Brazilian Journal

of Chemical
Engineering

ISSN 0104-6632
Printed in Brazil
www.abeq.org.br/bjche

Vol. 31, No. 02, pp. 355 - 363, April - June, 2014
dx.doi.org/10.1590/0104-6632.20140312s00002415

PRODUCTION OF FERMENTABLE SUGARS BY

COMBINED CHEMO-ENZYMATIC HYDROLYSIS
OF CELLULOSIC MATERIAL FOR BIOETHANOL
PRODUCTION
M. Idrees1, A. Adnan1, S. A. Bokhari2 and F. A. Qureshi3*
1

Department of Chemistry, GC University, Katchery Road, Lahore 54000, Pakistan.

E-mail: [email protected];[email protected]
2
Department of Biosciences, COMSATS Institute of Information Technology, Park Road,
Chak Shahzad Campus, Islamabad 45600, Pakistan.
E-mail: [email protected]
3
Office of Research, Innovation and Commercialization, COMSATS, Institute of Information
Technology, Phone: + 923218415890, Chak Shahzad, Park Road, Islamabad 45600, Pakistan.
*
E-mail: [email protected]
(Submitted: November 29, 2012 ; Revised: September 10, 2013 ; Accepted: September 16, 2013)

Abstract - To change the recalcitrant nature of the lignocellulosic material for maximum hydrolysis yield, a
comprehensive study was done by using sulphuric acid as an exclusive catalyst for the pretreatment process.
The enzymatic digestibility of the biomass [Water Hyacinth: Eichhornia crassipes] after pretreatment was
determined by measuring the hydrolysis yield of the pretreated material obtained from twenty four different
pretreatment conditions. These included different concentrations of sulphuric acid (0.0, 1.0, 2.0 and 3.0%), at
two different temperatures (108 and 121 C) for different residence times (1.0, 2.0 and 3.0h).The highest
reducing sugar yield (36.65 g/L) from enzymatic hydrolysis was obtained when plant material was pretreated
at 121 C for 1.0 h residence time using 3.0% (v/v) sulphuric acid and at 1:10 (w/v) solid to liquid ratio. The
total reducing sugars obtained from the two-stage process (pretreatment + enzymatic hydrolysis) was 69.6g/L.
The resulting sugars were fermented into ethanol by using Saccharomyces cerevisiae. The ethanol yield from
the enzymatic hydrolyzate was 95.2% of the theoretical yield (0.51g/g glucose), as determined by GS-MS,
and nearly 100% since no reducing sugars were detected in the fermenting media by TLC and DNS analysis.
Keywords: Eichhornia crasspies; Lignocellulosic; Water hyacinth; Saccharomyces cerevisiae; Ethanol;
Fermentation.

INTRODUCTION
Lignocellulosic biomass is an abundant, inexpensive and readily available source of fermentable
sugars (Ho et al., 1998). For the last few decades, the
conversion of these resources into glucose and other
reducing sugars has been considered as an attractive
route for production of ethanol or other valuable chemicals (Curreli et al., 1997; Gaspar et al., 2005). A wide
array of biomass sources, including agricultural
*To whom correspondence should be addressed

residues such as corn stover, wheat and rice straw

and forestry residue; industrial residues such as pulp
and paper processing waste and energy crops such as
switchgrass have been employed as biomass source.
However, unlike starch, which contains homogenous
and easily hydrolyzed biopolymers, lignocellulosic
plant matter contains cellulose (23-53%), hemicellulose (20-35%), and polyphenolic lignin (10-25%).
Glucose, obtained from lignocellulosic material, is
usually expected to be a renewable source, which

356

M. Idrees, A. Adnan, S. A. Bokhari and F. A. Qureshi

can be efficiently converted into fuels, foods, and

other valuable chemicals (Huber et al., 2006; Klemm
et al., 2005; Fan et al., 1987; Zhang and Lynd, 2004;
Ragauskas et al., 2006; Davda and Dumesic, 2005).
Cellulosic conversion into glucose therefore is a key
process which needs to be further studied.
The selection of the biomass source is of great
importance from a technical and economical point of
view. Ethically, biomass should not compete with
the food crops, and thus waste biomass or crops with
low commercial value, such as agricultural waste or
weeds, are preferred for such types of processes for
producing valuable chemicals. Furthermore, it is important to select a source that requires less fertilizer,
has a high growth rate and is preferably available the
whole year, as is the case with water hyacinth. Water
hyacinth could be an excellent biomass feedstock for
further conversions and utilization (Girisuta et al.,
2008).
Water hyacinth is a fast growing aquatic weed
present in water reservoirs such as large lakes, rivers,
shallow ponds, wetlands and marshes (Naseema et
al., 2004). It can double its mass within 8-10 days
and a single plant can produce up to 3000 offspring
in 50 days (Verma et al., 2003). Because of its capacity for exponential increase in the biomass, this
weed needs constant vigilance by farmers and canal
irrigation personnel. Because of its enormous amount,
some uses have been suggested such as composting,
cattle feed, biogas plant resources, paper and pulp
industry, furniture making, and waste water treatments (Gunnersson and Petersen, 2007). However,
there is no reported utilization of this weed on a large
industrial scale and it still continues to be jeopardy
for farmers and water management authorities as it
blocks water flow in irrigation and drainage canals,
channels and streams. This weed also makes aquatic
recreational activity difficult and is potentially unsafe in lakes, thus causing potential hurdles to
tourism and related industries. In an attempt to
address such problems, we propose to use it as a raw
material for the extraction of fermentable sugars for
value-added chemicals by optimizing and studying
the two-stage hydrolysis process using commercial
cellulase enzyme.
MATERIALS AND METHODS
Chemicals
3,5-Dinitrosalicylic acid and analytical grade
phenol were received from Fluka Chemie, D(+)xylose(GPR) was obtained from BDH (England) and

L-(+)-arabinose from Sigma Aldrich. Concentrated

sulphuric acid (9597 wt.%) and D(+)-glucose were
obtained from Panreac and -naphthol was purchased from Merck (Darmstadt, Germany). Distilled
water was used to prepare the various solutions.
AccelleraseTM1500 (Cellulase) having multiple
enzyme activities; exoglucanase, endoglucanase, hemicellulase and beta-glucosidase as reported by the manufacturer, was obtained from Genencore International
(U.S.A).
Water Hyacinth
Fresh and healthy water hyacinth plants were collected from a natural pond near Shahdrah, Lahore
(Punjab, Pakistan), during December. They were
thoroughly washed with tap water several times to
remove adhering dirt. Samples of stem, petiole and
leaf of the fresh plant were selected as the substrate
for saccharification. These parts were dried in an
oven at 105 C for 20 min and subsequently chopped
into small pieces (~1-2 cm) and blended to small
particles (~3-5 mm).
Pretreatment of the Water Hyacinth
The powdered dry plant material was used for
pretreatment under different conditions. Twenty five
grams of water hyacinth powder were mixed separately with 0.0, 1.0, 2.0 and 3.0% H2SO4 solution in
1:10 w/v% ratio, in 500 mL flasks. The flasks were
autoclaved (CL-40L (ALP Co, Ltd. Tokyo, Japan) at
two different temperatures (108 C and 121 C at a
pressure of 0.11 MPa) for different time intervals
(1.0, 2.0 and 3.0h). The solutions in the flasks were
cooled and filtered with Whatman filter paper. The
residue was washed with distilled water 3-5 times to
bring the pH at 4.8. The residue was dried at 105 C
for 20 min and weighed.
Enzymatic Hydrolysis of Pretreated Material
The enzymatic hydrolysis was carried out in 250
mL glass flasks using solid biomass residue obtained
after each pretreatment condition. A specific volume
of enzyme (0.2 mL/g dry weight of biomass) was
used for hydrolysis. Five grams of pretreated dry
powder of water hyacinth were added in each flask
separately. The pH of the reaction mixture was set at
4.8 by adding 100mL of 0.1M acetate buffer solution. The flasks were kept in an orbital shaker for
48.0 hours at 50 C and 160 rpm. At regular time intervals, sample were taken from each flask and kept
in boiling water to inactivate the enzyme. Each

Brazilian Journal of Chemical Engineering

Production of Fermentable Sugars by Combined Chemo-Enzymatic Hydrolysis of Cellulosic Material for Bioethanol Production

sample was filtered on Whatman filter paper and

subsequently analyzed. Each experiment was performed in duplicate.

357

concentration of ethanol (v/v) in the sample was determined, which was converted to (w/v) by multiplying it by 0.79 (specific gravity of ethanol at 20.0 C).

Ethanol Fermentation
RESULTS AND DISCUSSION
Commercial Bakers yeast (Saccharomyces cerevisiae) obtained from a local market, was used for
the ethanol fermentation. Inoculum was prepared by
transferring yeast cells (1.0g/100mL) into 250 mL
flasks containing 50.0 mL of culture medium containing 10.0 g/L yeast extract, 20.0 g/L peptone, and
20.0 g/L glucose and subsequently incubating at
30.0 C for 24.0 h. This was used to inoculate the
fermentation medium. Cellulosic hydrolyzate, obtained from enzymatic hydrolysis, was supplemented
with 1.0 g/L yeast extract, 2.0 g/L (NH4)2SO4 and 1.0
g/L of MgSO4. The inoculum-to-solution ratio of
1:10 was used for fermentation purposes. Samples
for glucose and ethanol analysis were taken at the
beginning and end of a 24.0 h fermentation process.
Analysis of Reducing Sugars and Ethanol
The amount of the reducing sugars was determined using 3,5-dinitrosalicylic acid (DNS) reagent
by the Ghose method (1987). Identification of monomeric sugars was done with thin layer chromatography (TLC) by using alpha-naphthol as locating reagent and a water: acetonitrile mixture (85:15) as
eluting solvent, as used by Beom et al. (2009) and
outlined in Idrees et al. (2013). The hydrolysis yield
was calculated on the basis of pretreated solid biomass used for enzymatic hydrolysis. Statistical analysis was done with Graph Pad Prism 5.
After centrifuging the liquid from the fermentation media for 10 min, ethanol was quantified in the
supernatant with the help of GC-MS (GCMS-QP2010
of Shimadzu) using a DB-5 capillary column (diameter 0.25 mm, length 30.0 m and thickness 0.25 m).
Nitrogen was used as carrier gas with flow rate of
1.41 mL/min. The temperature program was: temperature maintained at 40 C for 1.0 min, then raised
to 44.0 C at 15.0 C/min and at 1.0 C/min up to
50.0 C, then continuously increased to 250.0 C at
25.0 C/min and finally held at 250.0 C for 2.0 min.
The ion source temperature was 200.0 C. Data was
obtained in the scan mode in the mass range of 30120 m/z after injecting 2.0 L of sample. Fragment
ions 31 m/z and 45 m/z were used for identification
and quantification of ethanol, respectively. The calibration curve was obtained from 0.1, 0.2, 0.3 up
to 1.0% v/v concentration of ethanol in HPLC grade
water and their peak areas. From this, the

Water hyacinth has a high cellulosic content

(40.0-65.0%) (Malik, 2007; Nigam, 2002) with extremely high growth rate (140 ton/ha. year, dry wt.)
and have been considered as a prospective source for
production of ethanol and other fuels (Girusta et al.,
2008; Abraham et al., 1996; Sherma et al.,1999;
Singhal and Rai, 2003). To obtain the maximum
enzymatic hydrolysis for the production of ethanol, a
pretreatment step which consumes cheap chemicals
is necessary for process economy. The pretreatment
has the capability to decrease the crystallinity of the
cellulose and hemicellulose, remove the lignin content and avoid the production of potential inhibitors
for fermenting organisms. Dilute acid pretreatment
using sulphuric acid below 4.0% was considered to
be an economical method, providing higher hydrolysis yield among the different physicochemical
pretreatments (Esteghlalian et al., 1997). The process
has been conducted in the temperature range of
100 C - 200 C with pressure 15Psi to 75Psi for different time intervals (Gangulya et al., 2012). We
have used sulphuric acid for pretreatment to enhance
the hydrolysis yield at different temperatures and
pressures for varying times. AccelleraseTM 1500 was
used at specific concentrations to investigate the enzymatic hydrolysis performance of cellulose and
hemicellulose present in the treated water hyacinth
plant. The conversion of lignocellulosic material into
reducing sugars and liquid fuel (ethanol) has been
achieved by using three sequential steps: acid pretreatment, enzymatic hydrolysis and yeast fermentation.
Effect of Biomass Concentration on Hydrolysis
Yield
The effect of biomass concentration was investigated on the enzymatic hydrolysis yield and amount
of reducing sugars by using different quantities of
pretreated biomass with fixed concentration of the
enzyme (0.5 mL enzyme). With the increase in substrate concentration, the amount of the reducing sugars increases while the hydrolysis yield decreases,
showing an opposite variation trend (Fig. 1). At low
concentration (5.0 g/L) of substrate, the hydrolysis
yield is maximum corresponding to 96.0%, which
decreases to 39.0% when the quantity of biomass
increased to 125.0 g/L (Fig. 1). The low hydrolysis

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358

M. Idrees, A. Adnan, S. A. Bokhari and F. A. Qureshi

yield at high substrate loading was due to two reasons, the lower enzyme to substrate ratio and inhibition of end product feedback caused by the high concentration of reducing sugar produced during hydrolysis (Wen et al., 2004). The effect of the substrate
on the amount of the reducing sugars and hydrolysis
yield was significant (p value<0.0001) with R2 value
0.9909. The optimum biomass concentration was
determined from the amount of the reducing sugars
and the hydrolysis yield. At 40.0 g/L solid mass the
hydrolysis yield is 74.0%, which decreases at 70.8%
when the substrate amount increases to 50.0 g/L and
58.0% with 75.0 g/L substrate. So fifty grams per
liter substrate is effective for enzymatic hydrolysis.

and 3.0 h of pretreatment, the reducing sugars

produced from hemicellulose (Ackerson et al., 1981;
Taherzadeh and karimi, 2007) were converted into
furfural (Fig. 2B). The production of furfural depends on the acid concentration and temperature
(Gonzales, 1986). During enzymatic hydrolysis, the
quantity of reducing sugars increases with increase
in time of pretreatment for 1.0% and 2.0% acid and
decreases with 3.0% acid due to the lower quantity
of hemicellulose present in the pretreated biomass.
The maximum amount of reducing sugars (hydrolysis yield) was obtained from water hyacinth when it
was pretreated with 1.0% acid for 3.0 h and 3.0%
acid for 1.0 h (Fig. 3(a), (b)).
Effect of Acid Concentration on Hydrolysis

Figure 1: Effect of substrate concentration on the

enzymatic hydrolysis at fixed ratio of enzyme.
Effect of Time and Temperature on Pretreatment
The effect of the time and temperature on the pretreatment and enzymatic hydrolysis was prominent.
The amount of cellulosic residue left after each pretreatment was different for varying pretreatment
conditions (Table 1). At high temperature and longer
residence time, the maximum hydrolysis of the hemicellulose was observed in the pretreatment steps,
which resulted in decreasing the remaining polysaccharides in the biomass. After pretreatment with
3.0% acid at low temperature (108 C), the solid
residue left was higher (50.32%) which have more
hemicellulose content while at high temperature (121
C) the residue was less (35.45%), due to maximum
hydrolysis of hemicellulose into component sugars.
During sulphuric acid pretreatment glucose, arabinose and xylose were obtained from the hydrolyzate,
which contains glucose as major component as
shown by TLC. Previously it was claimed that the
acid hydrolyzate of water hyacinth plant contains
xylose as major component (Nigam, 2002). At 121 C

Cellulose related polysaccharides are considered

to be a major component of water hyacinth (Malik,
2007; Nigam, 2002; Mukherjee and Nandi 2004;
Ingole and Bhole, 2002). The plant body contains
26.3 wt% C-6 sugars such as glucose (19.8%) and galactose (6.5%) and 20.5 wt% C-5 sugars with 11.5 wt%
xylose and arabinose (Girisuta et al., 2008; Aswathy
et al., 2010). The pretreatment conditions had a significant influence on the amount of sugars released
during pretreatment step and enzymatic hydrolysis.
In acid pretreatment the cellulose and hemicellulose
hydrolyzed into reducing sugars. The hydrolysis of
hemicellulose increases with the increase in concentration of acid used for pretreatment, which also results in more of a decrease in residual mass (Table 1).
The reduction in mass of water hyacinth increased
from 2.62 % to 64.55% when the acid concentration
was increased from 0.0 to 3.0% during pretreatment.
This reduction in weight during pretreatment was
due to removal of metal oxides (Girisuta et al., 2008),
lignin and hydrolysis of hemicellulose. Pretreatment
of water hyacinth with sulphuric acid yields a mixture of sugars (glucose, xylose, arabinose), with
glucose as the major component (Fig. 2A and 2C),
which exactly corresponds to the results of acid
hydrolysis of water hyacinth leaves by Girisuta et al.
(2008). The amounts of the reducing sugars obtained after enzymatic hydrolysis, when pretreatment
was done with 0.0, 1.0, 2.0 and 3.0% sulphuric acid
at 121 C for 1.0h, were 1.15+0.12, 30.38+0.77,
31.85+0.8 and 36.68+0.82 g/L respectively. Similarly, 1.77+0.07, 35.29+1.4, 32.03+1.09 and 26.26+1.19
g/L of reducing sugars were obtained from water
hyacinth biomass when pretreatment was done with
0.0, 1.0, 2.0 and 3.0% sulphuric acid at 121 C for 3.0 h
(Table 1). The sugars obtained during enzymatic hydrolysis were almost pure glucose (Fig. 5A and 5B).

Brazilian Journal of Chemical Engineering

Production of Fermentable Sugars by Combined Chemo-Enzymatic Hydrolysis of Cellulosic Material for Bioethanol Production

The effect of the acid concentration on the amount

of the reducing sugars was significant (P<0.0001)
with R2 value 0.9709 calculated from ANOVA
analysis using Graph PadPrism5. It was clear that the
hydrolysis yield increases with the increase in acid
concentration with short pretreatment time and

359

decreases with long pretreatment time. The 3.0%

acid pretreatment gave a higher amount of reducing
sugars when pretreated for 1.0 h and less when
treated for 3.0 h. This was due to the complete
hydrolysis of the hemicellulose and charring of the
remaining cellulose in the pretreatment step.

Table 1: The reaction conditions, biomass residue and amount of fermentable sugars obtained after
enzymatic hydrolysis.
Acid Conc. Pretreatment
Time
(%)
(h)

0.0

1.0

2.0

3.0

1
1
2
2
3
3
1
1
2
2
3
3
1
1
2
2
3
3
1
1
2
2
3
3

Temperature
(C)

Decrease in
Biomass
(%)

Biomass
Residue
(%)

108
121
108
121
108
121
108
121
108
121
108
121
108
121
108
121
108
121
108
121
108
121
108
121

2.64
16.18
5.04
26.44
25.32
35.85
47.19
57.03
50.26
59.05
51.43
61.37
48.84
61.33
49.12
63.33
50.15
61.54
47.94
61.97
48.98
63.26
49.68
64.55

97.36+1.12
83.82+1.71
94.96+3.78
73.56+0.88
74.68+2.46
64.15+1.97
52.81+1.17
42.97+0.94
49.74+1.11
40.95+0.13
48.57+0.93
38.63+0.24
51.16+1.96
38.67+0.64
50.88+0.66
36.67+1.16
49.85+0.70
38.46+0.86
52.06+0.45
38.03+1.96
51.02+1.26
36.74+0.24
50.32+0.84
35.45+1.11

Reducing
Sugars g/L
(Pretreatment
step)
0.57+0.23
2.42+0.58
0.81+0.34
2.99+2.01
3.15+1.32
5.21+1.28
23.41+1.37
31.29+0.96
21.72+0.89
29.03+2.14
25.47+1.22
27.25+2.40
21.83+1.34
32.76+0.96
20.12+2.11
31.31+1.58
24.38+1.76
29.71+2.80
28.58+1.36
33.47+2.10
29.19+1.59
32.76+1.16
26.93+2.11
29.43+1.73

Reducing
Sugars g/L
(Enzymatic
Step)
1.04+0.14
1.150+.12
0.95+0.08
1.62+0.19
1.45+0.21
1.76+0.07
28.80+0.93
30.36+0.77
29.97+1.61
32.38+1.01
31.60+0.80
35.30+1.40
29.12+1.13
31.85+0.85
30.75+1.40
29.10+0.90
29.59+1.19
32.03+1.09
30.75+1.83
36.68+0.82
31.30+1.06
31.67+0.94
29.25+0.83
26.26+1.19

Enzymatic
Hydrolysis
Yield
(%)
2.1
2.31
1.9
3.24
2.9
3.55
57.61
60.77
59.8
64.72
63.48
70.56
58.24
63.58
61.6
57.8
59.56
63.98
61.51
73.4
62.6
63.34
58.6
52.7

Experiments were done in duplicate. The average values and standard deviation are shown.

Figure 2: Reducing sugars in the acid hydrolyzate obtained during pretreatment (TLC
images) A: 108 C for 3.0h, B: 121 C for 3.0h and C:121 C for 1.0h,W: water, G: glucose,
X: xylose, Ar: arabinose, F:furfural, D: Effect of sulphuric acid and NaOH on glucose and
xylose(a:Acid, b: NaOH).
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360

M. Idrees, A. Adnan, S. A. Bokhari and F. A. Qureshi

Figure 3: (a), (b). Effect of acid concentration and time of pretreatment on enzymatic hydrolysis (%).
Time Course of Enzymatic Hydrolysis
Enzymatic hydrolysis was done with a fixed
amount of the cellulase enzyme and the reaction carried out for 48.0 h. The reducing sugar concentrations were determined at regular intervals, (12.0 h)
starting from 0 h during hydrolysis. The amount of
the sugars increased gradually and reached 0.734 g/g
of cellulosic material, which corresponds to a 73.4%
hydrolysis yield after 48.0 h, as shown in Fig. 4. The
percentage of hydrolysis was calculated from the
amount of reducing sugars and the amount of pretreated biomass used in enzymatic hydrolysis. The
graph showed that, with the passage of time, the
amount of reducing sugars increased and after 48.0 h
there was an insignificant increase in the amount of
sugars observed in some experiments. During hydrolysis, in the first 24.0 h more sugars were obtained and then sluggishly increased and reached a
maximum at 48.0 h. Previously 71.3% enzymatic
saccharification efficiency was reported by Aswathy
et al., (2010) with NaOH pretreated water hyacinth
and 60.2% by Mishima et al., (2008).
Most Effective Pretreatment Condition
Acids such as sulphuric acid, hydrochloric acid or
nitric acid (Patel et al., 1993) and bases like NaOH
or NH3 (Zhao et al., 2007; Xu, 2007) can be used efficiently for pretreatment of biomass at different
temperatures for maximum enzymatic hydrolysis.
Among them, NaOH pretreatment could provide
higher enzymatic saccharification as compared to
acids (Zhao et al., 2007; Aswathy et al., 2010). During
acidic or basic pretreatment, there occurred a loss in
the biomass weight (Jiele et al., 2010; Wang et al.,
2009) due to hydrolysis of hemicellulose and
removal of lignin (Blasi et al., 1999; Lin et al.,
2010).

Figure 4: Time course of enzymatic hydrolysis when

biomass was pretreated at 121 C with different concentrations of sulphuric acid (0%, 1%, 2%, and 3%).

Brazilian Journal of Chemical Engineering

Production of Fermentable Sugars by Combined Chemo-Enzymatic Hydrolysis of Cellulosic Material for Bioethanol Production

Water hyacinth contains hemicellulose as major

component (48.0%) with only 3.5-4.6% of lignin
(Nigam, 2002), which was removed during the
pretreatment step (decrease in biomass weight Table
1). For obtaining the maximum benefit of pretreatment the sugars produced during pretreatment should
be stable. The effect of both H2SO4 and NaOH on
pure glucose and xylose at 100 C was checked. NaOH
degraded these sugars, while in acidic media no
change was observed for glucose and xylose (Fig.
2D). So the use of H2SO4 at low temperature for pretreatment was a better choice for obtaining the maximum amount of fermentable sugars in two steps,
pretreatment and enzymatic hydrolysis step. The
pretreatment of water hyacinth with 3.0% sulphuric
acid at 121 C for 1.0 h was found to be the most
optimal condition as the subsequent enzyme hydrolysis showed maximum 73.4% yield. This pretreatment
also provided 33+2.1 g/L of reducing sugars per
100 gm of biomass during pretreatment step, which
is close to the results obtained by Abraham et al.
(2006) by using 10.0% sulphuric acid at 121 C for
30 min, which was also available for fermentation
(Masami et al., 2008). Nigam (2002) obtained 0.51 g/g
reducing sugars with 3.0% sulphuric acid when the
pretreatment time was 1.5 h. The pretreatment with
1.0 and 2.0% sulphuric acid for 3.0 h at 121 C
provided 27.25+ 2.4 and 29.19+2.8 g/L of
reducing sugars per 100.0 g of biomass, along with

361

70.56 and 63.98% enzymatic hydrolysis yields,

respectively.
Ethanol Production
Two types of enzymatic hydrolyzate, one obtained from 1.0% acid and other from 3.0% acid
treated biomass, were used for ethanol production.
These two hydrolyzates have 36.65 g/L and 35.7 g/L
fermentable sugars which converted into 18.25 and
17.33 g/L ethanol, equivalent to 95.2% of the theoretical yield of the glucose, which is 0.51 g ethanol/
g of glucose. After twenty four hours, sugars were
entirely fermented into ethanol, which was confirmed through DNS analysis and TLC results (Fig.
5C). Sornvoraveat and Kongkiattikajorn (2010) obtained a 96.07% ethanol yield from fermentation of
enzymatic hydrolyzate of water hyacinth and Chen et
al. (2007) obtained a 94.0% yield from corncob enzymatic hydrolyzate in 18.0 h, by using Saccharomyces
cerevisiae. Nigam (2002) and Magdum et al., (2012)
reported 18.0 g/L and 19.2g/L of ethanol from the
acid hydrolyzate of water hyacinth leaves, respectively. Sulphuric acid produced pure cellulose after
hydrolysis of the hemicellulose during pretreatment.
Enzymatic saccharification of this cellulose produced pure glucose (Fig. 5) which converted completely into ethanol through fermentation by using
commercial yeast.

Figure 5: Monomeric sugars after enzymatic hydrolysis: A Enzymatic hydrolysis after pretreatment at 108 C for 1h, B enzymatic
hydrolysis after pretreatment at 121 C for 1.0 h and C after fermentation (W: water, G: glucose, X: xylose, Ar: arabinose, FM: fermentation media).

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362

M. Idrees, A. Adnan, S. A. Bokhari and F. A. Qureshi

CONCLUSIONS
Chemo-enzymatic hydrolysis of the water hyacinth yielded reducing sugars in two steps: (a)
Pretreatment step yielded 33.0% hydrolysis; (b) Enzymatic hydrolysis yielded 73.4%. Multiple enzyme
activities of AccelleraseTM1500 converted the cellulose and cellubiose completely into pure glucose.
The amounts of glucose obtained from enzymatic
hydrolysis of pretreated water hyacinth with 3.0%
and 1.0% acid were 36.65 and 35.7 g/L, which gave
18.25 and 17.33 g/L of ethanol with commercial
bakers yeast respectively. The two-step hydrolysis
process of water hyacinth, pretreatment with sulphuric acid followed by Accellrase 1500 hydrolysis,
is a suitable method for achieving high recovery of
fermentable sugars and high ethanol conversion yield.
ACKNOWLEDGEMENT
We gratefully acknowledge the financial support
from the Higher Education Commission Islamabad,
Pakistan, and Genencor International Inc. for providing enzyme samples. We also extend our gratitude to
Dr. Farooq Anwar for his professional guidance.
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Brazilian Journal of Chemical Engineering Vol. 31, No. 02, pp. 355 - 363, April - June, 2014