Sci.Int.(Lahore),24(1),41-45,2012
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41
DILUTE SULFURIC ACID: A CHEAP ACID FOR OPTIMIZATION
OF BAGASSE PRETREATMENT
Asma Manzoor1, 3, *Zia-Ullah Khokhar1, 2, Athar Hussain3, Uzma1, 3, Sh. Asrar Ahmad4,
Qurat-ul-Ain Syed1 and Shahjhan Baig1
1
Food & Biotechnology Research Center PCSIR Labs, Ferozpur Road Lahore, 54600 Pakistan
2
Institute of Biochemistry and Biotechnology, Punjab University, Lahore, Pakistan
3
Government College University, Lahore, Pakistan
4
Division of Science & Technology, University of Education, Township, Lahore, Pakistan
*
(Corresponding Author E-mail: zia2_khokhar@hotmail.com , Cell # 0092-300-7432748)
ABSTRACT: Pretreatment is an important tool in cellulose conversion process. It breaks the lignin seal
and disrupts the crystalline structure of cellulose to make it more accessible for enzymes, used for
conversion of carbohydrate polymers into fermentable sugars. Dilute sulphuric acid is normally used as
impregnating agent as it effectively hydrolyzes hemicellulose into monomeric sugars (xylose, arabinose,
galactose, glucose, and mannose) and oligomers. In the present study, the effect of different parameters i.e.
Time and concentration of impregnating agent was studied for optimization of conditions for sugar cane
bagasse delignification. The temperature and biomass concentration were kept constant i.e. 121°C and 1kg
respectively. Delignification of sugar cane bagasse was assessed, at 30, 60, 90, 120, 150 and 180 minutes
time intervals using different concentrations sulphuric acid ranging 0.5-5% as impregnating agent per 10 g
dry matter. Control was also run on varying time period and concentration of sulphuric acid. After
processing, the effectiveness of various pretreatment conditions were assessed by estimation of weight
reduction after pretreatment, lignin content, cellulose content and concentration of total and reducing
sugars released in hydrolyzates. Results showed that efficient delignification, maximum weight loss and
highest total and reducing sugars were obtained at 180 minutes in 4% sulfuric acid. There was about 82%
delignification in 4% sulphuric acid pretreatment at 180 minutes, which may result in higher production of
ethanol.
INTRODUCTION
Biofuels are considered as one of the important alternatives
to fossil fuel. Biofuel prepared from renewable biomasses
provide strategic, social and environmental benefits [1].
Biomass, like animal and human waste, trees, shrubs, yard
waste, wood products, grasses, and agricultural waste such
as wheat straw, corn stover, rice straw, grasses and cotton
stalk, is considered as potential renewable resources [2].
Nowadays edible parts of food crops such as sugar cane
juice, corn etc are also used for bioethanol production.
However, it creates an undesirable competition between
food supply and biofuel production [3]. Cellulosic substrates
can be used as an alternative to feedstock for production of
bioethanol. The cellulose substrates are heterogeneous
carbohydrate polymer and occupy 55-75% of total
carbohydrate weight.
Biomass can be processed either chemically or biologically
by breaking the chemical bonds to extract energy in the form
of biofuels such as bioethanol, biodiesel, and methane. By
pretreatment we can alter the structural and chemical
composition of lignocellulosic biomass to facilitate rapid and
efficient hydrolysis of carbohydrates to fermentable sugars
[4]. Acid pretreatment involves the use of sulfuric, nitric, or
hydrochloric acids to remove hemicellulose components and
expose cellulose for enzymatic digestion [5, 6].
Sugar cane, one of the major lignocellulosic biomass, is
most important industrial crop in Pakistan and occupies 5th
position in area used for crop production [7]. Sugar cane
bagasse, obtained after juice extraction, consist of 40-50%
cellulose, 25% hemicellulose and 25% lignin. It is
speculated that 75% of bagasse could be converted to
fermentable sugars by thermal, chemical and/or enzymatic
hydrolysis [8-10]. All the three components present in
lignocellolosic biomass (bagasse) forms a very complex
structure whereas lignin consisting of monomeric subunit act
as a cement to hold in place to cellulose fibrils and
hemicellilose backbone, resulting in a strong and
impermeable structure. Due to this strong packaging of
hemicellulose, an effective, reliable strategy is required to
break the lignin seal and expose crystalline cellulose.
Pretreatment of lignocellulosic biomass makes it more
vulnerable to cellulose enzyme to fermentable sugars.
Different pretreatment strategies differ in their mechanism of
action and the degree of cellulose hydrolysis. Hence, the
choice of an effective pretreatment strategy relies on the cost
effectiveness and the rate of cellulose hydrolysis or lignin
removal. Pretreatment with in organic acids has received
considerable research attention over the year [11-16].
Sulfuric acid removes hemicelluloses and releases
oligosaccharides and monosaccharides by the breakdown of
cellulose. Dilute sulfuric acid at moderate temperature
effectively removes most of the hemicelluloses and recovers
as dissolved sugars. It may be advantageous by producing
soluble carbohydrates and partially hydrolysed cellulose that
are readily usable by cellulytic organisms and yeast as well
[17]. In the present study, the chemical pretreatment of
bagasse with dilute sulfuric acid was optimized.
Biochemical analysis of bagasse was done to determine the
effectiveness of the developed strategy and its usefulness in
bioethanol production by yeast.
MATERIALS AND METHODS
Substrate: Lignocellulosic Biomass (Sugar cane bagasse)
obtained from Shakar Gunj Sugar Mills (Pvt.) Ltd, Jhang
was kept under shade in open air for 2 weeks and then dried
in oven at 50ºC for 48 hours. The dried substrate was
chopped into 3 mm mesh size in a Hammer better mill,
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pulverized and then washes in hot water to remove residual
sugars [18].
Preparation of Bagasse for Pretreatment: The dry sugar
cane bagasse was weighed to 4kg and stored at room
temperature. 10 g of baggase were added in the individual
flasks and soaked in 0.5-5% sulfuric acid solution in
duplicate to submerge the sugar cane residue and allowed to
soak for 24 hours. After soaking, the mixture was filtered.
The residue was rinsed with distilled water for 30 minutes
thrice. The residue was oven dried at 100-105ºC and stored
for further chemical hydrolysis.
Chemical Pretreatment of Bagasse: The bagasse was
pretreated with various concentrations of sulphuric acid at
different time intervals (0 to 3 hrs), keeping the temperature
and mass of bagasse constant. 10 gm of Bagasse was taken
in separate conical flasks and different concentrations of
sulphuric acid (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,
4.5%, and 5%) were added. Bagasse was autoclaved at 121
ºC at 30 minute, 1hr, 1.5hr, 2hr, 2.5hr and 3hrs intervals.
Autoclaved samples were filtered and pure hydrolyzate was
stored at 4ºC for analysis of sugar contents. The remaining
residue was washed till neutral pH and oven dried at 100105ºC. This solid residue was kept for biochemical analysis.
Hydrolysates for fermentation were prepared by taking
500gm of each dry milled lignocellulosic biomass, mixed
with 5M sulphuric acid solution and kept in the digester for
one hour for both concentrations. After required intervals of
time, water was added in the Erlenmeyer flasks for dilution
of acid and by filtration digested samples were recovered
and washed with distilled water til the neutral pH. Then
dried at 60◦C overnight using the method described by Fan et
al [19] and [20]. The dried hydrolysate was stored in sterile
polypropylene bags for further use.
Weight Loss of Bagasse after Pretreatment: Sugar cane
bagasse was treated with different concentrations of
Sulphuric acid 0.5-5% at different time interval 30-180 mins
respectively, then autoclaved at 121ºC, filtered it, washed till
the pH was neutral and dried in an oven at 100-105ºC for
over night finally the weight of bagasse was reduce after
pretreatment was measured by using electric balance.
Sci.Int.(Lahore),24(1),41-45,2012
[22]. The total sugar concentrations were measured by using
spectrophotometer at 470 nm.
Reducing Sugars Analysis: reducing sugars in un-treated
and treated raw material were measured by DNS method
using glucose as a standard [23].
Statistical Analysis: Analysis of variance (ANOVA) was
applied. Statistical significance was adjusted at p < 0.05.
Analysis was done by using SPSS.
RESULTS AND DISCUSSION
In the present study, biochemical analysis of sugar cane
bagasse after acid pretreatments was done to achieve
maximum delignification which results in digestion of
biomass sample and production of fermentable sugars and
ethanol production. Lignin is a factor affecting the
enzymatic hydrolysis of biomass. Hence, removal of lignin
lowers the enzyme requirement [24]. Lignocellulosic
biomass can not be saccharified by enzyme to high yields
without pretreatment, mainly because of the lignin in plant
cell wall forms a barrier against enzymatic attack [25]. An
ideal chemical pretreatment would reduce the lignin
contents, crystallinity of cellulose and increased the surface
area [26] and separation of the structural linkage between
lignin and carbohydrate, increasing hydrolysis of cellulose.
Pretreatment is however, crucial for ensuring good ultimate
yield of sugars from both polysaccharides i.e. cellulose and
hemicellulose of the biomass. It has been seen that
enzymatic hydrolysis without pretreatment yield less than
20% while pretreatment rises sugar yield over 90% [27]. The
biodegradability of lignocellulosic biomass is limited by
several factors like crystallinity of cellulose, available
surface area, and lignin content [28]. Dilute sulphuric acid
used as a catalyst for hemicellulose and lignin solubilization
at low concentration (0.05%-5%). The acid treatment
minimized the formation of degradation products and
maximize sugars yield at the end of the process [29].
BIOCHEMICAL AND ANALYTICAL ANALYSIS
Determination of lignin: Van Soest and Wine [21]
procedure was applied for the determination of lignin
content. The % lignin was determined by using formula:
(Wt. of digested material) – (Wt. of ash)
% lignin =
X 100
Weight of sample taken
Determination of Cellulose: Cellulose content was
determined by using standard procedure demonstrated by
Van soest and Wine [21]. The % cellulose was determined
by using formula:
(Wt. of digested material) – (Wt. of ash)
% cellulose =
X 100
Weight of sample taken
Total sugar Analysis: The carbohydrate/sugar contents of
un-treated and pretreated raw materials were measured using
Phenol Sulphuric acid method just as Pettersson and Porath
Fig 1 Weight loss (gms) of baggase at different concentrations of
sulphuric acid treatment for 30 min (closed square), 60 min (open
square), 90 min (closed circle), 120 min (open circle), 150 min
(closed triangle) and 180 min (open triangle).
Sci.Int.(Lahore),24(1),41-45,2012
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Fig 2 Lignin (%) of baggase at different concentrations of sulphuric
acid treatment for 30 min (closed square), 60 min (open square), 90
min (closed circle), 120 min (open circle), 150 min (closed
triangle) and 180 min (open triangle).
Fig 3 Delignification (%) of baggase at different concentrations of
sulphuric acid treatment for 30 min (closed square), 60 min (open
square), 90 min (closed circle), 120 min (open circle), 150 min
(closed triangle) and 180 min (open triangle).
Fig 4. Cellulose (%) of baggase at different concentrations of
sulphuric acid treatment for 30 min (closed square), 60 min (open
square), 90 min (closed circle), 120 min (open circle), 150 min
(closed triangle) and 180 min (open triangle).
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Fig 5 Total sugars (%) of baggase at different concentrations of
sulphuric acid treatment for 30 min (closed square), 60 min (Open
Square), 90 min (closed circle), 120 min (open circle), 150 min
(closed triangle) and 180 min (open triangle).
Fig 6 Reducing sugar (%) of baggase at different concentrations of
sulphuric acid treatment for 30 min (closed square), 60 min (Open
Square), 90 min (closed circle), 120 min (open circle), 150 min
(closed triangle) and 180 min (open triangle)
Weight loss: The maximum weight loss of bagasse (47.7%)
occurred on 4% sulfuric acid at 121ºC for 180 minutes (Fig
1). The finding is in agreement with previous study, which
reported that the amount of weight loss of chemical
pretreatment of lignocellulosic residue was due to lignin
removal [30]. Furthermore, greater weight loss indicates
more lignin loss and cellulose digestibility.
Lignin: Fig 2 shows that 4.4% lignin remained in the
biomass, when bagasse was pretreated with 4% of sulfuric
acid at 121ºC for 180min. The previous study showed that
20% lignin remained in bagasse when it was treated for 15
min with 10% sulfuric acid at 121ºC [17]. The present study
shows that maximal lignin can be removed by treating sugar
cane baggase in lower concentrations of sulphuric acid for
longer period of time. The present pretreatment conditions
are more economical and can be used at commercial scale.
Delignification: The maximum delignification was 82% at
121ºC on 4% of sulfuric acid for 180 minutes as shown in
Fig 3. Rocha et al., [31] demonstrated that when bagasse is
pretreated in sulfuric acid for production of bleached pulp.
There is about 99.6% delignification, in the processing of
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crude pulp to bleached pulp. Our results are better than
Khokhar et al [26] who reported 74% delignification of
wheat straw with 1.5% sulphuric acid for 75 mints at 121ºC
and 15 lb pressure while maximum weight loss was 49.9%.
Our result is also better than Irfan M. et al (2010) who
reported 81% delignification of bagasse with 3.5 % Sodium
Sulphite [34].
Cellulose: The present study indicates that digestibility of
cellulose increased by increasing the treatment time at a
particular concentration Fig 4. 12.08% cellulose was
remained in the bagasse residue after treating with 4%
sulfuric acid at 121ºC for 180 min. Two grass species
creeping wild rye and jose tall wheatgrass in 1.4% w/w
dilute sulphuric acid 165°C 8 min loss 59 % jose tall
wheatgrass, cellulose hydrolysis 9-12% [32]. 0.5-1 %
sulphuric acid at 140-190 °C effectively removes and
recovers most of the hemicellulose to almost 100 % [33].
However, contrary to our findings, Han and Callihan [17]
reported that the digestibility of cellulose was maximum
when the bagasse was treated for 15 min with 50% of
sulfuric acid at 121ºC, followed by dilution of acid to 1%
and heating for 1h, at 121ºC. About 20% of cellulose was
remained in the sugar cane bagasse.
Total sugar:In this study, the bagasse treatment with 3.5%
Sulphuric acid at 121ºC for 180 minutes resulted in
production of 265.04 mg/ml of total sugars Fig 5. Previous
study has reported that the total sugar production was
maximum (23%), when the bagasse was treated for 15 min
with 50% sulfuric acid at 121ºC, followed by dilution of acid
to 1% and heating for 15 min at 121ºC [17]. The present
study demonstrates that the decreased sulphuric acid
concentrations increases total sugar production. The data
further suggest that 3.5% sulphuric acid not only increases
total sugar release but also reduces the economic cost needed
for biofuel production through wastes.
Reducing sugar: The bagasse treated with 2.5% Sulphuric
acid at 121ºC for 180 minutes resulted in production of
85.96 mg/ml of reducing sugars (Fig 6). Han and Callihan
[17] reported maximum production (19.2%) of reducing
sugar when the bagasse was treated for 15 min with 50% of
sulfuric acid at 121ºC, followed by dilution of acid to 1%
and heating for 2 h, at 121ºC. Khokhar et al (2010)
pretreated wheat straw with 1.5% sulphuric acid for 75 mints
at 121ºC and 15 lb pressure and reported that maximum total
phenols 10.89 mg/ml, reducing sugars 42.99 mg/ml and total
sugars 65.34 mg/ml [25-26].
the industrial processing of bioresources may produce raw
materials for bioethanol production. The biofuels produced
through biotechnological processes can be the excellent
alternative to fossil fuels and hence decrease air pollution
and save a lot of foreign exchange which is need of the day.
ACKNOWLEDGMENTS
Authors are thankful to the Ministry of Science and
Technology (MoST) and Higher Education Commission
(HEC), Govt. of Pakistan. Authers are also thankful to
laboratory staff for their helping co-operation.
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CONCLUSION
The present study demonstrates that a more effective and
economical delignification of biomass can be done in acidic
medium. Chemical pretreatment is most advanced and
effective way because it decreases the cellulose crystallinity,
increases accessible surface area for enzymes and removal
of lignin from lignocellulosic biomass and ethanol
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Under these pretreatment conditions 82% delignification
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achieved our objectives successfully. Further research is
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Sci.Int.(Lahore),24(1),41-45,2012
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