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KHALISANNI BIN KHALID

1.0 TITLE OF THE RESEARCH

Isolation and Characterisation of Lignocellulolytic Enzymes from Fermented Rice Straw

2.0 INTRODUCTION

Enzymes are among the most important products obtained for human needs through microbial

sources. A large number of industrial processes in the areas of industrial, environmental and

food biotechnology utilize enzymes at some stage or the other. Current developments in

biotechnology are yielding new applications for enzymes. Solid state fermentation (SSF) holds

tremendous potential for the production of enzymes. SSF is the process of cultivating

microorganisms, usually fungi, on solid substrates in the absence of free water. It has emerged

as a potential technology for the production of microbial products such as feed, fuel, food,

industrial chemicals and pharmaceutical products. Its application in bioprocesses such as

bioleaching, biobeneficiation, bioremediation, biopulping and others has offered several

advantages. Utilisation of agro-industrial residues as substrates in SSF processes provides an

alternative avenue and value-addition to these otherwise under- or non-utilised residues. Crop

residues which are mainly lignocellulosic will be very suitable substrates for this fermentation.

White-rot fungi, such as Pleurotus sajor caju, are normally employed because they possess

lignocellulose degrading properties. Lignocellulose degradation during fermentation is

mediated by extracellular enzymes comprising lignin modifying enzymes (LME) and a variety

of other exoenzymes. Ruminants can utilise cellulose and hemicellulose as energy sources but

access to these polysaccharides can be limited by lignin. SSF therefore affords the possibility

of improving the access to them by utilising the lignocellulolytic activity of white-rot fungi. In

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addition, the SSF of crop residues can yield a variety of enzymes of agricultural and industrial

importance.

3.0 PROBLEM STATEMENTS

Crop residues such as rice straw, rice husks and palm press fibre are abundant in Malaysia but

remain relatively unutilised. High in lignocellulose, i.e.: cellulose (30-35%), hemicellulose

(20-35%), lignin (10-25%), they have an unrealised potential as animal feeds if the

lignocellulose can be effectively degraded. There were two major methods of lignocellulose

degradation, either by chemical reaction or enzymatic degradation. It is well known that some

microorganism has degrading properties as their survival techniques in poor-nutrient

conditions, in which that they will secrete enzymes to get nutrient supplement. These

lignocellulolytic enzymes can also function as degrading agent for several xenobiotic

compounds, as well as anti-oxidant agent. Biological degradation of lignocellulose by white-

rot fungi using the technique of SSF is a promising but poorly researched biotechnology in

Malaysia

4.0 GOAL OF THE RESEARCH

The objective of the study is to produce value-added products, enzymes and animal feeds from

the solid-state fermentation of rice straw by the white-rot fungus, Pleurotus sajor-caju. The

spent fermented waste will be analysed for its nutritional value as animal feed. The

extracellular fungal enzymes will be extracted from the spent fermented waste and assayed for

cellulase, xylanase and laccase activity. The crude extract that shows highest activity for any

of the three enzymes will be used for enzyme isolation. The isolated enzyme will then be

concentrated, purified and tested for its lignocellulolytic activity against commercial substrate

that imitate real crop residues and industrial waste-water in lab-scale system. The research can

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potentially generate commercially viable products from the presently unutilised and abundant

crop residues.

5.0 OBJECTIVES OF THE RESEARCH

The objectives of the research are:

1. To assay for lignocellulolytic enzymes activity from rice crop residues fermented with

non-toxic fungi, Pleurotus sajor-caju.

2. To isolate, purify and characterise the enzyme from the crude extract.

3. To estimate the nutritional value of the fermentation spent waste.

6.0 SCOPE OF THE RESEARCH

1. To set up a lab-scale solid state fermentation on Malaysian rice crop residues to produce

a viable, value added product.

2. To analyse the fermented rice straw nutritional value for conversion into animal feed.

7.0 LIMIT OF THE RESEARCH

1. The solid state will be carried out for rice straw botanical fractions of the selected

varieties that show best enzyme activity and nutritional value.

2. Downstream processing will only involved the enzyme extraction, assays, purification

and characterisation.

3. Analysis on nutritional properties of rice straw will estimate the difference and

improvement of nutritional value before and after fermentation.

8.0 RESEARCH METHODOLOGY / DESIGN

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1. Fermentation by Pleurotus sajor caju will be conducted for 7, 14 and 21 days in the dark

at optimal temperatures using the rice straw fractions (leaf, stem and sheath) as

substrates.

2. The fermented spent waste will be analysed for its chemical composition and in vitro

digestibility before the extraction of extracellular fungal enzymes.

3. Enzymes will be extracted from the spent waste with deionised water in an orbital

shaker at 3000 rpm for 24 hours in the ratio (w/v) of 1: 4. The supernatant will be

recovered by centrifugation at 7000 rpm for 10 minutes followed by filtration.

4. Cellulase, xylanase and laccase will be assayed by standard spectrophotometric methods

(König et al., 2002; Ride, 1980) using cellulose, oat spelts xylan and syringaldazine

(Sigma-Aldrich) respectively as substrate.

5. Enzyme with the highest activity will be isolated from the crude extract followed by

enzyme purification and physical properties characterisation.

9.0 LITERATURE REVIEW

Solid-state fermentation is the process of cultivating microorganisms, usually fungi, on solid

substrates in the absence of free water. In fermenting lignocellulolytic crop residues, the

primary objective is to enrich the residue with fungal biomass and generate a product with an

improved digestibility. Improving also the protein content is dependent on the substrate,

source and level of nitrogen supplementation, high levels of inorganic sources generally

suppressing lignin degradation (Villas-Boas et al., 2002) whereas low levels of organic

sources showed no adverse effect (Vadiveloo, 2003).

Lignocellulose degradation during fermentation is mediated by extracellular enzymes directly

involved in the degradation of lignin (lignin modifying enzymes, LME) and auxiliary enzymes

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which themselves are unable to degrade lignin but are involved in feedback circuits which link

ligninolysis with cellulose and hemicellulose degradation. The auxiliary enzymes include

glyoxal oxidase (GLOX) and superoxide dismutase for intracellular production of H2O2, a co-

substrate in LME, as well as glucose oxidase, aryl alcohol oxidase and cellobiose

dehydrogenase (Leonowicz et al., 1999).

The main LME are oxidoreductases, two types of peroxidases, manganese peroxidase (MnP,

E.C. 1.11.1.13) and lignin peroxidase (LiP, E.C. 1.11.1.14) and a phenol oxidase, laccase (Lac,

E.C. 1.10.3.2). These LME are produced in multiple isoforms during secondary metabolism,

often induced by limited nutrient levels of C and N. A third group of peroxidases, versatile

peroxidase, have been described in species of Pleurotus and Bjerkandera and these can

oxidise both Mn2+ as well as phenolic and non-phenolic aromatic compounds (Heinfling et al.,

1998). Laccases, produced by almost all white-rot fungi, oxidise and decarboxylate phenolic

and methoxy-phenolic acids (Wesenberg et al., 2003). Apart from the LME and auxiliary

enzymes, a variety of other exoenzymes, cellulases, xylanases, amylases, glucoamylases,

lipases, proteases and pectinases are secreted to facilitate fermentation (Cohen and Hadar,

2001).

Fungus-substrate specificity demonstrated during the fungal fermentation of three fibrous

substrates, sago fibre, rice straw and sawdust supplemented with organic or inorganic sources

of nitrogen (Vadiveloo, 2003) suggests variation in the complex of enzymes secreted by the

fungus to degrade lignocellulose. The activity of lignocellulose enzymes in aqueous extracts of

the spent waste can be estimated by continuous spectrophotometry with a variety of substrates.

These are well documented for laccase (Ride, 1980), cellulase and xylanase (König et al.,

2002).

Biological systems can be environmentally mild and have the potential to totally degrade the

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pollutants. Xenobiotic degradation by the lignin degrading system of the white rot fungi has

several advantages compared to bacterial processes. For instance, the process is unspecific,

able to degrade mixtures of chemicals, which often occur at farms where several pesticides are

used. Most of the studies on degradation of organic pollutant compounds by white rot fungi

have been carried out using liquid cultures in the laboratory. However, the use of such cultures

on a larger scale has several limitations. They are expensive, sensitive and difficult to manage,

always requiring qualified personnel making them unsuitable for application on farms. An

interesting alternative is the use of solid substrate fermentation (SSF), which mainly uses

lignocellulose as substrate/support material for the fungi (Castillo, 1997).

10.0 SIGNIFICANCE AND BENEFITS OF THE RESEARCH

Open up possibilities for commercial production of enzyme mixtures and conversion of crop

residues into animal feeds.

11.0 LIST OF REFERENCES

1. Castillo M.d.P. (1997). Degradation of pesicides by Phanerochaete chrysosporium in

solid substrate fermentation. Doctor's dissertation, Swedish University of Agricultural

Sciences, Sweden.

2. Cohen, R and Hadar, Y (2001). The roles of fungi in agricultural waste conversion. In:

'Fungi in bioremediation'.G M Gadd (Editor). Cambridge University Press, pp 305-334

3. Heinfling, A, Ruiz-Duenas, F J, Martinez, M J, Bergbauer, H, Szewzyk, U and

Martinez, A T (1998). A study on reducing substrates of manganese oxidising

peroxidases from Pleurotus eryngii and Bjerkandera adusta. FEBS Lett, 428 : 141-146

4. König, J, Grasser, R, Pikor, H and Vogel, K (2002). Determination of xylanase, β

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-glucanase, and cellulase activity, Anal Bioanal Chem. 374 : 80-87

5. Leonowicz, A, Matuszewska, A, Luterek, J, Ziegenhagen, D, Wojtas-Wasilewska, Cho,

N-S, Hofrichter, M and Rogalski, J (1999). Biodegradation by white-rot fungi. Fungal

Genet Biol, 27 : 175-185

6. Ride, J. P. (1980). The effect of induced lignification on the resistance of wheat cell

walls to fungal degradation. Physio Plant Path, 16 : 187-196

7. Vadiveloo, J (2003). Solid-state fermentation of fibrous residues. J Anim Feed Sci, 12 :

665-676

8. Villas-Boas, S G, Esposito, E and Mitchell, D A (2002). Microbial conversion of

lignocellulolytic residues for production of animal feeds. Anim Feed Sci Technol, 98:

1-12

9. Wesenberg, D, Kyriakides, I and Agathos, S N (2003). White-rot fungi and their

enzymes for the treatment of industrial dye effluents. Biotechnol Adv, 22 : 161-187

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