food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85

Contents lists available at ScienceDirect

Food and Bioproducts Processing

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

Simultaneous liquefaction, saccharification and

fermentation at very high gravity of rice at pilot
scale for potable ethanol production and distillers
dried grains composition

Son Chu-Ky a,∗ , Thi-Hoan Pham a , Kim-Lien T. Bui a , Tien-Thanh Nguyen a ,

Kim-Dang Pham b , Hoai-Duc T. Nguyen a , Hong-Nga Luong a , Viet-Phu Tu a ,
Thanh-Hang Nguyen a , Phu-Ha Ho a , Thanh-Mai Le a
a School of Biotechnology and Food Technology (SBFT), Hanoi University of Science and Technology (HUST),
1 Dai Co Viet Hai Ba Trung, Hanoi 10000, Vietnam
b Faculty of Animal Science (FAS), Vietnam National University of Agriculture (VNUA), Gia Lam, Hanoi 10000,

Vietnam

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

Article history: In this work, a simultaneous liquefaction, saccharification, and fermentation (SLSF) process
Received 7 June 2015 at very high gravity (VHG) of broken rice for potable ethanol production was developed at
Received in revised form 30 pilot scale. The SLSF–VHG process was performed in a unique fermenter, at 30 ◦ C. Rice flour
September 2015 (RF) was dissolved in tap water to reach 311.5 g/l dry matter and then the mixture was simul-
Accepted 15 October 2015 taneousely liquefied, saccharified, and fermented. Thanks to the addition of a raw starch
Available online 24 October 2015 hydrolyzing enzyme containing a mixture of alpha-amylase and gluco-amylase (Stargen 002
at 991.8 GAU/kg RF), gluco-amylase (Amigase Mega L at 0.035% w/w), protease (Fermgen at
Keywords: 600 SAPU/kg RF), active dry yeast Saccharomyces cerevisiae (Red Ethanol at 3.5 × 107 cells/ml),
Simultaneous liquefaction KH2 PO4 (4.8 mM), and urea (16.0 mM). Under these conditions, the SLSF–VHG process fin-
Saccharification and Fermentation ished after 120 h and achieved an ethanol content of 17.6% v/v corresponding to 86.3% of
(SLSF) the theoretical ethanol yield. We scaled up this SLSF process at very high gravity at pilot
Very high gravity (VHG) scale (25 l) and achieved an ethanol content of 17.0% v/v corresponding to a yield of 83.2%
Rice of the theoretical ethanol yield. Rice-based distillers dried grains (DDG) was produced from
Potable ethanol the whole stillage of SLSF process at very high gravity by being plate-filtered and dried. The
Distillers dried grains obtained DDG had high contents of crude protein (47.5%) and fibers (15.8%). Our results sug-
gest, the SLSF under VHG condition of broken rice as well as the recovery of protein-rich
DDG could have a great potential for the ethanol and animal feeding industry in Vietnam.
© 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction ethanol production as it possesses many benefits. The major

rice consumers are food and drink industries, such as pasta,
In Vietnam, potable ethanol is widely produced from cane and bread industries, beer and other liquor distilleries, as well
sugar molasses, rice and cassava. Among these raw materi- as pharmaceutical industry (Gang et al., 2007). Over 20 years,
als, rice is particularly considered a suitable one for potable the total production of rice in Vietnam increased twofold and

∗
Corresponding author. Tel.: +84 4 3868 0119; fax: +84 4 3868 2470.
E-mail address: son.chuky@hust.edu.vn (S. Chu-Ky).
http://dx.doi.org/10.1016/j.fbp.2015.10.003
0960-3085/© 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
80 food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85

reached 43.4 million tons in 2013. Vietnam exported about The ethanol content achieved 17.2% and 16.5% v/v correspond-
8 million tons of rice annually (General-Statistic-Office-of- ing to 86.1% and 83.6% of the theoretical ethanol yield at lab
Vietnam, 2013). Besides being consumed as food, rice is often and pilot scales, respectively.
transformed into different products. One of these is ethanol In another work, Poonsrisawat et al. (2014) investigated
whose productivity will be 800 million liters in 2015. the viscosity reduction of cassava for ethanol fermentation at
Most ethanol distilleries in Vietnam use conventional pro- very high gravity by using cell wall degrading enzymes from
cesses for the production of potable ethanol which basically Aspergillus aculeatus. Cassava root mash was adjusted to 32%
involves an energy-consuming liquefaction (95–105 ◦ C), sep- (w/w) dry matter and was pretreated with 0.5% (w/w) viscosity
arate saccharification (60–62 ◦ C), and fermentation (30–32 ◦ C) reducing enzyme preparation and incubated at 45 ◦ C, pH 5.0
of starch slurry. Liquefaction and saccharification require for 2 h. The pretreated cassava mash was liquefied by 0.03%
the starch granules to be extensively gelatinized at high (w/w) thermostable ␣-amylase (Liquozyme SCDS) in a biore-
temperature. This is an energy-intensive process requir- actor at pH 5.5–6.0, 85 ◦ C for 2 h. The liquefied mash was then
ing the addition of heat energy to starch granule slur- simultaneously saccharified by 0.06% (w/w) gluco-amylase
ries, until the gelatinization temperature of the starch (Spirizyme Fuel) or low-temp amylase (in-house enzyme pre-
is exceeded. The whole process requires a high-energy pared from Aspergillus strain) was added at 0.35 IU/g of raw
input, important investment thus increases the production starch and fermented by Saccharomyces cerevisiae at pH 4.5,
cost. 32 ◦ C for 96 h. The ethanol content reached 19.65% v/v cor-
With biotechnological advances in recent years, a new gen- responding to 87.55% yield with thermal process and only
eration of liquefying enzymes such as thermostable starch 17.54% v/v corresponding to 75.33% with non-thermal process,
hydrolyzing ␣-amylase is produced by a genetically modified respectively.
strain of Bacillus licheniformis. These enzymes can reach liq- In another research related to VHG technology with rice
uefaction step efficiently at lower temperature (Gang et al., flour, the optimized process was applied to study the effects
2007; Wang et al., 2007). New blends of enzymes produced of some key factors that influence ethanol production such
by controlled fermentation of genetically modified strains as gravity, particle size, initial pH, fermentation temperature,
of B. licheniformis and Trichoderma reesei are also available to time, and enzyme concentration. Under optimized conditions,
perform saccharification more effectively (Gang et al., 2009). high ethanol concentration (greater than 15%) and high starch
Stargen, capable of hydrolyzing raw starch, contains alpha- utilization ratio (c.a. 90%) were obtained (Yingling et al., 2011).
amylase and gluco-amylase synthetized by Aspergillus niger However, the investigation on VHG technology with broken
and Aspergillus kawachi. These enzymes are adsorbed on the rice at a larger scale than that of laboratory has still been
surface of starch grain and induce holes on this surface where limited.
glucose is released. The capability of hydrolyzing raw starch In ethanol industry, fermentation produces a co-product,
depends on the nature of starch. When the starch contains a so-called dried distiller’s grain with solubles (DDGS). Since
a high quantity of amylopectin, the enzymatic hydrolysis is only starch and sugars are converted into ethanol, non-
performed easily (Wang et al., 2007). fermentable components in cereal grains are concentrated by
Simultaneous Liquefaction, Saccharification, and Fermen- a factor of more than two in DDGS (Monceaux and Kuehner,
tation process (SLSF) or no-cook process has been recently 2009). Currently, the majority of the DDGS has been used as an
introduced to increase ethanol yield and to save energy and ingredient for livestock feed. In dry-grind processes, the fer-
investment cost (Gohel and Duan, 2012; Kelsall and Piggot, mentation beer is distilled and therefore ethanol is recovered.
2009). Alpha-amylase, gluco-amylase are added to the slurry, The non-volatile components then leave this step as a prod-
concomitantly with yeasts. The SLSF is conducted in a unique uct called whole stillage. The whole stillage contains the fiber,
bioreactor, at a unique pH and at ambient temperature. The fats, protein, other unfermented components of the grain,
presence of yeast along with enzymes minimizes the sugar and yeast cells. Whole stillage is usually centrifuged to pro-
accumulation in the vessel. Since the sugar is produced slowly duce a liquid fraction (thin stillage) and a solids fraction (wet
during starch breakdown, higher rates, yields and concentra- distillers’ grains). The remaining thin stillage is concentrated
tions of ethanol are possible with the use of SLSF (Robertson through multiple effect evaporators to produce syrup called
et al., 2006; Xu and Duan, 2010). condensed distillers’ solubles (CDS) (Monceaux and Kuehner,
A recent work conducted by Xu and Duan (2010) showed 2009). While wet distillers’ grains, syrup, or the combination of
that the use of new enzymes for ethanol production with- both (wet distillers’ grains with solubles, WDGS) can be sold
out heating at very high gravity was achievable with sorghum. as an animal feed, the combination of wet distillers’ grains
Indeed, the temperature control in combination with enzy- and syrup is often dried to produce dried distillers’ grains
matic hydrolysis using raw starch hydrolyzing enzyme with solubles (DDGS) in order to greatly lengthen its shelf-life.
(Stargen) could significantly improve the efficiency of fermen- Indeed, the composition of DDGS has been of great interest to
tation process. Ethanol reached up to 20% v/v after 90 h of researchers in the area of animal science, ethanol producers,
fermentation, with the use of commercial dry yeast and a and especially to the animal feeding industry as the major-
sorghum concentration up to 35% dry matter. ity of this has been sold as feed ingredients for livestock (Liu,
Nguyen et al. (2014) developed a simultaneous saccharifi- 2011).
cation and fermentation (SSF) process of cassava flour at very In this work, we aimed at developing SLSF processes of
high gravity (VHG). Indeed, cassava flour was resuspended broken rice at very high gravity (>300 g/l dry solid) either
in water to reach 311.5 g/l dry matter, and then the mix- at laboratory or pilot scales by utilizing enzymes capable of
ture was liquefied at 80 ◦ C for 90 min by using a mixture of hydrolyzing raw starch at ambient temperature. Other objec-
alpha-amylase and beta-glucanase. SSF of liquefied mash of tives were to recover rice-based distillers dried grains (DDG)
cassava was performed at 30 ◦ C with the simultaneous addi- obtained from ethanol by-products of SLSF-VHG process and
tion of gluco-amylases, active dry yeast, urea, and KH2 PO4 . to determine the main composition of rice-based DDG for ani-
Under these conditions, the SSF process finished after 72 h. mal feeding usage.
 food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85 81

Table 1 – Characteristics of enzyme products used in this work.

No. Enzyme products Nature Optimal pH Optimal temperature (◦ C) Activity

1 Stargen 002 Alpha-amylase and gluco-amylase 4.0–4.5 20–40 570 GAU/ga

2 Amigase Mega L Gluco-amylase 4.0–4.5 55–60 –
3 Fermgen Protease 4.0–5.0 28–35 1000 SAPU/gb
4 Optimash TBG Beta-glucanase 4.5–6.0 75–85 5625 U/gc

a
: GAU: Gluco-Amylase Unit defined by Dupont (one Gluco-amylase Unit (GAU) is the amount of enzyme that liberates one gram of reducing
sugars calculated as glucose per hour from soluble starch substrate under the conditions of the assay).
b
SAPU: Spectrophotometric Acid Protease Units defined by Dupont (one SAPU is the amount of enzyme activity that liberates one micromole
of tyrosine per minute from a casein substrate under conditions of the assay).
c
U: Unit defined by Dupont (one unit of beta-glucanase activity is defined as the quantity of enzyme which produces reducing sugars equivalent
to one micromole of dextrose per minute from barley beta-glucan under standard assay conditions).

2. Materials and methods with stirring at 50 rpm during the first 24 h. Four SLSF pro-
cesses were performed and differentiated as follows:
2.1. Microorganism
• SLSF1: alpha-amylase and gluco-amylase (Stargen 002) at
Commercial active dry yeast Saccharomyces cerevisiae (Red the dosage of 992 GAU/kg RF.
Ethanol), kindly provided by Leaf Technologies (France), was • SLSF2 was similar to SLSF1 with an addional protease (Fer-
used in this work. Active dry yeast was hydrated in tap water mgen) at the dosage of 600 SAPU/kg RF which was added to
at 35 ◦ C for 15 min prior the addition to the rice slurry. conduct SLSF.
• SLSF3 was similar to SLSF2 with an additional gluco-
2.2. Materials amylase (Amigase Mega L) at the dosage of 0.035% w/w,
which was added to conduct SLSF.
Broken rice was purchased in Yen My district, Thanh Hoa • SLSF4 was similar to SLSF3 with an additional beta-
province, Vietnam. Broken rice was ground into rice flour to glucanase (Optimash TBG) at the dosage of 3234 U/kg RF
the size minor than 0.3 mm, and stored at dry and cool place which was added to conduct SLSF.
at laboratory condition. Starch content of the rice flour used
in this work was 81 ± 1% and its humidity was 11 ± 1%. 2.4. Simultaneous liquefaction, saccharification and
Different commercial enzyme products provided by fermentation at very high gravity (VHG) at lab scale
Dupont (previously known as Genencor – A Danisco Division)
were used in this work including Stargen 002 (contain- In this part, due to the very high gravity condition of the rice
ing A. kawachi alpha-amylase expressed in T. reesei and slurry (311.5 g/l), a higher supplement of urea (16.0 mM) and
a gluco-amylase from T. reesei), Optimash TBG (containing KH2 PO4 (4.8 mM) and a higher dosage of yeast (Red Ethanol at
beta-glucanase from Talaromyces emersonii), and Fermgen (con- 3.50 × 107 cells/ml) were added to the rice slurry to better per-
taining protease from T. reesei). Amigase Mega L (containing form ethanol fermentation. Two SLSF processes at very high
gluco-amylase from A. niger) was provided by DSM – Food Spe- gravity at lab scale (311.5 g/l dry matter) (namely SLSF3–VHG
cialties – Beverage Ingredients. The main properties of these and SLSF4–VHG) were adapted from SLSF3 and SLSF4, respec-
enzyme products are presented in Table 1. tively, with some modifications as follows: addition KH2 PO4
(4.8 mM), higher dosage of urea (16.0 mM), and higher dosage
2.3. Simultaneous liquefaction, saccharification and of active dry yeast (Red Ethanol at 3.50 × 107 cells/ml).
fermentation (SLSF) at laboratory scale
2.5. Simultaneous liquefaction, saccharification and
Four SLSF processes developed in this work are shown in fermentation at very high gravity (SLSF–VHG) at pilot
Table 2. Indeed, rice flour (RF) was mixed with tap water scale
in a Sartorius 2-liter fermenter to achieve a concentra-
tion of 186.9 g/l dry solid in a final volume of 1 l. For all The SLSF at very high gravity was scaled up to the pilot scale
four investigated processes (SLSF1, 2, 3, 4), alpha-amylase, (namely SLSF3–VHG-p) based on the results obtained with
gluco-amylase, protease, beta-glucanase, active dry yeast Red SLSF3–VHG at lab scale as described in 2.4. The pilot scale
Ethanol (1.75 × 107 cells/ml), and urea (6.7 mM) were added experiment SLSF3–VHG-p was carried out in a total volume
simultaneously into the mixture. The SLSF was conducted of 25 l under the same conditions for SLSF3–VHG.

Table 2 – Differentiation for SLSF processes used in this work.

Ingredients used SLSF1 SLSF2 SLSF3 SLSF4 SLSF3–VHG SLSF4–VHG

Rice (g/l dry solid) 186.9 186.9 186.9 186.9 311.5 311.5
Stargen 002 (GAU/kg) 992 992 992 992 992 992
Fermgen (SAPU/kg) – 600 600 600 600 600
Amigase Mega L (% w/w) – – 0.035 0.035 0.035 0.035
Optimash TBG (U/kg) – – – 3234 – 3234
Yeast (cells/ml) 1.75 × 107 1.75 × 107 1.75 × 107 1.75 × 107 3.50 × 107 3.50 × 107
Urea (mM) 6.7 6.7 6.7 6.7 16 16
KH2 PO4 (mM) – – – – 4.8 4.8
82 food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85

2.6. Preparation of rice-based distillers dried grains

The distillers wet grains (DWG) was separated from the whole
stillage after SLSF at very high gravity by using plate filter.
Rice-based DWG (with moisture of 54%) was then dried in a
chamber dryer at 90 ◦ C for 1 h, then at 80 ◦ C for 4 h to obtain
rice-based distillers dried grains (DDG) which were ready for
compositional analysis.

2.7. Analytical procedures

Reducing sugar and total sugar were measured according to

the methods described by Le Thanh et al. (2007). Ethanol
was distilled from fermentation beer, and then ethanol
concentration was determined by using an ethanol ebul-
liometer (Dujardin-Salleron, France). Glucose, glycerol, lactic
acid, acetic acid, and ethanol were determined by using High
Performance Liquid Chromatography (HPLC) as described by
Nguyen et al. (2014). The viscosity of rice mash during SLSF
was measured by using Elcometer RV1 Rotational Viscome- Fig. 1 – Evolutions of residual sugar and ethanol
ter (Elcometer, UK) with rotation speed at 100 rpm (Nguyen concentration of the four investigated SLSF processes.
et al., 2014). Rice-based DDG were analyzed for moisture (AOAC
927.05), protein (AOAC 991.20), crude fibers (AOAC 993.21), fats as protease, gluco-amylase and beta-glucanase were added.
(AOAC 991.36), and ash (AOAC 930.30). Our results were in accordance with those obtained by Gohel
and Duan (2012) in which the ethanol content reached max-
2.8. Statistical analysis imally 12.07% v/v when 25% dry solid of broken rice (68.45%
starch) was treated with a combination of Stargen 002 and
The mean values and standard deviations were calculated Fermgen. Indeed, the use of acid fungal protease (Fermgen)
from three independent experiments. hydrolyzed the proteins present in the grains into peptides and
amino acids crucial for yeast growth. Furthermore, it has been
3. Results and discussion reported that protease plays a key role not only in hydrolyzing
the protein matrices in the kernel that binds the various frac-
3.1. Simultaneous liquefaction, saccharification and tions, which degrades “hard-to-hydrolyze” starch, but also in
fermentation (SLSF) at lab scale accelerating ethanol production rates and resulting in higher
ethanol yield for grain based substrates as compared to those
According to the recommendation of the manufacturer without protease (Duan et al., 2007). When more enzymes
(Dupont), Stargen 002 containing a mixture of alpha–amylase such as additional gluco-amylase and/or beta-glucanase were
and gluco-amylase at the dosage of 992 GAU/kg together with added, the ethanol content did not increase any more, indicat-
active dry yeast Red Ethanol (1.75 × 107 cells/ml), urea (6.7 mM) ing the ethanol yield could reach its limit. Nevertheless, the
were added to the rice slurry (at 186.9 g/l dry solid) to con- duration of both SLSF3 and SLSF4 decreased significantly and
duct SLSF1 (Table 2). SLSF1 finished after 144 h and achieved was around 72 h. It is interesting to note that we could reduce
an ethanol content of 10.6% v/v equivalent to a yield of 86.6%. SLSF duration significantly by 24 h, therefore increase produc-
However, the duration of SLSF was too long (about 144 h or tivity. The reduction in process duration could be explained
6 days). To increase the yield and to shorten the SLSF dura- by the fact that gluco-amylase from selected classical strain
tion, we adopted the SLSF process by using two enzyme of A. niger (in Amigase Mega L) permitted total hydrolysis
products which were a mixture of alpha-amylase and glu- of dextrins to fermentable glucose, from all types of starch,
coamylase (Stargen 002), and protease (Fermgen) according therefore more sugar was released and available for fermen-
to the process developed by Gohel and Duan (2012) (SLSF2). tation. The concentrations of glucose, glycerol, lactic acid and
An additional glucoamylase (Amigase Mega l) (SLSF3) and acetic acid being equal to 0.28 g/l, 0.65 g/l, 4.90 g/l, and 0.37 g/l,
both the additional gluco-amylase (Amigase Mega L) and respectively, remained low at the end (72 h) of SLSF, indicating
the beta-glucanase (Optimash TBG) (SLSF4) were also used. that fermentation deviation and/or bacterial contamination
Our hypothesis was that the yield would increase and the did not occur. Narendranath and Brey (2009) have reported the
duration would decrease thanks to the additional activity of stress began at 0.05% acetic acid and 0.2% lactic acid, although
gluco-amylase and beta-glucanase which would increase the the minimal inhibitory concentration were 0.6% and 2.5%,
hydrolysis of residual starch in broken rice according to the respectively, for the two acids. In our work, the concentration
recommendation of the manufacturer. The results (sugar and of acetic (0.37 g/l) and lactic (0.65 g/l) acids remained to a great
ethanol concentrations) are presented in Fig. 1. All three SLSF extent lower than these stressful values. A trial to reduce SLSF
processes (SLSF2, SLSF3, and SLSF4) achieved an ethanol con- duration was conducted by adding another beta-glucanase in
centration of 10.8% v/v which was equivalent to an ethanol order to reduce the viscosity and degrade “hard-to-hydrolyze”
yield of 88.5%. However, the duration of each process was dif- starch into sugar. However, the viscosity did not change signif-
ferent. SLSF2 finished after 100 h and both SLSF3 and SLSF4 icantly during SLSF (Table 4) and this tentative did not reduce
finished at the same time (after 72 h). It is worth noting that the the process duration.
ethanol content increased from 10.6% to 10.8% v/v for all three To sum up, the SLSF was applied and adapted success-
processes SLSF2, SLSF3, and SLSF4 when more enzymes such fully (with broken rice as substrate) by using a combination of
 food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85 83

Table 3 – Evolutions of the concentrations of maltose, glucose, lactic acid, glycerol and ethanol during Process SLSF3–VHG
(at lab scale) and SLSF3–VHG-p (at pilot scale).
Time (h) Glucose (g/l) Lactic acid (g/l) Glycerol (g/l) Ethanol (% v/v)

Lab scale Pilot scale Lab scale Pilot scale Lab scale Pilot scale Lab scale Pilot scale

0 0.70 ± 0.04 0.34 ± 0.14 0 0 0 0 0 0

24 0.45 ± 0.07 0.54 ± 0.05 0.32 ± 0.03 0.07 ± 0.05 5.06 ± 0.08 3.79 ± 0.63 7.93 ± 0.09 6.80 ± 0.56
49 0.51 ± 0.05 0.48 ± 0.24 0.15 ± 0.02 0.20 ± 0.02 5.99 ± 0.07 6.95 ± 0.48 12.83 ± 0.15 10.23 ± 0.25
73 0.38 ± 0.06 0.59 ± 0.11 0.39 ± 0.05 0.29 ± 0.05 5.92 ± 0.05 6.74 ± 0.41 14.97 ± 0.17 13.74 ± 0.12
96 1.13 ± 0.11 0.48 ± 0.23 0.45 ± 0.05 0.34 ± 0.06 6.45 ± 0.03 6.88 ± 0.22 16.2 ± 0.08 16.27 ± 0.04
120 1.30 ± 0.03 0.07 ± 0.05 1.08 ± 0.07 0.34 ± 0.10 10.48 ± 0.10 6.71 ± 0.45 17.60 ± 0.07 17.00 ± 0.09

different enzymes (alpha-amylase, gluco-amylase, protease, SLSF4–VHG, respectively, and compared with those of the SLSF
and beta-glucanase). The duration of the process could be without and with beta-glucanase for SLSF3 and SLSF4, respec-
changed and reduced significantly when using appropriate tively (Table 4). It was noted that no significant change in
enzymes, which could allow more flexibility for ethanol pro- viscosity was observed during these processes at different dry
ducers. solids. This result could be explained by the fact that no gela-
tinization occurred in our process; therefore, beta-glucanase
3.2. Simultaneous liquefaction, saccharification and did not reduce significantly the viscosity of the mash. In addi-
fermentation at very high gravity at lab scale. tion, glucose, lactic acid concentrations remained at low levels
(Table 3), which demonstrated the advantages of the SLSF pro-
In this part, SLSF were performed under very high grav- cess that a low concentration of reducing sugar could decrease
ity (311.5 g/l) condition. Two processes SLSF3–VHG and the osmotic pressure on yeast and reduce risk of contamina-
SLSF4–VHG were adopted from SLSF3 and SLSF4, respec- tion (Thomas et al., 1996).
tively, with some modifications such as addition of KH2 PO4 In our work, no pretreatment step was performed. Different
(4.8 mM), higher dosage of urea (16.0 mM) and higher dosage enzymes were used in order to decrease viscosity during liq-
of Red Ethanol active dry yeast (3.50 × 107 cells/ml) as the yeast uefaction. In our best process (Process SLSF3–VHG), ethanol
required more nutrients under very high gravity condition concentration was 17.6% v/v corresponding to 86.3% of the
(Ingledew, 2009). The evolutions of residual sugar and ethanol theoretical ethanol yield, which was similar to the average
concentration during SLSF under VHG condition are presented yield achieved in the current ethanol factories in Vietnam.
in Fig. 2. For both processes under VHG condition, the ethanol Therefore, SLSF3–VHG was chosen to scale up at the pilot scale
contents reached high values of 17.6% v/v which corresponded to examine its efficiency.
to a yield of 86.3%. Both processes SLSF3–VHG and SLSF4–VHG
finished over 120 h (equivalent to 5 days). The contents of 3.3. Simultaneous liquefaction, saccharification and
residual sugar (6.5 g/l) and starch (3.8 g/l) remained low, indi- fermentation at very high gravity at pilot scale
cating that the fermentation finished. However, the durations
of both processes SLSF3–VHG and SLSF4–VHG (120 h) were The main goal of the pilot experiment was to seek gaps and
longer than those of SLSF3 and SLSF4. Indeed, beta-glucanase problems that were not significantly noticed at the laboratory
(Optimash TBG) was added into the rice mash (in SLSF4–VHG) scale, and to check the maintenance of ethanol yield after
with the hypothesis that beta-glucanase could reduce the SLSF process. According to the results at lab scale, SLSF3–VHG
mash viscosity, and therefore to accelerate the hydrolysis was scaled up to the pilot scale (25 l/batch) as described in
of raw starch. Beta-glucanase has been generally reported 2.5. Fig. 2 shows the evolutions of ethanol concentration, and
to be used to reduce the mash viscosity after gelatinization residual sugars during SLSF processes at both laboratory and
before conducting the simultaneous saccharification and fer- pilot scales. After 120 h of SLSF, the ethanol content reached
mentation with cassava as substrate in some previous works 17.0% v/v equivalent an ethanol yield of 83.2%. The ethanol
(Nguyen et al., 2014; Poonsrisawat et al., 2014; Srichuwong yield was lower than that obtained at laboratory scale (17.6%
et al., 2009). Nguyen et al. (2014) used a beta-glucanase (in Opti- v/v). However, the glycerol content at pilot scale (6.71 g/l) was
mash TBG) to reduce significantly the viscosity of the cassava lower than that at the laboratory scale (10.48 g/l). During SLSF
mash during liquefaction from 400 cp to 290 cp. The viscosity at the pilot scale, the content of glucose (<0.60 g/l), glycerol
values measured during SLSF processes at very high gravity (<7.00 g/l), and lactic acid (<0.40 g/l) remained lower than
without and with beta-glucanase for Process SLSF3–VHG and the minimal inhibitory concentrations of those metabolites

Table 4 – Viscosity of the rice mash during SLSF and SLSF-VHG.

Time (h) Mash viscosity (cp)

SLSF processes SLSF processes at very high gravity

SLSF3 SLSF4 SLSF3–VHG SLSF4–VHG

0 179.3 ± 1.2 180.3 ± 0.9 212.3 ± 2.1 216.0 ± 1.6

24 161.0 ± 2.2 163.0 ± 0 174.3 ± 2.1 175.7 ± 2.1
48 151.3 ± 1.2 149.7 ± 1.7 145.7 ± 1.7 147.0 ± 1.6
72 150.0 ± 0.8 148.3 ± 2.1 147.0 ± 0.8 145.0 ± 0.8
96 148.0 ± 0.8 145.3 ± 1.2 146.3 ± 1.2 146.7 ± 1.7
144 146.7 ± 1.2 144.0 ± 2.8 149.0 ± 2.2 147.7 ± 1.7
84 food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85

achieved an ethanol content of 17.0% v/v corresponding to a

yield of 83.2% of the theoretical ethanol yield. Rice-based DDG
from the whole stillage of SLSF process at very high gravity had
significantly higher contents of crude protein (47.5%) and fiber
(15.8%) in comparison with those of corn-based DDGS. These
promising results suggest that the SLSF under VHG condition
of broken rice could have a great potential for the ethanol and
animal feeding industry in Vietnam.

Acknowledgment

This work was supported by Vietnamese Ministry of Education

and Training (grant B2013.01.07DA) and Ministry of Science
and Technology (grant 15/2014/HD-NDT). We thank Dupont,
DSM and Leaf Technologies (Lesaffre) for kindly providing us
with technical support, enzymes and yeast samples, respec-
tively. We thank Dr Tuan-Anh Nguyen and Xuan-Bach Cao
for their technical assistance in fermentation experiments.
Fig. 2 – Evolutions of residual sugar and ethanol
We also thank Chinh-Nghia Nguyen for technical support to
concentration of the three VHG processes (Process
prepare the manuscript.
SLSF3–VHG and SLSF4–VHG at laboratory scale and Process
SLSF3–VHG-p at pilot scale).

References
Table 5 – Main composition of rice-based distillers dried
grains.
Duan, G., Dunn-Coleman, N., Lantero, O.J., Pilgrim, C.E., Shetty,
Composition Content (%) J.K., 2007. Acid fungal protease in fermentation of insoluble
starch substrates. Patent WO2006073843 A3.
Moisture 8.9 ± 0.8
Gang, D., Xu, S., Zhou, J., Tok, S.K., Shetty, J.K., 2007.
Protein 47.5 ± 2.2
Non-conventional process for ethanol production,
Crude fiber 15.8 ± 0.9
Proceedings of the 4th International Conference on Starch
Ash 1.5 ± 0.1
Technology Starch Update 2007, Bangkok, Thailand.
Fats 10.7 ± 0.3
Gang, D., Xu, S., Zhou, J., Tok, S.K., Shetty, J.K., 2009. Novel
enzymatic processes for ethanol production from cassava
feedstock, Proceedings of the 5th International Conference on
mentioned by Narendranath and Brey (2009). It is worth
Starch Technology Starch Update 2009, Bangkok, Thailand.
noting that the evolutions of residual sugars, ethanol content
General-statistic-office-of-Vietnam, 2013. Rice production in
(Fig. 2), glucose, lactic acid, and glycerol during SLSF at very Vietnam.
high gravity stayed similar to those observed at the labora- Gohel, V., Duan, G., 2012. No-cook process for ethanol production
tory scale, which indicated no deviation of metabolites was using Indian broken rice and pearl millet. Int. J. Microbiol.
observed when scaling up from lab scale to pilot scale. 2012, 1–9.
Ingledew, W.M., 2009. Yeast stress in the fermentation process.
In: Ingledew, W.M., Kelsall, D.R., Austin, G.D., Kluhspies, C.
3.4. Rice-based DDG compositions
(Eds.), The Alcohol Textbook. , 5th edition, pp. 115–126.
Kelsall, D.R., Piggot, R., 2009. Grain milling and cooking for
The distillers wet grains (DWG) was separated from the whole alcohol production: Design for the options in dry milling. In:
stillage after SLSF at very high gravity at pilot scale by using Ingledew, W.M., Kelsall, D.R., Austin, G.D., Kluhspies, C. (Eds.),
plate filter. Rice-based DWG was then dried in a chamber dryer The Alcohol Textbook. , 5th edition, pp. 161–176.
to obtain rice-based distillers dried grains (DDG). The main Le Thanh, M., Nguyen Thi, H., Pham Thu, T., Nguyen Thanh, H., Le
Lan, C., 2007. Analytical Methods in Fermentation Technology.
composition of rice-based DDG was analyzed and the results
Science and Technology Publishing House, Hanoi, Vietnam.
are presented in Table 5. Rice-based DDG had higher contents
Liu, K., 2011. Chemical composition of distillers grains: a review. J.
of protein (47.5%) and fiber (15.8%) in comparison with those of Agric. Food Chem. 59, 1508–1526.
corn-based DDGS (Liu, 2011). The high content of crude protein Monceaux, D.A., Kuehner, D., 2009. Dryhouse technologies and
in rice-based DDG could result in high nutritional addition for DDGS production. In: Ingledew, W.M., Kelsall, D.R., Austin,
animal feeding and be a promising resource for ingredients G.D., Kluhspies, C. (Eds.), The Alcohol Textbook. , 5th edition,
in animal feeding industry, as well as reduce significantly the pp. 303–322.
Narendranath, N.V., Brey, S., 2009. Bacterial contamination and
import of DDGS in Vietnam.
control. In: Ingledew, W.M., Kelsall, D.R., Austin, G.D.,
Kluhspies, C. (Eds.), The Alcohol Textbook. , 5th edition, pp.
4. Conclusion 337–356.
Nguyen, C.-N., Le, T.-M., Chu-Ky, S., 2014. Pilot scale simultaneous
In this work, we successfully developed SLSF processes under saccharification and fermentation at very high gravity of
cassava flour for ethanol production. Ind. Crops Products 56,
VHG condition (311.5 g/l dry matter) of rice for ethanol produc-
160–165.
tion at laboratory scale by using appropriate combination of
Poonsrisawat, A., Wanlapatit, S., Paemanee, A., Eurwilaichitr, L.,
enzymes (alpha-amylase and two gluco-amylases, protease). Piyachomkwan, K., Champreda, V., 2014. Viscosity reduction
The ethanol content achieved 17.6% v/v corresponding to of cassava for very high gravity ethanol fermentation using
86.3% of the theoretical ethanol yield at laboratory scale. This cell wall degrading enzymes from Aspergillus aculeatus.
nocook process was performed at the pilot scale (25 l) and Process Biochem. 49, 1950–1957.
 food and bioproducts processing 9 8 ( 2 0 1 6 ) 79–85 85

Robertson, G.H., Wong, D.W., Lee, C.C., Wagschal, K., Smith, M.R., Wang, P., Singh, V., Xue, H., Johnston, D.B., Rausch, K.D.,
Orts, W.J., 2006. Native or raw starch digestion: a key step in Tumbleson, M.E., 2007. Comparison of raw starch hydrolyzing
energy efficient biorefining of grain. J. Agric. Food Chem. 54, enzyme with conventional liquefaction and saccharification
353–365. enzymes in dry-grind corn processing. Cereal Chem. 84, 10–14.
Srichuwong, S., Fujiwara, M., Wang, X., Seyama, T., Shiroma, R., Xu, H., Duan, G., 2010. Effect of temperature on the no cook, very
Arakane, M., Mukojima, N., Tokuyasu, K., 2009. Simultaneous high gravity ethanol fermentation process. Sheng Wu Gong
saccharification and fermentation (SSF) of very high gravity Cheng Xue Bao 26, 330–334.
(VHG) potato mash for the production of ethanol. Biomass Yingling, B., Zongcheng, Y., Honglin, W., Li, C., 2011. Optimization
Bioenergy 33, 890–898. of bioethanol production during simultaneous
Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1996. Practical and saccharification and fermentation in very high-gravity
theoretical considerations in the production of high cassava mash. Antonie van Leeuwenhoek Int. J. Gen. Mol.
concentrations of alcohol by fermentation. Process Biochem. Microbiol. 99, 329–339.
31, 321–331.