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Effect of alkaline pretreatment on

delignification of wheat straw
a

Umar Asghar , Muhammad Irfan , Mehvish Iram , Zile Huma ,

a

Rubina Nelofer , Muhammad Nadeem & Quratulain Syed

a

Food and Biotechnology Research Center (FBRC), PCSIR

Laboratories Complex, Ferozpur Road, Lahore 54600, Pakistan
Published online: 06 Oct 2014.

To cite this article: Umar Asghar, Muhammad Irfan, Mehvish Iram, Zile Huma, Rubina
Nelofer, Muhammad Nadeem & Quratulain Syed (2014): Effect of alkaline pretreatment on
delignification of wheat straw, Natural Product Research: Formerly Natural Product Letters, DOI:
10.1080/14786419.2014.964712
To link to this article: http://dx.doi.org/10.1080/14786419.2014.964712

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Natural Product Research, 2014

http://dx.doi.org/10.1080/14786419.2014.964712

Effect of alkaline pretreatment on delignification of wheat straw

Umar Asghar, Muhammad Irfan*, Mehvish Iram, Zile Huma, Rubina Nelofer,
Muhammad Nadeem and Quratulain Syed
Food and Biotechnology Research Center (FBRC), PCSIR Laboratories Complex, Ferozpur Road, Lahore
54600, Pakistan

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(Received 27 July 2014; final version received 31 August 2014)

This study was conducted to analyse structural changes through scanning electron
microscopy (SEM) and Fourier transform infrared (FTIR) after alkaline pretreatment of
wheat straw for optimum steaming period. During the study, 2 mm size of substrate was
soaked in 2.5% NaOH for 1 h at room temperature and then autoclaved at 1218C for
various steaming time (30, 60, 90 and 120 min). Results revealed that residence time of
90 min at 1218C has strong effect on substrate, achieving a maximum cellulose content
of 83%, delignification of 81% and hemicellulose content of 10.5%. Further SEM and
FTIR spectroscopy confirmed structural modification caused by alkaline pretreatment
in substrate. Maximum saccharification yield of 52.93% was achieved with 0.5%
enzyme concentration using 2.5% substrate concentration for 8 h of incubation at 508C.
This result indicates that the above-mentioned pretreatment conditions create
accessible areas for enzymatic hydrolysis.
Keywords: alkaline pretreatment; wheat straw; SEM; FTIR

1. Introduction
Over the few decades, the production of bioethanol has attracted the researchers and proved that
ethanol can be used as an alternate fuel. Several countries have used bioethanol blend with
gasoline (Hahn-Hagerdal et al. 2006). Pure ethanol can also be exploited in vehicles after some
modifications in their engines (Ibrahim 2012). The production of biofuel from lignocellulosic
biomass is more feasible and economical than fossil fuel (Ibrahim 2012). Various types of
lignocellulosic biomass are carbohydrates (cellulose and hemicelluloses) being used for the
production of bioethanol. This study investigated wheat straw, which is produced as a byproduct of wheat after cultivation. A small amount of wheat straw is used for livestock but large
amounts are burned in fields causing global warming (Han et al. 2012). In general, wheat straw
contains 35 45% cellulose, 20 30%, hemicellulose and 8 15% lignin. The conversion of
lignocellulosic biomass to ethanol is performed in four steps, pretreatment, hydrolysis of
cellulose, fermentation and distillation. The pretreatment is a crucial step to breaking down
lignocellulosic biomass by removing lignin to expose cellulose content for proper application of
saccharifying enzyme. Several method of pretreatment has been used for maximum degradation
of lignin (Kim & Holtzapple 2006).
Among these methods, alkali pretreatment especially by NaOH has advantages over acid
pretreatment to expose maximum cellulosic contents. The main characteristic of alkaline
pretreatment is that it modified the structure of cellulose fibre, making it thermodynamically
stable than the control fibre. Pretreated biomass after saccharification is employed in

*Corresponding author. Email: mirfanashraf@yahoo.com, irfan.biotechnologist@gmail.com

q 2014 Taylor & Francis

U. Asghar et al.

2. Result and discussion

Before pretreatment, wheat straw was subjected to compositional analysis on dry basis weight
(w/w) and the contents of cellulose (45%), hemicelluloses (21.30%) and lignin (19%) were
determined. This study provides the information about the effect of 2.5% sodium hydroxide
(alkaline solution) at various steaming times (30, 60, 90 and 120 min) on structural changes that
take place in wheat straw. As reported earlier, the alkaline pretreatment as well as under steam
pressure for various times removes maximum lignin and also solubilises hemicelluloses (Irfan
et al. 2010). The result of earlier study showed that maximum cellulose and delignification was
achieved at 2.5% sodium hydroxide (Nadeem et al. 2013).
Figure 1 represents the effect of 2.5% sodium hydroxide and also the observed effect of
steaming time on substrate. Initially, the wheat straw was kept at 1218C steam under pressure for
30 min, where 67% cellulose, 17.30% hemicelluloses and 63.5% delignification were achieved.
In the same way, the substrate was pretreated for 60 min the cellulose (77.51%) and
delignification (72.5%) increased but reduction was observed in hemicelluloses (14.6%) as
compared with 30 min steam under pressure. Moreover, the steaming time was further increased
where the substrate was treated for 90 min, resulting in the maximum cellulose content of 83%
and delignification of 81%, whereas the hemicelluloses decreased further to 10.50%. The
reduction in hemicelluloses and highest value of cellulose indicate the removal of
hemicelluloses from wheat straw. The previous study also reported that reduction of
Delignification

Hemicellulose

25

80

20

60

15

40

10

20

0
Control

30

60
Steam Time (min)

Figure 1. Comparison of control and pretreated wheat straw.

90

120

Hemicellulose (%)

Cellulose

100
Cellulose, delignificaion (%)

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fermentation process to produce bioethanol (Zhang et al. 2010). Moreover, fermented material
used in distillation for removing excess water from ethanol. The main objective of this study was
maximum exposure of cellulose and maximum removal of lignin content from wheat straw after
alkaline pretreatment. Alkaline pretreatment has stronger effects on cleavage of ester bonds
which are present in lignin and hemicelluloses (Modenbach 2013). Recently, the major problem
in bioethanol production from lignocellulosic biomass is the high cost of fermentation process
based on various steps (Gupta 2008). However, a cheaper and abundantly available substrate
having high cellulosic content play an important role to overcome such issues involved in
commercial production of bioethanol.
In this study, we report on the changes in physical properties and cellulose microstructure of
the pretreated wheat straw. In continuation of our research work reported earlier (Irfan et al.
2010, 2011) regarding the delignification of pretreated plant biomass for ethanol production, this
work is conducted to enhance the delignification of wheat straw by various steaming time.

Downloaded by [39.34.153.200] at 09:54 10 October 2014

Natural Product Research

hemicelluloses and lignin content indicates the effect of sodium hydroxide and steam explosion
(Rahnama et al. 2013). Furthermore, when the substrate was treated for 120 min, a reduction in
cellulose contents (76%) was observed. These results expressed that treatment at 1218C for
120 min has some harsh effect on lignocellulosic biomass as compared with 90 min which may
degrade the lignin, hemicelluloses and also solubilised cellulose contents.
Scanning electron microscopy (SEM) was done to study the morphological changes in
untreated and treated biomass. Results revealed that (Figure 2) alkaline pretreatment caused
pores in substrate. These pores were created by dissolution of lignin and hemicellulose content.
These morphological changes in pretreated substrates revealed that these pores provide surface
area for the penetration of cellulase enzyme to enhance the saccharification process. Our findings
were consistent with previous reports (Irfan et al. 2011; Khokhar et al. 2014). The previous work
showed that alkaline 2% NaOH degrades the linkage between lignin and hemicelluloses,
increasing the surface area and making cellulose more accessible for enzymatic hydrolysis.
Further structural modification was studied by Fourier transform infrared (FTIR)
spectroscopy. FTIR analysis revealed changes in pretreated and untreated wheat straw (Figure
3). The peaks at 3336 cm21 expressed to hydroxyl group (OH) which is present in control
wheat straw and after treatment, this band was stretched and appeared as 3315.26 cm21. The
intensity of hydroxyl group (ZOH) decreased which indicated the effect of 2.5% NaOH on
wheat straw. The earlier work reported that the peak 3334 cm21 represents the absorption of
ZOH group of alcoholic hydroxyl (Irfan et al. 2011). Moreover, the second peak at 1316 cm21
corresponds to lignin which was observed in untreated wheat straw while this peak disappeared
in treated (2.5% NaOH) wheat straw. These results proved that alkali pretreatment effectively
removes the lignin content. This peak formation was similar to the work of Han et al. (2012). The
previous work reported that the peak at 1315 cm21 indicated hemicelluloses in control sample.
The peak at 1030 , 1034 cm21 linked with CZO, CvO, CZCZO band stretching present in
control as well as pretreated which indicates the band of cellulose. Zhang et al. (2013) reported
that the band at 1032 cm21 was associated with polysaccharide.
The pretreated substrate (wheat straw) was evaluated for enzymatic hydrolysis by
commercial cellulases. Enzymatic hydrolysis was optimised at 508C, pH 5.0 by changing the
incubation time, enzyme concentration and substrate concentration. Figure 4(A) describes the
effect of incubation time on saccharification process. Results revealed that maximum
saccharification was obtained at 8 h of incubation period. Further increase or decrease in
incubation period beyond this limit resulted in the decline in saccharification. Han et al (2012)
reported maximum enzymatic hydrolysis at 30 h of incubation period at 508C. Saliu and Sani
(2012) obtained maximum sugars (7.63 mg/mL) from alkali-treated corncobs hydrolysed with

Figure 2. SEM of wheat straw: (A) control and (B) pretreated substrate. The red circles (in B) represent the
morphological changes of pretreated wheat straw (visible as holes) which indicates the degradation of
lignin and hemicelluloses.

U. Asghar et al.

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Figure 3. FTIR spectrum of untreated and treated (2.5% NaOH) wheat straw.

cellulases of Aspergillus niger after 48 h of hydrolysis time period. Furthermore, enzyme

concentration (CMCase activity of 2900 IU/mL and filter paper activity of 1500 FPU/mL) in
50 mL of citrate buffer was optimised by varying the concentration from 0.5% to 3%. Results
(Figure 4(B)) showed that maximum saccharification was achieved at 0.5% enzyme
concentration. High concentration (3%) of enzyme did not increase the saccharification yield.
Substrate concentration is also another important factor in enzymatic reactions due to the
limited availability of enzyme active sites. Concentration of substrate (wheat straw) was also
optimised by changing from 0.5% to 3%. From this experiment, it was observed that 2.5%
substrate concentration gave better saccharification rate (52.93%) as shown in Figure 4(C).
Han et al. (2012) reported that 3% concentration of substrate was optimum for maximum
saccharification.

Saccharification (%)

Natural Product Research

60
e

50

40
30

c
b

20
10

0
4
6
8
Incubation time (h)

10

80
60

40

c
b

20
0
0.5

1.5

2.5

Enzyme Conc. (%)

C

Saccharification (%)

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Saccharification (%)

60
50
40
30
20
10
0

f
d

c
b
a

0.5

1.5

2.5

Substrate Conc. (%)

Figure 4. Optimisation of saccharification process at pH 5.0, 508C. (A) Incubation time, (B) enzyme
concentration, (C) substrate concentration.

3. Materials and methods

3.1. Lignocellulosic biomass
Wheat straw was used as lignocellulosic biomass, which was procured from the local market of
Lahore city, Pakistan. The biomass (sample ID WS001/2014) was washed and dried to remove
dust particles and then chopped to powder form (2 mm) with hammer mill.
3.2. Pretreatment of substrate
The chopped sample of wheat straw was soaked in 2.5% of NaOH solution at the ratio of 1:10
(solid/liquid) for 1 h at room temperature. Later, the samples were autoclaved at 1218C for
various time periods (30, 60, 90 and 120 min). Then, the samples were filtered and solid residues
were washed up to neutrality (Irfan et al. 2010).
3.3. Compositional analysis
The cellulose content was estimated by using the method of Gopal and Ranjhan (1980). Lignin
content was estimated by using the method described by Milagres (1994). Hemicellulose was
estimated by using the method of Goering and Van Soest (1975).

U. Asghar et al.

3.4. SEM of wheat straw

Samples of untreated and treated wheat straw were oven-dried at 508C for 1 h, and thick layers
were supported in the sample holder fixed on a carbon ribbon. This assembly was maintained in a
vacuum-desiccator until the analysis. The SEM type S-3700 microscope (Hitachi, Tokyo, Japan)
was used for observing the bagasse fibres in both treated and untreated samples (Irfan et al.
2011).
3.5. FTIR spectroscopy

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FTIR was used to check the chemical changes in treated and untreated samples. Mixture of
sample and KBr (5% sample: 95% KBr) was passed into a disk for FTIR spectroscopy
measurement. The spectra were recorded with 32 scans in the frequency range of 4000
400 cm21 with a resolution of 4 cm21 (Irfan et al. 2011).
3.6. Enzymatic hydrolysis of substrate
In 500 mL flask, 50 mL citrate buffer pH 5 and 1 mL of commercial cellulase enzyme (CMCase
activity of 2900 IU/mL and filter paper activity of 1500 FPU/mL) with 2% pretreated wheat
straw was incubated in a shaking water bath at 508C with agitation speed of 140 rpm for 8 h.
After termination of enzymatic hydrolysis, the material was centrifuged at 10,000 rpm for
10 min. The supernatant was removed for sugar content analysis. Saccharification (%) was
calculated using the following formula (Alam et al. 2004):
Saccharification %

Reducing sugars mg=mL

100:
Substrate used mg=mL

3.7. Statistical analysis

Statistical analysis was done by using computer-based software SPSS. The difference in values
was indicated in the form of probability ( p , 0.05) values.
4. Conclusion
At this concentration, 81% cellulose, 83% delignification and 10.50% hemicelluloses were
achieved. During the study, reduction of lignin and hemicelluloses indicates the effect of
physiochemical pretreatment and the enhanced surface area for enzymatic hydrolysis. The
pretreated substrate showed maximum saccharification with 0.5% enzyme concentration using
2.5% substrate concentration for 8 h of incubation at 508C.
Funding
The authors would like to thank the Ministry of Science and Technology (MoST), Islamabad, Pakistan for
providing the financial support to carry out this research work under PSDP project entitled Production of
Bioenergy from Plant Biomass.

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