GERF Bulletin of Biosciences December 2011, 2(2):29-31

Abstract

Introduction

*Corresponding author: dev.biochem@gmail.com

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Short Communication

Copyright © 2011 Green Earth Research Foundation

Hydrolysis of wood saw dust by combined chemical pretreatment andenzymatic methods for lignocellulosic saccharification

Devendra Kumar1*, Kaushlesh K. Yadav2 and Munna Singh1

1Department of Botany, Lucknow University, Lucknow-226007, India2Department of Biotechnology, Dr. Ram Manohar Lohia Avadh University, Faizabad- 224001, India

Keyword: Wood saw dust (WSD), Aspergillus fumigatus, Acid hydrolysis, Enzymatic hydrolysis. Cellulose, Hemi-cellulose.

Wood saw dust (WSD) after lignocellulosic saccharification by different hydrolysis methods is more efficient for ethanolproduction as, its contains cellulose and hemi-cellulose at higher levels 65% (w/v) and 35% (w/v), respectively. Celluloseand hemicellulose account for about a quarter of whole biomass in all land plants. A pretreatment method using chemicalhydrolysis and enzymatic conversion from starch into fermentable sugars was investigated. The WSD was hydrolyzed at1.69 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 acidicnature. Optimum conditions for lignocellulosic saccharification is discussed in this paper.

High lignocellulosic agri-horticulture biomasses areemployed as alternative bio-energy (resource) to fossilenergy sources via lignocellulosic saccharification (Vintilaet al., 2010). Two processes used to convert cellulose andhemicellulose into biofuel (ethanol) are enzymatic and acidhydrolysis (Akin-Osanaiye et al., 2005; Chandel et al., 2007;Jurcoane et al., 2009; Karmakar et al., 2011). The mostcommonly adopted technique is acid hydrolysis (Badger etal., 2002). Acidic hydrolysis is an effective method used forlignocelluloses raw material pretreatment in saccharificationwhich could change into ethanol. Although acids arepowerful agents used for biomass hydrolysis, concentratedacids are toxic, erosive and hazardous. Handling higherconcentrations of acid requires reactors that are resistant toerosion in raw material pretreatment. Diluted acid hydrolysisespecially sulfuric acid has been successfully developed forpretreatment of cellulosic materials.

Another method of hydrolysis is enzymatic hydrolysis.Enzymes are naturally occurring plant proteins that result incertain 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, baseand enzymatic methods in hydrolysis of saw dust isinvestigated.

The WSD was collected in saw dust from Kakori industrialarea. The samples were brought to room temperature washedwith 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 atroom temperature treatment for 24 hr. The hydrolysates wereseparated to obtain any suspended or unhydrolysatedmaterials and was neutralized by 2 N NaOH and 1 N H2SO4solution for analytical processing then autoclaved at 121­ºCand 15 lbs pressure for 15 min (Nat Steel Equipment Pvt. Ltd,India).

Enzymatic hydrolysis

The WSD 25% (w/v) were hydrolyzed with various fungalenzymes from (106 spores) of Aspergillus fumigatus,Rhizopus, Trichoderma viridae and Aspergillus wenti) withextra cellular enzymes (i.e. α-amylase, glucoamylase,cellulase and pectinase) were used in the experiment. Thehydrolyate was separated by centrifugation at 12,000 rpm atroom temperature.

Materials and Methods

Sample collection

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GERF Bulletin of Biosciences 2011, 2(2):29-31 30

Table1: Effect of wood saw dust (WSD) fungal enzymeand chemical treatment for saccharification.

Hydrolysis of wood saw dust waste (WSD)

Sugar gm%

Aspergillus fumigatus Rhizopus Trichoderma viride Aspergillus wenti Aspergillus fumigatus+ HCl (1N) Rhizopus+ HCl (1N) Trichoderma viride+ HCl (1N) Aspergillus wenti+ HCl (1N)

0.024±0.001 0.026±0.005 0.022±0.002 0.119±0.136 0.990±0.001 0.893±0.001 0.025±0.002 0.029±0.003

Values are presented as mean + standard deviation (n=3)

Enzymatic and Chemical treatment

Autoclaved gm % (w/v)

Un-Autoclaved gm% (w/v)

Control Aspergillus fumigatus NaOH (1N) H2SO4(1N) HCl(1N)

0.60±0.05 1.69±0.01 0.57±0.02 5.52±0.05 4.67±0.08

0.07±0.03 1.32±0.04 0.072±0.02 1.1±0.02 0.86±0.01

Table 2: Effect of wood saw dust (WSD) fungal enzymeand chemical treatment for saccharification for bio-energyproduction

Values are presented as mean + standard deviation (n=3)

0.03 0.05 0.13

0.41

0.72

1.72

2.20

0.02 0.251.30

4.075.09

7.00

11.00

0.00

0.50

1.00

1.50

2.00

2.50

0 1 3 5 7 17 21

Tim e Interval (hour)

Hyd

roly

sis

of E

nzym

e (m

g/m

l)

-2.00

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Hyd

roly

sis

with

aci

d (m

g/m

l)

Enzym e Su lfuric acid

Fig1: Effect of enzyme and sulfuric acid on hydrolysis ofwood saw at dust different time interval.

Reducing sugar quantification by DNS method

One gm of 3, 5 Dinitro salicylic acid (DNS) was mixedwith 20 ml of 2 N NaOH. Thirty gm of sodium potassiumtartrate was added and volume was made up to 100 ml.Substrate (0.4 ml) was taken in a fresh tube and 0.1 ml ofenzyme was added into it, then 1 ml of 3, 5 DNS was mixed inthe solution and kept in boiling water bath for 10 min. Thesamples were with drawn and cooled under running tap water.Ten ml of distilled water was added and reading was taken at546 nm (Jurcoane et al., 2009).The amount of reducing sugarwas determination as per method described by Sadasivamand Manickam (1996).

Results and Discussion

Enzyme hydrolysis from several fungal strains was tested.It was found that the values of the reducing sugars obtainedfrom the WSD are shown in Table 1. T. viride producedenzymes showed lowest value (0.022±0.002 g/l) forhydrolysis as well as a saccharification and maximumsaccharification was observed (0.119±0.136 g/l) with A. wentiigenerated microbial enzyme.

Treatment with 1 N H2SO4 after A. fumigatus extracellularenzymatic hydrolysis showed higher value (0.99±0.001g/l).It increases 24% more than enzymatic saccharification. Mostlignocellulosic wastes, due to the presence of cellulosecrystallinity, the chemical attack on the cellulose is retarded(Mosier et al., 2002). Therefore, chemical pretreatment wasnecessary to increase the susceptibility of lignocellulose forhydrolysis reaction. Chemical treatment may accelerate therate of reaction and the extent of cellulose hydrolysis(Najafpour et al., 2007).

By comparison of enzyme and chemical hydrolysis, it wasfound that autoclaved enzyme treatment followed bysulphuric acid hydrolysis resulted in maximum saccharifica-tion (5.52±0.05 g/l) in Table 2. It was approximate increase of5% than unautoclaved but sodium hydroxide showed no

significant effect for saccharification in horticulture waste.Earlier (Nzelibe et al., 2007) also reported that sulfuric acidhydrolysis was better than alkaline hydrolysis. Perhaps WSDwaste might have high cellulose and hemicellulose contentsand low lignin content. Enzyme is placed beneath the networkof lignin and hemicellulose components. Pretreatment orhydrolysis with sulphuric acid might have removed andhydrolysed hemicellulose to their monomeric constituentand lignin hemicellulose cellulose interactions partiallydisrupted. Compared to acid hydrolysis 11.0±0.75 g/l wasfound better than enzyme hydrolysis (2.20±0.08 g/l) in Fig.1.This showed acid hydrolysis significantly (P<0.01) enhancedsaccharification of saw dust waste. Increasing theirconcentration (1, 3 and 5 N) sulfuric acid lowered hydrolysis(7.7±0.1 g/l) at unautoclaved condition but maximumhydrolysis was found same concentration (1 N sulfuric acid)at autoclaved condition (23.4375±0.2 g/l) and 5 N sulfuricacid does not shows any significant result for hydrolysiscompared to low acid concentration (1N and 3 N). As clearlystated by the numbers, the sugar concentration wasincreased with an increase in the acid concentration thatwas 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

Reference

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The more hydrogen ions formed in the solution, the morerapid the hydrolysis process occurred (Mosier et al., 2002).Aonla pomace was used as strong hydrolyser because itwas acidic in pH (>2) which help saccharification of wood.The WSD hydrolyzed with extracellular enzyme, dilutesulfuric acid (1 N) and aonla pomace waste as hydrolyserproduced sugars, 3.28, 23.11 and 2.61 g/l, respectively. It’sshowed 11.29% hydrolysis compared to dilute sulfuric acid.

Conclusion

This study revealed that WSD was hydrolyzed at 1.69 g/l, using a A fumigatus extracted crude culture filtrate at pH5.0, 30 ºC in acetate buffer 50 mM, while when using 1 Nsulfuric 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 alsosuggested that aonla pomace waste could be used ashydrolyser.