GERF Bulletin of Biosciences
December 2011, 2(2):29-31
*Corresponding author: firstname.lastname@example.org
Copyright © 2011 Green Earth Research Foundation
Hydrolysis of wood saw dust by combined chemical pretreatment and
enzymatic methods for lignocellulosic saccharification
Devendra Kumar1*, Kaushlesh K. Yadav2 and Munna Singh1
1Department of Botany, Lucknow University, Lucknow-226007, India
2Department of Biotechnology, Dr. Ram Manohar Lohia Avadh University, Faizabad- 224001, India
Keyword: Wood saw dust (WSD), Aspergillus fumigatus, Acid hydrolysis, Enzymatic hydrolysis. Cellulose, Hemi-
Wood saw dust (WSD) after lignocellulosic saccharification by different hydrolysis methods is more efficient for ethanol
production as, its contains cellulose and hemi-cellulose at higher levels 65% (w/v) and 35% (w/v), respectively. Cellulose
and hemicellulose account for about a quarter of whole biomass in all land plants. A pretreatment method using chemical
hydrolysis and enzymatic conversion from starch into fermentable sugars was investigated. The WSD was hydrolyzed at
1.69 g/l, using a crude culture filtrate Aspergillus fumigatus at pH 5.0 and 30ºC in acetate buffer 50 mM, while 23.3 g/
l was with 1 N sulfuric acid (H2SO4) treatment. Aonla pomace waste was used as substitute to acid because of high acidic
nature. Optimum conditions for lignocellulosic saccharification is discussed in this paper.
High lignocellulosic agri-horticulture biomasses are
employed as alternative bio-energy (resource) to fossil
energy sources via lignocellulosic saccharification (Vintila
et al., 2010). Two processes used to convert cellulose and
hemicellulose into biofuel (ethanol) are enzymatic and acid
hydrolysis (Akin-Osanaiye et al., 2005; Chandel et al., 2007;
Jurcoane et al., 2009; Karmakar et al., 2011). The most
commonly adopted technique is acid hydrolysis (Badger et
al., 2002). Acidic hydrolysis is an effective method used for
lignocelluloses raw material pretreatment in saccharification
which could change into ethanol. Although acids are
powerful agents used for biomass hydrolysis, concentrated
acids are toxic, erosive and hazardous. Handling higher
concentrations of acid requires reactors that are resistant to
erosion in raw material pretreatment. Diluted acid hydrolysis
especially sulfuric acid has been successfully developed for
pretreatment of cellulosic materials.
Another method of hydrolysis is enzymatic hydrolysis.
Enzymes are naturally occurring plant proteins that result in
certain chemical reaction. However, for enzymes to work,
they must obtain access to the molecules to be hydrolyzed
(Baig et al., 2004). A combined strategy involving acid, base
and enzymatic methods in hydrolysis of saw dust is
The WSD was collected in saw dust from Kakori industrial
area. The samples were brought to room temperature washed
with distilled water and used in the experiment.
Acid -base hydrolysis
The WSD 25% (w/v) was hydrolyzed with 100 ml (1:2 w/
v) of of various concentrations of H2SO4, HCl and NaOH at
room temperature treatment for 24 hr. The hydrolysates were
separated to obtain any suspended or unhydrolysated
materials and was neutralized by 2 N NaOH and 1 N H2SO4
solution for analytical processing then autoclaved at 121ºC
and 15 lbs pressure for 15 min (Nat Steel Equipment Pvt. Ltd,
The WSD 25% (w/v) were hydrolyzed with various fungal
enzymes from (106 spores) of Aspergillus fumigatus,
Rhizopus, Trichoderma viridae and Aspergillus wenti) with
extra cellular enzymes (i.e. α-amylase, glucoamylase,
cellulase and pectinase) were used in the experiment. The
hydrolyate was separated by centrifugation at 12,000 rpm at
Materials and Methods
GERF Bulletin of Biosciences 2011, 2(2):29-31 30
Table1: Effect of wood saw dust (WSD) fungal enzyme
and chemical treatment for saccharification.
Hydrolysis of wood saw dust
Aspergillus fumigatus+ HCl (1N)
Rhizopus+ HCl (1N)
Trichoderma viride+ HCl (1N)
Aspergillus wenti+ HCl (1N)
Values are presented as mean + standard deviation (n=3)
gm % (w/v)
Table 2: Effect of wood saw dust (WSD) fungal enzyme
and chemical treatment for saccharification for bio-energy
Values are presented as mean + standard deviation (n=3)
01357 17 21
Time Interval (hour)
Hydrolysis of Enzyme (mg/ml)
Hydrolysis with acid (mg/ml)
Enzym eSulfuric acid
Fig1: Effect of enzyme and sulfuric acid on hydrolysis of
wood saw at dust different time interval.
Reducing sugar quantification by DNS method
One gm of 3, 5 Dinitro salicylic acid (DNS) was mixed
with 20 ml of 2 N NaOH. Thirty gm of sodium potassium
tartrate was added and volume was made up to 100 ml.
Substrate (0.4 ml) was taken in a fresh tube and 0.1 ml of
enzyme was added into it, then 1 ml of 3, 5 DNS was mixed in
the solution and kept in boiling water bath for 10 min. The
samples were with drawn and cooled under running tap water.
Ten ml of distilled water was added and reading was taken at
546 nm (Jurcoane et al., 2009).The amount of reducing sugar
was determination as per method described by Sadasivam
and Manickam (1996).
Results and Discussion
Enzyme hydrolysis from several fungal strains was tested.
It was found that the values of the reducing sugars obtained
from the WSD are shown in Table 1. T. viride produced
enzymes showed lowest value (0.022±0.002 g/l) for
hydrolysis as well as a saccharification and maximum
saccharification was observed (0.119±0.136 g/l) with A. wentii
generated microbial enzyme.
Treatment with 1 N H2SO4 after A. fumigatus extracellular
enzymatic hydrolysis showed higher value (0.99±0.001g/l).
It increases 24% more than enzymatic saccharification. Most
lignocellulosic wastes, due to the presence of cellulose
crystallinity, the chemical attack on the cellulose is retarded
(Mosier et al., 2002). Therefore, chemical pretreatment was
necessary to increase the susceptibility of lignocellulose for
hydrolysis reaction. Chemical treatment may accelerate the
rate of reaction and the extent of cellulose hydrolysis
(Najafpour et al., 2007).
By comparison of enzyme and chemical hydrolysis, it was
found that autoclaved enzyme treatment followed by
sulphuric acid hydrolysis resulted in maximum saccharifica-
tion (5.52±0.05 g/l) in Table 2. It was approximate increase of
5% than unautoclaved but sodium hydroxide showed no
significant effect for saccharification in horticulture waste.
Earlier (Nzelibe et al., 2007) also reported that sulfuric acid
hydrolysis was better than alkaline hydrolysis. Perhaps WSD
waste might have high cellulose and hemicellulose contents
and low lignin content. Enzyme is placed beneath the network
of lignin and hemicellulose components. Pretreatment or
hydrolysis with sulphuric acid might have removed and
hydrolysed hemicellulose to their monomeric constituent
and lignin hemicellulose cellulose interactions partially
disrupted. Compared to acid hydrolysis 11.0±0.75 g/l was
found better than enzyme hydrolysis (2.20±0.08 g/l) in Fig.1.
This showed acid hydrolysis significantly (P<0.01) enhanced
saccharification of saw dust waste. Increasing their
concentration (1, 3 and 5 N) sulfuric acid lowered hydrolysis
(7.7±0.1 g/l) at unautoclaved condition but maximum
hydrolysis was found same concentration (1 N sulfuric acid)
at autoclaved condition (23.4375±0.2 g/l) and 5 N sulfuric
acid does not shows any significant result for hydrolysis
compared to low acid concentration (1N and 3 N). As clearly
stated by the numbers, the sugar concentration was
increased with an increase in the acid concentration that
was applicable to the acid, catalyzed the hydrolysis process.
The catalyst activity was proportional to H+ concentration.
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31 GERF Bulletin of Biosciences 2011, 2(2):29-31
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The more hydrogen ions formed in the solution, the more
rapid the hydrolysis process occurred (Mosier et al., 2002).
Aonla pomace was used as strong hydrolyser because it
was acidic in pH (>2) which help saccharification of wood.
The WSD hydrolyzed with extracellular enzyme, dilute
sulfuric acid (1 N) and aonla pomace waste as hydrolyser
produced sugars, 3.28, 23.11 and 2.61 g/l, respectively. It’s
showed 11.29% hydrolysis compared to dilute sulfuric acid.
This study revealed that WSD was hydrolyzed at 1.69 g/
l, using a A fumigatus extracted crude culture filtrate at pH
5.0, 30 ºC in acetate buffer 50 mM, while when using 1 N
sulfuric acid at a temperature of 121ºC for 20 min, was 23.3 g/
l but in 5 N there was no significant effect. This study also
suggested that aonla pomace waste could be used as