I= OPTIMIZATION OF CELLULASE SYNTHESIS BY LOCALLY ISOLATED TRICHODERMA SPECIES USING AGRICULTURAL BY-PRODUCTS AS SUBSTRATES
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Title of Thesis
OPTIMIZATION OF CELLULASE SYNTHESIS BY LOCALLY ISOLATED TRICHODERMA SPECIES USING AGRICULTURAL BY-PRODUCTS AS SUBSTRATES

Author(s)
Ikram-Ul-Haque
Institute/University/Department Details
University Of The Punjab
Session
1992
Subject
Botany
Number of Pages
230
Keywords (Extracted from title, table of contents and abstract of thesis)
cellulase synthesis, trichoderma species, trichoderma aspergilllus, penicillium, cellulose hydrolysis, acetate buffer, cellulose enzymes

Abstract
The present study is concerned with the optimization of the cultural conditions of locally isolated mould cultures for the production of cellulose enzymes by both surface culture and submerged culture methods in conical flasks and stirred fomenter. The mould cultures after their isolation and screening were identified and found to be the species of trichoderma, Aspergilllus, penicillium, Rhizohus. The cultures of trichoderma viride GCB-20, Aspergillus niger GCB-5 however were best producer of cellulases. The agricultural byproducts such as wheat bran, bagasse, rice straw and wheat straw etc. were evaluated as substrate by both mould culture for the production of enzymes. Of all the substrates tested, however, wheat, bran was found to be an ideal substrate providing all the nutrients for the synthesis of cellulases. The parameters in solid state fermentation such as depth of wheat bran and its partial replacement with other agricultural by-products such as wheat straw, rice straw or bagasse, moisture content i.e. ratio of wheat bran to water, selection of diluents, extraction of enzymes and rate of fermentation were optimized.

The porosity of wheat bran substrate after moistening which water (1 : 1) was more suitable for the maintenance of aerobic condition during fermentation. Further increase production. Effect of the concentration of cellulose powder on enzyme formation was also studied. The enzyme activity was maximum in the presence of 1.0% w/v cellulose powder, 144 hours after spore inoculation (10.0 unit/m). Further increase in cellulose concentration resulted in decreasing the production of cellulases. The crude cellulase enzyme was also used for the saccharifivation of filter paper. Paper pulp, cotton fiber and CMC. The percentage decomposition was maximum in the presence of CMC (60%) and it was decreased in the order, filter paper, paper pulp, cotton.

Effect of the concentration of cellulase on the hydrolysis of cmc (1.0%) was also investigated. The hydrolysis of cmc was increased with increasing of cellulase units in the form of reducing sugar (glucose), being 1.27 mg/ml for one enzyme unit. The data show near linearity, and this means that the increase in enzyme concentration gave a linear relationship with it‚€™s activity. Effect of CMC concentration on the rate of enzymic hydrolysis was also studied; it was found that increase of cmc concentration resulted in more reducing sugar up to 1.6%, beyond which no more sugar was liberated. The rate of cmc decomposition followed similar trend, reaching the maximum rate at 0. 8 ‚€“ 1.2% and then gradually decreased. This study indicates that the increase of cmc concentration more than 1.27 causes a relative decrease in substrate decomposition.

The delignified by solvent or alkali treatment was also examined for the production of cellulose degrading enzymes by Trichoderma viride GCB-20 in flasks. The amount of enzyme cellulase produced was greatly enhanced due to the removal of lignin material. The hydrolysis of the delignified, rice straw, wheat straw or bagasse by the enzyme celluloses was also investigated. The percentage decomposition was decreased in the order of bagasse, rice straw.

The production of cellulases in stirred fermenter (10 1) by Trichoderma viride GCB-20 using wheat bran and newspaper pulp was also investigated. Both the substrates were suspended in mineral salt solution with CMC. The rate of enzyme production was faster in comparison with shake flasks. The fermentation reached maximum 48 hrs after inoculation. That is , the better supply of oxygen in the fermenter was the reason for earlier the completion of the fermentation. The effect of the rated of aeration (250, 500, 1000 c .c /1/min.) on enzyme formation was also investigated. The production of enzyme was maximum when the rate of aeration was 1000 cc/1/min.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
159.82 KB
2 1 Introduction 1
171.51 KB
3 2 Review Of Literature 17
489.12 KB
  2.1 Chemistry Of Cellulose 19
  2.2 Chemistry Of Cellulase 23
  2.3 Microbial Sources Of Cellulases 27
  2.4 Production Of Cellulase 33
  2.5 Pretreatment 50
  2.6 Cellulose Hydrolysis 52
4 3 Materials And Methods 55
186.36 KB
  3.1 Isolation Of Cellulose Decomposing Fungi 55
  3.2 Classification And Identification Of Fungal Isolates 56
  3.3 Preparation Of Inoculum 56
  3.4 Fermentation Procedure 60
  3.5 Solid State Fermentation 60
  3.6 Preparation Of Enzyme Extract From Solid State Fermented Mash 60
  3.7 Submerged Fermentation 61
  3.8 Chemical Treatment 63
  3.9 Stirred Fermenter 65
  3.10 Sodium Citrate Buffer 65
  3.11 Acetate Buffer 65
  3.12 Standard Curve Of Glucose 66
  3.13 Assay 66
  3.14 Analysis 69
5 4 Results 71
1912.46 KB
  4.1 Isolation And Selection Of Cellulase Producing Mould Cultures 71
  4.2 Selection Of Substrate 74
  4.3 Rate Of Fermentation Of Cellulose Degrading Enzyme 77
  4.4 Effect Of The Ratio Of Wheat Bran To Diluent 79
  4.5 Selection Of Diluents 79
  4.6 Effect Of Depth On The Production Of Cellulolytic Enzymes 82
  4.7 Extraction Of Enzymes 84
  4.8 Effect Of Partial Replacement Of Wheat Bran With Other Agricultural By ‚€“Products 86
  4.9 Submerged Fermentation 90
  4.10 Production Of Cellulases By Aspergillus Niger Gcb-5 By Solid State Fermentation 97
  4.11 Effect Of Various Diluents Of Enzyme Synthesis By Aspergillus Niger 100
  4.12 Extraction Of Enzyme By Different Solutions 100
  4.13 Effect Of Partial Replacement Of Wheat Barn On Enzyme Synthesis 103
  4.14 Submerged Fermentation By A. Niger Gcb-5 108
  4.15 Selection Of Substrate 108
  4.16 Effect Of Different Concentration Of Wheat Bran In Mineral Salt Solution On The Production Of Cellulolytic Enzyme By A. Niger Gcb-5 110
  4.17 Effect Of Suspending Wheat Bran In Different Solution 112
  4.18 Rate Of Enzyme Synthesis Of A. Niger In Shake Flask 112
  4.19 Cellulase Production In Synthetic Basal Medium 116
  4.20 Effect Of Carbon Sources On Cellulase Induction 116
  4.21 Effect Of Cellulose Concentration On The Enzyme Production 119
  4.22 Effect Of Cellulase Preparation On Different Kinds Of Cellulose 119
  4.23 Effect Of Cellulase Concentration On Hydrolysis Of Cmc 120
  4.24 Effect Of Cmc Concentration On Enzymatic Velocity 124
  4.25 Production Of Cellulases By Trichoderma Viride Gcb-20 Using Delignified Bagasse 126
  4.26 Chemical Treatment 126
  4.27 Cellulase Production In Basal Medium Containing Treatment Bagasse 126
  4.28 Production Of Cellulases In The Stirred Fermenter 137
  4.29 Effect Of Aeration 140
6 5 Discussion 144
205.82 KB
7 6 References 159
749.61 KB