Iran J Sci Technol Trans Sci
DOI 10.1007/s40995-017-0284-z
RESEARCH PAPER
Effect of KOH Pretreatment on Lignocellulosic Waste to be Used
as Substrate for Ethanol Production
Umar Asghar1 • Muhammad Irfan2 • Muhammad Nadeem1 • Rubina Nelofer1
Quratulain Syed1
Received: 5 September 2015 / Accepted: 20 September 2015
Ó Shiraz University 2017
Abstract In present study, Leptochloa fusca (Kallar grass)
and Gossypium herbarium (cotton stalk) were treated with
various concentrations of KOH (1, 1.5, 2, 2.5 and 3%) for
different soaking periods (6, 12, 24 and 48 h) to expose
maximum cellulosic content. The results showed that
maximum cellulose exposure and delignification was
increased with the increase in concentration of alkali and
soaking time. However, maximum cellulose content (Kal-
lar grass 49.01 ± 0.1%, cotton stalk 56.12 ± 0.1%) was
achieved with 2.5% KOH at 24 h and a decline was
observed as further increase in concentration of KOH (3%).
Moreover, maximum delignification (Kallar grass 66.2%,
62.6 cotton stalk) was observed at 3% KOH after 48 h of
soaking period. However, these results showed adverse
effect of higher concentration of alkali on holocellulose.
The cellulosic content of Kallar grass and cotton stalk after
pretreatment at suitable conditions indicated that these
substrates could be used as promising substrates for sac-
charification process to the produced second generation
biofuel.
Keywords Leptochloa fusca  Gossypium herbarium 
Pretreatment  Sugars
& Muhammad Irfan
Irfan.biotechnologist@gmail.com; m.irfan@uos.edu.pk
1 celluloses or cellulose (Sun and Cheng 2002). Kallar grass
Food and Biotechnology Research Center (FBRC), Pakistan
Council of Scientific and Industrial Research (PCSIR)
Laboratories Complex, Ferozpure Road, Lahore, Pakistan
2
Department of Biotechnology, University of Sargodha,
Sargodha 40100, Pakistan
•
1 Introduction
Natural resources of crude oil are decreasing due to
increasing demand; therefore, there is a need to find a
substitute for fossil fuel. Bioethanol (biofuel) which is
produced from lignocellulosic biomass can be used as an
alternative resource (Singh and Trivedi 2013). Biomasses
obtained from various sources like forestry, agriculture
waste (wheat straw and rice straw) can produce billions of
liters of biofuel. Such types of biofuel do not increase the
release of CO2 in the environment (Marina et al. 2014).
For the conversion of lignocellulosic biomass into bio-
fuel, it is necessary that cellulose be accessible for enzy-
matic hydrolysis (Dawson and Boopathy 2008). The
cellulose and hemicelluloses are wrapped with lignin;
therefore, pretreatment is an important step to break down
lignin to expose cellulose content for proper application of
saccharifying enzyme to convert saccharified material
(sugar) into ethanol during fermentation. A number of
pretreatment methods have been used. These include steam
explosion, alkaline, dilute acid, and wet oxidation, etc.
Among these pretreatment methods, alkaline pretreatment
has received considerable attention because it removes
lignin and degrades acetyl groups and various uronic acid
substitutions on hemicelluloses of the biomass (Modenbach
2013).
The mechanism of dilute alkaline solution causes
swelling of biomass which leads to degradation of lignin.
During pretreatment, the hydroxide ion attacks on carbon
ester bonds which are present between lignin and hemi-
and cotton stalk have rich sources of cellulose content and
are easily available in Pakistan; therefore, the present
research work was carried out to exploit these abundantly
available resources for biofuel production in future.
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2 Materials and Methods 2.3 Analytical Methods

2.1 Substrate Collection After treatment, dried residues and filtrate of first washing
obtained from processed sample were analyzed as follows.
The substrates, Kallar grass, and cotton stalk were col- The solid residual part of sample was used for cellulose
lected from remote area of Punjab, Pakistan. Both the estimation and lignin estimation. The cellulose content was
substrates were sun dried, chopped, and preserved in estimated by the method of Gopal and Ranjhan (1980).
polyethene bags at room temperature. Lignin content was estimated by the method as described
by Milagres (1980). Delignification was calculated using
2.2 Pretreatment of Substrate the following formula (Irfan et al. 2014)
Lu  Lt
2.2.1 Physical Pretreatment Delignification ð%Þ ¼  100;
Lu

The chopped materials were dried at 80 °C in an oven and where Lu = Lignin (untreated sample), and Lt = Lignin
then pulverized up to 2 mm mesh size with hammer beater (treated sample).
mill (PX-MFC 90 D-Kinematics). The crushed sample was The liquid part after filtration was used for estimation of
stored at room temperature in polythene bags for further reducing sugar, total sugar, and total phenol. The DNS
study. method was used for estimation of reducing sugar (Miller
1959). Total sugar content in filtrate was estimated as
2.2.2 Chemical Pretreatment described by Dubois et al. (1959). Total phenolic com-
pounds in filtrate were determined by the method of Car-
In chemical pretreatment, 10 g chopped sample of each ralero et al. (2005).
substrate (2 mm size) was treated with 100 ml (1:10 solid
liquid ratio) solution having various concentrations (1,1.5, 2.4 Statistical Analysis
2, 2.5, and 3%) of KOH in separate flask and soaked at
room temperature and washed with distilled water five to All the data were statistically analyzed using Microsoft
six times to get pH 7.0. The washed residues were dried in Excel program and the values mentioned were the mean of
an oven at 105 °C and further analyzed for the estimation triplicates.
of cellulose and lignin contents.
Total Sugar Reducing sugar Total Phenol

25 10
Total Sugar Reducing sugar Total Phenol 9
20 25
18 20 8

Total Phenol (mM)

Total phenol (mM)

7
Sugars (mg/ml)

16 20
Sugars (mg/ml)

14 15 6
12 15 5
10
10 4
8 10
6 3
4 5 5 2
2 1
0 0 0 0
1 1.5 2 2.5 3 1 1.5 2 2.5 3
KOH Conc. (%) KOH Conc. (%)

Cellulose Lignin Delignification

Cellulose Lignin Delignification
50 30
50 50
Lignin, Delignification (%)

45
Lignin, Delignification (%)

45 45 25
40
40 40
35
Cellulose (%)
Cellulose (%)

35 35 20
30 30 30
25 25 25 15
20 20 20
15 15 15 10
10 10 10 5
5 5 5
0 0 0 0
1 1.5 2 2.5 3 1 1.5 2 2.5 3
KOH Conc. (%) KOH Conc. (%)

Fig. 1 Effect of 6 h soaking with different concentration of KOH on Fig. 2 Effect of 6 h soaking with different concentration of KOH on
degradation products of Kallar grass degradation products cotton stalk

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Total Sugar Reducing sugar Total Phenol Total Sugar Reducing sugar Total Phenol
30 30
20 25

Total Phenol (mM)

18 25 25
16 20

Sugars (%)
Total Phenol (%)
20 20
14
Sugars (%)

12 15 15 15
10 10 10
8 10
6 5 5
4 5 0 0
2 1 1.5 2 2.5 3
0 0 KOH Conc. (%)
1 1.5 2 2.5 3
KOH Conc. (%)
Cellulose Lignin Delignification
60 70

Lignin, Delignification (%)

Cellulose Lignin Delignification
50 50 50 60

Lignin, Delignification (%)

45 45 50

Cellulose (%)
40 40 40
40
Cellulose (%)

35 35
30
30 30 30
25 25 20
20
20 20
15 15 10 10
10 10 0 0
5 5 1 1.5 2 2.5 3
0 0
1 1.5 2 2.5 3 KOH Conc. (%)
KOH Conc. (%)
Fig. 5 Effect of 24 h soaking with different concentration of KOH on
Fig. 3 Effect of 12 h soaking with different concentration of KOH on degradation products of Kallar grass
degradation products of Kallar grass
pretreatment, the substrates were analyzed for cellulose and
Total Sugar Reducing sugar Total Phenol lignin contents. About 27.0% cellulose and 25.0% lignin
30 25 were estimated in Kallar grass while 40.0% cellulose and
25 19.0% lignin were found in cotton stalk, respectively.
Total Phenol (mM)
Sugars (mg/ml)

20
20
15 These substrates were soaked at various concentrations (1,
15 1.5, 2, 2.5 and 3%) of KOH for different time intervals (6,
10
10 12, 24 and 48 h) in separate flasks. During study it was
5 5
observed that increase in the concentration of potassium
0 0
1 1.5 2 2.5 3
hydroxide at different soaking time periods increased the
KOH Conc. (%) cellulose contents along with delignification. Earlier study
Cellulose Lignin Delignification
reported that pretreatment of wheat straw with KOH gave
50 60 maximum efficiency of 76.3%, which was achieved during
Lignin, Dlignification (%)

45
50
40 fermentation (Tutt et al. 2012).
Cellulose (%)

35 40
30 Figure 1 represents 6 h soaking of Kallar grass and
25 30
20 Cotton stalk (Fig. 2) at various concentrations (1, 1.5, 2,
15 20
10
2.5 and 3%) of KOH. Maximum cellulose was observed in
10
5 Kallar grass 43.12 ± 0.1 and 44.12 ± 0.2% in cotton stalk
0 0
1 1.5 2 2.5 3 at 2.5% concentration of KOH. Thereafter, a further
KOH Conc. (%) increase in concentration of KOH (3%) resulted in decline
in cellulose content of both substrates. But, on the other
Fig. 4 Effect of 12 h soaking with different concentration of KOH on
degradation products of cotton stalk hand, maximum delignification in Kallar grass was
20 ± 0.3 and 23.6 ± 0.4% and in cotton stalk was
3 Results and Discussion achieved at 3% KOH, respectively. This might happen due
to the bad effect of high concentration (3%) of KOH on
Lignocellulosic biomass is a natural abundant source of holocelluses which resultantly decreased the cellulose
cellulose contents. Pretreatment of lignocellulosic biomass content. A similar finding at 2% concentration of alkali
with various methods to explore maximum cellulosic (NaOH) was reported in earlier investigations, where 79%
content is the main requirement for its proper implication. of lignin removal was achieved (Kataria and Ghosh 2014).
In present study, Kallar grass and cotton stalk were treated The previous study was reported that 70.7% delignification
with potassium hydroxide (KOH). It was noted that KOH was achieved in bagasse with 2.5% KOH at autoclaving
has significant effect on these substrates. Before time of 45 min (Nadeem et al. 2013).

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Total Sugar Reducing sugar Total Phenol Total Sugar Reducing sugar Total Phenol
40 30
35 30
35 25
30

Total Phenol (mM)

25

Total Phenol (mM)

30
Sugars (mg/ml)

25 20

Sugars (%)
20 25
20 20 15
15
15 15
10
10
10 10
5 5
5 5
0 0 0 0
1 1.5 2 2.5 3 1 1.5 2 2.5 3
KOH Conc. (%) KOH Conc. (%)

Cellulose Lignin Delignification

Cellulose Lignin Delignification
70 60 50 70

Lignin, Delignification (%)

Lignin, Delignification (%)
45
60 50 60
40
50
Cellulose (%)

40 35 50

Cellulose (%)
40 30 40
30 25
30 30
20 20
20 15 20
10 10 10
10
0 0 5
1 1.5 2 2.5 3 0 0
KOH Conc. (%) 1 1.5 2 2.5 3
KOH Conc. (%)

Fig. 6 Effect of 24 h soaking with different concentration of KOH on

Fig. 8 Effect of 48 h soaking with different concentration of KOH on
degradation products of cotton stalk
degradation products of cotton stalk

cellulosic contents were achieved at 24 h soaking and

Total Sugar Reducing sugar Total Phenol
35 30 reduction was observed at 48 h. The filtrate contained total
30 sugar (17.4 mg/ml), reducing sugar (1.5 mg/ml), and total
Total Phenol (mM)

25
Sugars (mg/ml)

25 phenol (26 mg/ml) (Irfan et al. 2011). After that, both

20
20
15
15 substrates (Kallar grass and cotton stalk) were further
10 soaked for 12 h, it was found that cellulose content and
10
5
5 delignification increased up to 45.01 ± 0.1, 42.4 ± 0.2%
0 0
1 1.5 2 2.5 3 in Kallar grass, 45.12 ± 0.1, and 44.52 ± 0.1% in cotton
KOH Conc. (%) stalk as compared to 6 h as shown in Figs. 3 and 4.
Cellulose Lignin Delignification
Later, the maximum cellulose 49.01 ± 0.1% in Kallar
50 80 grass (Fig. 5) and 56.12 ± 0.1% in cotton stalk (Fig. 6)
Lignin, Delignification (%)

45 70
40 was observed with 2.5% concentration of KOH for 24 h
60
Cellulose (%)

35 soaking time. Figures 7 and 8 express 48 h soaking period

30 50
25 40 where a decline was observed in cellulose content while the
20 30
15 delignification increased as compared to 24 h soaking.
20
10 Therefore, 48 h soaking period liberated maximum amount
5 10
0 0 of total sugar, reducing sugar, and phenolic compounds.
1 1.5 2 2.5 3
24.23 ± 0.5 and 3.760 ± 0.2 mg/ml was observed in
KOH Conc. (%)
Kallar grass while (27.81 ± 0.5, 2.831 ± 0.2 mg/ml) in
Fig. 7 Effect of 48 h soaking with different concentration of KOH on total sugar and reducing sugar was observed in cotton stalk,
degradation products of Kallar grass respectively, which represents the degradation of hemi-
celluloses. The observation of total phenol up to
The total sugar, reducing sugar, and total phenol were 25 ± 0.6 mg/ml Kallar grass and 22.92 ± 0.6 mg/ml in
determined in filtrate. The values of total sugar cotton showed that lignin component has broken down into
(21.78 ± 0.2), reducing sugar (1.81 ± 0.1), and total phenolic components. Another work reported that 2%
phenol (8.92 ± 0.2) were increased in filtrate at 3% KOH KOH solution removes maximum lignin at different tem-
concentration as compared to 2.5% KOH. These values perature in corn fiber (Boonyisa et al. 2012).
further suggest that high concentration of KOH not only
degraded lignin but also solubilized cellulose contents
during pretreatment. Earlier study reported that maximum

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4 Conclusion
The present study was investigated to expose maximum
cellulosic contents of Leptochola fusca (Kallar grass) and
Gossypium herbarium (cotton stalk) by treating these
substrates with various concentrations of KOH under var-
ious soaking periods. The treatment was found to be a more
effective 24 h soaking period as compared to 48 h. Maxi-
mum cellulose and delignification (49.01 and 56.4%) in
Kallar grass was achieved while in cotton stalk the cellu-
lose and delignification 56.12 and 52.10% was achieved
with concentration of 2.5% KOH. All these findings indi-
cated that chemical pretreatment with 2.5% KOH was
effective for enzymatic hydrolysis.
Acknowledgements The authors would like to thank ‘‘Fermentation
Biotechnology Lab’’ PCSIR Lab Complex Lahore, Pakistan.
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