Journal of Bioscience and Bioengineering

VOL. 107 No. 5, 488 – 493, 2009

www.elsevier.com/locate/jbiosc

Simultaneous non-thermal saccharification of cassava pulp by multi-enzyme

activity and ethanol fermentation by Candida tropicalis

Ukrit Rattanachomsri, Sutipa Tanapongpipat, Lily Eurwilaichitr, and Verawat Champreda⁎

Enzyme Technology Laboratory, Bioresource Technology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand
Science Park, Paholyothin Road, Klong Luang, Pathumthani 12120, Thailand

Received 1 July 2008; accepted 22 December 2008

Cassava pulp, a solid by-product from starch processing, is a promising and underused biomass that can be converted to
biofuels and other value-added bio-products. In this study, an alternative cassava pulp saccharification process, which utilizes
the multi-activity enzyme from Aspergillus niger BCC17849 and obviates the need for a pre-gelatinization step, was developed.
The crude multi-enzyme composed of non-starch polysaccharide hydrolyzing enzyme activities, including cellulase, pectinase
and hemicellulase act cooperatively to release the trapped starch granules from the fibrous cell wall structure for subsequent
saccharification by raw starch degrading activity. A high yield of fermentable sugars, equivalent to 716 mg glucose and 67 mg
xylose/g of cassava pulp, was obtained after 48 h incubation at 40 °C and pH 5 using the multi-enzyme, which was greater than
the yield obtained from the optimized combinations of the corresponding commercial enzymes. The multi-enzyme
saccharification reaction can be performed simultaneously with the ethanol fermentation process using a thermotolerant
yeast Candida tropicalis BCC7755. The combined process produced 14.3 g/l ethanol from 4% (w/v) cassava pulp after 30 h of
fermentation. The productivity rate of 0.48 g/l/h is equivalent to 93.7% of the theoretical yield based on total starch and
cellulose, or 85.4% based on total fermentable sugars. The non-thermal enzymatic saccharification process described is more
energy efficient and yields more fermentable sugar than the conventional enzymatic process. Furthermore, the process is
applicable for production of various bio-products of economic importance.
© 2009, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Aspergillus niger; Candida tropicalis; Cassava pulp; Ethanol; Non-starch polysaccharide hydrolyzing enzyme; Raw starch degrading
activity]

In recent years, there has been an increasing trend towards more
efficient utilization of agro-industrial by-products for conversion to
a range of value-added bio-products, including biofuels, biochem-
icals and biomaterials (1, 2). Bioprocessing of agro-industrial resi-
dues can help solve environmental problems associated with their
disposal and reduce dependence on petroleum resources. The
exploration of novel, efficient bioprocesses for underused biomasses
is thus at the forefront of biotechnological research and industrial
application.
Cassava (Manihot esculenta) is one of the most important crops in
terms of its production, ranked as the sixth most important world food
crop (3). Cassava pulp is a fibrous by-product of the cassava processing
industry, and is generally used as low-value animal feed (4).
Cassava pulp contains about 50–70% starch on a dry weight basis
and 20–30% fibers, which are composed mainly of cellulose and other
non-starch polysaccharides (2). Owing to its rich organic nature and
low ash content, combined with the ease of its hydrolysis, low
collection cost and lack of competition with other industrial uses,
cassava pulp is an ideal substrate for the microbial production of
value-added products.
⁎ Corresponding author. Tel.: +66 2564 6700x3473; fax: + 66 2564 6707.
E-mail address: verawat@biotec.or.th (V. Champreda).
Bioprocessing based on microbial conversion is an important
approach for the production of value-added products from cassava
pulp. In conventional bioprocessing of cassava pulp, the pulp is
subjected to high temperature pre-gelatinization in the presence of a
thermostable α-amylase to release the trapped starch granules,
followed by saccharification with glucoamylase (2). Cassava pulp
hydrolysate has been used for the production of various fermentation
products, including citric acid by Candida lipolytica (2), fumaric acid
by Rhizopus strains (5, 6) and L-(+)-lactic acid by Lactobacilli (7, 8).
However, the conventional saccharification process is energy-inten-
sive and does not release fermentable sugars from the non-starch
component of the substrate. The development of a more efficient
enzymatic saccharification process is thus of great interest for the
biotechnological utilization of this feedstock.
Direct saccharification of starch-based substrates without pre-
gelatinization using raw starch degrading (RSD) amylolytic enzymes
is considered to be a promising alternative to conventional sacchar-
ification. RSD amylolytic enzymes of fungal origin, in particular As-
pergillus spp. and Rhizopus spp., have been characterized and used for
industrial applications (9). Efficient multi-enzyme hydrolysis pro-
cesses applying RSD amylolytic activity in combination with other
hydrolytic enzymes to raw starch or starch-based feedstocks have
been developed. For example, the synergistic action of two glucoa-
mylases with the addition of α-amylase, pullulanase and protease on

1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2008.12.024
VOL. 107, 2009
the hydrolysis of corn solids was studied (Lantero, O. J. Jr., Shetty J.K.,
US patent application 20050100996, 2004). The application of multi-
polysaccharide degrading enzymes containing lignocellulolytic, pec-
tinolytic and amylolytic activities was also reported for the treatment
of starch and non-starch polysaccharide components in complex
plant-derived animal feed (10). The trapped nature of starch granules
in cassava pulp hinders their extraction and presents a challenge for
direct saccharification of this feedstock without pre-gelatinization.
One solution to this problem was reported by Sriroth et al., who
showed that pretreatment of primary cassava pulp with a mixture of
cellulase and pectinase resulted in the increased release of secondary
starch with physicochemical properties similar to those of the primary
starch (11). From this work, we were inspired to develop a novel
multi-enzyme process for simultaneous starch release and sacchari-
fication under non-thermal conditions as a cost-effective alternative
for cassava pulp bioconversion.
In this study, a process involving direct hydrolysis and sacchari-
fication of cassava pulp without a pre-gelatinization step was
developed based on the simultaneous action of multi non-starch
polysaccharide hydrolyzing enzymes and RSD amylolytic activity. The
composite enzyme activities were analyzed, and the reaction para-
meters were investigated with the multi-enzyme preparation from a
selected strain of A. niger. The optimized conditions were applied in
ethanol production using simultaneous saccharification and fermen-
tation (SSF) with the thermotolerant, glucose and xylose-utilizing
yeast, Candida tropicalis. This non-thermal saccharification process is
promising for application in bioconversion of cassava pulp to biofuels
and other value-added products with improved fermentable sugar
yield and energy efficiency.
MATERIALS AND METHODS
Substrate and enzymes Cassava pulp was obtained from the Cassava and
Starch Technology Research Unit, BIOTEC, Bangkok, Thailand. The pulp was dried at
50 °C, ground and sieved through a mesh 10–100 (2 mm–150 μm). Cassava pulp
composition was analyzed according to the AOAC standard methods. Commercial
cellulase from Trichoderma reesei (Cellulclast® 1.5 L), pectinase from Aspergillus niger
(Pectinex Ultra SP-L), β-glucosidase from A. niger (Novozym® 188), thermostable α-
amylase from Bacillus licheniformis (Termamyl® 120 L), and glucoamylase from A. niger
(AMG 300 L) were from Novozymes (Bagsvaerd, Denmark). The β-glucanase/
hemicellulase from Trichoderma reesii (Optimash BG) was obtained from Genencor
(Danisco A/S, Copenhagen, Denmark).
Microorganism strains A. niger BCC17849 and C. tropicalis BCC7755 were
obtained from the BIOTEC Culture Collection, Thailand (www.biotec.or.th/bcc) and
maintained on potato dextrose agar (PDA) and YM agar, respectively.
Multi-activity enzyme production A. niger BCC17849 was grown under
aerobic conditions in a 50 ml submerged culture in 250-ml conical flasks at 25 °C
with shaking at 200 rpm. An inoculum was prepared from the fungal culture on PDA by
excising four agar pieces covered with a profuse mycelial mat using a cork borer No. 2,
and inoculated into 50 ml of the medium. In enzyme production studies, the fungus was
cultivated in agro-industry substrates, including different combinations of 4% wheat
bran, 3% soybean meal, 4% rice bran, 4% cassava pulp, 3% sugarcane bagasse and 0.5%
pectin from citrus, or in MXP (2% soybean meal, 1.5% wheat flour, 1% cellulose avicel,
0.5% maltodextrin 15, 1% birchwood xylan, 0.3% bacto peptone and 0.5% pectin from
citrus in distilled water) at 25 °C for 3 d with shaking at 200 rpm. The optimized
medium, WSP, contained 4% wheat bran, 3% soybean meal and 0.5% pectin. The starter
culture was grown at 25 °C for 3 d with 200 rpm shaking and inoculated at 10% (v/v)
into a fresh 50 ml WSP medium in a 250-ml flask. The culture was further incubated for
3 d under the same conditions. The crude enzyme (culture filtrate) was obtained by
filtration through a 0.5 mm diameter mesh, clarified by centrifugation at 7500 × g for
15 min and filtered through a 0.45 μm nitrocellulose membrane (Sartorius, Goettingen,
Germany). The enzyme was concentrated by ultrafiltration on a Minimate tangential
flow filtration system using a Minimate TFF capsule with 10 kDa MWCO membrane
(Pall, Easthills, NY, USA).
Enzyme activity assay Polysaccharide degrading activities were analyzed
based on the amount of liberated reducing sugars using the 3,5-dinitrosalisylic acid
(DNS) method (12). The 100 μl reactions contained the appropriate dilution of enzyme
in 50 mM sodium acetate buffer, pH 5.0 and 1% of the corresponding substrate: pectin
from citrus for pectinase activity, birchwood xylan for hemicellulase activity,
carboxymethyl cellulose for CMCase activity, soluble starch for α-amylase activity,
and raw cassava starch for RSD activity. The reaction was incubated at 40 °C for 30 min
and the amount of reducing sugars was determined from the absorbance mea-
NON-THERMAL SACCHARIFICATION OF CASSAVA PULP 489
surements at 540 nm and interpolated from a standard curve of the corresponding
sugar. One international unit (IU) was defined as the amount of enzyme that pro-
duced 1 μmole of reducing sugar in 1 min. β-Glucosidase activity was determined
using p-nitrophenyl-β-D-glucopyranoside (PNPG) as the substrate (13). The filter
paper unit (FPU) for cellulase was analyzed according to the standard method (14).
Enzymatic hydrolysis of cassava pulp The 5 ml reactions contained 4% cassava
pulp in 50 mM sodium acetate buffer, pH 5.0 with commercial enzyme mixture,
consisting of different combinations of cellulase (10 FPU/g cassava pulp), pectinase
(14 IU/g based on polygalacturonase), β-glucosidase (6 IU/g), hemicellulase (13 IU/g
based on xylanase), glucoamylase (20 or 255 IU/g based on RSD activity), and α-
amylase (55 IU/g based on soluble starch) or with an appropriate dilution of multi-
enzyme from A. niger BCC17849 (1× enzyme loading contained 1.3 FPU/g cellulase,
21 IU/g pectinase, 28 IU/g hemicellulase, and 12 IU/g RSD activity). The reaction was
incubated at 40 °C for 48 h with rotary shaking at 200 rpm. Samples were collected at
time intervals for analysis. The total amount of reducing sugar released was analyzed by
the DNS method. Sugar composition was analyzed on a high performance liquid
chromatograph, Waters 2360 equipped with a differential refractometer, Waters 410
using a SugarPak column (Waters, Milford, MA, USA). Deionized water was used as a
mobile phase at a flow rate of 1 ml/min.
Ethanol fermentation by SSF The 10 ml fermentation mixtures contained 4%
cassava pulp in 50 mM sodium acetate buffer, pH 5.0 with 1× enzyme loading of the
multi-enzyme from A. niger BCC17849 plus 5 ml of C. tropicalis BCC7755 overnight
starter culture in basal medium (0.25% yeast extract, 0.5% peptone, 0.1% KH2PO4, 0.03%
MgSO4.7H2O, 0.2% NH4Cl) with an OD600 of 0.6 (approximately 10 CFU) in a 20-ml
closed vial. The reaction was incubated at 40 °C with rotary shaking at 200 rpm for 48 h.
Samples were taken at time intervals for ethanol analysis on a gas chromatograph
equipped with a flame ionization detector (GC-17A, Shimadzu, Kyoto, Japan) using a
pre-packed 6′ × 1/8″-Porapak Q column (Showa Denko K.K., Kawasaki, Japan) based on
Templeton (15). Helium was used as the carrier gas with a flow rate of 25 ml/min.
RESULTS AND DISCUSSION
Synergistic enzyme activities in non-thermal saccharification
of cassava pulp The chemical composition of cassava pulp is
dependent on the starch processing technology used. The cassava pulp
used in this study was analyzed and found to be composed mainly of
starch and fibrous material containing cellulose and hemicellulose,
which are sources of fermentable sugars (Table 1), in addition to other
components e.g., pectin, protein and lignin (11). The cassava pulp was
dried below the starting gelatinization temperature of cassava starch
to preserve the physical characteristics of the starch granules (16).
Enzymatic saccharification of cassava pulp is distinct from raw
cassava starch or cassava root homogenate because of the trapped
nature of raw starch granules and higher non-starch polysaccharide
content in pulp. The cooperative action of an array of polysaccharide
hydrolyzing enzymes is thus essential for efficient degradation of this
substrate. For the first stage, multi-enzymatic activities involved in
non-thermal cassava pulp saccharification were investigated syste-
matically using different commercial enzyme combinations (Table 2).
Enzymatic hydrolysis by individual non-starch polysaccharide hydro-
lyzing enzymes (C, P, B, and H) at high enzyme loading released
relatively low levels of reducing sugar, which were derived from
decomposition of the cell wall components. The combined action of
cellulase and pectinase yielded more reducing sugar (242 mg/g
cassava pulp) owing to the efficient hydrolysis of the non-starch
fibrous structure and the cementing pectin containing compounds
(11). The addition of commercial β-glucanase/hemicellulase to the
enzyme mix did not appreciably increase the fermentable sugar
yield. This might be due to the presence of high hemicellulase side
TABLE 1. Compositional analysis of cassava pulp
Component % Content (dried basis)
Starch (enzyme method) 60.10 ± 0.09
Neutral detergent fiber (NDF) 23.04 ± 0.07
Acid detergent fiber (ADF) 18.46 ± 0.16
Lignin 2.83 ± 0.06
Cellulose a 15.63
Hemicellulose b 4.58
a
ADF-lignin.
b
NDF-ADF.
490 RATTANACHOMSRI ET AL. J. BIOSCI. BIOENG.,

TABLE 2. Direct non-thermal enzymatic saccharification of cassava pulp by combinations of enzyme activities
Enzyme Enzyme loading a Glucose Xylose Reducing sugars
(mg/g) (mg/g) (mg/g)
Cel (FPU/g) Pec (IU/g) BGL (IU/g) Hemicel (IU/g) RSD (IU/g)

C 10 + − 12 − 84 0 109
P − 14 + ++ − 61 48 124
B + + 6 + + 205 0 193
H ++ + ++ 13 − 30 4 37
C+P 10 14 ++ 12 − 132 52 242
C+P+B 10 14 6 12 + 285 59 475
C+P+H 10 14 ++ 25 − 132 54 251
C+P+B+H 10 14 6 25 + 293 60 484
AMG + + − + 255 212 0 206
C + P + B + AMG 10 14 6 12 255 343 52 492
C + P + B + AMG + Amy b 10 14 6 12 255 391 47 571
Ctrl (-Enz) − − − − − 0 0 4
Liquefaction/saccharification c + + − + 10 538 0 735
Acid hydrolysis 1M H2SO4 d − − − − − 650 68 767

Reactions contained 4% cassava pulp in 50 mM sodium acetate buffer, pH 5.0 with different combinations of commercial enzymes: C: Celluclast® 1.5 L; P: Pectinex Ultra SP-L; B:
Novozym® 188; H: Optimash BG; AMG: AMG 300 L; Amy: Termamyl® 120 L. The reactions were incubated at 40 °C with rotary shaking at 200 rpm for 30 h. Enzyme loading as FPU or
IU per g substrate is indicated: — Cel: cellulase activity; Pec: pectinase as polygalacturonase activity; BGL: β-glucosidase activity; Hemicel: hemicellulase as xylanase activity; RSD:
raw starch degrading activity of glucoamylase.
a
Only the major activity of the respective commercial enzymes was used in determination of the enzyme unit (see methods section). Minor activity/major activity ratio: ++
(5–20%); + (1–5%); − (no significant activity).
b
α-amylase was added at 55 IU/g cassava pulp based on unit activity on soluble starch.
c
Liquefaction of 4% cassava pulp with 0.15% (v/w) Termamyl® 120 L, in 50 mM sodium acetate buffer, pH 5.0 containing 0.02% CaCl2 at 120 °C for 15 min, followed by
saccharification at 40 °C with 0.5% (v/w) AMG 300 L for 48 h (8).
d
Acid hydrolysis reaction contained 10% cassava pulp in 1 M H2SO4 and heated at 120 °C for 30 min.

activity of the commercial cellulase. In contrast, addition of a com-
mercial β-glucosidase led to a marked increase in reducing sugar
yield. The higher reducing sugar yield could be due to the presence of
strong raw starch-hydrolyzing side activity of the commercial
enzyme preparation from A. niger, in addition to the possible direct
effect from the β-glucosidase activity on cellulose-derived substrates.
Saccharification of the substrate by excess glucoamylase (255 IU/g)
led to a relatively low reducing sugar yield, despite reports of raw
cassava starch hydrolyzing activity for glucoamylase preparation
from Aspergillus strains (17, 18). Further reduction of glucoamylase
loading (20 IU/g) resulted in only a slight decrease on the sacchari-
fication yield (199 mg/g), while increasing glucoamylase did not show
any effect on reducing sugar yield, suggesting excessive use of
glucoamylase under these experimental conditions. It should be
noted that the relative glucoamylase activity on cassava pulp was
approximately 32% of that on the extracted raw cassava starch based
on the released reducing sugar yield using the same RSD unit (20 IU/g)
of glucoamylase under the same conditions. This indicated the lower
accessibility of the enzyme to the trapped starch granules in cassava
pulp.
As suggested from the substrate's chemical components and
structure, the combination of the multiple non-starch polysaccharide
degrading enzymes and the enzymes with strong RSD activity led to
a respective increase in fermentable sugar yield (132, 285 and
343 mg glucose/g for C + P, C + P + B and C + P + B + AMG,
respectively). This suggested the cooperative action of multi-non
starch polysaccharide hydrolyzing activities in degradation of the
fibrous cell wall structure together with the amylolytic RSD activity
acting on the accessible released starch granules. Saccharification
yield was further increased by the addition of an endo-acting
bacterial α-amylase to the reaction with the maximum reducing
sugar yield of 571 mg/g cassava pulp, equivalent to 391 mg/g for
glucose and 47 mg/g for xylose, though the bacterial enzyme had a
relatively low activity on raw cassava starch. Further systematic
optimization of the enzyme units in the active enzyme mix led to an
optimal reaction with reduced enzyme loading (1.3 FPU/g cellulase,
1.5 IU/g hemicellulase, 17 IU/g pectinase, 1.5 IU/g β-glucosidase,
20 IU/g RSD activity with the addition of 55 IU/g bacterial α-
amylase based on soluble starch, equivalent to 0.8 IU/g RSD activity).
This resulted in an increased reducing sugar yield of 809 mg/g of
cassava pulp after 48 h (see below). The results thus indicate that
efficient saccharification of cassava pulp with no pre-gelatinization is
possible by the combined actions of multiple non-starch polysac-
charide hydrolyzing enzymes and RSD amylolytic enzymes.

FIG. 1. Enzyme inducibility profile of A. niger BCC17849. The fungal isolate was cultivated by submerged fermentation in different substrates at 25 °C for 3 d with shaking at 200 rpm.
MXP: modified Mex-1 medium; CP: cassava pulp; S: soybean meal; B: sugarcane bagasse; P: pectin from citrus; RB: rice bran; W: wheat bran. ⁎Cellulase activity as CMCase.
VOL. 107, 2009
Saccharification of cassava pulp by multi-enzyme from A.
niger Fungal isolates from the BIOTEC culture collection (Thailand)
were screened for production of the target multi-enzyme activities
suitable for non-thermal saccharification. Among the fungal isolates,
A. niger BCC17849 was selected because of its high levels of cellulase
(CMCase), pectinase, hemicellulase and RSD activities under the
experimental conditions at 40 °C and pH 5. Aspergillus spp. are a
known source of diverse, industrially important enzymes for hydro-
lysis of complex agro-industrial substrates (19). With the long recog-
nized roles of Aspergillus spp. in the biotechnological industry, it
would of interest to develop large-scale production of the multi-
enzyme from the isolate BCC17849.
The inducibility of the target enzyme activities was investigated in
a semi-synthetic medium (MXP) and in complex media in submerged
fermentation (Fig. 1). The greatest level of induction of target enzyme
activities was observed for fermentation in wheat bran and soybean
meal supplemented with pectin, which was selected as the optimal
multi-enzyme production medium. Analysis of medium composition
revealed that the addition of synthetic pectin resulted in an
approximately 20% increase of pectinase activity, while no induction
of other target composite enzyme activities was observed. Further
optimization of the medium, as shown by others (10), could lead to
increased production of the multi-enzyme.
Non-thermal saccharification of cassava pulp with the multi-
enzyme preparation from A. niger BCC17849 at the optimal loading
(1×) led to higher fermentable sugar yield compared with other
enzyme treatments, including the optimized mixture of commercial
enzymes over the 48 h reaction period (Fig. 2). The use of the multi-
enzyme resulted in the rapid release of fermentable sugars, reaching
716 mg of glucose and 67 mg of xylose/g of cassava pulp (dry
weight), equivalent to 91.2% of the theoretical yield of the available
glucose from starch and cellulose, and 97.2% of the theoretical yield
of the available xylose based on acid hydrolysis, indicating the almost
complete saccharification of the substrate had occurred. The saccha-
rification of cassava pulp using the multi-enzyme was superior to the
conventional liquefaction/saccharification process using only amylo-
lytic enzymes (8) as indicated by an increased fermentable sugar
FIG. 2. Time-dependent enzymatic saccharification of cassava pulp by multi-enzyme activity. Reactions contained 4% cassava pulp in 50 mM sodium acetate buffer, pH 5.0 and
incubated at 40 °C with shaking at 200 rpm using 1× multi-enzyme from A. niger BCC17849, or combinations of commercial enzymes: C + P + B + AMG + Amy (optimized unit:
1.3 FPU/g cellulase, 1.5 IU/g hemicellulase, 17 IU/g pectinase, 1.5 IU/g β-glucosidase, 20 IU/g RSD activity of glucoamylase, and 55 IU/g α-amylase), C + P and AMG units are detailed
in Table 2. Inlet: glucose and xylose yields from cassava pulp saccharification using 1× multi-enzyme from A. niger BCC17849.
NON-THERMAL SACCHARIFICATION OF CASSAVA PULP 491
yield (Table 2). In the multi-enzyme process, the increase in sugar
yield was due to hydrolysis of both raw starch and cellulose to
glucose, together with the release of xylose from hemicellulose. The
non-thermal multi-enzyme reaction also yielded more fermentable
sugar than the acid hydrolysis process due to the loss of sugars as
dehydration by-products after exposure to strong acid at high
temperature (Table 2) (20, 21).
Based on the analysis of composite enzyme activities in cassava
pulp saccharification as discussed above, the high saccharification
efficiency of the multi-enzyme could be due to the cooperative action
of the composite multiple non-starch polysaccharide hydrolyzing
enzymes (including cellulase, pectinase and hemicellulase) which
saccharify the cellulosic component of the pulp and release the starch
granules from the fibrous cell wall structure. The released raw starch
granules are then hydrolyzed by the action of RSD activity in the
multi-enzyme, resulting in nearly complete saccharification of the
substrate. The higher saccharification efficiency of the multi-enzyme
compared with combinations of commercial enzymes could be due to
the complex composition and specificities of composite enzyme
activities in the multi-enzyme preparation, and partially to the strong
RSD activity of the fungal strain selected specifically on cassava starch.
Further experimental study is needed in order to elucidate the
cooperative actions of the composite enzyme activities in the multi-
enzyme in detail.
Reaction parameter analysis revealed that the multi-enzyme
worked optimally in the temperature and pH ranges of 37–50 °C
and pH 4–5, respectively (Fig. 3), which is in accordance with the
optimal working conditions of the A. niger composite enzyme
activities reported previously (9). The saccharification yield did not
change with increasing cassava pulp loading from 4% to 12% when
keeping the enzyme unit to substrate ratio constant, nor with
increasing particle size of the cassava pulp (particle size ranging
from mesh 10 to 18 (2 mm–1 mm), mesh 18–100 (1 mm–150 μm) and
bmesh 100 (b150 μm)), suggesting that physical pretreatment of the
substrate is not required (data not shown). When thermal sterilization
of the substrate is not performed, the mildly acidic and relatively high
temperature optimal working conditions for the multi-enzyme
492 RATTANACHOMSRI ET AL.
FIG. 3. Effects of temperature and pH on saccharification of cassava pulp by the multi-enzyme from A. niger BCC17849. Reactions contained 4% cassava pulp in 50 mM buffer at the
corresponding temperature and pH with 1× multi-enzyme. (A) Optimal working temperature in 50 mM sodium acetate buffer, pH 5.0; (B) optimal pH at 40 °C.
suppress the growth of contaminating microorganisms. The multi-
enzyme process would also be applicable for simultaneous sacchar-
ification and fermentation using thermophilic microorganisms.
Ethanol fermentation The thermotolerant yeast C. tropicalis
BCC7755 was selected for its ethanol fermentation capability at
relatively high temperature and mild acidity. The use of C. tropicalis for
ethanol fermentation from a range of substrates has been investigated,
in addition to its conventional utilization in xylitol production (22). C.
tropicalis was reported to ferment ethanol from glucose and xylose
(23). The yeast also produces glucoamylase and ferments starch
slowly to ethanol in the absence of extra α-amylases, suggesting its
potential for ethanol fermentation from starch-based substrates (24).
Together with its ability to tolerate and decompose phenols and
polyphenols generated during the pretreatment of lignocellulosic
biomass (21), C. tropicalis is considered an attractive system for
production of ethanol and other compounds from renewable plant-
derived biomass.
The applicability of the developed non-thermal saccharification
process was demonstrated in the simultaneous cassava pulp sacchar-
ification and ethanol fermentation using the multi-enzyme and C.
tropicalis BCC7755 at 40 °C and pH 5 as shown in Fig. 4. Fermentation
of cassava pulp in the absence of the multi-enzyme preparation
resulted in very low ethanol production, suggesting an inability of the
yeast to effectively digest raw starch granules trapped in the fibrous
structure solely by glucoamylase activity. Addition of the multi-
FIG. 4. Effect of the multi-enzyme from A. niger BCC17849 on simultaneous
saccharification and ethanol fermentation by C. tropicalis BCC7755. Reactions contained
4% cassava pulp in 50 mM sodium acetate buffer, pH 5.0 with different multi-enzyme
loading in 0.5× basal medium. Control: no enzyme.
J. BIOSCI. BIOENG.,
enzyme at 0.4× loading to the fermentation mixtures led to a marked
increase in ethanol production. Higher ethanol yields were achieved
with more enzyme loading. The maximum ethanol yield of 14.3 g/l
was reached after 30 h of fermentation with 1× multi-enzyme. This
yield corresponds to a productivity rate of 0.48 g/l/h, equivalent to
93.7% of the theoretical yield based on the total available glucose from
starch and cellulose, or 85.3% based on the total fermentable sugar
(glucose and xylose).
The optimization of the SSF process showed that the optimal
fermentation conditions were 40 °C and pH 4.5–5, which is in
accordance with the optimal working conditions for the multi-
enzyme and the ethanol fermenting capability of C. tropicalis (24).
Under these experimental conditions, almost all glucose released from
cassava pulp saccharification was efficiently converted to ethanol
while less than 20% of the available xylose was assimilated (Fig. 5). A
similarly slow conversion of xylose was also observed in control
reactions using 4% xylose as the sole carbon source, in which a small
amount (0.97 g/l) of ethanol was produced after prolonged
fermentation for 72 h (data not shown). The result thus indicated
that almost all ethanol produced under the optimized experimental
conditions was mainly from glucose released from saccharification of
FIG. 5. Simultaneous saccharification and ethanol fermentation profile of C. tropicalis
using cassava pulp as the substrate under the optimized conditions. Fermentation
reaction contained 4% cassava pulp in 50 mM sodium acetate buffer, pH 5.0 with 1×
multi-enzyme loading in 0.5× basal medium. The reaction was incubated at 40 °C with
rotary shaking at 200 rpm. The pH of the fermentation medium is shown.
VOL. 107, 2009
raw starch and cellulose. The ethanol yield obtained with this process
was comparable to that of C. tropicalis using liquefied corn soluble
starch as substrate in the presence of exogenous α-amylase (24). In
addition, this process is much faster compared with a previous study
on the application of crude glucoamylase preparation from A. niger for
fermentation of cassava root homogenate with Saccharomyces
cerevisiae (25). Application of the multi-enzyme in fermentation
also led to the reduction of solid content, thus allowing for greater
solid substrate loading in fermentation. The greater substrate load
would lead to increased ethanol concentration, and therefore improve
the efficiency of downstream processing.
In respiratory yeast, the metabolism of glucose and xylose is tightly
regulated by dissolved oxygen concentration, as reflected by the redox
potential. In the presence of mixed sugars, glucose represses xylose
metabolism and is preferentially assimilated until it is consumed
below the sub-repressive levels (22). While conversion of glucose to
ethanol occurs under a wide redox potential range, the shift between
ethanol and xylitol production from xylose in C. tropicalis is dependent
on the redox balance of the cells (23). The low redox potential in
limited oxygen conditions leads to the favoring of the reduction of
xylose to xylitol relative to ethanol production. The optimization of
the process conditions, e.g., by using a potentio-stat control system to
achieve the optimal redox potential would lead to more efficient
xylose assimilation and thus improve ethanol yield. Further process
development on fermentation conditions, including media optimiza-
tion, oxygen concentration, and reactor design is needed to maximize
ethanol productivity. Work to optimize the process using a bioreactor
is in progress.
In conclusion, an improved enzymatic process using the multi-
enzyme from A. niger for efficient saccharification of cassava pulp with
no pre-gelatinization has been developed. The multi-enzyme process
has the advantages of reduced energy consumption, as elevated
temperatures are not required, and improved fermentable sugar yield
compared with the conventional process. The non-thermal sacchari-
fication process is applicable for fermentation and biocatalytic reac-
tions using cassava pulp as a feedstock for the production of various
bio-products, including biofuels, biochemicals and bioplastics of
economic importance. The application of the multi-enzyme with
fermentation using thermotolerant microbes for biotechnological
conversion of this important agro-industrial biomass is promising.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Kuakoon Piyachomkwan,
Cassava and Starch Technology Research Unit, BIOTEC, for cassava pulp
composition analysis and valuable suggestions. Manuscript proof-
reading by Dr. Piyanun Harnpicharnchai, Dr. Darin Kongkasuriyachai,
and Dr. Philip Shaw is appreciated. This work was supported by a
research grant from the National Science and Technology Develop-
ment Agency (Grant number BT-B-02-CM-BC-5101).
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