Approach of Cassava Waste Pretreatments for Fuel Ethanol Production in Thailand

Approach of Cassava Waste Pretreatments
for Fuel Ethanol Production in Thailand

Teerapatr Srinorakutara*, Lerdluk Kaewvimol and
La-aied Saengow





Cassava waste can be utilized to produce ethanol due to its containing cellulose and
hemi-cellulose at levels of 24.99 and 6.67 % (w/w) respectively. A cassava pretreatment
method using enzymatic and acid hydrolysis for converting starch into fermentable sugars
was investigated. The cassava waste was hydrolyzed by using 0.2-5.0 M sulfuric acid
(H2SO4) at a temperature of 60-120oC for 30 min. It was found that the maximum reducing
sugar obtained at 6.1% (w/v) using a cassava waste to acid ratio of 1:2 (w/v) at 0.6 M H2SO4
and 120oC. While the maximum reducing sugar obtained in this study was at 6.2% (w/v),
using a mixture of cellulase and pectinase at pH 4.5, 28oC for 1 hr followed by ?-amylase at
pH 5.5, 100oC for 2 hrs and finally glucoamylase at pH 4.5, 60oC for 24 hrs. This is the
optimum condition for cassava waste pretreatment using enzymatic hydrolysis. It was also
found that the maximum ethanol production using Saccharomyces cerevisiae TISTR 5596
from enzymatic hydrolyzed sugar at initial reducing sugar 8.92% (w/v) was 3.62% (w/v) at
24 hrs of fermentation, corresponding to a 91% theoretical yield.

Key words: Cassava waste, cellulosic pretreatment, ethanol production and
Saccharomyces cerevisiae












Biotechnology Department, Thailand Institute of Scientific and Technological Research (TISTR), 35 Moo 3,
Technopolis, Klong 5, Klong luang, Pathumthani 12120, Thailand.
*Correspondence to: e-mail: teerapatr_tistr@yahoo.com

---

???????????????????????????????????????????????????????????

??????? ????????? ?????????? ???????? ?????????? ??????? (2549)
?????????????????????? ????????????????????? 31(1)



??????????????? ?????????????????? ??????????????? ?????? 24.99 ??? 6.67 ???
???????????? ????????? ??????????????????????????????????????????????????????????????
?????????????? pretreatment ???????????????????????????????????????????????????????????
??????? ???????????????????????????????????????????????????????? 0.2-5.0 ?????? ??????
??????????????? 60-120OC ???????? 30 ???? ?????????????????????????????? 0.6 ?????? ???
??????????????? 120OC ????????????????????????????????????? 1:2 ?????????????????????????
?????? 6.1 ????????????????????? ????????????????????????????????????????? ???????????
???????????????????????????????????? ??????????? 28OC ?????? pH 4.5 ???????? 1 ???????
?????????????????????????? ??????????????? 100OC ?????? pH 5.5 ???????? 2 ??????? ???
??????????????????? ??????????? 60OC ?????? pH 4.5 ???????? 24 ??????? ?????????
???????????????? ????????????????????? ?????? 6.2 ????????????????????? ??????????????????????
???????????????????????????????????? ???????????????????? ?????? 8.92 ??????????????
??????? ??????????????????? Saccharomyces cerevisiae TISTR5596 ????????????? 10 ????
???????????????????????? ?????? 3.62 ????????????????????? ???????????? 24 ???????
???????????????????????? 91

????????? ??????????????? ??????????????? ?????????????? Saccharomyces cerevisiae

---

INTRODUCTION
Cassava is a major raw material used in
cassava grated waste (CGW) biomass. At
many industries in Thailand. Production of
120oC for 30 min and using a high
MSG (Monosodium glutamate) and other concentration of H2SO4 (1-5M) hydrolysis was
amino acids, sweeteners, ethanol, makes use of
achieved but with excessive charring or
cassava. The cassava wastes left over from
dehydration reactions. A 60% process
these production processes are abundant and
efficiency was achieved with 0.3 M H2SO4
still contain a high amount of starch content.
with ethanol yield at 3.5%(v/v) after yeast
Most cassava wastes can be used as animal feed
fermentation. Acid hydrolysis of cellulosic
due to its high content of protein and other
pyrolysate to glucose and its fermentation to
nutrients which are necessary for animal ethanol were investigated by Zhisheng and
growth. In addition, cassava wastes can be
Hongxun.(4,5) The maximum glucose yield of
used to produce ethanol. Use of cassava waste
17.4% was obtained by hydrolysis with

as raw material in ethanol production not only
0.2 mol/l H2SO4 using autoclave at 121oC for
reduces waste material created from the cassava
20 min. The fermentation by Saccharomyces
starch industry, but also lowers the cost of
cerevisiae of a hydrolysate medium containing
ethanol production.(1)
95.8% glucose, resulted in about a 47%
Two technologies used to convert cellulose
increase in ethanol production as compared to
and hemicellulose to fuel ethanol are acid and
its present strain, which was fermented using
enzymatic hydrolyses. The most common is
commercial glucose. The reducing sugar was
acid hydrolysis.(2)
measured at 69.96% from 2.5% cassava pulp
Acidic hydrolysis is an effective method
at 135oC after 90 min.(6)
used for raw material pretreatment in ethanol
Another method of hydrolysis is
production. In previous research, a high enzymatic hydrolysis. Enzymes are naturally
concentration of acid has been used to treat
occurring plant proteins that cause certain
waste materials that contain cellulose.
chemical reaction to occur. However, for
Although acids are powerful agents used for
enzymes to work, they must obtain access to
biomass hydrolysis, concentrated acids are the molecules to be hydrolyzed. Eva-Lena
toxic, erosive and hazardous. Handling high
Jakobsson(7) reported that an enzymatic
concentrations of acid requires reactors that pretreatment at 190oC and 10 min resulted in
are resistant to erosion in raw material the best over all yield for glucose. A hundred
pretreatment. Diluted acid hydrolysis has been
grams of wheat straw yielded 42.6 g glucose
successfully developed for pretreatment of and 22 g xylose.
cellulose materials. Diluted sulfuric acid
In the present paper, the optimum
(H2SO4) can achieve significant results. Agu condition for acid and enzymatic hydrolyses of
et al.(3) reported on the effect of a combined
cassava waste was investigated.
heat treatment and acid hydrolysis study on

Table 1. The chemical composition of cassava waste.

Composition
Cassava waste %(w/w)

Sample 1
Sample 2
Sample 3
Moisture 78.16 79.50
82.74
Protein 1.82 2.03
2.31
Fat 0.09
0.20
0.16
Ash 1.61
2.38
2.05
Fiber 10.61
14.35
14.56
Starch 69.90
61.84
64.36
Note: Cyanide content in cassava waste = 154 mg/kg dry weight and
cyanide content after acid hydrolysis = 4.54 mg/kg dry weight

---

MATERIALS AND METHODS
Sample collection
Cassava waste was tendered by Sanguan
Ethanol concentration was measured by using
Wongse industries company limited, and ebulliometer.
Nakornratchasrima, Thailand in


a frozen form. The samples were thawed to
Ethanol Production
room temperature before performing the
Reducing sugar obtained from hydrolysis
experiment.
of cassava waste was fermented using
Acid hydrolysis
Saccharomyces cerevisiae TISTR 5596 in 10L
fermenter at 30oC for 60 hrs. Samples of

Cassava waste (50 g) was hydrolyzed
fermented broth were collected every 6 hrs for
with 100 ml (1:2 w/v) of various concentrations
cell number, reducing sugar, pH, and ethanol
of H2SO4 at 60-120oC. The hydrolysates were
concentration analysis.
separated to obtain any suspended or

unhydrolysated materials. Each sample was
RESULTS AND DISCUSSIONS
neutralized by 2 M NaOH solution for Composition of cassava waste
analytical processing.

Cassava waste was analyzed for its
Enzymatic hydrolysis
composition as shown in Table 1. The major

component, starch, was approximately 65.37%.
Cassava wastes (8, 10, and 12 g) were
The second highest component was fiber at
hydrolyzed with various enzymes (i.e.
?
13.17%. The composition of the cellulose in
-amylase, glucoamylase, cellulase and cassava waste is presented in Table 2. It can be
pectinase) which were generous gifts from
seen from Table 2 that holo-cellulose and
Genencor International Company. Several alpha-cellulose, which are composed of C6 and
hydrolysis conditions, including cellulase 15 unit
C5 sugars respectively, account for the majority
per gram cassava at pH 4.8, 40oC for 20 min,
of cellulose found. For ethanol production from
pectinase 4.7 unit per gram cassava at pH 3.5,
cassava waste, C6 and C5 sugars were studied.
20oC for 30 min, mixture of cellulase and

pectinase at pH 4.5, 28oC for 1 hr, ?-amylase
Table 2. The cellulosic composition of
24.02 unit per gram cassava at pH 5.5, 100oC
cassava waste.
for 2 hrs, and glucoamylase 0.66 unit per gram

cassava at pH 4.5, 60oC for 24 hrs, were tested.
Composition
The hydrolysates were separated by centrifuge
(%w/w)
at 9,000 rpm for analysis.(8)
Holo-cellulose
100.00
Analytical method
- Alpha-cellulose
73.31
- Pentosan
26.69
The AOAC method(9) was employed in
the determination of the composition of cassava
waste. The main components were starch,
Acid hydrolysis of cassava waste
protein, fiber, ash, fat and moisture.
In our first step, cassava wastes were
The reducing sugar content in the hydrolyzed in a 60oC waterbath for 30 min.
hydrolysates was determined quantitatively by
Other samples were autoclaved at 100oC,
using the Nelson Somogyi method as outlined
110oC, and 120oC for 30 min at various H
by Agu et al.(3) After mixing the samples with
2SO4
concentrations (0.2-5.0 M). The oligomers and
the assay reagent, the absorbance was measured
cellulosic materials in cassava waste were
at 520 nm against the appropriate blank converted into glucose. The results showed
solution by using Spectrophotometer model
cassava waste in a 60oC waterbath for 30 min
Uvikon-xs. The amount of reducing sugar
provided a lower sugar content than cassava
released was colorimetrically determined.
waste hydrolyzed by autoclaving at 100oC,
A calibration curve was obtained using
110oC and 120oC. The maximum reducing
D-glucose as standard.
sugar at 6.09% (w/v) recovered from cassava
Cell number was determined by pore
waste was detected after pretreatment with 0.6
plate in yeast malt agar and a haemacytometer.(8)
M H2SO4 for 120oC for 30 min (Figure 1).

---

7
7



6
6


5
5


4
S
4


% R 3
%RS 3


2

2


1
1


0
0

0.2
0.4
0.6
0.8
1
2
3
4
5

40
60
80
100
120
140
Concentration of H2SO4(M)




Tempe rature (oC )

Figure 1. Effect of acid concentration on Figure 2. Percentage of reducing sugar at
hydrolysis of cassava waste to
various temperatures of acid
reducing sugar (RS) at various
hydrolysis at (?) 0.6 M; ( ) 3 M;
temperature for 30 min.(?) 60oC;
( ) 4 M H2SO4
(×) 100oC; ( ) 110oC; ( ) 120oC.



At higher concentrations of H
The increase in temperature from 60oC to
2SO4 than
0.6 M, the reducing sugar was lower than
110oC resulted in an increased amount of
6.09%. This suggests that less reducing sugar in
reducing sugar from 0.5 to 6.1%w/v
the solution may be derived from dehydrating
particularly when using 3.0 and 4.0 M H2SO4.
or oxidizing by sulfuric acid on hydrolytic
The reducing sugar decreased about 1%w/v
products. The solution obtained in this study
when increasing temperature from 110oC to
also showed various colors. Similar results 120oC. However, the cassava waste pretreated
of acid hydrolysis of CGW biomass has been
using 0.6 M H2SO4 gave an increase in sugar
reported by Agu et al.(3) At high acid level from 0.2 to 6.2%w/v when performing the
concentration used for CGW biomass experiment at all temperatures from 60oC to
hydrolysis, a lot of charring or browning or
120oC (Figure 2).
dehydrating reactions occurred to a varying
Figure 3 shows the consequence of acid
degree. Other chemical reactions reported in
hydrolysis time at 0.6 M H2SO4, 120oC on
previous studies include the formation of reducing sugar content. The reducing sugar
furfural from xylose.(10) Furfural was reported
content in the early phase of hydrolysis rapidly
to inhibit activities of some glycolytic enzymes,
increased, but after 30 min the reducing sugar
particularly dehydrogenases in S.cerevisiae for
contents were almost constant. It was
ethanol production.(11)
previously found(6) that the highest content of
After comparison of acid concentration
reducing sugar was also produced from an acid
and temperature for hydrolysis, time for acid
hydrolysis of 2.5 w/v cassava pulps at 135oC
hydrolysis was investigated. The effect of time
and 90 min reaction time. However, increasing
for hydrolysis was studied at the maximum
hydrolysis time and temperature may increase
reducing sugar of each temperature.
the processing costs.

---

7
Figure 4 shows the effect of carbohydrate
6
concentration on reducing sugar production.
5
The reducing sugar in the early phase of
hydrolysis rapidly increased and mostly showed
S 4
maximum level at 7 hrs except for 12%
%R 3
carbohydrate showing a maximum level at

2
8 hrs. After 7-8 hrs of hydrolysis, the reducing
sugar decreased. The maximum level of
1
reducing sugar obtained at 11% of carbohydrate
0
concentration at 7 hours is shown in Figure 5.
15
30
45
60
Time (min)

For the same amount of cassava waste, %
reducing sugar by acid hydrolysis gave the

same level as enzyme hydrolysis, i.e. about
Figure 3. Percentage of reducing sugar from 6.2% reducing sugar. However, we used
0.6 M H2SO4 120oC at various time
enzyme pretreatment for the ethanol production
of acid hydrolysis.
process because of the unwanted browning

reaction of the product and the higher cost of
Enzymatic hydrolysis of cassava waste
acid hydrolysis.

The cassava waste was hydrolyzed using

four enzymes, cellulase, pectinase, ?-amylase and



glucoamylase and the hydroysis produced the

maximum reducing sugar (Table 3). This was

similar to the result of Supavadee.(12) The

14
reducing sugar obtained using the mixture of all

four enzymes was higher than when using
12

mixtures of three enzymes (cellulase, ?-amylase

10
and glucoamylase or pectinase, ?-aulase and
8
glucoamylase).
% RS
4
6


2

14



0 7
8
9
10
11
12

12

10

Carbohydrate in cassava residue (%)



8

RS



6
%

Figure 5. The maximum reducing sugar


at different amounts of
4




carbohydrate.




2



0




0
2
4
6
8
10




Time (hrs)



Figure 4. The amount of reducing sugar

at different concentrations of

carbohydrate by four enzyme

treatment. (?) 7%; ( ) 8%;

( ? ) 9%; ( ) 10%; ( ) 11%;

(?) 12%.

---

Table 3. Effect of different enzymatic treatments on percentage of reducing sugar.

Cassava residue
%Reducing sugar concentration
(g, dry weight)
by different enzymes
aA
Ce
Ce Pe Pe Ce Ce
Glu
aA
aA
Pe
Pe
Glu
Glu
aA
Glu
8
2.91
3.11
0.13 3.26 0.19 3.39 0.32
10
3.16
3.68
0.21 3.35 0.26 3.86 0.43
12
4.23
4.74
0.25 4.53 0.30 4.98 0.52
Note: aA: a-amylase, Glu: Glucoamylase, Ce: Cellulase, and Pe: Pectinase.


Ethanol production
Ethanol production derived from enzymatic
production obtained at 24 hours of fermentation
hydrolysate containing 8.92% (w/v) reducing
was 3.62% (w/v), corresponding to 91% of
sugar was done in 10L fermenter using S.
theoretical yield and after that the level of
cerevisae TISTR 5596. The maximum ethanol
ethanol remained constant (Figure 6).



100
6

,
)
l
80
5
H

/
l
)
,
p
/m
U

/l)
4
F
a
r (g
g
60
/
v), 5 CFU/ ml. C
g

u
(
3
,
w
5
s
s
e
(

c
o
40
e
r x 10
2
l
(%
e
ll x
c
i
ng
l
u

G
no
C
20

e
du
1
G
t
ha
R
E
O
L

0
0

0
10
20
30
40
50
60

Time (hours)
Cell numb

Reducing S Re
ugar ducing suga
Glucosre Glucose
Cell number x105

Ethanol
pH
Cellx5

Ethanol
pH



Figure 6. Ethanol production from enzymatically pretreated cassava waste by using
S.cerevisae TISTR 5596 in 10L fermenter.


The results showed a shorter production
because it is not necessary to import expensive
time when compared with the results of enzymes, and the microorganism hydrolysis
Theppanya(13) who reported that 4.21%w/v method is friendly to the environment.
ethanol production from cassava waste

hydrolysate at 54 hours.
CONCLUSIONS
Ethanol production derived from acid
Dilute sulfuric acid was used to pretreat
hydrolysate and enzymatic hydrolysate and also
cassava waste in the production of reducing
from microorganism hydrolysate (not earlier
sugar. This study shows that the hydrolysate at
mentioned) have been under investigation. 0.6 M H2SO4, at 120oC for 30 min gave
Among the three hydrolysis methods, the use of
the maximum reducing sugar at 6.1% (w/v).
microorganisms is probably the best one to
For a shorter hydrolysis time, a higher acid
pursue for fuel ethanol production in Thailand
concentration must be used.

---

The use of the mixture of cellulase and
5. Yu, Z. and Zhang, H. (2004) “Ethanol
pectinase at pH 4.5, at 28oC for 1 hr followed
fermentation of acid hydrolyzed cellulosic
by ?-amylase at pH 5.5, at 100oC for 2 hrs and
pyrolysate with Saccharomyce cerevisae”
finally glucoamylase at pH 4.5, at 60oC for 24
Bioresource Technology. 93, 199-204.
hrs was the optimum condition for enzymatic
6. Yoonan, K., Kongkittikajorn, J. and
cassava waste pretreatment in this study and
Rattanakanokchai, K. (2004) “Ethanol
also gave the maximum reducing sugar at 6.2%
production from acid hydrolysates of
(w/v).
cassava pulps using fermentation by
The maximum ethanol production obtained
Saccharomyces cerevisae” School of
from enzymatic hydrolysate containing 8.92%
bioresources and technology, King
(w/v) reducing sugar using S. cerevisae TISTR
Mongkut’s University of Technology
5596 in 10L fermenter at 24 hrs was 3.62%
Thonburi, Bangkok, Thailand.
(w/v), corresponding to 91% of theoretical
7. Jakobsson, E. (2003) “Optimization of the
yield.
pretreatment of wheat straw for production

of bioethanol. Department of Chemical
ACKNOWLEDGMENT
Engineering” Lund University.

The author is grateful to Associate 8. Suesat, C.(2003) “Utilization of waste from
Professor Wichien Kitpreechavanit (Faculty of
cassava starch plant for ethanol production”
Science, Kasetsart University, Bangkok,
Thesis of Master degree, Kasetsart
Thailand) for valuable discussion.
University, Bangkok, Thailand.

9. Association of Official Analytical Chemists
REFERENCES
(AOAC) (1990) “Official Method of
1. Akpan, I., Uraih, N., Obuekwe, C. O. and
Analysis. 15th edition The Association of
Ikenebomeh, M. J. (2004) “Production of
Official Agricultural Chemists, Verginia”
ethanol from cassava waste” Acta Biotech-
10. Delegenes, J. P., Moletta, R. and Navarro, J.
nologica 8(1) 39-45.
M. (1990) “Acid hydrolysis of wheat straw
2. Badger, P. C. (2002) “Ethanol from

and process consideration for ethanol
cellulose: A general review. In: J. Janick
fermentation by Pichia stipitis Y7124”
and A. Whipkey (eds.), Trends in new
Process Biochemistry. 25, 132-135.
crops and new uses” ASHS Press, 11. Banerjee, N., Bhatnagar, R. and Viswanathan,
Alexandria, VA. 17–21.
L. (1981) “Inhibition of glycolysis by
3. Agu, R. C., Amadife, A. E., Ude, C. M.,
furfural in Saccharomyces cerevisae”
Onya, A., Ogu, E. O., Okafor, M. and
European Journal of Applied Microbiology
Zejiofor, E. (1997) “Combined heat
and Biotechnology. 11, 226-228.
treatment and acid hydrolysis of cassava
12. Desaro, S. (2000) “Enzymatic hydrolysis of
grate waste (CGW) biomass for ethanol
cassava residue in ultrafiltration reactor”
production” Waste Management. 17(1),
Thesis of Master degree, Chulalongkorn
91-96.
University.
4. Yu, Z. and Zhang, H. (2003) “Pretreatment
13. Charocrnrat, T. (2001) “Batch Fermentation
of cellulose pyrolysate for ethanol
of Ethanol Production from Cassava Waste”
production by Saccharomyces cerevisae,
Thesis of Master degree, Kasetsart
Pichia sp. YZ-1 and Zymomonas mobilis”
University, Bangkok, Thailand.
Biomass and Bioenergy 24, 257-262.

---