Biomass and Bioenergy 23 (2002) 237 – 243

Enzymatic saccharication of pretreated sunower stalks

Sanjeev K. Sharma ∗ , Krishan L. Kalra, Harmeet S. Grewal
Department of Microbiology, College of basic sciences and Humanities, Punjab Agricultural University,
Ludhiana 141 004, Punjab, India
Received 29 October 2001; received in revised form 27 March 2002; accepted 27 March 2002

Abstract

The sunower stalks were pretreated by steam explosion (at 1:05 kg=cm2 for 0:5–1:5 h) and sodium hydroxide (0:25–
1:5% w=v NaOH at 1:05 kg=cm2 for 0:5–1:5 h) using solid : liquid ratio of 0:05 g=ml and subsequently saccharied enzymat-
ically. Steam explosion at 1:05 kg=cm2 pressure for 1:5 h was found to be the optimum pretreatment. Maximum enzymatic
saccharication of 57.8% was observed by treating 5% (w=v) pretreated sunower stalks with T. reesei Rut-C 30 cellulase
◦
(25 FPU=g) at 50 C, pH 5.0 for 72 h. ? 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Sunower stalks; Enzymatic saccharication; Steam explosion; Sodium hydroxide pretreatment; Cellulase

1. Introduction
cellulose to enhance the conversion of cellulose to glu-
cose. The commonly used methods for breakdown of
Lignocellulosic materials such as agricultural
cellulose to glucose are acid and enzymatic hydrol-
residues, food processing wastes, wood, municipal
ysis. Each method has its advantages and disadvan-
solid wastes and wastes from pulp and paper industry
tages, but the overriding factor in the long run must be
are considered as low cost and abundant raw materials
low energy requirement and low pollution. Enzymatic
for bioconversion into sugars which can be fermented
hydrolysis is not only energy sparing, because of the
to fuel ethanol.
relatively mild reaction conditions but also avoids the
In lignocellulosic materials cellulose, a linear
use of toxic and corrosive chemicals.
polymer of glucose is associated with hemicellulose
Various crop residues like wheat straw, rice straw,
and surrounded by lignin seal. Lignin, a complex
corn stalks and cobs, groundnut shells, etc., have been
three-dimensional polyaromatic matrix prevents en-
used for ethanol production but there is no report to
zymes and acids from accessing some regions of
the best of our knowledge on utilization of sunower
the cellulose polymers. Crystallinity of the cellulose
stalks for ethanol production. This crop was cultivated
further impedes acid and enzymatic hydrolysis [1,2].
in an area of 2.2 million hectares with production of
The pretreatment of lignocellulosics is primarily
1.50 million metric tons in India in 1998 [3]. This
employed to increase the accessible surface area of
result in huge quantity of sunower stalks annually
which do not nd any suitable end use and are gen-
∗ Corresponding author. Pzer Limited, 178-178A, Industrial erally burnt in the elds causing environmental pollu-
Area, Phase-I, Chandigarh 160 002, India. Tel.: +91-172-650578;
fax: +91-172-655178.
tion. Therefore, sunower stalks, as lignocellulosics,
E-mail addresses: sanjeev.sharma@pzer.com (S.K. Sharma), aFord a renewable and low-cost raw material for the
klkalra1@rediFmail.com (K.L. Kalra). production of fermentable sugars.

0961-9534/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 1 - 9 5 3 4 ( 0 2 ) 0 0 0 5 0 - 8
238 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243
The main objective of our work is to nd the optimal
conditions for the pretreatment and enzymatic saccha-
rication of sunower stalks and ultimately to ferment
the sugars to ethanol. In this paper, we report on the
optimization of pretreatment and enzymatic sacchari-
cation of sunower stalks.
2. Materials and methods
2.1. Materials
The sunower stalks used in the present study were
collected from the experimental farm of Department of
Plant Breeding, Punjab Agricultural University, Lud-
hiana, India. The stalks were washed 2–3 times with
water to remove extraneous matter. The sun dried
stalks were chopped into 2–3

pieces with the help
of an electric chopper and further dried in oven at
◦
70 C to constant weight. Oven dried sunower stalks
were then ground (40 mesh) with electric grinder. The
ground substrate was stored at room temperature till
further use.
2.2. Microorganisms
T. reesei Rut-C 30 NRRL 11460 used in the present
study for cellulase production was procured from the
ARS Patent Culture Collection, United States Depart-
ment of Agriculture, Peoria, IL, USA. Culture was
◦ ◦
maintained on PDA slants at 40 C and subcultured
fortnightly.
2.3. Enzyme production
The cellulase was produced by T. reesei Rut-C
30 under submerged batch conditions using Andreotii
[4] basal medium supplemented with 1% cellulose.
One hundred milliliter of basal medium was dispensed
into each of 250 ml Erlenmeyer asks containing 1 g
cellulose. The asks were autoclaved at 1:05 kg=cm2
for 20 min, cooled to room temperature and inocu-
lated with 10 ml of fungal culture pregrown on GYE
medium. Flasks were then placed on rotary shaker
◦
(200 rpm) at 28 C for 8 days. After incubation culture
broth was ltered and unpuried culture ltrate was
used as cellulase enzyme in further studies. The cul-
ture ltrate had a lter paper activity of 1:05 IU=ml,
a CMCase activity of 4:62 IU=ml and a cellobiase
activity of 0:42 IU=ml as measured by the methods
suggested by Mandels et al. [5].
2.4. Analytical methods
Moisture, crude fat and ash analysis were conducted
according to AOAC procedures [6]. Protein was deter-
mined by the Kjeldahl method. Cellulose content was
determined by the method of Crampton and Maynard
[7]. Hemicellulose and lignin were determined by
the methods described by Goering and Vansoest [8]
and reducing sugars were determined by the DNS
method [9].
2.5. Pretreatment of sun5ower stalks
Powdered substrate was subjected to physical
(steam explosion) and chemical pretreatments prior
to enzymatic saccharication. Steam explosion was
performed in a vertical pressure-cooker-type auto-
clave at 1:05 kg=cm2 for 0.5, 1.0 and 1:5 h followed
by sudden depressurization by fully opening the
steam exhaust valve of autoclave. Sodium hydrox-
ide (0:25–1:5% w=v) pretreatment of substrate was
◦
carried out in an autoclave at 121 C for 0.5, 1.0 and
1:5 h [10]. Solid : liquid ratio in both steam explosion
and sodium hydroxide treatment was maintained at
0:05 g=ml. In both cases solids recovered by ltration
were repeatedly washed with distilled water until
wash water turned to pH 7.0 and oven dried at 60 C.
2.6. Enzymatic sacchari8cation
The steam exploded pulp of sunower stalks ob-
tained after pretreatment was saccharied using crude
culture ltrate of T. reesei Rut-C 30 in 0:1 M citrate
buFer (pH 4.8) in stoppered Erlenmeyer asks. The
◦
asks were shaken at 150 rpm at 50 C. The initial
solid : liquid ratio used was 4 g=100 ml. The enzyme
substrate ratios studied were 5–25 FPU=g as a little
increase in hydrolysis eLciency has been reported
for higher enzyme concentrations [11,12]. Samples
were withdrawn after intervals of 12 h, centrifuged
at 5000 rpm for 20 min and the supernatant was
analyzed for reducing sugars. To determine eFective-
ness of diFerent pretreatments enzyme substrate
ratio was maintained at 10 FPU=g and substrate was
 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243 239

saccharied for 48 h. Other saccharication condi- 280

tions were optimized using enzyme concentration of
25 FPU=g of substrate. EFect of substrate concentra-
◦
tion (4:0–8:0% w=v), temperature (40–60 C) and pH 275

Reading sugars (mg/g)

(4:0–6:0) on enzymatic saccharication of sunower
stalks was also studied. The extent of saccharication
of sunower stalks was calculated using the factor 270
0.9x (reducing sugar concentration obtained=potential
sugar concentration in the pretreated substrate
subjected to hydrolysis) [10]. Experiments were
265
performed in triplicates and results are analyzed by
complete randomized factorial design and average
values were represented.
260
0.5 1 1.5
Autoclaving Time (hrs) at 1.05 kg/cm²
3. Results
Fig. 1. EFect of steam explosion on the enzymatic hydrolysis of
3.1. Physical and chemical characteristics of sunower stalks. Incubation time 48 h.
sun5ower stalks

Sunower stalks used in the present study were

characterized for physical and chemical components. 200

The moisture content (on wet weight basis) was 190


9.20%. The ash (%), cellulose (%), hemicellulose 180
(%), lignin (%), crude fat (%) and protein (%) con- 
Reading sugars (mg/g)

170  
tents were 4.57, 38.50, 33.50, 17.50, 1.95 and 2.00 
 

on dry weight basis, respectively. Rest 1.98% were 160

the hot water and organic solvent extractives. 150 



140 
3.2. Pretreatment of sun5ower stalks by physical   0.25% (w/v) NaOH
130
and chemical methods 


 0.50% (w/v) NaOH
120  0.75% (w/v) NaOH
 1.00% (w/v) NaOH
Fig. 1 shows the eFect steam explosion on substrate 110  1.25% (w/v) NaOH
susceptibility for enzymatic saccharication. With
1.50% (w/v) NaOH
100
the increase in autoclaving time from 0.5 to 1:5 h at 0.5 1 1.5
1:05 kg=cm2 , enzymatic hydrolysis has been continu- Autoclaving Time (hrs) at 1.05 kg/cm²

ously improved being maximum at 1:05 kg=cm2 for

1:5 h (reducing sugars 277:60 mg=g). The autoclaving
time at a pressure of 1:05 kg=cm2 could not be in-
creased beyond 1:5 h due to the technical limitations
of the autoclave used in the present studies.
Fig. 2 depicts the eFect of sodium hydroxide con-
centration (0:25–1:50% w=v) alongwith autoclaving
(0:5–1:5 h at 1:05 kg=cm2 ) on the enzymatic sac-
charication of sunower stalks. It was observed
that sodium hydroxide at 0.5% concentration at
1:05 kg=cm2 for 1:5 h was more eFective pretreatment
as compared to the other concentrations and time
Fig. 2. EFect of sodium hydroxide alongwith autoclaving
at 1:05 kg=cm2 on the enzymatic hydrolysis of sunower stalks.
Incubation time 48 h.
combinations for subsequent enzymatic hydrolysis of
the substrate (reducing sugars 185:5 mg=g).
It can be concluded that steam explosion at
autoclaving pressure of 1:05 kg=cm2 for 1:5 h is the
best pretreatment for sunower stalks among the
performed experiments.
240 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243

3.3. Chemical characteristics of the pretreated

34:9 ± 1:15
40:8 ± 1:56
44:8 ± 1:32
47:0 ± 1:56
48:6 ± 1:61
49:7 ± 2:14
substrate

Data represent averages of triplicates. Sacchari8cation conditions: Enzyme source-T. reesei Rut-C 30 culture ltrate, incubation temperature: 50 C, RS—reducing
Optimally pretreated sunower stalks (with steam

S
at 1:05 kg=cm2 pressure for 1:5 h followed by sudden

25

260:3 ± 2:52
308:4 ± 2:69
338:3 ± 2:78
355:4 ± 2:14
367:5 ± 1:98
375:7 ± 2:70
depressurization) have been analyzed for chemi-
cal components. Pretreated sunower stalks contain
51.0% cellulose, 17.0% hemicellulose and 14.6%

RS

◦
lignin. Therefore, by comparison to the chemical
components in the untreated stalks it is clear that

33:8 ± 1:12
39:5 ± 0:83
44:0 ± 1:23
45:6 ± 1:89
47:3 ± 1:95
48:1 ± 1:57
pretreatment solubilized 12.57% cellulose, 66.31%
hemicellulose and 44.94% lignin. Extraction yield
(fraction of sunower stalks recovered after pretreat-

S
20
ment) was 66.0%.

255:4 ± 3:03
298:2 ± 2:41
332:7 ± 1:98
344:7 ± 2:70
357:0 ± 2:14
363:6 ± 2:69
EFect of cellulase concentration and incubation period on the enzymatic saccharication of pretreated sunower stalks

Enzyme concentration (FPU=g of substrate)

RS
3.4. Enzymatic sacchari8cation

sugars (mg=g), S—saccharication (%) and ( )—transformed degree values of percentage saccharication.
31:8 ± 1:61
37:1 ± 1:45
41:3 ± 1:98
43:1 ± 1:26
44:2 ± 1:53
45:0 ± 1:45
Enzymatic saccharication of pretreated sunower
stalks was carried out by culture ltrate of T. reesei
Rut-C 30. The various parameters, viz., enzyme con-
S
15
centration, incubation period, substrate concentration,
240:5 ± 2:61
280:6 ± 3:03
312:4 ± 2:16
325:3 ± 1:85
334:1 ± 1:98
340:3 ± 2:70
hydrogen ion concentration and temperature were op-
timized to achieve maximum saccharication of the
pretreated sunower stalks.
RS
27:1 ± 0:57
32:1 ± 1:19
34:5 ± 1:42
36:7 ± 1:53
37:9 ± 1:26
38:5 ± 1:53
3.4.1. E9ect of enzyme concentration and
incubation period on the rate of sacchari8cation
S

The eFect of T. reesei Rut-C 30 enzyme (concen-

10

trations 5–25 FPU=g of substrate) and the incubation

204:5 ± 3:70
242:4 ± 2:14
260:5 ± 2:78
277:0 ± 3:13
286:5 ± 1:98
290:7 ± 2:70

period (12–72 h) on the hydrolysis of sunower stalks

has been studied and results are presented in Table 1.
RS

Sunower stalks after saccharication with 5 FPU=g

enzyme for 12 h yielded 112:63 mg=g reducing sugars
14:9 ± 1:43
20:4 ± 1:19
23:0 ± 1:61
24:1 ± 1:98
25:2 ± 2:06
26:2 ± 1:45

with a corresponding saccharication of 14.91%. The

level of reducing sugars signicantly (P ¡ 0:05) in-
creased to 375:70 mg=g with 49.73% saccharication
S

by increasing the enzyme concentration to 25 FPU=g

5

and incubation period to 72 h. The initial increase in

112:6 ± 2:16
154:4 ± 2:98
172:6 ± 2:52
182:3 ± 3:03
190:4 ± 2:69
198:1 ± 3:14

the enzyme dose from 5 to 10 FPU=g led to nearly

1:3–2:0-fold increase in the amount of released sugars.
RS

However, thereafter the increase observed was less.

Likewise the rate of hydrolysis was fast up to 36 h and
Incubation
period (h)

then slowed down, resulting in comparatively lower

Table 1

rate of hydrolysis between 36 – 48, 48– 60 and 60–72 h

12
24
36
48
60
72

of incubation.
 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243 241

440 450 60
 57 
420 55
400

400  50


Reading sugars (mg/g)

Reading sugars (mg/g)

52
350
380 45

360  300 40
47

340 35
250 
320 42 30
200 
300 25

280 37 150 20
4 5 6 7 8 40 44 48 52 56 60
Substrate Concentration % (w/v) Temperature (°C)

Fig. 3. EFect of substrate concentration on the enzymatic sac- Fig. 4. EFect of temperature on the enzymatic saccharication
charication of pretreated sunower stalks. Enzyme concentration of pretreated sunower stalks. Enzyme concentration 25 FPU=g,
◦
25 FPU=g, incubation period 72 h, temperature 50 C, pH 4.8. incubation period 72 h, substrate concentration 5% (w=v), pH 4.8.

3.4.2. E9ect of substrate concentration on the

480
enzymatic sacchari8cation 60.5
The results of the eFect of substrate concentration 450

(4:0–8:0% w=v) on enzymatic saccharication are 420

shown in Fig. 3. The rate of saccharication increased
Reading sugars (mg/g)

390 50.5
up to substrate concentration of 5.0%. Further increase 360
in the substrate concentration decelerated the rate of
330
hydrolysis. Maximum saccharication of 56.5% was
300  40.5
achieved at substrate concentration of 5.0%.
270

3.4.3. E9ect of temperature on enzymatic 240 

30.5
sacchari8cation 210
The saccharication of pretreated sunower stalks 180
◦
was carried out at temperature ranging from 40 C to 
◦ 150 20.5
60 C. Fig. 4 indicate that the initial hydrolysis rate in- 4 4.5 5 5.5 6
creased with increasing hydrolysis temperature. Max- pH
◦
imum saccharication (56.4%) was observed at 50 C
Fig. 5. EFect of pH on the enzymatic saccharication of pretreated
with corresponding reducing sugars 426:2 mg=g. De- sunower stalks. Enzyme concentration 25 FPU=g, incubation pe-
creased saccharication was observed at temperatures ◦
riod 72 h, substrate concentration 5% (w=v), temperature 50 C.
higher than the optimum.

3.4.4. E9ect of pH on enzymatic sacchari8cation saccharication was obtained at pH values lesser or

The saccharication of pretreated sunower stalks
was carried out at a range of pH values (4:0–6:0) and
results are presented in Fig. 5. Maximum sacchari-
cation of 57.8% was observed at pH 5.0 with corre-
sponding reducing sugars of 436:6 mg=g. Decreased
production of reducing sugars as well as percent
higher than optimum.
In view of the results obtained it can be concluded
that optimum saccharication of sunower stalks
could be achieved by treating 5% (w=v) pretreated
stalks with 25 FPU=g T. reesei Rut-C 30 cellulase at
◦
50 C, pH 5.0 for 72 h.
242 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243
4. Discussion
Ground sunower stalks (40 mesh) were pretreated
and then saccharied by the T. reesei Rut-C 30 cellu-
lase. Reducing sugars thus formed have the potential
for conversion to bioethanol. Steam explosion pre-
treatment by autoclaving at a pressure of 1:05 kg=cm2
for 1:5 h followed by sudden depressurization was
found to be the best among diFerent time and pressure
combinations tried. Other workers [13–15] have also
observed signicant increase in enzymatic hydrolysis
of the cellulose substrate by pretreatment with steam
explosion method. Pretreatment with 0:5% (w=v)
sodium hydroxide at an autoclaving pressure of
1:05 kg=cm2 for 1:5 h was more eFective among dif-
ferent concentration and time combinations tried for
subsequent enzymatic hydrolysis of the substrate.
Results are similar to the conclusions of diFerent
authors [10,16]. A sodium hydroxide concentration
of 1% and steam pressure of 1 kg=cm2 for 1 h has
been reported as optimum for delignication of water
hyacinth [17]. At higher pretreatment severities it is
likely to have a better access to the ber. This should
be tested since the optimum obtained in this work is
at the highest severity experiment.
Among two types of pretreatments studied steam
explosion was found to be the best for subsequent
enzymatic hydrolysis of sunower stalks. This might
be due to less lignin content in the sunower stalks
as sodium hydroxide basically acts as delignica-
tion agent. Also at higher temperatures sodium hy-
droxides might cause important material loss [18].
Petreatment of sunower stalks by steam explosion
under optimized conditions solubilized 12.57%
cellulose, 66.31% hemicellulose and 44.94% lignin.
Extraction yield obtained was 66.0%. According
to Szczodrak [11] hydrothermal action favor the
pentosan degradation giving a 93.5% loss of this
component after the autohydrolysis reaction of wheat
straw. Dekker and Wallis [19] reported that autohy-
◦
drolysis of sunower seed hulls at 200 C for 5 min,
followed by explosive debration solubilized 78% of
the total hemicellulose and 85% of the pectic sub-
stances. The remaining exploded pulp was reported
to be highly susceptible to hydrolysis by cellulase.
Enzymatic saccharication of the pretreated sun-
ower stalks was carried out by T. reesei Rut-C 30
cellulase. Enzyme concentration of 25 FPU=g of the
substrate and incubation time of 72 h resulted in the
maximum saccharication (49.73%) of the substrate.
The saccharication rate at the start of incubation
period was higher and then it slowed down. This
behavior might be due to the decrease in the extent
of adsorbed enzyme, transformation of the structure
of cellulose into less digestible form and inhibition
of the enzyme action by the accumulated hydrolysis
products [20]. The initial (1:3–2:0-fold) increase in
the reducing sugars with increase in enzyme dose
from 5 to 10 FPU=g and slow increase in reducing
sugars afterwards, might be due to the less adsorption
eLciency for higher enzyme concentrations than for
diluted ones [10]. Saturation of the cellulose surface
with enzyme might be the other reason behind it.
Slow rate of saccharication after 60 h has also been
reported earlier [12,21,22].
The substrates concentration of 5.0% (0:05 g=ml)
resulted in the maximum saccharication. Further
increase in substrate concentration decelerated the
rate of hydrolysis. Substrate concentrations over
0:05–0:075 g=ml have been shown to cause substrate
inhibition both with pure cellulose and pretreated lig-
nocellulosic materials [10,20,23]. However, substrate
concentration of 6% [24] and 10% [22] has also been
found adequate to release optimum reducing sugars.
Stirring diLculties, reduction of the aqueous movable
phase and end product inhibition can hinder the enzy-
matic hydrolysis of pretreated lignocellulosic residues
at higher substrate concentration [11,20].
Most suitable temperature for enzymatic hydrolysis
◦
of sunower stalks was found to be 50 C. At temper-
atures lower or higher than this less saccharication
was observed. Reduced saccharication at higher tem-
perature could be attributed to the thermal inactivation
of endoglucanase I and cellobiohydrolase I [25,26].
◦
The temperature of 50 C was also found optimum for
enzymatic saccharication of diFerent lignocellulosic
materials [11,27–29]. However, a hydrolysis temper-
◦
ature of 40 C was found optimum for high glucose
yield and low enzyme deactivation [21] A pH value of
5.0 was found to be optimum for enzymatic saccha-
rication of sunower stalks. Decreased saccharica-
tion was observed at pH values lesser or higher than
the optimum. This might be due to the requirement of
the cellulase enzyme for specic hydrogen ion con-
centration in the reaction mixture for eLcient perfor-
mance. The results with respect to optimum pH are
 S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243
in close agreement with those of the earlier workers
[11,27,28,30].
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