Industrial Crops and Products 44 (2013) 488495

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Industrial Crops and Products
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Alkali and enzymatic delignication of sugarcane bagasse to expose cellulose
polymers for saccharication and bio-ethanol production
Muhammad Asgher, Zanib Ahmad, Haz Muhammad Nasir Iqbal

Industrial Biotechnology Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan
a r t i c l e i n f o
Article history:
Received 31 July 2012
Received in revised form
30 September 2012
Accepted 3 October 2012
Keywords:
Sugarcane bagasse
Alkali pretreatment
Ligninolytic pretreatment
P. ostreatus IBL-02
Bio-ethanol
a b s t r a c t
To expand the range of natural resources researchers have been re-directing their interests in biomass
based bio-fuels, which can be obtained from lignocellulosic biomass. Sugarcane bagasse was pretreated
with alkali and ligninolytic enzymes extract produced from Pleurotus ostreatus IBL-02 to depolymerize
lignin and expose the cellulose polymers. The de-lignied bagasse was used as substrate for bio-ethanol
production in sequential saccharication and fermentation by an indigenous strain of Saccharomyces cere-
visiae. Alkali treatment (4% NaOH) and ligninolytic enzymes extract (25 mL) treatment caused 48.7 and
33.6% de-lignication of sugarcane bagasse, respectively. The de-lignied residues were treated with
indigenously produced crude cellulase extract from Trichoderma harzaianum that resulted in 69.2 and
72.9% cellulose hydrolysis of alkali and ligninolytic enzymes pretreated bagasse, respectively. S. cerevisea
was grown on the hydrolyzates to produce ethanol at 37

C and pH 5.5 that produced 18.2 and 16.3 g/L

ethanol using alkali and enzyme pretreated substrates, respectively. For maximum ethanol production,
different parameters like fermentation time period, pH, temperature, substrate level and inoculum sizes
were optimized using both alkali and enzymes pretreated substrates. Under optimum conditions ethanol
production of 32.45 g/L and 28.15 g/L was obtained from alkali and ligninolytic enzymes treated sugarcane
bagasse, respectively. In conclusion, the results obtained after high-performance liquid chromatography
(HPLC) analysis suggesting ligninolytic pretreatment as a promising tool for bio-ethanol production in
sequential saccharication and fermentation. Enzymatic treatment of waste biomass could be of partic-
ular interest, since it seems an eco-friendly approach for bio-ethanol production.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Increasing costs of fossil fuels and their greenhouse gases
emission effects are creating a dire need to explore cheaper and
environment friendly bio-fuels. Presently, ethanol as well as other
bio-fuels produced from plant biomass is an alternative to fossil
fuels. Ethanol is valuable in many respects, ranked as industrial
solvent and employed in the preparation of medicines, resins,
avoring extracts, perfumes, varnishes, and shellac. Ethanol is a
natural fuel that burns cleaner andcauses less environmental prob-
lems than petroleum. This enables preparation of gasolineethanol
blends resultingingasohol for internal combustionengines that has
higher octane value and replaces lead in gasoline (Ghorbani et al.,
2011).
Currently, bio-ethanol is produced on industrial scale from
sucrose and starch; however, these bio-ethanol production sys-
tems pose concerns about competitionwithfood and feed supplies.
Although this ethanol is produced at a competitive cost, the raw

Corresponding author. Tel.: +92 41 9200161x3312.

E-mail address: nasir pk99@hotmail.com(H.M.N. Iqbal).
material supply will not be sufcient to meet the increasing
demand for fuel ethanol (Galbe and Zacchi, 2007). Ethanol made
from lignocellulosic biomass is attractive as renewable liquid fuel
for transportation. Lignocelluloses materials are most promising
feedstock as natural, abundant, and renewable resource and can
potentially provide a long term sustainable fuel supply (Alonso
et al., 2008; Gohet al., 2010). However, large-scaleeconomical com-
mercial production of fuel ethanol from lignocellulosic materials
has still not been implemented.
For designing fuel ethanol productionprocesses, the assessment
of the utilization of different feed stocks (i.e. sucrose containing,
starchy materials, lignocellulosic biomass) is required consider-
ing the big share of raw materials in ethanol costs (Cardona
and Snchez, 2007). The transformation of biological resources as
energy-rich crops or lignocellulosic biomass requires pretreatment
of the feedstock for fermenting organisms to convert them into
ethanol (CardonaandSnchez, 2007). Thecost-effectiveconversion
of various types of lignocellulosic biomass to fermentable sugars
withas little toxic inhibitory byproducts as possible remains a chal-
lenge for the production of cellulosic ethanol (Dagnino et al., 2013).
The lignin component of the biomass material poses pre-treatment
challenges because of its nonproductive binding and inactivation
0926-6690/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2012.10.005
M. Asgher et al. / Industrial Crops and Products 44 (2013) 488495 489
of cellulases. Pre-treatment of lignocellulosic materials to remove
ligninandhemicellulose cansignicantlyenhance the hydrolysis of
cellulose(Goshadrouet al., 2011). Asolutiontothis problemmay be
additional enzymatic hydrolysis by complex containing, enzymes
like ligninolytic (laccase, MnP, LiP) and cellulases (Pal et al., 2013).
Inindustrial scale ethanol productionfromlignocellulosic residues,
the alkali pretreatment of substrates for removal of lignin barrier
is one of the bottle neck problems because it substantially adds to
the overall production cost and also contributes to environmental
issues. There is a dire need to develop a cheaper biological process
for delignication of lignocellulosic biomass.
White rot fungi (WRF) are the most efcient degraders of
lignin (Iqbal et al., 2011), and are probably also the most suit-
able organisms to be utilized in an industrial process that requires
delignication of lignocellulosic substrates (Asgher et al., 2012a).
Ligninperoxidase (LiP) E.C. 1.11.1.14, manganese peroxidase (MnP)
E.C. 1.11.1.13 and laccase E.C. 1.10.3.2 are the major ligninolytic
enzymes of WRF that are directly involved in the degradation
of lignin in lignocellulosic substrates (Asgher and Iqbal, 2011;
Bandounas et al., 2011; Iqbal and Asgher, in press). There are sev-
eral advantages of using WRF enzyme extracts for delignication
of plant biomass including the following:
i. The ligninolytic enzyme extracts from WRF also contain other
accessory enzymes and mediators required for the action of
these enzymes for complete degradation of lignin
ii. The enzyme extracts may also contain cellulase enzymes that
can simultaneously hydrolyze the exposed cellulose bers to
someextent, thus facilitatingthesubsequent actionof cellulases
during saccharication of de-lignied biomass
iii. The ligninolytic and celluloytic enzyme extracts can be used in
a novel simultaneous pretreatment, saccharication and fer-
mentation (SPSF) process conguration that can save time,
energy and environment.
The present study involved the use of ligninolytic enzymes
extract from P. ostreatus IBL-02 for de-lignication of sugarcane
bagasse, followed by saccharication using cellulases extract from
T. harzianumand bio-ethanol production by S. cerevisea.
2. Materials and methods
2.1. Chemicals and solid substrates
All the chemicals used were of analytical grade and mainly pur-
chased fromSigmaAldrich (USA), Merck (Germany) and Scharlau
(Spain) and used as such without further purication. The agro-
industrial wastes, i.e. wheat straw and sugar cane bagasse were
obtained froma local fruit market in Faisalabad, Pakistan. The col-
lected substrates were crushed into pieces, sun and oven dried (at
60

C), ground to ne particle size (40mmmesh size) and stored in

airtight plastic jars to avoid free moisture effect.
2.2. Production of ligninolytic enzymes and their assays
2.2.1. Preparation of fungal inoculum
An indigenous fungal strain P. ostreatus IBL-02 was obtained
from National Fungal Culture Collection Pakistan (NFCCP; Strain-
IBL-02) and used to produce ligninolytic enzymes. Aqueous spore
suspension of P. ostreatus IBL-02 was prepared by growing the fun-
gus in inoculummediumfor 5 days. The inoculummediumwas the
Kirks basal mediumsupplemented with 1%sterile glucose solution
(Tien and Kirk, 1988). The mediumwas adjusted at pH 4.5 using M
HCl/MNaOHand autoclaved (121

C) for 15min. A loop full culture

of P. ostreatus IBL-02 was transferred to the sterilized mediumand
theaskwas incubatedfor 3days at 30

Ctoget 10
6
10
8
spores/mL.
2.2.2. Production of ligninolytic enzymes under optimum
conditions
The pre-optimized solid state fermentation medium of wheat
straw was used for the production of ligninolytic enzyme (Aslam
and Asgher, 2011). The ligninolytic production medium contain-
ing 5g substrate was moistened with Kirks basal salts medium
(the main constituents of the salt media were: (NH
4
)
2
SO
4
, 10g/L;
KH
2
PO
4
, 4g/L; MgSO
4
7H
2
O, 0.5g/L and CaCl
2
, 0.5g/L). The asks
were autoclaved, inoculated with 5mL of the fungal inoculumand
incubated at 30

C for 5 days. To the fermented biomass 100mL dis-

tilled water was added and the ask was kept in shaker at 120rpm
for 30min (Iqbal et al., 2011). The contents were ltered and l-
trates were centrifuged at 3000g for 5min. The supernatant was
assayed for ligninolytic enzymes and used as enzymes extract for
delignication of sugarcane bagasse.
2.2.3. Ligninolytic enzymes assays
Lignin peroxidase (LiP) activity was measured by the method
of Tien and Kirk (1988), following the H
2
O
2
dependent oxidation
of veratryl alcohol to veratraldehyde at 25

C. Reaction mixture
contained 4mM veratryl alcohol (1mL) at 25

C in (1mL) 100mM
tartarate buffer of pH 3, and the reaction was initiated by 0.2mM
H
2
O
2
(0.5mL). Absorbance of reaction mixture was monitored at
310nm (
310
=9300) (Iqbal et al., 2011). Manganese peroxidase
(MnP) was assayed by the method of Wariishi et al. (1992), at 25

C
by the H
2
O
2
dependent oxidation of oxidized manganicmalonate
complex at 270nm (
270
=11,570). Reaction mixture contained
1mL of 1mM manganese sulfate in 1mL sodium malonate buffer
(50mM; pH4.5) and100L of enzyme sample andthe reactionwas
initiated by the addition of H
2
O
2
to a nal concentration of 0.1mM
(Asgher and Iqbal, 2011). Laccase activity was determined by
monitoringtheoxidationof 2,2

-azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid(ABTS) ina reactionmixture containing(1mL) 1mM
ABTS in (1mL) 0.1M sodium acetate buffer (pH 4.5) with a 50L
enzyme sample (Wolfenden and Willson, 1982). The oxidation was
followed at 25

C and 420nm(
420
=36,000) (Asgher et al., 2012b).
2.3. Cellulase production and their assays
2.3.1. Cellulase production under optimumconditions
Sugarcane bagasse was used as substrate for the production of
cellulase enzymes by T. harzianumunder pre-optimizedliquidstate
fermentation conditions. For preparation of inoculumT. harzianum
was grown in Vogels medium at pH 4.5. The inoculated ask was
incubated at 28

C for three days to get homogenous inoculumhav-

ing 10
7
10
8
spores/mL. For the production of cellulases in liquid
state fermentation, the fungus was grown in 500mL Erlenmeyer
ask containing 5g sugarcane bagasse in 100mL Vogels medium.
The ask was sterilized, inoculated (2mL inoculum) and it was
incubated for 5 days at 28

C. The contents of the ask were ltered

and the ltrate was used as crude cellulase extract for sacchari-
cation of alkali and ligninolytic pretreated sugarcane bagasse.
2.3.2. Cellulase assays
Endo 1,4- glucanases was assayed using carboxymethyl cel-
lulose (CMC) as substrate and 3,5-dinitrosalisylic acid (DNS) as
coupling reagent by the method of Gadgil et al. (1995). The assay
mixture contained 0.1mL of enzyme solution with 1mL of 1% CMC
and 1mL of 0.1Mcitrate buffer of pH 4.8. The assay tube was incu-
bated for 30min at 50

C and the reaction was then terminated by

adding DNS reagent. The reaction mixtures were boiled for 15min
and then cooled in ice. The absorbance was measured at 540nm
against reagent blank. The released glucose was determined by
490 M. Asgher et al. / Industrial Crops and Products 44 (2013) 488495
Table 1
Pretreatment of sugarcane bagasse with varying doze levels of ligninolytic
enzymes.
a
S. No. Substrate (g) Ligninolytic
extract (mL)
Malonate
buffer (mL)
1 30 10 190
2 30 15 185
3 30 20 180
4 30 25 175
5 30 30 170
a
pH 4.5; temperature, 35

C, time, 48h.
comparingthe absorbance withstandardglucose solution. One unit
of enzyme activity was dened as the amount of glucose (mol)
released by 1mL of enzyme solution per min.
2.3.3. Exo-glucanase assay
Exo 1,4- glucanase was assayed according to the method of
Deshpande et al. (1984), using 1% salicin as reaction substrate with
DNS as coupling reagent. The reaction mixture contained 0.1mL
of enzyme extract with 1mL of 1% salicin and 1mL of 0.1M suc-
cinate buffer of pH 5. The mixture was incubated for 30min at
50

C and the reaction was then terminated by adding DNS reagent

(2mL). The reaction mixtures were heated for 15min in a boiling
water bath followed by cooling in ice. The absorbance was mea-
sured at 540nmagainst reagent blank. One unit of enzyme activity
was dened as the amount of glucose (mol) released by 1mL of
enzyme solution per min.
2.3.4. -Glucosidase assay
-Glucosidase activity was determined by the method of
Gielkens et al. (1999). The assay mixture contained 0.1mL
of enzyme extract with 1mL of 4mM p-nitrophenyl--d-
glucopyranoside solution (standard) in 0.1Msodiumcitrate buffer
of pH 5. The assay mixture was incubated for 30min at 40

C
followed by the addition of 2mL of 1mM Na
2
CO
3
solution as
reaction stopper. The release of p-nitrophenol was measured spec-
trophotometrically at 400nm against reagent blank. One unit of
enzymeactivitywas denedas theamount of p-nitrophenol (mol)
released per mL of enzyme solution per min.
2.4. Delignication of sugarcane bagasse
Sugarcane bagasse was pre-treatedwithvarying concentrations
of NaOHand varying volumes of crude ligninoytic enzymes extract
for de-lignication.
2.4.1. Pretreatment with ligninolytic enzymes
Varying volumes of enzyme extract containing LiP, MnP and lac-
case was applied at different doze levels (Table 1) for 48h at 35

C.
The volume of each ask containing 30g substrate was made to
200mL mark with 0.5mM sodium malonate buffer of pH 4.5. The
pretreatedsugarcanebagassewas washedwithdistilledwater until
pH 5.5 was attained.
2.4.2. Alkali pretreatment
Sugarcane bagasse (30g) was mixedin200mL solutions of vary-
ing concentrations (15%, w/v) of NaOH (Table 2) and subjected to
thermal treatment at 121

C for 30min in autoclave. The pretreated

substrate was washedwithdistilledwater andthenwithdilute acid
solution (HCl) until pH 5.5 was attained.
2.5. Determination of lignin and percent de-lignication
Untreated as well as alkali and ligninolytic enzymes treated
bagasse was analyzed for lignin content by the method described
Table 2
Pretreatment of sugarcane bagasse with varying concentrations of NaOH.
a
S. No. Substrate (g) Concentration of
NaOH (% w/v)
Total volume of
NaOH (mL)
1 30 1 200
2 30 2 200
3 30 3 200
4 30 4 200
5 30 5 200
a
Temperature, 121

C; time 30min.
by Johnson et al. (1961), with minor modications. The sample was
dissolved in 10mL 25% (w/v) acetyl bromide in redistilled glacial
acetic acid99% by heating at 70+0.1

C in water bath for 30min.

The sample was stored in a special digester tube with a notched
glass stopper. After 30min the dissolved sample was transferred
into a 200mL volumetric ask containing 5mL mixture of acetic
acid and caustic soda in 1:1 ratio. Interfering substances were
removedbyadding0.2ghydroxylamine hydrochloride. The sample
was diluted to 15mL volume with 99% acetic acid, and absorbance
was read at 280nm. The lignin content of treated and untreated
samples was calculatedusingstandardfactor calculatedfromlignin
standard curve constructed following the same protocol.
2.6. Saccharication of de-lignied biomass
2.6.1. Hydrolysis of cellulose polymers by cellulase extract
The saccharication of alkali and ligninolytic enzymes
pretreated/de-lignied cellulosic residue was carried out by
crude cellulase extract from T. harzianum produced under opti-
mum conditions. Erlenmeyer ask (500mL) containing 20g
de-lignied residue was mixed with 30mL cellulase extract and
volume was made to 200mL mark with 0.1M citrate buffer of pH
4.8. The ask was incubated at 37

C in orbital shaker at 150rpm

for 24h.
2.6.2. Cellulose determination
Celluose content of de-lignied as well as cellulase treated
biomass was determined by the method of David (1969), with
minor modications to investigate the extent of cellulose hydrol-
ysis. The de-lignied biomass was homogenized before and after
cellulase treatment in a warring blender and centrifuged for 5min
at 3000g. The supernatant was decanted and discarded. To the
pellet, added 3.0mL acetic/nitric reagent and mixed well on Vortex
mixer. With a marble on the top to reduce evaporation and cre-
ate a reuxing action, incubated the tubes in boiling water bath for
30min. The contents were centrifuged for 5min at 5000g after
incubation and supernatant was decanted and 10mL 67% H
2
SO
4
(V/V) was added to the residue, mixed well on Vortex mixer and
allowed to stand for 1h. Diluted 1100mL with distilled water
and centrifuged if any precipitate or turbidity was present. Placed
1.0mL of this dilution in a 150mm 18mmscrewcap type culture
tube and added 4.0mL distilled water in it. Tubes were ice cooled in
crushedice and10mL coldanthrone reagent was addedby layering
with a pipette and mixed well on Vortex mixer. Placed a marble on
top of each and placed the tubes in a boiling water bath for 16min
and cooled in ice bath for 23min. Tube was allowed to stand at
roomtemperature for 510min and absorbance was measured on
spectrophotometer at 620nmagainst reagent blank.
2.6.3. Glucose estimation
Glucose estimationwas performedbythe methodof Gadgil et al.
(1995). The hydrolyzate (1mL) was incubated for 30min with 2mL
of 0.1M citrate buffer of pH 4.8 at 50

C. DNS reagent (3mL) was

added and test tube was kept in boiling water bath for 15min
and then cooled in ice. After cooling to room temperature, the
M. Asgher et al. / Industrial Crops and Products 44 (2013) 488495 491
absorbance was measured at 540nm against reagent blank. This
absorbance was multiplied with standard factor computed from
standard curve constructed for known concentrations of glucose to
get glucose concentration in mol/mL.
2.7. Ethanol production
2.7.1. Yeast inoculumpreparation
A pure culture of S. cerevisiae was obtained from Shakarganj
Sugar Mills (Pvt.) Limited, Jhang, Pakistan. Inoculum medium was
prepared in 500mL ask with a working volume of 100mL. The
contents of themediumwere: dextrose, 6%; peptone, 0.5%andyeast
extract, 0.5%. The medium was sterilized at 121

C (15lb/in.
2
) for
15min. S. cerevisiae (10g) was added to the inoculummediumand
the ask was incubated at 37

C at 120rpm for 18h (Rani et al.,

2010).
2.7.2. Ethanol fermentation protocol
Two different sets of triplicate Erlenmeyer asks (500mL)
containing 5g/100mL hydrolyzates from alkali and ligninolytic
enzymes pretreatedsugarcanebagassewereinoculated(3mL yeast
inoculum) and fermented at 37

C for 72h in shaking incubator

(150rpm). After 72h, the asks from both sets were ltered and
the ltrates were centrifuged at 3000g to get more clarity. The
clear supernatants were collected and analyzed for ethanol.
2.7.3. Ethanol analysis by HPLC
An HPLC systemwas used to estimate ethanol concentration in
the fermentedsamples, according to the methodology describedby
Sipos et al. (2010). The Shimadzu HPLC system was used, consist-
ing on an isocratic pump, vacuum degasser, autosampler, column
and refractive index detector. The column used was a fermentation
monitor column (150mm7.8mm, 5m), (BioRad). 5mM aque-
ous H
2
SO
4
was used as the mobile phase at a 0.8mL/min owrate
with a column temperature of 60

C. In comparison to the crude

sample a standard ethanol sample was used for product estimation
in the samples for all HPLC analysis.
2.8. Optimization of fermentation parameters for ethanol
production
Fermentation parameters including fermentation time, pH,
temperature, substrates level and inoculum sizes were optimized
using alkali and enzymes based pretreated hydrolyzates. Classical
optimization strategy was adopted; varying one variable at a time
and keeping the previously optimized factors at optimumlevel.
2.8.1. Fermentation time
To optimize the fermentation time, the asks (500mL) each
containing 5g/100mL hydrolyzates from alkali and enzymes pre-
treated sugarcane bagasse were set to pH5.5, sterilized, inoculated
and fermented at 37

C for different time periods, viz. 24, 48, 72

and 96h in shaking incubator (150rpm). Triplicate asks were har-
vested at each fermentation time and analyzed for ethanol.
2.8.2. Fermentation mediumpH
Fermentation media containing 5g/100mL hydrolyzates were
adjusted to varying pH levels (pH 3, 4, 5, 6 and 7) using M
HCl/NaOH), inoculated with yeast and allowed to ferment for stip-
ulated fermentation time period (72h).
2.8.3. Incubation temperature
To determine the optimum temperature for the growth of S.
cerevisiae and ethanol production the triplicate asks containing
5g/100mL hydrolyzates were adjusted to pH 5 (optimum), inoc-
ulated and subjected to ethanol fermentation for 72h at varying
temperatures (2550

C)
2.8.4. Substrate level
To investigate the effect of substrate level on ethanol produc-
tion, varying levels of hydrolyzates both fromalkali and enzymatic
pretreated substrate (2.5, 5, 7.5, 10 and 15g/100mL) were used.
The triplicate asks were inoculated (5mL inoculum) with yeast
and subjected to fermentation for optimum time period (72h) at
optimumpH (pH 5) and temperature (37

C).
2.8.5. Yeast inoculumsize
To determine the optimum inoculum level that gives the best
ethanol production by S. cerevisiae, the triplicate asks were inoc-
ulated with varying volumes of yeast inoculums (15mL) and
processed for 72h at 37

C under optimumconditions.
2.9. Statistical analysis
All the experimental data was conducted in triplicate and
presented as meanstandard error (SE). Statistical analysis was
performed by analysis of variance (ANOVA) using the statistical
software MINITAB windows version and the probability values
(p0.05) were considered as a statistically signicant difference.
The means and standard errors of means (MeanS.E) were com-
puted for each treatment and S.E values have been displayed as
Y-error bars in gures.
3. Results and discussion
3.1. Ligninolytic production by P. ostreatus IBL-02
P. ostreatus IBL-04 was grown on wheat straw in solid state
fermentation for the production of ligninolytic extract under pre-
optimized conditions (Aslam and Asgher, 2011). The enzyme
extract contained 355.55.28, 4992.84 and 5171.05IU/mL of
LiP, MnP and laccase, respectively.
3.2. Cellulase production by T. harzianum
The production of cellulases is a key factor in the hydrolysis of
cellulosic materials andit is essential to make the process economi-
cally viable. Cellulase productionfromT. harzianumwas carried out
under pre-optimized conditions (Ahmed et al., 2009). The fungus
produced 53.51.24, 41.31.31 and 46.81.43U/mL of endoglu-
canase, exoglucanase and -glucosidase respectively. The enzyme
extract was used for saccharication of alkali and enzyme pre-
treated bagasse.
3.3. Pretreatment for delignication, saccharication and
fermentation
Pretreatment refers to complete or partial degradation of lig-
nocellulosic biomass to expose cellulose polymers for convenient
cellulose hydrolysis into sugars by cellulase enzyme action. Ligni-
nolytic and alkali treatments were used for delignication of
sugarcane bagasse before hydrolysis by cellulases.
3.3.1. Pretreatment with ligninolytic enzymes extract
Different volumes of the ligninolytic extract were used for
enzymatic delignication of sugarcane bagasse containing 35.5%
cellulose and 21.3% lignin. The ligninolytic treated sugarcane
bagasse residues were hydrolyzed using cellulase extract from T.
harzianum. The extent of delignication(%), cellulose hydrolysis (%)
and sugars released (g/100mL) were determined. The hydrolyzates
492 M. Asgher et al. / Industrial Crops and Products 44 (2013) 488495
Table 3
Delignication of sugarcane bagasse by varying volumes of ligninolytic enzymes extract fromP. ostreatus IBL-02, cellulose hydrolysis by T. harzianumcellulase extracts and
ethanol production by S. cerevisiae.
Lignininolytic extract
(mL)
Delignication
(%)
Cellulose hydrolysis
(%)
Glucose (g/L) Ethanol
(g/L)
Before fermentation After fermentation
5 5.2 32.4 27.7 22.5 4.0
10 9.4 37.5 30.6 21.3 6.2
15 12.4 50.1 33.4 16.5 8.5
20 19.7 63.5 41.7 13.8 12.2
25 33.5 72.3 42.8 15.9 16.6
were then used as feed stocks for ethanol fermentation by S. cere-
visiae. After ligninolytic treatment the lignin content of sugarcane
bagasse reduced and maximum delignication of 33.5% deligni-
cation was observed when 25mL ligninolytic enzymes extract was
used (Table 3). After hydrolysis of ligninolytic pretreated residue
with cellulase extract (30mL) from T. harzianum for 24h at 50

C,
the cellulose was converted into glucose and cellulose content
reduced to 7.6% (69.2% cellulolysis) with concomitant release of
35.9g/100mL glucose. Using the hydrolyzate prepared after 25mL
ligninolytic extract and 30mL cellulase extract treated bagasse,
maximum ethanol production by Saccharomyces cerevisiae was
16.6g/L.
Cellulose can be converted to bio-fuel by a multistep process
that includes pre-treatment, enzymatic hydrolysis, and fermenta-
tion(Xiao et al., 2012). For ethanol production, complete enzymatic
hydrolysis of cellulose to glucose requires a complex of endoglu-
canase, exoglucanse and -glucosidase enzymes (Shaikh et al.,
2011). Previously, Adsul et al. (2005) also used bagasse waste for
delignication purposes, followed by hydrolysis by cellulase and
xylanase enzymes. The cellulose conversionobtainedafter 72hwas
in the same range as those reported earlier for other raw materi-
als (Rosgaard et al., 2007). In an earlier study Chen and Jin (2006)
reported that the best temperature for saccharication of cellulose
by callulases in SSF systemwas 40

C.
3.3.2. Pretreatment with NaOH
Different concentrations of NaOH were used for
pretreatment/de-lignication of sugarcane bagasse and lignin
contents of treated and untreated bagasse were determined.
Maximum de-lignication (48.7%) was caused by 5% NaOH treat-
ment for 24h followed by 4% NaOH (Table 4). The de-lignied
residues were then treated for 24h at 50

C with cellulase extract

(30mL) obtained fromT. harzianum. After cellulase treatment, the
cellulose was hydrolyzed and by 69.2% with concomitant release
of 49.8g/100mL glucose. However, maximumethanol production
of 18.6g/L by S. cerevisiae was observed when 4% NaOHpretreated,
followed by 30mL cellulase extract treatment was applied. The
effectiveness of different pre-treatment methods including alkali,
acid and chlorite pre-treatment of lignocellulosic feedstocks for
improving the saccharication of cellulose has been evaluated
by Gupta et al. (2011). Mild alkali treatment conditions caused
complete conversion of cellulose I to cellulose II, which is a more
stable formwith antiparallel chain structure in NaOH solution (Xu
et al., 2012). In another recent study, Liu et al. (2012) reported that
an alkaline pre-treatment can improve the cellulose hydrolysis by
modifying the cellulose crystalline structure.
3.4. Optimization of fermentation parameters for ethanol
production
Sugarcane bagasse biomass residues obtained after pre-
treatment with 25mL ligninolytic extract and 4% NaOH were
selected as best hydrolyzates for ethanol production in sequential
saccharication and fermentation. Both substrates were used for
optimizationof somefermentationparameters andtheresults have
been discussed under respective sub-headings.
3.4.1. Fermentation time period
Tooptimize the fermentationtime for ethanol productionbythe
yeast using alkali and ligninolytic treated biomass, fermentation
was allowed at pH 5.5 and 37

C for different time periods. Ethanol

production using both substrates increased with time and maxi-
mum ethanol productionof 18.3and17.1g/100mL was observedin
72h (Fig. 1) using alkali and ligninolytic treated sugarcane bagasse,
respectively. Ballesteros et al. (1991) identiedandreportedS. cere-
visiae strain that had optimumethanol productivity at 37

C in 70h
and Ko et al. (2009) noted maximum ethanol yield after 96h fer-
mentation. S. cerevisiae, Candida tropicalis and their co-culture have
been reported to produce maximal ethanol concentration of 27, 23,
21g/L (w/v), respectively using 200g/l (w/v) dry corn corbs after
96h of fermentation (Latif and Rajoka, 2001).
3.4.2. Fermentation mediumpH
Hydrolyzates (10g) from alkali and ligninolytic pretreatment
were inoculated (4mL yeast inoculums) and subjected to fermen-
tation (150rpm) at varying pH for optimum fermentation time
(72h) (Fig. 2). The maximumethanol production of 27.45g/dL was
obtainedat pH5usingalkali pretreatedsubstratewhilethefermen-
tation of enzyme pretreated substrate yielded 24.23g/dL of ethanol
at same pH. Statistical analysis revealed signicant (p<0.05) effect
of pH on ethanol production. Comparison of treatment means
showed signicant difference between mean values of ethanol
production with varying pH. Zhu et al. (2006) reported that S. cere-
visiae produces 31.1g/L ethanol at pH 5.3 which is near to the
present results. If the pH is eliminated from the model using the
Fig. 1. Effect of fermentation time on ethanol production by S. cerevisiae using alkali
and ligninolytic pre-treated sugarcane bagasse.
M. Asgher et al. / Industrial Crops and Products 44 (2013) 488495 493
Table 4
Delignication of sugarcane bagasse by varying concentrations of NaOH, cellulose hydrolysis by T. harzianumcellulase extract and ethanol production by S. cerevisiae.
Concentration of alkali (NaOH)
(%)
Delignication by NaOH
(%)
Cellulose hydrolysis
(%)
Glucose (g/L) Ethanol
(g/L)
Before fermentation After fermentation
1 9.7 32.4 21.7 18.5 5.0
2 23.2 37.5 30.3 15.2 11.2
3 30.3 50.1 37.9 17.5 15.2
4 45.4 63.5 46.4 15.8 18.6
5 48.7 69.2 49.8 17.9 16.7
Fig. 2. Effect of mediumpH on ethanol production by S. cerevisiae using alkali and
ligninolytic pre-treated sugarcane bagasse.
optimization of the enzymatic hydrolysis it affects the response
variables (Vsquez et al., 2007). The yeast displays good perfor-
mance in a slightly acid pH range in which contamination of the
fermentation mediumby bacteria is uncommon.
3.4.3. Incubation temperature
To determine the optimumtemperature that gives best ethanol
production from S. cerevisiae 5g hydrolyzates (optimum pH 5)
from alkali and ligninolytic treated bagasse were inoculated and
subjected to ethanol fermentation for 72h at varying tempera-
tures. Themaximumethanol productionof 32.45g/dL was obtained
at 35

C using alkali pretreated substrate while the fermentation

of ligninolytic pretreated substrate yielded 28.15g/dL of ethanol
at 35

C (Fig. 3). Statistical analysis revealed signicant (p0.05)

effect of temperature on ethanol production as mean ethanol
Fig. 3. Effect of temperature on ethanol production using alkali and ligninolytic
pre-treated sugarcane bagasse.
production with different temperatures varied signicantly. In a
recent study Manikandan and Viruthagiri (2010) used Aspergillus
niger and yeast S. cerevisiae in a co-culture fermentation and noted
best ethanol yield at 30

C. In line with our ndings, Ballesteros

et al. (1991) used 27 different species of S. cerevisiae and observed
that 37

C temperature was optimumfor ethanol production by all

strains
3.4.4. Substrate concentration
To optimize the substrate concentration different levels of
alkali and ligninolytic treated bagasse (g/100mL) were used for
ethanol fermentation. Maximum ethanol production of 22.42g/L
was obtained using 5.7g/100mL of alkali pretreated substrate
whileoptimumproductionof 20.25g/Lethanol was producedusing
7.5g/100mL of ligninolytic treated substrate (Fig. 4). Statistical
analysis revealed signicant (p0.05) effect of substrate level on
ethanol production. Comparison of treatment means showed sig-
nicant difference between mean ethanol productions under all
treatments. Substrate concentration is one of the main factors that
affect the yield and initial rate of enzymatic hydrolysis of cellulose
and fermentation. An increase of substrate concentration in lower
range normally results in an increase of the sugar yield that subse-
quently enhances the ethanol yield (Cheung and Anderson, 1997).
However, high substrate concentrations can cause substrate inhi-
bition, which sustainably lowers the rate of ethanol fermentation
(Xinet al., 2010). Highsubstrate concentrationalso causes decrease
in hydrolysis yield due to product inhibition, and the extent of sub-
strate inhibition is dependent on the ratio of the total substrate to
total enzyme loaded (Xin et al., 2010; Wang et al., 2011).
3.4.5. Yeast inoculumsize
Yeast inoculum density is very important parameter towards
industrial exploitation of ethanol fermentation. Therefore, varying
inoculum(10
7
10
8
cells/mL) levels were usedfor inoculationof the
optimumfermentation medium. Maximumethanol production of
Fig. 4. Effect of substrate level on ethanol production using alkali and ligninolytic
pre-treated sugarcane bagasse.
494 M. Asgher et al. / Industrial Crops and Products 44 (2013) 488495
Fig. 5. Effect of yeast inoculum size on ethanol production using alkali and ligni-
nolytic treated sugarcane bagasse under optimumconditions.
44.50 and 39.35g/L was noted in the fermentation asks receiving
4mL yeast inoculum using alkali and ligninolytic treated sub-
strate, respectively (Fig. 5). Statistical analysis revealed signicant
(p0.05) effect of varying inoculumsize on ethanol production as
all treatment means showed signicant difference. As described in
literature (DAmore et al., 1989), the hydrolysis rate and level of
ethanol produced increases with the increases in inoculumsize. A
strong interaction between sugar concentration and inoculumsize
in high-cell-density cultures can lead to increases in the levels of
ethanol yield (Laluce et al., 2009). Yeast inoculumsize has a signi-
cant effect for ethanol production (Turhan et al., 2010) and there is
signicant (p0.05) difference betweenvarying levels of inoculum
in terms of ethanol production rate.
4. Conclusions
In summary, pretreatment of sugarcane bagasse by ligninolytic
enzymes extract showed commendable performance when com-
pared with NaOH pretreatment. However, cellulose hydrolysis
in case of enzymatic treatment was higher than alkali pretreat-
ment, suggesting that ligninolytic extract may contain cellulase
activities that caused some cellulose breakdown simultaneously
with lignin degradation. Alkali pretreatment gave better ethanol
production than ligninolytic pretreatment but the difference
was non-signicant. More efcient ligninolytic enzymes can be
developed using advanced molecular approach that may be the
economical and environment friendly future catalysts for biomass
delignication. In conclusion, the enzymatic treatment of waste
biomass could be of particular interest, since it seems an eco-
friendly approach to carrying out waste biomass treatment and
concomitant glucose production that can be further used for
bio-ethanol production. By adopting this proposedapplicationpro-
cedure that favors greenchemistry technology, the burdenof waste
management/treatment can be reduced signicantly.
Acknowledgments
The present study was a part of the research project focused
on development of ligninolytic enzymes for industrial applications.
The nancial support for this project by Higher Education Commis-
sion, Islamabad, Pakistan is thankfully acknowledged.
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