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Product Code: 00008056

No of Pages: 48

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Bioconversion of waste materials into useful products is very topical not only to protect natural resources, but also to limit environmental pollution. In this study, waste paper was enzymatically hydrolysed using crude enzyme from Trichoderma viridae for alcohol and biomass production. The T. viridae was maintained on sabouraud Dextrose Agar (SDA) slants. Spores from fully grown slants were dislodged into sterile saline containing 0.05% (v/v) Tween 80. The spores suspension was transferred into an enriched medium with carboxymethyl cellulose (CMC) as sole carbon source to induce cellulase synthesis. After seven days of fermentation, the filtrate of the medium was centrifuged and the obtained supernatant was used as the crude enzyme source. The waste paper was ground into powder and pretreated at 1210C for 15 minutes. The crude enzyme was transferred to the pretreated paper and the mixture was incubated at 370C for 72 hours for hydrolysis to take place. The sugar content and the specific gravity of hydrolysate were 6.94% and 1.033g/cm3 respectively. The hydrolysate sugar content was optimized with 67g of sugar (sucrose) and the sugar content rose to 21.54% while the specific gravity rose to 1.114g/cm3. After 7 days of fermentation of the hydrolysate with Baker’s yeast, the sugar content and specific gravity dropped to 2.59% and 1.005g/cmrespectively while the pH reduced from 5.6 to 3.1. The temperature fluctuated between 290C and 300C with biomass production and alcohol yield of 1.99g/l and 89.0% respectively. Optimizing enzymatic hydrolysis parameters and proceeding to secondary distillation could give a better yield.



Title Page i

Certification ii

Dedication iii

Acknowledgements iv

Table of contents v

List of tables viii

List of figures ix

Abstract x


1.1 Introduction 1

1.2 Aims and Objectives 3


2.1 Constituents of paper 4

2.1.1 Cellulose 4

2.1.2 Hemicellulose 4

2.1.3 Lignin 5

2.2 Alcohol production from cellulosic materials 5

2.3 Pretreatment 7

2.3.1 Physical pretreatment 7 High pressure steaming 7 Hydrothermal pretreatment 8

2.3.2 Chemical Pretreatment 8 Dilute acid pretreatment 8 Hydrogen peroxide pretreatment 8 Lime pretreatment 9 Ammonia fiber explosion (AFEX) 9 Organosolv pretreatment 9

2.4 Enzymatic hydrolysis and Microbial Cellulases 10

2.5 Ethanol fermentation 11

2.6 Biomass 11


3.1 Materials 12

3.2 Methods 12

3.2.1 Processing of Waste paper 12

3.2.2 Pretreatment of waste paper 12

3.3 Media preparation and sterility test 12

3.4 Microorganism and inoculums preparation 13

3.5 Submerged fermentation for cellulase production 13

3.6 Enzymatic hydrolysis of pretreated waste paper 14

3.6.1 Sugar analysis 14

3.6.2 Measurement of soluble liquid 15

3.6.3 Optimization of sugar content 15

3.7 Alcohol fermentation 15

3.7.1 Determination of pH and temperature 16

3.7.2 Determination of Titratable Acidity 16

3.7.3 Determination of Biomass production 17

3.7.4 Alcohol content 17

3.8 Biomass separation 17

3.9 Distillation 18





5.1 Discussion 30

5.2 Conclusion 31

5.3 Recommendation 31




Tables Titles Pages


1 Physicochemical properties of the hydrolysate  before and after sugar optimization 20

2 Changes in the physicochemical properties of the hydrolysate during alcohol fermentation 21

3 Qualities of the produced ethanol 22












1 Cellulose Ethanol Production 6

2 Changes in sugar content during fermentation 23

3 Changes in Specific Gravity during fermentation 24

4 Changes in pH during fermentation 25

5 Changes in the alcohol content during fermentation 26

6 Changes in Biomass during fermentation 27

7 Changes in temperature during fermentation 28

8 Changes in Titratable Acidity during fermentation 29





1.1 Introduction

Waste as described by Babatola, (2008) is any material or substance lacking direct value to the user and so must be disposed off. Solid wastes are non-liquid and non-gaseous and consist of organic matter (biodegradable) and inorganic matter (non-biodegradable). Each day in different cities and towns, municipal solid wastes are continually being generated. This include food wastes, market wastes yard wastes plastic containers product packing materials and other miscellaneous solid waste from residential commercial and institutional sources. Waste was an early problem of mankind and a growing one that is of major concern to every nation of the world. The management of waste have proved to be a vital factor affecting the quality of the environment. According to Rixin, (2014), only about one-third of the municipal solid waste is recycled, leaving the remaining two-third to be disposed off in landfills or incinerated. These methods of municipal solid waste disposed are economically and ecologically expensive and inefficient. They create air, soil and water pollution as well as mountains of unprocessed waste and they fail to utilize materials that can be recycled into useful products.         

Waste paper constitutes a considerable share of municipal solid waste (Giada et al., 2010). Paper materials can be classified as multipurpose commodities. They provide a medium of presenting information, can act as protective agents, serve as packaging material, to name but a few of their uses. Used paper products are recycled to a limited extent, suggesting that a significant part of municipal solid waste is not useful as recycled products or cannot be recycled due to destruction after its use.

Ethanol is derived from renewable plant resources through a process known as alcoholic fermentation (Bibi and Aslam, 2014). It is used both as a disinfectant and an antiseptic in medical laboratories. Ethanol has wide spread use as solvent of substances intended for human contact including: perfumes, flavours, colouring, and medicines (Pranavya et al., 2015). It is also used in the manufacture of detergent, adhesives, pharmaceutical dyes, pesticides, resins and antifreeze agents. Ethanol can be blended with gasoline as an octane enhancer (10%-85%); used as a blend in flexible fuel vehicles (up to 85%) and used a gasoline substitute in a modified internal combustion engine (Prasad, 2014). Ethanol can be produced from starch or cellulose and hemicelluloses that originate from many sources of biomass These includes; sugar cane, corn, sugar beet, cassava, rice straw, switch grass, wheat, barley, molasses as well as agricultural waste and waste paper. Ethanol production involves two key processes, first the breaking down of starch or cellulose and hemicelluloses portion of the biomass using acid, base or enzymes into simple sugar such as glucose through a process called saccharification (hydrolysis of polysaccharide), second, fermentation of the simple sugars by microorganisms such as yeast to produce ethanol.

The enzymatic hydrolysis of cellulosic materials involves the use of the enzymes cellulase. Cellulases are collectively named as class of variable multienzymes complexes (Van Wyk, 2002). They are produced as soluble enzymes which are diffused from the cells, or are organized as cellulosomes. The soluble enzymes are (i) β-1,4-endoglucanase or endocellulase that clears internal-1,4-glycosidic bonds, (ii) cellobiohydrolase or exocellulase that removes cellobiose from non-reducing terminal of cellulose and (iii) β-glucosidase that hydrolyzes cellobiose to β-D-glucose. (Vynios et al., 2009). All the activities act synergistically.

Paper materials are principally composed of the compounds; cellulose, hemicelluloses and lignin (Van Wyk, 2002). The major constituent of paper product is cellulose a homopolysaccharide made up of long chains of glucose linked by β-1,4-glycosidic bonds. Waste paper therefore, can be utilized in production of ethanol as glucose can be produced after enzymatic hydrolysis of the cellulose by the use of cellulase (Vladimir et al., 2014). This results to conversion of waste into useful product and consequently reduces the total amount of municipal solid wastes.


This research is aimed at;

Ø producing alcohol (ethanol) and biomass using cheap substrate such as waste paper.

Ø  converting waste (waste paper) into useful product (alcohol).

Ø  finding an alternative way of waste (waste paper) disposal.

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