1. GERF Bulletin of Biosciences
December 2011, 2(2):29-31
Short Communication
Hydrolysis of wood saw dust by combined chemical pretreatment and
enzymatic methods for lignocellulosic saccharification
Devendra Kumar1*, Kaushlesh K. Yadav2 and Munna Singh1
1
Department of Botany, Lucknow University, Lucknow-226007, India
2
Department of Biotechnology, Dr. Ram Manohar Lohia Avadh University, Faizabad- 224001, India
Abstract
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.
Keyword: Wood saw dust (WSD), Aspergillus fumigatus, Acid hydrolysis, Enzymatic hydrolysis. Cellulose, Hemi-
cellulose.
Introduction
Materials and Methods
High lignocellulosic agri-horticulture biomasses are
employed as alternative bio-energy (resource) to fossil Sample collection
energy sources via lignocellulosic saccharification (Vintila
et al., 2010). Two processes used to convert cellulose and The WSD was collected in saw dust from Kakori industrial
hemicellulose into biofuel (ethanol) are enzymatic and acid area. The samples were brought to room temperature washed
hydrolysis (Akin-Osanaiye et al., 2005; Chandel et al., 2007; with distilled water and used in the experiment.
Jurcoane et al., 2009; Karmakar et al., 2011). The most
commonly adopted technique is acid hydrolysis (Badger et Acid -base hydrolysis
al., 2002). Acidic hydrolysis is an effective method used for
lignocelluloses raw material pretreatment in saccharification The WSD 25% (w/v) was hydrolyzed with 100 ml (1:2 w/
which could change into ethanol. Although acids are v) of of various concentrations of H2SO4, HCl and NaOH at
powerful agents used for biomass hydrolysis, concentrated room temperature treatment for 24 hr. The hydrolysates were
acids are toxic, erosive and hazardous. Handling higher separated to obtain any suspended or unhydrolysated
concentrations of acid requires reactors that are resistant to materials and was neutralized by 2 N NaOH and 1 N H2SO4
erosion in raw material pretreatment. Diluted acid hydrolysis solution for analytical processing then autoclaved at 121­ºC
especially sulfuric acid has been successfully developed for and 15 lbs pressure for 15 min (Nat Steel Equipment Pvt. Ltd,
pretreatment of cellulosic materials. India).
Another method of hydrolysis is enzymatic hydrolysis. Enzymatic hydrolysis
Enzymes are naturally occurring plant proteins that result in
The WSD 25% (w/v) were hydrolyzed with various fungal
certain chemical reaction. However, for enzymes to work,
enzymes from (10 6 spores) of Aspergillus fumigatus,
they must obtain access to the molecules to be hydrolyzed
Rhizopus, Trichoderma viridae and Aspergillus wenti) with
(Baig et al., 2004). A combined strategy involving acid, base
and enzymatic methods in hydrolysis of saw dust is extra cellular enzymes (i.e. α-amylase, glucoamylase,
investigated. cellulase and pectinase) were used in the experiment. The
hydrolyate was separated by centrifugation at 12,000 rpm at
room temperature.
*Corresponding author: dev.biochem@gmail.com
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2. GERF Bulletin of Biosciences 2011, 2(2):29-31 30
Reducing sugar quantification by DNS method
Table 2: Effect of wood saw dust (WSD) fungal enzyme
One gm of 3, 5 Dinitro salicylic acid (DNS) was mixed and chemical treatment for saccharification for bio-energy
with 20 ml of 2 N NaOH. Thirty gm of sodium potassium production
tartrate was added and volume was made up to 100 ml.
Enzymatic and Autoclaved Un-Autoclaved
Substrate (0.4 ml) was taken in a fresh tube and 0.1 ml of gm % (w/v) gm% (w/v)
Chemical treatment
enzyme was added into it, then 1 ml of 3, 5 DNS was mixed in Control 0.60±0.05 0.07±0.03
the solution and kept in boiling water bath for 10 min. The Aspergillus fumigatus 1.69±0.01 1.32±0.04
samples were with drawn and cooled under running tap water. NaOH (1N) 0.57±0.02 0.072±0.02
Ten ml of distilled water was added and reading was taken at H2SO4(1N) 5.52±0.05 1.1±0.02
546 nm (Jurcoane et al., 2009).The amount of reducing sugar HCl(1N) 4.67±0.08 0.86±0.01
was determination as per method described by Sadasivam
and Manickam (1996). Values are presented as mean + standard deviation (n=3)
Results and Discussion
2 .5 0 1 4 .0 0
Enzyme hydrolysis from several fungal strains was tested.
Hydrolysis of Enzyme (mg/ml)
2 .2 01 2 .0 0
Hydrolysis with acid (mg/ml)
2 .0 0 1 1 .0 0
It was found that the values of the reducing sugars obtained 1 0 .0 0
1 .7 2
from the WSD are shown in Table 1. T. viride produced 1 .5 0 8 .0 0
7 .0 0
enzymes showed lowest value (0.022±0.002 g/l) for 6 .0 0
5 .0 9
hydrolysis as well as a saccharification and maximum 1 .0 0 4 .0 7 4 .0 0
saccharification was observed (0.119±0.136 g/l) with A. wentii 0 .7 2
2 .0 0
generated microbial enzyme. 0 .5 0 1 .3 0
0 .2 5 0 .4 1
0 .02 0 .0 0
0 .1 3
Treatment with 1 N H2SO4 after A. fumigatus extracellular 0 .0 0 0 .03 0 .0 5 -2 .0 0
0 1 3 5 7 17 21
enzymatic hydrolysis showed higher value (0.99±0.001g/l).
Tim e Inter va l (hour)
It increases 24% more than enzymatic saccharification. Most
En zym e Su lfu ric a cid
lignocellulosic wastes, due to the presence of cellulose
crystallinity, the chemical attack on the cellulose is retarded Fig1: Effect of enzyme and sulfuric acid on hydrolysis of
(Mosier et al., 2002). Therefore, chemical pretreatment was wood saw at dust different time interval.
necessary to increase the susceptibility of lignocellulose for
hydrolysis reaction. Chemical treatment may accelerate the significant effect for saccharification in horticulture waste.
rate of reaction and the extent of cellulose hydrolysis Earlier (Nzelibe et al., 2007) also reported that sulfuric acid
(Najafpour et al., 2007). hydrolysis was better than alkaline hydrolysis. Perhaps WSD
waste might have high cellulose and hemicellulose contents
Table1: Effect of wood saw dust (WSD) fungal enzyme and low lignin content. Enzyme is placed beneath the network
and chemical treatment for saccharification. of lignin and hemicellulose components. Pretreatment or
hydrolysis with sulphuric acid might have removed and
Hydrolysis of wood saw dust Sugar gm% hydrolysed hemicellulose to their monomeric constituent
waste (WSD) and lignin hemicellulose cellulose interactions partially
Aspergillus fumigatus 0.024±0.001 disrupted. Compared to acid hydrolysis 11.0±0.75 g/l was
Rhizopus 0.026±0.005 found better than enzyme hydrolysis (2.20±0.08 g/l) in Fig.1.
Trichoderma viride 0.022±0.002 This showed acid hydrolysis significantly (P<0.01) enhanced
Aspergillus wenti 0.119±0.136 saccharification of saw dust waste. Increasing their
Aspergillus fumigatus+ HCl (1N) 0.990±0.001 concentration (1, 3 and 5 N) sulfuric acid lowered hydrolysis
Rhizopus+ HCl (1N) 0.893±0.001 (7.7±0.1 g/l) at unautoclaved condition but maximum
Trichoderma viride+ HCl (1N) 0.025±0.002 hydrolysis was found same concentration (1 N sulfuric acid)
Aspergillus wenti+ HCl (1N) 0.029±0.003 at autoclaved condition (23.4375±0.2 g/l) and 5 N sulfuric
Values are presented as mean + standard deviation (n=3) acid does not shows any significant result for hydrolysis
By comparison of enzyme and chemical hydrolysis, it was compared to low acid concentration (1N and 3 N). As clearly
found that autoclaved enzyme treatment followed by stated by the numbers, the sugar concentration was
sulphuric acid hydrolysis resulted in maximum saccharifica- increased with an increase in the acid concentration that
tion (5.52±0.05 g/l) in Table 2. It was approximate increase of was applicable to the acid, catalyzed the hydrolysis process.
5% than unautoclaved but sodium hydroxide showed no The catalyst activity was proportional to H+ concentration.
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3. 31 GERF Bulletin of Biosciences 2011, 2(2):29-31
The more hydrogen ions formed in the solution, the more hydrolysis of pretreated palm oil lignocellulosic
rapid the hydrolysis process occurred (Mosier et al., 2002). wastes. IJE Transactions. 20(2): 147-156.
Aonla pomace was used as strong hydrolyser because it 9. Nzelibe HC and Okafoagu CU (2007). Optimization
was acidic in pH (>2) which help saccharification of wood. of ethanol production from Garcinia kola (bitter
The WSD hydrolyzed with extracellular enzyme, dilute kola) pulp agrowaste. Afr. J. Biotechnol. 6(17):
sulfuric acid (1 N) and aonla pomace waste as hydrolyser 2033-2037.
produced sugars, 3.28, 23.11 and 2.61 g/l, respectively. It’s 10. Sadasivam S and Manickam A (1996). Biochemical
showed 11.29% hydrolysis compared to dilute sulfuric acid. Methods, New Age. International Publishers (P)
Ltd., New Delhi, India.
Conclusion 11. Vintila T, Dragomirescu M, Croitoriu V, Vintila C,
Barbu H and Sand C (2010 ). Saccharification of
This study revealed that WSD was hydrolyzed at 1.69 g/ lignocellulose using different cellulases. Romanian
l, using a A fumigatus extracted crude culture filtrate at pH Biotechnol. Lett. 15(4): 5498-5504.
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
hydrolyser.
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