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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 a CMCase activity of 4:62 IU=ml and a cellobiase
conditions for the pretreatment and enzymatic saccha- activity of 0:42 IU=ml as measured by the methods
rication of sunower stalks and ultimately to ferment suggested by Mandels et al. [5].
the sugars to ethanol. In this paper, we report on the
optimization of pretreatment and enzymatic sacchari- 2.4. Analytical methods
cation of sunower stalks.
Moisture, crude fat and ash analysis were conducted
according to AOAC procedures [6]. Protein was deter-
2. Materials and methods mined by the Kjeldahl method. Cellulose content was
determined by the method of Crampton and Maynard
2.1. Materials [7]. Hemicellulose and lignin were determined by
the methods described by Goering and Vansoest [8]
The sunower stalks used in the present study were and reducing sugars were determined by the DNS
collected from the experimental farm of Department of method [9].
Plant Breeding, Punjab Agricultural University, Lud-
hiana, India. The stalks were washed 2–3 times with 2.5. Pretreatment of sun5ower stalks
water to remove extraneous matter. The sun dried
stalks were chopped into 2–3 pieces with the help Powdered substrate was subjected to physical
of an electric chopper and further dried in oven at (steam explosion) and chemical pretreatments prior
◦
70 C to constant weight. Oven dried sunower stalks to enzymatic saccharication. Steam explosion was
were then ground (40 mesh) with electric grinder. The performed in a vertical pressure-cooker-type auto-
ground substrate was stored at room temperature till clave at 1:05 kg=cm2 for 0.5, 1.0 and 1:5 h followed
further use. by sudden depressurization by fully opening the
steam exhaust valve of autoclave. Sodium hydrox-
2.2. Microorganisms 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
T. reesei Rut-C 30 NRRL 11460 used in the present 1:5 h [10]. Solid : liquid ratio in both steam explosion
study for cellulase production was procured from the and sodium hydroxide treatment was maintained at
ARS Patent Culture Collection, United States Depart- 0:05 g=ml. In both cases solids recovered by ltration
ment of Agriculture, Peoria, IL, USA. Culture was were repeatedly washed with distilled water until
◦ ◦
maintained on PDA slants at 40 C and subcultured wash water turned to pH 7.0 and oven dried at 60 C.
fortnightly.
2.6. Enzymatic sacchari8cation
2.3. Enzyme production
The steam exploded pulp of sunower stalks ob-
The cellulase was produced by T. reesei Rut-C tained after pretreatment was saccharied using crude
30 under submerged batch conditions using Andreotii culture ltrate of T. reesei Rut-C 30 in 0:1 M citrate
[4] basal medium supplemented with 1% cellulose. buFer (pH 4.8) in stoppered Erlenmeyer asks. The
◦
One hundred milliliter of basal medium was dispensed asks were shaken at 150 rpm at 50 C. The initial
into each of 250 ml Erlenmeyer asks containing 1 g solid : liquid ratio used was 4 g=100 ml. The enzyme
cellulose. The asks were autoclaved at 1:05 kg=cm2 substrate ratios studied were 5–25 FPU=g as a little
for 20 min, cooled to room temperature and inocu- increase in hydrolysis eLciency has been reported
lated with 10 ml of fungal culture pregrown on GYE for higher enzyme concentrations [11,12]. Samples
medium. Flasks were then placed on rotary shaker were withdrawn after intervals of 12 h, centrifuged
◦
(200 rpm) at 28 C for 8 days. After incubation culture at 5000 rpm for 20 min and the supernatant was
broth was ltered and unpuried culture ltrate was analyzed for reducing sugars. To determine eFective-
used as cellulase enzyme in further studies. The cul- ness of diFerent pretreatments enzyme substrate
ture ltrate had a lter paper activity of 1:05 IU=ml, ratio was maintained at 10 FPU=g and substrate was
S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243 239
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
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
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
of incubation.
S.K. Sharma et al. / Biomass and Bioenergy 23 (2002) 237 – 243 241
440 450 60
57
420 55
400
400 50
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.
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
in close agreement with those of the earlier workers [16] Ishihara M, Toyama S. Enzymatic degradability of chemically
[11,27,28,30]. pretreated bagasse. Science bulletin, vol. 37. Okinawa:
College of Agriculture, University of the Ryukyus, 1990. p.
2733.
[17] Motwani M, Seth R, Daginawala HF, Khanna P. Microbial
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