Biomass and Bioenergy 27 (2004) 399 – 402

Short communication
Fermentation of enzymatic hydrolysate of sunower hulls for
ethanol production and its scale-up
Sanjeev K. Sharma 1 , Krishan L. Kalra∗ , Gurvinder S. Kocher
Department of Microbiology, College of Basic Sciences & Humanities, Punjab Agricultural University, Ludhiana 141004, Punjab, India

Received 8 August 2003; received in revised form 8 March 2004; accepted 30 March 2004

Abstract

Pretreated sunower hulls hydrolyzed with Trichoderma reesei Rut C 30 cellulase showed 59.8% sacchari4cation. En-
zymatic hydrolysate concentrated to 40 g=l reducing sugars was fermented with Saccharomyces cerevisiae var. ellipsoideus
under optimum conditions of time (24 h), pH (5.0), temperature (30◦ C) and inoculum size, and it showed a maximum ethanol
yield of 0:454 g=g. Ethanol production scaled up in 1 and 15 l fermentors under optimum conditions revealed maximum
ethanol yields of 0.449 and 0:446 g=g, respectively.
? 2004 Elsevier Ltd. All rights reserved.

Keywords: Sunower hulls; Enzymatic sacchari4cation; Fermentation; Ethanol

All over the world, various crop residues like wheat density, thus occupying large storage areas. The
straw, rice straw, cotton stalks, corn stalks and cobs, present study was thus carried out to utilize sunower
groundnut shell, etc. have been used for ethanol pro- hulls as substrate for ethanol production.
duction [1,2]. However, little e=ort has been made to The sunower hulls were removed from the sun-
utilize sunower residue as a substrate for ethanol pro- ower seeds obtained locally, oven dried at 70◦ C to
duction [3]. constant weight and ground to 40-mesh size. They
Sunower is cultivated over a large area with pro- were pretreated by sodium hydroxide 0:5% w=v at
duction of more than 1.5 million metric tons in India. an autoclaving pressure of 15 psi for 1:5 h and 4l-
This results in large amounts of sunower hulls and tered resulting in an extraction yield of 57.6%. The
stalks during industrial processing of sunower seeds. solids recovered were repeatedly washed with dis-
Sunower hulls have little commercial value and be- tilled water, oven dried (60◦ C to constant weight) and
come a disposal problem owing to their low bulk used as substrate for enzymatic sacchari4cation [4].
Pretreated sunower hulls contained 53.0% cellulose,
17.5% hemicellulose and 11.40% lignin on dry weight
∗ Corresponding author. Tel.: +91-161-2401960x330. basis.
E-mail addresses: pisces sanjeev@redi=mail.com The cellulase production was carried out by Tri-
(S.K. Sharma), klkalra1@redi=mail.com (K.L. Kalra).
1 Present address: Reliance Life Sciences Pvt. Ltd., Reliance coderma reesei Rut-C30 NRRL 11460 as was done
Industries Ltd., SSO, Annexe-IV, Moti Khavdi, Jamnagar 361140, earlier [3] and the crude culture 4ltrate was as-
Gujarat, India. sayed for cellulase, 4lter paper activity (FPA) and

0961-9534/$ - see front matter ? 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2004.03.005
400 S.K. Sharma et al. / Biomass and Bioenergy 27 (2004) 399 – 402

carboxymethylcellulase activity (CMCase) by the The e=ects of various parameters, viz fermen-
method of Mandels et al. [5] and cellobiase activity tation time (12–60 h), temperature (26–34◦ C),
by the method of Srivastava et al. [6] and used for pH (3.5–5.5) and inoculum size (2–6% v=v) were
sacchari4cation of pretreated sunower hulls. One optimized under the shake ask culture conditions
unit of enzyme was de4ned as the amount of enzyme (Table 1).
capable of producing one micromole (M) of reduc- Maximum ethanol yield of 0:452 g=g was obtained
ing sugars in one minute. The T . reesei Rut-C 30 after 24 h (Table 1) corresponding to a fermenta-
NRRL 11460 produced maximum cellulase after 8 d tion eLciency of 88.63%. The optimum temperature
of incubation at 28◦ C. The maximum FPA, CMCase for the fermentation of enzymatic hydrolysate of sun-
and cellobiase activities were found to be 1.05, 4.62 ower hulls was found to be 30◦ C. Ethanol yield of
and 0:42 U=ml, respectively. 0:453 g=g was obtained from enzymatic hydrolysate
The residues of sunower hulls obtained after pre- when fermented at 30◦ C for 24 h. The e=ect of ini-
treatment were sacchari4ed using crude culture 4ltrate tial pH value ranging from 3.5 to 5.5 on fermenta-
of T . reesei Rut-C 30 in 0:1 M citrate bu=er (pH 5.0) tion of enzymatic hydrolysate of sunower hulls re-
in stoppered Erlenmeyer asks at 50◦ C for 72 h under vealed maximum ethanol yield of 0:455 g=g at a pH
shaking conditions (150 rpm). The solid–liquid ratio of 5.0. There was a little variation in the ethanol yield
was maintained at 0:05 g=ml. Enzyme and substrate and hence fermentation eLciency at a pH range of
concentration used were 25 FPU=g and 5% (w/v), re- 4.0–5.0. A pH value lesser than 4.0 and higher than
spectively [4,7]. 5.0 resulted in sharp decrease in the ethanol yield.
Sacchari4ed mash was spun at 5000 rpm for 20 min The fermentation of enzymatic hydrolysate of sun-
and supernatant was analyzed for reducing sugars [8]. ower hulls at di=erent inoculum levels of the yeast
The enzymatic hydrolysate was concentrated by evap- (2–6% v=v) showed an inoculum level of 3% (v/v)
oration to 40 g=l of reducing sugars in a water bath with 2:1 × 108 cells=ml as optimum for maximum
and used as a substrate for ethanol fermentation by ethanol of 0:454 g=g corresponding to a fermentation
S.cerevisiae var. ellipsoideus. The extent of sacchari- eLciency of 89.02%. (Table 1). With the increase in
4cation was calculated as

Reducing sugar concentration obtained

Sacchari4cation(%) = ×0:9×100:
Potential sugar concentration in the pretreated substrate subjected to hydrolysis
Sacchari4cation of the pretreated sunower hulls
under optimized conditions (temperature 50◦ C, inoculum size from 3% to 6% (v/v), ethanol yield and
pH 5.0, substrate concentration 5:0% w=v and en- fermentation eLciency decreased, being 0:424 g=g
zyme concentration 25 FPU=g) yielded 468:4 mg=g and 83.14%, respectively at inoculum level of 6%
reducing sugars with 59.8% sacchari4cation, thus (v/v).
producing 26:98 g reducing sugars from 100 g of Earlier, 36 h fermentation time at 8% v=v inocu-
ground sunower hulls [3]. lum size has been reported optimum for ethanol
A 24 h old culture of S. cerevisiae var. ellipsoideus production from enzymatic hydrolysate of water hy-
at 3% (v/v) containing 2:1 × 108 cells=ml was inocu- acinth by S. cerevisiae [11]. While, an incubation
lated in 50 ml (in 250 ml Erlenmeyer asks) of fer- period of 24 h has been found to be optimum for
mentation medium made in enzymatic hydrolysate [9] production of ethanol by S. cerevisiae from acid and
containing 40 g=l reducing sugars. The fermentation enzymatic hydrolysate of agricultural residues [12].
was carried out at 30◦ C under stationary conditions Reduced yield with increase in temperature beyond
and ethanol was estimated [10] periodically. 30◦ C might be due to the inactivation of the enzymes
The fermentation eLciency (%) was calculated as involved in ethanol production pathways. Slininger

Ethanol yield obtained(Yp=s )

× 100:
Theoretical maximum ethanol yield from sugar substrate
 S.K. Sharma et al. / Biomass and Bioenergy 27 (2004) 399 – 402 401

Table 1
E=ect of fermentation parameters on ethanol production from enzymatic hydrolysate of sunower hulls by S. cerevisiae var. ellipsoideus

Fermentation Ethanol yield Fermentation Fermentation Ethanol yield Fermentation

parameter (Yp=s ) eLciency parameter (Yp=s ) eLciency
(g/g) (%) (g/g) (%)

Time (h) pH
12 0.209 40.98 3.5 0.177 34.70
24 0.452 88.63 4.0 0.326 63.92
36 0.426 83.53 4.5 0.426 83.53
48 0.392 76.86 5.0 0.455 89.22
60 0.360 70.59 5.5 0.406 79.61

Temperature Inoculum size

(◦ C) (% v/v)
26 0.395 77.45 2 0.402 78.82
28 0.429 84.12 3 0.454 89.02
30 0.453 88.82 4 0.445 87.25
32 0.438 85.88 5 0.436 85.49
34 0.407 79.80 6 0.424 83.14

Substrate—enzymatic hydrolysate of sunower hulls containing 40 g=l reducing sugars.

Table 2
Ethanol production from enzymatic hydrolysate of sunower hulls in 1 and 15 l fermentors by S. cerevisiae var. ellipsoideus

Fermentation time (h) Ethanol yield (Yp=s )(g=g) Fermentation eLciency (%)

1 l fermentor 15 l fermentor 1 l fermentor 15 l fermentor

6 0.113 0.111 22.16 21.76

12 0.272 0.274 53.33 53.73
18 0.449 0.446 88.04 87.45
24 0.436 0.437 85.49 85.68
30 0.430 0.428 84.31 83.92
36 0.423 0.412 82.94 80.78

Fermentation conditions: Substrate - Enzymatic hydrolysate of sunower hulls containing 40 g=l reducing sugars; Temp. - 30◦ C;
pH - 5.0; Agitation - 150 rpm; Aeration - for 4rst 10 h at 1 l min−1 .

et al. [13] reported that optimum fermentation rates used to maintain pH), temperature 30◦ C, aeration
with P. tannophilus were obtained at 32◦ C. They sug- 1 l min−1 and dissolved oxygen of 30%. Aeration was
gested that at high temperatures either the enzyme is stopped after 10 h and samples were collected asepti-
not induced and/or once formed the enzyme degrades cally every 6 h for estimation of ethanol. The results
rapidly. presented in Table 2 revealed similar ethanol yield
Ethanol production was studied under optimized and fermentation eLciencies of 0:449 g=g, 88.04%
conditions in two fermentors of capacity 1 and 15 l and 0:446 g=g, 87.45% after 18 h of fermentation in
according to the method of Chung and Lee [9] using 1 and 15 l fermentors, respectively.
working volumes of 0.6 and 10 l, respectively. The Comparatively similar ethanol yield and fermen-
medium was inoculated aseptically with a 24 h old tation eLciencies were observed in 1 and 15 l fer-
culture of S. cerevisiae var. ellipsoideus at 3% (v/v) mentors after 18 h of fermentation. In one of our
containing 2:1 × 108 cells=ml. earlier studies [4], we reported ethanol yield of
The fermentation conditions were maintained at 0:439 g=g and 0:437 g=g after 18 h fermentation
pH 5.0 (2 N ammonium hydroxide and 1 N HCL were of enzymatic hydrolysate of sunower stalks by
402 S.K. Sharma et al. / Biomass and Bioenergy 27 (2004) 399 – 402

S. cerevisiae in 1 and 15 l fermentors, respectively. [3] Sharma SK. Sacchari4cation and bioethanol production
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