pH

Optimal temperature ( C)



Spezyme Alpha
Optimash TBG
Distillase ASP
Amigase Mega L




13,775 AAU/ga
5,625 U/gb
580 GAU/gc

AAU: Alpha Amylase Unit dened by Dupont (One AAU unit of bacterial alpha-amylase activity is the amount of enzyme required to hydrolyze 10 mg of starch per minute
under specied conditions).
U: Unit dened by Dupont (one unit of beta-glucanase activity is dened as the quantity of enzyme which produces reducing sugars equivalent to 1 mol of dextrose
per minute from barley beta-glucan under standard assay conditions).
GAU: GlucoAmylase Unit dened by Dupont (One Glucoamylase Unit (GAU) is the amount of enzyme that liberates 1 g of reducing sugars calculated as glucose per hour
from soluble starch substrate under the conditions of the assay).

Nevertheless, VHG technology causes also some inconvenience,

including the high viscosity of starch paste after liquefaction,
which leads to the resistance to solidliquid separation, difculties in handling process, incomplete hydrolysis of starch to
fermentable sugars and lower fermentation efciency (Ingledew
et al., 1999; Srikanta et al., 1992). Therefore, the success of its
application depends on the preparation of mash with low viscosity. For instance, in order to reduce starch pastes viscosity,
sweet potato was pretreated in a VHG process by using cell-wall
degrading enzymes such as cellulases, pectinase, hemi-cellulases
and viscosity reduction enzyme (xylanase). As a result, the ethanol
yield was achieved approximately 90% of the theoretical ethanol
yield (Srichuwong et al., 2009; Zhang et al., 2010, 2011). Thomas
et al. (1993) reported that in VHG (dissolved solids 300 g/l) of wheat
mash fermentation at 20 C for 200 h, maximal nal ethanol concentration of 23.8% v/v was obtained.
In another approach to VHG technology with cassava, optimization has been applied to study the effects of some key factors
that inuence ethanol production such as gravity, particle size, initial pH, liquefaction and fermentation temperature, liquefaction
time and enzyme concentration. Under optimized conditions, high
ethanol concentration (greater than 15%) and high starch utilization ratio (c.a. 90%) were obtained (Yingling et al., 2011b). However,
the investigation on VHG technology with cassava at a larger scale
than that of laboratory has still been limited.
In this work, our approach is to develop cost-effective ethanol
processes which are based on: (i) decreasing energy consumed by
utilizing enzymes which are capable of hydrolyzing raw starch at
lower temperatures; (ii) saving equipment investment and increasing ethanol yield by using SSF process of cassava our under VHG
condition. This work aimed to develop SSF processes under VHG
condition of cassava our at lab and pilot scales for ethanol production.
2. Materials and methods
2.1. Microorganism
Commercial active dry yeast Saccharomyces cerevisiae (Ethanol
Red), kindly provided by Fermentis (France), was used in this study.
Dry yeast was hydrated in tap water at 38 C for 20 min prior to
addition to the liqueed mash of cassava our.
2.2. Materials
Cassava our was obtained in Tuyen Quang province (North
Vietnam). After thoroughly dried, cassava chips were ground into
cassava our to the size minor than 0.3 mm, and stored at dry and
cool place in the lab. Starch content of the cassava our used in this
work was 77 1% and its humidity was 11 1%.
Different kinds of commercial enzyme products kindly provided
by Dupont (previously known as GenencorA Danisco Division)

were used in this work including Spezyme Alpha (containing

alpha-amylase from Bacillus licheniformis), Optimash TBG (containing beta-glucanase from Talaromyces emersonii) and Distillase
ASP (containing glucoamylase from Bacillus licheniformis and Trichoderma reesei). Amigase Mega L (containing glucoamylase from
Aspergillus niger) was provided by DSM Food Specialties Beverage Ingredients. Properties of these enzyme products are presented
in Table 1.
2.3. Simultaneous saccharication and fermentation (SSF) at lab
Three SSF processes at VHG were developed in this work (Fig. 1).
Cassava our (CF) was mixed with tap water in 2-l fermentor to
achieve a concentration of 315.4 g/l dry solid in a nal volume of
1 l. For all three investigated processes, the liquefaction step was
conducted at 80 C and stirred at 200 rpm for 90 min at pH 5.5. After
liquefaction, the mash was cooled to room temperature (30 C)
before subsequent SSF. The SSF of liqueed cassava mash was performed at 30 C in a 2-l fermentor, with the simultaneous addition
of glucoamylase, active dry yeast (Ethanol Red at 1.5 107 cells/ml),
urea (12 mM) and KH2 PO4 (4 mM). During the rst 8 h of SSF, the
fermentation broth was agitated every hour for 5 min at 120 rpm to
ensure homogenization. After this period, the SSF was conducted
under static condition and nished after 72 h. In our work, three
SSF processes were performed and differentiated as follows:
- SSF1 process: alpha-amylase (Spezyme Alpha) at the dosage of
3,532 AAU/kg CF was added to the cassava slurry under VHG
condition, and glucoamylase (Distillase ASP) at the dosage of
540 GAU/kg CF was added to conduct SSF.
- SSF2 process was similar to SSF1 process with only one modication as follows: additional beta-glucanase (Optimash TBG) at
the dosage of 2,812 U/kg CF was added to the cassava slurry under
VHG condition during liquefaction to reduce viscosity of liqueed
mash of cassava.
- For SSF3 process, both alpha-amylase (Spezyme Alpha) at
3,532 AAU/kg CF and beta-glucanase (Optimash TBG) at
2,812 U/kg CF were added for liquefaction. For SSF, besides
using glucoamylase (Distillase ASP) at 540 GAU/kg CF, additional
glucoamylase (Amigase Mega L) at 0.035% w/w was added to
improve the efcacy of hydrolyzing residual starch in the slurry.
2.4. Simultaneous saccharication and fermentation (SSF) at
pilot scale
The SSF under VHG condition was upgraded to the pilot scale
based on the results obtained with SSF3 process which was conducted at the lab scale as described in Section 2.3. The pilot scale
experiment was carried out in a total volume of 100 l using a double jacket reactor (200 l) for liquefaction and a fermentor (200 l) for
SSF, respectively. As the same for SSF3 process, both alpha-amylase


C.-N. Nguyen et al. / Industrial Crops and Products 56 (2014) 160165

Cassava flour

Tap water

Mixture of
(315.4 g/L)
Spezyme Alpha
(3,532 AAU/kg

Liquefaction at
800C for 90 min


Distillase ASP
(540 GAU/kg


Optimash TBG
(2,812 U/kg

Saccharification and
Fermentation (SSF)
at 300C for 72 h

Hydrated in
water at 380C
for 20 min

Amigase Mega L
(0.035 % w/w)

Ethanol Red
(1.5x107 cells/ml)

(4 mM)



Fig. 1. Three investigated SSF processes at VHG of cassava our for ethanol production: SSF1 process: Spezyme Alpha + Distillase ASP; SSF2 process: Spezyme Alpha + Distillase
ASP + Optimash TBG; SSF3 process: Spezyme Alpha + Distillase ASP + Optimash TBG + Amigase Mega L.

(Spezyme Alpha) at 3,532 AAU/kg CF and beta-glucanse (Optimash

TBG) at 2,812 U/kg CF were added for liquefaction, For SSF, two glucoamylases in Distillase ASP at 540 GAU/kg CF and in Amigase Mega
L at 0.035% w/w were added to the liqueed mash to improve the
efcacy of hydrolyzing residual starch. The SSF under VHG condition at pilot scale was performed at 30 C. During the rst 8 h of
SSF, the fermentation broth was agitated every hour for 5 min at
120 rpm to ensure homogenization. After this period, the SSF was
conducted under static condition and nished after 72 h.

2.5. Analytical procedures

To measure reducing sugar, fermentation beer was ltrated,
then reducing sugar was determined by using the DNS (3,5-dinitro
salicylic acid) method (Miller, 1959). Residual sugar was measured
by the same method after acid hydrolysis (HCl 2% for 120 min at
100 C) of the fermentation beer. Ethanol was distilled from fermentation beer, and then ethanol concentration was determined
by an ethanol ebulliometer (Dujardin-Salleron, France). Maltose,
glucose, acetic acid and lactic acid were determined by using
High Performance Liquid Chromatography (HPLC) (Agilent 1200
series, Agilent Technologies, Germany) equipped with an Aminex
HPX 87H column (Bio-Rad, Hercules, USA) at a pressure of 52 bar
with H2 SO4 10 mM as eluent according to the providers instruction. All reagents used for HPLC analysis were of analytical grade.

Concentrations were calculated by means of standard curves

related to individual concentration to peak area. The viscosity of
starch slurry after liquefaction was measured by using Elcometer
RV1 Rotational Viscometers (Elcometer, UK) with rotation speeds
at 100 rpm and 50 rpm according to the providers instruction.
Dextrose Equivalent (DE) values were estimated as DE = [reducing
sugars] 100/[total dry matter] (Shariffa et al., 2009).
2.6. Statistical analysis
The mean values and standard deviation were calculated from
three independent experiments. The signicance of the difference
between the mean values was determined using the analysis of
variance (ANOVA). The condence interval for a difference in the
means was set at 95% (P 0.05) for all comparisons.
3. Results and discussion
3.1. Liquefaction
Liquefaction step aimed to convert the starch into maltodextrins
at high temperature and to reduce the viscosity of starch slurry
by using thermo-stable alpha-amylases. In VHG process, starch
slurry viscosity during liquefaction plays an important role, which
can decrease enzyme efcacy in starch hydrolysis, thus reducing

C.-N. Nguyen et al. / Industrial Crops and Products 56 (2014) 160165

Table 2
Viscosity of liqueed mash of cassava our after liquefaction in SSF1 and SSF2
Mash viscosity (cp)

SSF1 process (with addition of Spezyme Alpha)

SSF2 process (with addition of both Spezyme
Alpha and Optimash TBG)

Rotation speed (rpm)



24 h

36 h

48 h

60 h

72 h

50 rpm




Maltose (g/l)
Glucose (g/l)
Lactic acid (g/l)
Ethanol (% v/v)












Sugar concentration (g/l)

Ethanol concentration (% v/v)




Time (hours)


Table 3
Evolutions of the concentration of maltose, glucose, lactic acid and ethanol during
SSF3 process.

100 rpm

the ethanol yield. Therefore, decreasing starch-paste viscosity is

prerequisite for conducting ethanol production at VHG. Different
methods have been previously used to resolve this problem. Indeed,
enzymes decreasing viscosity such as cellulase, hemi-cellulase,
pectinase (Srichuwong et al., 2009) or xylanase (Zhang et al., 2010,
2011) were added into the mash. In another work, Yingling et al.
(2011b) used gelatinization step with different enzyme doses, followed by autoclaving at 121 C for 15 min to completely breakdown
the starch and to avoid contamination. In the conventional method,
the liquefaction step is normally carried out at the boiling temperature (roughly 100 C), which demands a great deal of energy for
heating and maintaining the mash at the boiling temperature. In
our processes when the liquefaction step was performed at signicantly lower temperature (only 80 C), the energy was accordingly
saved that demonstrated one of the advantages of the SSF process
(Ingledew, 2009; Thomas et al., 1996).
In SSF1 process, an alpha-amylase, a liquefying enzyme
(Spezyme Alpha), was used in liquefaction step whereas a betaglucanase (Optimash TBG) was added in SSF2 process for viscosity
reduction. The efciency of this beta-glucanase was measured by
a reduction in starch slurry viscosity and improvement of ethanol
yield. The combination of two enzymes alpha-amylase and betaglucanase in SSF2 process signicantly decreased starch slurry
viscosity compared to SSF1 process where only one alpha-amylase
was added (270 cp compared to 340 cp at rotation speed of 100 rpm,
respectively) (Table 2). However, DE (Dextrose Equivalent) values
of these processes after liquefaction were not signicantly different
(12.2 and 11.9, respectively). Moreover, in SSF2 process, residual
sugar was lower and ethanol concentration was higher than those
in SSF1 process after 72 h fermentation (52.9 g/l and 14.8% v/v compared to 74.6 g/l and 13.6% v/v, respectively) (Fig. 2). According to
these results, it is likely that the use of beta-glucanase could lead
to a positive effect (viscosity reduction) on this technology. However, in SSF2 process, an ethanol concentration of 14.8% v/v was
insufciently high, which would require further improvement.



SSF1 ethanol concentration (% v/v)

SSF2 ethanol concentration (% v/v)

SSF3 ethanol concentration (% v/v)

SSF1 residual sugar (g/l)

SSF2 residual sugar (g/l)

SSF3 residual sugar (g/l)

Fig. 2. Evolutions of residual sugar and ethanol concentration of the three investigated SSF processes.

In a previous work, Srichuwong et al. (2009) studied the VHG

process of sweet potato at 28% dry matter. In order to decrease mash
viscosity, cell-wall degrading enzymes (cellulase, hemi-cellulase
and pectinase) have been used to decrease mash viscosity in a pretreatment step at 50 C for 50 min. As a result, the mash viscosity
was reduced from 300 cp to approximately 50 cp. In another work,
Zhang et al. (2010) added xylanase to the liqueed sweet potato
mash to reduce mash viscosity. After the treatment at 30 C for
90 min, the viscosity of mash decreased from 9,863.2 cp to 498.1 cp.
3.2. Simultaneous saccharication and fermentation (SSF)
In order to improve the ethanol yield in this study, another
glucoamylase (Amigase Mega L) was added to the mash to conduct SSF. In general, traditional brewing methods permit only 75
to 80% hydrolysis of starch present in the grain. According to the
producer, this glucoamylase permits total hydrolysis of dextrin to
fermentable glucose, for all types of starch. Indeed, in SSF3 process,
after 72 h of fermentation, ethanol concentration achieved 17.2%
v/v, which was equivalent to 86.1% of the theoretical ethanol yield,
while the residual sugar decreased to 17 g/l (Fig. 2). In comparison with the results obtained in SSF2 process (14.8% v/v for ethanol
concentration and 52.9 g/l for residual sugar), a signicant improvement was obtained using an additional glucoamylase (Amigase
Mega L). Moreover, with the exception of the rst 24 h of fermentation, it is noted that glucose, maltose and lactic acid concentrations
remained at low levels (Table 3), which demonstrated the advantages of the SSF process that a low concentration of reducing sugar
could decrease the osmotic pressure on yeast and reduce risk of
contamination (Thomas et al., 1996).
Srichuwong et al. (2009) have developed the VHG process of
sweet potato at dry matter of 28%. After 61.5 h, ethanol concentration of 16.6% v/v was achieved, which was equivalent to 89.7%
of theoretical yield. Zhang et al. (2011) reported that ethanol concentration of 16.3% v/v corresponding to 91.4% of the theoretical
ethanol yield achieved with an initial dry matter of 28%. In their
work, a pretreatment was carried out before fermentation to reduce
mash viscosity. In another study, Yingling et al. (2011b) identied gravity, particle size, initial pH, and fermentation temperature
as key factors that signicantly increased nal ethanol concentration for VHG processes of cassava. Moreover, Yingling et al.
(2011a) used the response surface methodology to study the VHG
of cassava mash at 33% dry matter. After model validation, the maximum ethanol concentration obtained at the optimal conditions of
hydrolysis was 17.96 0.63% while the maximum starch utilization ratio was 94.52 0.35%. In their research, the cassava mash
was gelatinized at 80 C for 15 min and liqueed at 75 to 77 C for
103108 min with the utilization of a high dosage of alpha-amylase.
In our work, no pretreatment step was performed. Different
enzymes were used in order to decrease viscosity during liquefaction. In our best process (SSF3 process), ethanol concentration
was 17.2% v/v corresponding to 86.1% of the theoretical ethanol
yield, which was similar to the average yield achieved in the current
ethanol factories in Vietnam. Therefore, SSF3 process was chosen
to scale up to pilot scale to examine its efciency. The ethanol productivities (which are equally important from an industrial point of

C.-N. Nguyen et al. / Industrial Crops and Products 56 (2014) 160165
















Sugar concentration (g/l)

Ethanol concentration (% v/v)




Time (h)


viscosity reduction. At lab scale, at dry matter of 28%, an ethanol

concentration of 16.3% v/v was achieved, which was equivalent to
an ethanol yield of 90%. However, in that work, the dry matter was
decreased to only 24% at pilot scale to maintain the ethanol yield at
90%. In our work, the dry matter has remained as high as that at pilot
scale (315.4 g/l or approximately 30% dry matter) when the process
was scaled up at pilot scale. Since the results at pilot scale were not
similar to those at lab scale, the process needs to be optimized to
improve the process efciency at pilot scale.
4. Conclusion


view) of the three investigated SSF processes were also calculated

and equal to 1.49, 1.62 and 1.88 g/l/h ethanol for SSF1, SSF2 and
SSF3 processes, respectively. The values of ethanol productivity of
SSF1, SSF2 and SSF3 processes were respectively 30, 41 and 64%
higher than that of the conventional ethanol process (1.15 g/l/h)
(Chu-Ky et al., 2009). These results were in agreement with those
obtained by Ingledew (2009), Srichuwong et al. (2009), Yingling
et al. (2011a,b) and emphasized the great advantage of VHG technology, namely increased ethanol productivity.

In this work, we have successfully developed SSF processes

under VHG condition (315.4 g/l dry matter) of cassava for ethanol
production at lab scale and pilot scale (100 l). The ethanol content achieved 17.2% v/v corresponding to 86.1% of the theoretical
ethanol yield at lab scale and decreased to 16.5% v/v corresponding to 83.6% of the theoretical ethanol yield at pilot scale. We
showed that combination of four enzymes (alpha-amylase, betaglucanase and two glucoamylases) led to a signicant reduction in
mash viscosity and to an increased ethanol yield. However, when
the process was scaled up to pilot scale, a decrease in ethanol yield
was observed (from 86.1% at lab scale to 83.6% at pilot scale, respectively). It is suggested that in order to improve the ethanol yield
at pilot scale, a pretreatment with additional viscosity-reducing
enzymes should be carried out. In addition, yeast growth conditions
should be also optimized and controlled.

3.3. Scale up of SSF at VHG of cassava our at pilot scale


The main objective of scaling-up was to identify problems that

were not signicantly noticed at lab scale, and to verify the maintenance of ethanol yield after fermentation. According to the results
at lab scale, SSF3 process was chosen to be scaled up to pilot scale.
Fig. 3 shows the evolutions of ethanol concentration, reducing and
residual sugars during SSF at pilot scale. After 72 h of SSF, the
ethanol content reached 16.5% v/v, which corresponded to 83.6% of
the theoretical ethanol yield and to 1.81 g/l/h of ethanol productivity. These values of ethanol yield and productivity were lower than
those obtained at lab scale (17.2% v/v and 1.88 g/l/h, respectively).
However, the content of residual sugars at pilot scale (6.9 g/l) was
lower than that at the lab scale (17.0 g/l). This result could be
explained by the fact that the yeast could have used in excess of
fermentable sugar under aerobic condition for its growth at the
beginning of SSF. Hence, the increased biomass would lead to a
decrease in the ethanol yield. In our pilot experiment, due to a larger
volume (100 l) than that at lab scale, it needed a longer period of
time to cool down the mash and to transfer the mash from the liquefaction reactor to the fermentor. The speed and the duration of
agitation should have been too high and long for the SSF process
at pilot scale even though these values were identical as those at
lab scale. Therefore, more oxygen could have been taken up into
the fermentation beer and the time for yeast growth could have
been longer than usual and leading to an increased yeast biomass
(Ingledew, 2009). It is suggested that the yeast pitching rate, culturing and aeration condition needed to be optimized and to be
controlled to improve the process efciency for ethanol production at pilot scale. The investigation on the VHG process for ethanol
production from sweet potato showed that high viscosity caused
resistance to solid-liquid separation and lower fermentation efciency (Srichuwong et al., 2009). In another work conducted by
Zhang et al. (2011), the VHG process with sweet potato was performed with 20 to 28% dry matter. After liquefaction at 85 C in
the presence of liquefying enzymes, the SSF was carried out at
30 C with the addition of saccharifying enzymes and xylanase for

This work was supported by Ministry of Education and Training and Ministry of Science and Technology of Vietnam. We thank
Dupont, DSM and Fermentis for kindly providing us with enzymes
and yeast samples, respectively. We also thank Dr Nguyen TienThanh for his technical assistance in HPLC analysis and Dr Ho
Phu-Ha for her revision of the English text.

Ethanol concentration (% v/v)

Reducing sugar (g/l)

Residual sugar (g/l)

Fig. 3. Evolutions of residual sugar, reducing sugar and ethanol concentration of SSF
process at pilot scale.

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