OPTIMIZING THE PRODUCTION OF BANANA PEEL PULP

USING CHEMICAL AND BIOLOGICAL METHODS

A MASTER’S THESIS
BY

AWAKE KEBEDE

DEPARTMENT OF ENVIRONMENTAL ENGINEERING

ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY

MAY 2019
OPTIMIZING THE PRODUCTION OF BANANA PEEL PULP USING

CHEMICAL AND BIOLOGICAL METHODS

By

AWAKE KEBEDE

A Thesis Submitted as a Partial Fulfillment for the Degree of Master of Science in

Environmental Engineering

to

DEPARTMENT OF ENVIRONMENTAL ENGINEERING

ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY

MAY 2019
DECLARATION
I hereby declare that this thesis entitled “Optimizing the Production of Banana Peel Pulp

Using Chemical and Biological Methods” was prepared by me, with the guidance of my

advisor. The work contained herein is my own except where explicitly stated otherwise in the

text, and that this work has not been submitted, in whole or in part, for any other degree or

professional qualification.

Author: Signature, Date:

Witnessed by:

Name of student advisor: Signature, Date:

ii
CERTIFICATE

This is to certify that the thesis prepared by Mr. Awake Kebede Tassew entitled “Optimizing

the Production of Banana Peel Pulp Using Chemical and Biological Methods” and submitted

as a partial fulfillment for the Degree of Master of Science complies with the regulations of the

University and meets the accepted standards with respect to originality, content and quality.

Singed by Examining Board:

External Examiner: Signature, Date:

Dr.Ing. Zebene Kiflie

Internal Examiner: Signature, Date:

Dr.Nuriligne Tefera

Chairperson: Signature, Date:

DGC Chairperson: Signature, Date:

College Dean/Associate Dean for GP: Signature, Date:

iii
ACKNOWLEDGMENT

First of all, I would like to thank the almighty God for giving me strength and patience
throughout my work. Moreover, it is my pleasure to appreciate and acknowledge the contribution
of some people, without whose support this research would have never come to an end. My
deepest gratitude goes to my advisor Mesfin Tafesse (Ph.D) for his constructive feedback,
valuable comments and continuous encouragements during the work.

It is also grateful to thank the Microbiology Department of Addis Ababa science and Technology
University for their permission to use the microbiology laboratory facilities, and all the workers
in that lab had been helping me while working with bio-bleaching analysis.

My special thanks go to Ethiopian pulp and paper factory Administration, Staff members and
laboratory assistants for allowing me to do experiments in their laboratory and for their Support
while doing laboratory experimental activities.
My gratefulness goes to all my families and my friends who were supporting and encouraging
me during my study.

iv
ABSTRACT
Wood is the primary raw material used to manufacture pulp and paper. But it causes
deforestation. So, choosing other alternatives like banana peel waste is the best solution,
because after using banana fruit the peel is useless and discarded. Therefore, it makes sense to
turn such waste into a useful product like pulp by using the concept “from waste to wealth’’.

In this thesis work, different laboratory experiments were run by varying the water content,
amount of peel and duration of drying of the solution to get the optimum point of production of
pulp from banana peel. The basic pulp characteristics were studied .As the duration of drying
time decreased, the moisture content decreased. But the strength of the pulp was increased due
to moisture. The average lignin content and cellulose content of banana peel sample were 10.45
% and 9.072 % respectively. In the first four consecutive experiments, the basic pulp
characteristics were undetectable. Surface sizing starch works as a binder, water holding agent,
and carrier for surface sizing chemicals and other functional additives. Experiments where
surface sizing starch used were better in terms of improved pulp characteristics .In the last three
experiments, the fiber freeness of the product was greater than 25 mL, which was best result. The
ash content of the pulp decreased as the time of drying and peels to water ratio decreased. In the
first four experiments ash content values were >10%. Burst strength and tensile strength value of
the pulp were improved.

The Second part of this thesis work emphasized on the utilization of crude cellulose enzyme
originally isolated from termite gut in pulp and paper processing. It was observed that highest
performance of enzyme activity was found to be at a 40 0C from the selected temperature ranges
(40 0C -70 0C). Similarly, the maximum enzyme activity was observed at a 6.0 pH followed by 7.0
pH.
Both chemical pulping method and bio bleaching method were applied. More bleached pulp was
observed by chemical method, while bio bleaching method was preferred in protecting the
environment from pollution.

Key words: Banana fibers

Bio-bleaching

v
 Table of contents
DECLARATION ............................................................................................................................ ii
CERTIFICATE .............................................................................................................................. iii
ACKNOWLEDGMENT................................................................................................................ iv
ABSTRACT .................................................................................................................................... v
LIST OF TABLES ......................................................................................................................... ix
LIST OF FIGURES ........................................................................................................................ x
LIST OF ACRONYMS ................................................................................................................. xi
CHAPTER ONE ............................................................................................................................. 1
1. INTRODUCTION ...................................................................................................................... 1
1.1 Background ........................................................................................................................... 1
1.2. Statements of the problem .................................................................................................... 3
1.3 Objectives .............................................................................................................................. 3
1.3.1 General Objective ........................................................................................................................ 3
1.3.2 Specific Objectives ...................................................................................................................... 3
1.4 Significance of the study ....................................................................................................... 4
CHAPTER TWO ............................................................................................................................ 5
2. LITERATURE REVIEW ........................................................................................................... 5
2.1 Review on pulp production ................................................................................................... 5
2.2 Raw materials ........................................................................................................................ 5
2.3 Banana Plant productions ...................................................................................................... 7
2.3.1 Banana Production in the world ................................................................................................... 7
2.3.2 Banana Production Trend in Ethiopia .......................................................................................... 8
2.4 Properties of Banana Peel ................................................................................................... 10
2.5 Starch ................................................................................................................................... 13
2.6 Properties and Characteristics of Banana Fiber .................................................................. 14
2.6.1 Characteristics of banana fibers ................................................................................................. 14
2.6.2 Potential of banana fiber ............................................................................................................ 15
2.7 Pulp Production Status in the world .................................................................................... 16

vi
 2.7.1 Worldwide pulp production ....................................................................................................... 16
2.8 Production process of pulp from banana peel ..................................................................... 16
2.9 Pulp Properties and Applications ........................................................................................ 19
2.9.1 Basic Pulp Properties ................................................................................................................. 19
2.9.2 Pulp application ......................................................................................................................... 20
2.10 Types of Pulping ............................................................................................................... 20
2.10.1 Mechanical pulping .................................................................................................................. 20
2.10.2 Chemical pulp .......................................................................................................................... 20
2.11 Bio-pulping and Bio-bleaching ......................................................................................... 25
2.11.1 Effect of pH on growth of cellulolytic bacteria ....................................................................... 27
2.11.2 Effect of temperature on growth of cellulolytic bacteria ......................................................... 27
2.12 Physico-chemical composition of banana peel ................................................................. 28
CHAPTERTHREE ....................................................................................................................... 31
3. MATERIALS AND METHODS .............................................................................................. 31
3.1. Study Site ........................................................................................................................... 31
3.2 Samples collection............................................................................................................... 31
3.3 Experimental set up ............................................................................................................. 31
3.4 Physico-chemical Composition of sample .......................................................................... 32
3.5 Preparation of raw materials ............................................................................................... 34
3.5.1 Effect of drying time and peel to water ratio ............................................................................. 34
3.5.2 Effect of surface sizing starch and bleaching chemical ............................................................. 35
3.6 Procedures for parameter characterization .......................................................................... 35
CHAPTER FOUR ......................................................................................................................... 38
4. RESULTS AND DISCUSSION ............................................................................................... 38
4.1 Physico-Chemical properties of Samples ............................................................................ 38
4.2 Effect of drying time and temperature ................................................................................ 39
4.3 Effect of surface sizing starch and bleaching chemical ...................................................... 40
4.4 Effects of the parameters interaction ................................................................................... 43
4.5 Screening the isolates for production of extracellular enzymes .......................................... 48
4.6 Effect of pH and Temperature on the growth of the cellulose degrading bacteria ............. 48
4.6.1 Growth at different pH ............................................................................................................... 48

vii
 4.6.2 Growth at different temperature ................................................................................................. 50
CHAPTER FIVE .......................................................................................................................... 52
5. CONCLUSION AND RECOMMENDATION ........................................................................ 52
5.1. Conclusion.......................................................................................................................... 52
5.2 Recommendations .......................................................................................................... 53
REFERENCES ............................................................................................................................. 54
APPENDICES .............................................................................................................................. 61

viii
 LIST OF TABLES

Table 2.1: Comparison of non-wood and wood resources for pulp and paper-making
Table 2.2: Minerals Composition of Banana peel
Table 2.3: Nutrient composition of banana peel
Table 2.4: Banana fiber properties
Table 2.5: Basic pulp Properties
Table 2.6: General Classification of pulping processes
Table 4.1: Physico-chemical characteristics of banana peel sample
Table 4.2: Pulp production yield from different parameters: ratio of peel to water, duration of
drying and ripeness of banana peel.

ix
LIST OF FIGURES

Figure 2.1: Banana exports by region 2009-2013 in million tones

Figure 2.2: Major banana production areas and market outlets covered by the survey 2014/15
Figure 2.3: Local banana cultivars grown in different parts of Ethiopia
Figure 2.4: Banana peel
Figure 2.5: Structure of a cellulose chain.
Figure 2.6 :World pulp production trend
Figure 2.7: Block diagram of pulp processing from banana peel waste
Figure 2.8: Bleaching of pulp
Figure 3.1: Laboratory experimental set up
Figure 3.2: pH and Termperature optimization of crude cellulase enzyme for biobleaching
activity
Figure 3.3: Sample preparation for biobleaching method
Figure 4.1: Result of experiment when peel to water ratio was a) 1:3 and b) 1:2
Figure 4.2: Result of experiment when peel to water ratio was a) 1:1.5 and b) 1:1
Figure 4.3: Result of experiment when peel to water ratio was 1:0.5
Figure 4.4:3D plot of duration of drying of banana peel interaction with peel to water ratio on ash
content of the product (design expert)
Figure 4.5:3D plot of duration of drying of banana peel interaction with peel to water ratio on
tearing resistance of the product (design expert)
Figure 4.6:3D plot of duration of drying of banana peel interaction with peel to water ratio on
burst strength of the product (design expert)
Figure 4.7: 3D plot of duration of drying of banana peel interaction with peel to water ratio on
tensile strength of the product (design expert)
Figure 4.8: Bleached pulp with different pH ranges.
Figure 4.9: Bleached pulp with different temperature ranges.

x
LIST OF ACRONYMS

ANOVA Analysis of variance

ASTM American Society for Testing Materials

CDB Cellulose degrading bacteria

CMS Carboxymethylcellulose

CFC Common Fund for Commodities

CSA Central Statistical Agency of Ethiopia

FAOSTAT Food and Agriculture Organization Statistical Division

IFC International Finance Corporation

TAPPI Technical Association of the Pulp and Paper Industry

TKN Total Kjeldahl Nitrogen

TS Total solid

VS Volatile solid

xi
CHAPTER ONE

1. INTRODUCTION

1.1 Background

The secret of pulp making remained in China for 650 years, until Arabs learned the art from
Chinese prisoners of war. The process was brought to Europe in the 12th century but the method
of making pulp from wood was lost along the way. Rags were used instead. A rag shortage in the
1850s, along with increasing demand for pulp, enticed European and American inventors to find
alternative supplies for making pulp. These inventors found mechanical and chemical methods
for efficiently making pulp from wood. Poplar was the wood of choice, and new mills began to
open near the source of this fiber (Chaudhury J.et al., 2013).

In the world wide, around 95% of all raw materials used by the paper industry to obtain cellulose
pulp consist of hardwood or softwood (Jaya B. et al., 2014).

In Ethiopia pulp is not produced but it is imported. Ethiopian pulp and papers Share Company
was one of the first paper manufacturing companies which was established and got legal
recognition in August 29, 1955 E.C. the shareholders of this companies were ministry of finance,
Ethiopian Development Bank, Ethiopian Investment Bank and Panser and Witmor Company
with a starting capital of 50,000 birr. When it started production in 1962 the total expenses were
22 million birr and at the same time its capital was pumped to 10 million birr. Currently the
shareholders of the company are Ethiopian government 70% and IFC 30%. During its
establishment the company produces paper product of 25 ton daily and 8,500 ton annually. In
1989 the company has increased its production capacity to ten thousands ton/annum.
This thesis involves basic raw material for paper production that is pulp from banana peel waste.
The purpose of pulping is to extract cellulose fibers from plant material, generally hardwood,
softwood trees or non-wood plant for papermaking. The most abundant component of the native
wood matrix is cellulose, a polysaccharide that is desired for paper production. The second most
abundant component of native wood is lignin, a complex polymer made of aromatic units.

Banana is one of the rhizomatous plants and currently grown in 129 countries around the world.
Banana ranks fourth as the world‟s most important starch crop after cassava and sweet potatoes.
Its yields of carbohydrates per unit area are very high. It is the fourth most widely-grown food
crop after rice, wheat and maize (Bates M. and Flordeliza A., 2010).
Approximately one-third of the bananas produced globally are grown in sub-Saharan Africa,
where the crop provides more than 25% of food energy requirements for more than 100 million
people. East Africa (including Burundi, Kenya, Rwanda, Tanzania, and Uganda) is the largest
banana producing and consuming region in Africa. Uganda is the world‟s second largest
producer after India, with a total of about 10.5 × 109 Kilogram per year (Wachira P.et al, 2013).
The average shift and productivity decline has been attributed to the increasing severity of
production constraints. Particularly the declining of soil fertility, pests and diseases are the major
causes for the reduction of banana production in some areas (CFC, 2004).
Currently banana in Ethiopia covers about 59.64% (53,956.16 hectares) of the total fruit area,
about 68.00% (478,251.04 tones) of the total fruits produced, and about 38.30% (2,574,035) of
the total fruit producing farmers. On the other hand, about 68.72% (37,076.85 hectares) hectares
of land covered by banana, about 77.53% (370,784.17 tones) of the banana produced and
22.38% (1,504,207) of the banana producers in Ethiopia are found in the Southern Nations
Nationalities and Peoples‟ Regional State (CSA, 2014).
Gamo-Gofa, Bench Maji and Sheka zones are among the major banana producing zones of the
Southern Nations Nationalities and Peoples‟ Regional State (SNNPRS), of which Gamo-Gofa
zone alone covers over 70% of the total banana marketed across the major market outlets in
Ethiopia (CFC, 2004).
In banana plantations, after the fruits are harvested, the trunks or stems will be wasted. Billion
tons of stems and leaves are thrown away annually. Such waste provides obtainable sources of
fibers, which leads to the reduction of other natural and synthetic fibers‟ production that requires
extra energy, fertilizer, and chemical. The properties of banana fiber are good absorbent, highly
breathable, quickly dry with high tensile strength.

As banana fiber can provide a wide variety of uses in textile and paper industry, the study of the
application of this locally and widely grown plant species for the sustainable development would
be beneficial (Jaya B. et al., 2014).

2
Banana peel is one example of non-wood fibers that will be used in this study as a raw material.
Banana peel usually considered as waste in the banana processing. Therefore, it makes sense to
turn such waste into a useful product like paper.

1.2. Statements of the problem

The banana peels waste causes emission of pollutant gases like CO2 and also gives growth to the
harmful fungi when stored in the environment after using. It has a negative impact on the
environment. The stored banana peel also causes unpleasant odor to the environment, causing
aesthetic problem even though they are biodegradable. Using wood for the manufacturing of
pulp and paper production causes deforestation. Since species of banana plant determines the
fiber properties of its constituent, it is difficult to identify the potential fiber. Another problem is
that Pulp is not produced in our country so far, it is imported and there is scarcity of paper in our
country since the demand increases from day to day.

1.3 Objectives
1.3.1 General Objective

The general objective of this study is to:

 Optimize the production of pulp derived from banana peel biomass using chemical and
biological methods.

1.3.2 Specific Objectives

The specific objectives of the study are:

 To determine cellulose and lignin content of the banana peel biomass.
 To apply chemical-bleaching techniques for production of banana peel pulp
 To determine physico-chemical characterization of banana peel pulp
 To conduct bio-bleaching (bacterial enzyme) method for production of banana peel pulp
 To identify banana peel for quality pulp production

3
1.4 Significance of the study

The principal interest in pulping non-woody raw materials is that, they provide pulp of excellent
quality for making specialty graded paper or constitute the sole affordable source of fibrous raw
material in some geographical areas. Rapid increase of competition for wood supplies coupled
with slowly rising cost of wood have generated renewed interest in the use of non-wood plant
fibers for paper-making in the highly industrialized countries. The use of agricultural wastes in
pulping and paper-based industries might be advantageous because it prevents the need for
disposal, which currently increases farming costs and causes environmental deterioration through
pollution and fires. The cellulose content of banana plant differ country to country, so this study
is helpful in investigating the cellulose content of banana plant in relation to producing quality
pulp.

Moreover, new substances from non-wood resources may allow modification of pulp and paper
properties to produce better quality paper products. The alternative fibers also encourage the
application of green technology in terms of generating new paper production from non-wood
resources to create more environmentally friendly processes. The study will also help the farmers
by providing them an opportunity to generate additional income through extracting or selling the
banana fruit. In addition, producing pulp from banana peel helps in saving foreign currency since
the pulp is imported so far.

This study is significant that it will be used as supportive idea to do further detailed researches
regarding to decrease the environmental impacts of the banana peel biomass and convert it to a
pulp.

4
CHAPTER TWO

2. LITERATURE REVIEW

2.1 Review on pulp production

Pulp is a fibrous material resulting from complex manufacturing processes that involve the
chemical and/or mechanical treatment of various types of plant material. Pulp is one of the most
abundant raw materials worldwide which are used predominantly as a major component in the
manufacture of paper and paperboard, and with increasing importance also in the form of a wide
variety of cellulose products in the textile, food, and pharmaceutical industries (Smook, 1992).

Historically, non-woody plants were major resources for pulp and paper production compared to
woody plant. In the 19th century, an insufficient supply of the traditional raw materials of cotton
and linen rags made it necessary to use wood to make paper (Bajpai et al., 2004). Today, wood
fiber either hardwood or softwood are the main raw materials used for the production of pulp and
paper. About 90-92% of the pulp and paper in the world are produced from wood, mostly in the
developed countries (Bajpai et al., 2004 and Jiménez et al., 2009) such as Canada and United
State of America, USA (Madakadze et al., 2010).
Wood and other plant materials like banana peel waste used to make pulp contain three main
components (apart from water): cellulose fibers (desired for papermaking), lignin (a three-
dimensional polymer that binds the cellulose fibers together) and hemicelluloses (shorter
branched carbohydrate polymers). The aim of pulping is to break down the bulk structure of the
fiber source, be it chips, stems or other plant parts, into the constituent fibers (Smook, 1992).

2.2 Raw materials

In the world wide, around 95% of all raw materials used by the paper industry to obtain cellulose
pulp consist of hardwood or softwood. The other raw materials used for this purpose are known
as “non-wood” materials. Although cellulose pulps are mainly obtained from woods, several
authors report that the production of pulp from non-wood resources has several advantages such
as easy pulping capability, excellent fibers for the special types of paper and high-quality
bleached pulp (Jaya B.et al.,2014).

5
Nowadays, in paper making industry, the environmental problems have brought forward the need
for cleaner technology where the new non-wood resources have been introduced to replace
traditional raw materials such as wood resources with non-wood resources. The cleaner
technology or green technology is applied to achieve increased production with minimum effect
especially on the environment and lessen the disposal cost, steadiness risks and resource cost
resulting in a declined burden on the natural environment and also increase the profits in pulp
and paper-based industries (Sridach, 2010b).
Selection Criteria
Banana peel is one example of non-wood fibers and usually discarded as an agricultural waste
from banana plantation. It is selected as a raw material for the reason that it‟s easily available,
lignin separates easily (low lignin content compared to wood fibers), time and energy saving
process. Banana peels contain up to 15% of cellulose (Porvika B. and Prashanthi B., 2014).
Table 2.1: Comparison of non-wood and wood resources for pulp and paper-making (Rousu et
al., 2002 and Kissinger et al., 2007).
Description Fiber resources
Wood Non-wood
Cycle growth Long growth cycles [X] Short cycle growth [√]
Cellulose content Higher cellulose content [√] Lower cellulose content depends on
the types of non-wood [X]
Lignin content Contain higher lignin content Contain lower lignin content [√]
[X]
Chemical uses Use a large volume of chemical Use a small amount of the chemical in
during pulping process [X] pulping process [√]
Time pulping Need long time for pulping Shorten time for pulping process [√]
process [X]
Cost operation Expensive due to the limitation Cheaper cost because the abundance
resources [X] resources [√]
Environmental Increase environmental problem Reduce environmental impact which
impact such as global warming and soil reduce the deforestation problem and
erosion [X] improve sustainable forestry [√]
Note: [√] = advantages and [X] = disadvantages

6
2.3 Banana Plant productions

Banana is a major fruit crop grown in many developed and developing countries. The production
distribution mainly lies in between 30˚ North and South latitudes at about 60˚ F or above winter
temperatures and 100 mm of average monthly rainfall distribution. Banana can also grow in frost
free cold areas (Bezuneh T.1975).

2.3.1 Banana Production in the world

Globally, banana is the fourth most important crop of the food market next to rice, wheat and
maize. This makes banana to be the prime leading fruit crop in terms of volume and value in the
world market (Woldu, Z. et al., 2015).
In 2013, the global banana export exceeded 17 million· tonnes, 6.5 percent increase as compared
with the year 2012 export rate, Latin America and the Caribbean being the leading suppliers to
the world market. Ecuador is the leading producer in the Latin and Dominican being the largest
in the Caribbean. For Asian exporters, mainly the Philippines (the leading exporter in the region
and the second largest supplier globally) has also supplied 2.7 million ·tonnes of banana to the
market in 2013 (Figure 2.1). In 2013, Africa had also showed a magnificent banana production
growth rate of 5.6% by exporting 665,000 tonnes with a major contribution from Cote d‟Ivoire
(the leading exporter in the region) and Cameroon (the second largest exporter in Africa). The
European Union, United States and the Russian Federation are the leading importers of banana in
the world (FAO, 2015).

7
. In the year 2010, the total banana world commerce valued at $8.05 billion.(Woldu, Z.,et
al.,2015)

Figure 2.1: Banana exports by region 2009-2013, million·tones, (FAO, 2015).

2.3.2 Banana Production Trend in Ethiopia

In Ethiopia, the modern banana production was started at the beginning of this century with the
establishment of the state farms and different plantations such as large scale sugarcane
plantations. The production system of the crop is mainly composed of 1) small plantations in
home gardens owned by small farmers-especially in the South-Western and Western provinces;
2) medium-sized plantations of not more than 10 ha supplying local consumers; and 3) relatively
large plantations above 20 ha developed to supply export markets (Bezuneh, T. 1975).
The export business of banana in Ethiopia dates back to 1961 where the country started at about
5000 tonnes. This figure, however, increased to 60,000 tonnes by the year 1972 when the
country exported to different countries of Europe, Asia and Africa. In 1975 the total production
of banana in the country has reached about 100,000 tonnes (Bezuneh, T. 1975, Berhe, K.,et
al.,2008).
Dessert banana is also the major fruit crop that is most widely grown and consumed in Ethiopia.
It is cultivated in several parts where the growing conditions are favorable. Especially in the
south and southwestern parts of the country, it is of great socioeconomic importance contributing

8
significantly to the overall wellbeing of the rural communities including food security, income
generation and job creation.

Figure 2.2: Major banana production areas and market outlets covered by the survey 2014/15
(Woldu, Z. et al., 2015).
The major commercial cultivars grown by small-scale growers in Ethiopia are Dwarf Cavendish,
Giant Cavendish, and Poyo, with Williams and Grand Naine recently coming into picture in
Gamo-Gofa zone and across the large-scale commercial farms of Ethiopia. Others like Robusta
and Butuzua are also among the recently released Cavendish banana cultivars released through
Melkasa Agricultural Research Center. The rest are less popular land races grown to a very
limited extent in certain localities across the country (Woldu, Z. et al., 2015).

9
Figure 2.3: Local banana cultivars grown in different parts of Ethiopia (Woldu, Z.,et al.,2015).

2.4 Properties of Banana Peel

Musa sapientum which is commonly called banana is an herbaceous plant of the family
Musaceae. It is known to have originated from the tropical region of Southern Asia and it is now
cultivated throughout the tropics. The plant is cultivated primarily for its fruits and to a lesser
extent for the production of fiber. It is also believed to be an ornamental plant (Akinyosoye,
1991).
The Musa sapientum grows up to a height of about 2-8 m with leaves of about 3.5m in length.
The stem which is also called pseudo stem produces a single bunch of banana before dying and
replaced by new pseudostem. The fruit grows in hanging cluster, with twenty fruits to a tier and
10
3-20 tiers to a bunch. The fruit is protected by its peel which is discarded as waste after the inner
fleshy portion is eaten (Akinyosoye, 1991).

Figure 2.4: Banana peel

Table 2.2: Minerals Composition of Banana peel (Anonymous, et al., 2001, 2005 & Ranzani, et
al., 1996)

Element Concentration(mg/g)
Potassium 78.1±6.58
Calcium 19.20±0.00
Sodium 24.30±0.12
Iron 0.61±0.22
Manganese 76.20±0.00
Bromine 0.04±0.00
Rubidium 0.21±0.05
Strontium 0.03±0.01
Zirconium 0.02±0.00
Niobium 0.02±0.00
Values are Mean ± SE

11
Table 2.3: Nutrient composition of banana peel (Anonymous, et al., 2001, 2005 & Ranzani,et al.,
1996)

Parameter Concentration
Moisture(%) 6.70±02.22
Ash(%) 8.50±1.52
Organic matter(%) 91.50±0.05
Protein(%) 0.90±0.25
Crude Lipid(%) 1.70±0.10
Carbohydrate(%) 59.00±1.36
Crude fiber(%) 31.70±0.25
Hydrogen cyanide(mg/g) 1.33±0.10
Oxalate(mg/g) 0.51±0.14
Phytate(mg/g) 0.28±0.06
Saponins(mg/g) 24.00±0.27
Values are Mean ± SE
Cellulose is a Carbohydrate composed of C, H, O with the chemical formula (C6H10O5)n n-
values varies with the different sources of cellulose. It is a linear and high molecular weight
polymer as well as natural, renewable, and biodegradable material. The cellulose fibers are
obtained as pulp after pulping process. It determines the character of the fiber and permits its use
in paper making (Jaya B. et al., 2014).

Cellulose linear polysaccharide assembled from glucose monomer units, and it is the main
constituent of plant cell walls. Along with several indigestible polysaccharides, cellulose
constitutes the main part of dietary fiber. Specifically cellulose is one of the components of
insoluble fiber. Cellulose shows a variable degree of polymerization, with anywhere from 1,000
to 14,000 glucose residues comprising a single cellulose polymer. Because of its high molecular
weight and crystalline structure, cellulose is insoluble in water and has a poor ability to absorb
water. Cellulose is derived from D-glucose units, which condense through β (1→4)-glycosidic
bonds. Cellulose is a straight chain (Gupta et al., 2012).

12
Figure: 2.5: Structure of a cellulose chain. The repeating unit, cellobiose, is indicated in brackets
while glucosidicoxygens are marked by a “β” (Brown et al., 1996).

Cellulase refers to a suite of enzymes produced chiefly by fungi, bacteria, and protozoans that
catalysecellulolysis (i.e. the hydrolysis of cellulose).Cellulases have a wide range of application
in numerous industries such as agriculture, pulp and paper, textile, food and beverage, animal
feed, detergent industry and bioconversion of cellulosic material into solvents such as ethanol
amongst others. Microorganism brings about biodegradation of cellulose in nature using
multienzyme complex Cellulaseenzymes,which can hydrolyze cellulose forming glucose and
other commodity chemicals(Aubert et al.,1987). Cellulase can be divided into three types:
endoglucanase (endo-1, 4β-D-glucanase); cellobiohydrolase or exoglucanase (exo-1, 4-β-D-
glucanase) and β-glucosidase (1, 4-β-Dglucosidase) (Gao,et al.,2008).
Lignin is highly polymerized substance. Its principal role is to form the middle lamella, the
intercellular material which cements the fibers together. The Lignin content in banana fruit is
much lower than that for woods (Jaya B. et al., 2014).

2.5 Starch

Starch is long chains of sugar molecules linked together like a chain. Starch is composed of the
sugar glucose. Glucose is a sugar molecule made up of carbon (C), hydrogen (H), and oxygen
(O) with a basic chemical formula of C6H12O6.Starch is composed of two kinds of
polysaccharides, amylose and amylopectin, exclusively composed of d‑glucose residues with
α‑(1→4) linkages in a linear amylose and α‑(1→4) linkages and ∼5% α‑(1→6) branch linkages
in amylopectin.

13
Starch is an important component of many paper grades. Starch consumption by weight in
papermaking and paper conversion processes ranks third after cellulose fiber and mineral
pigments. Starch is used as a flocculants and retention aid, as a bonding agent, as a surface size,
as a binder for coatings, and as an adhesive in corrugated board, laminated grades, and other
products. The major starch sources are corn, potato, waxy maize, wheat, and tapioca. Refined
starches are supplied in powder form or as slightly aggregated pearl starch. Unmodified (native)
starch is rarely used in the paper industry, except as a binder for laminates and in the corrugating
process. Most starches for use in paper making are specialty products that have been modified by
controlled hydrolysis, oxidation, or derivatization (James B. and Roy W., 2008).

2.6 Properties and Characteristics of Banana Fiber

2.6.1 Characteristics of banana fibers

 Banana fibers have great potentialities for paper making because of its high alpha-
cellulose and lignin content.
 These fibers have excellent tensile strength, they are strong fibers.
 They have low elongation and light weight
 They possess strong moisture absorption quality. It absorbs as well as release moisture
very fast.
 They are biodegradable and has no negative effect on environment hence categorized
eco-friendly fiber.
 Banana fibers also possess fire resistance property.

14
Table 2.4: Banana fiber properties (Lakhan S. and Tarunkanti B., 2013).

Fiber Properties Amount

Tenacity 29.98 g/denier

Fineness 17.25 denier

Moisture regain 13.0%

Elongation 6.54%

Total cellulose 81.80%

Alpha Cellulose 61.50%

Residual Gum 41.90%

2.6.2 Potential of banana fiber

 Banana fiber has great potentialities for paper making special demand of handmade
paper.
 As an eco-friendly substitute in textile industry in place of the environmentally hazardous
synthetic fibers.
 Being completely biodegradable and naturally occurring, the banana fiber products are
expected to be in great demand in the international markets as they pose no toxic effects
to man and environment.
 Boxes made from these boards can be used for transport of fruits and other materials‟
 Thicker varieties of banana paper can be used for making files, covers and packing
materials (Manish K.et al., 2011).

15
2.7 Pulp Production Status in the world
2.7.1 Worldwide pulp production

Globally the production of pulp is led by North America, which accounts for over one-third of
the pulp production and generates an excess supply of 5%. Europe and Asia follow, each with
close to one fourth of global pulp production. The overall production of pulp in 2014 equaled
178.5 million tons, with total consumption equaling 179.6 million tons (FAO,2016).

Figure 2.6: World pulp production trend (FAO, 2016).

2.8 Production process of pulp from banana peel

Process description

Raw material Preparation: -Here collection of the raw material which is non-wood (banana
peel waste) takes place. Particular measures are required during the storage period to reduce
losses caused by bio deterioration banana decay mainly depend on Storage duration, Climatic
and Storage conditions.

16
Drying-The banana peels are first peeled and dried in the sun. The extent of drying of the peel
depends on the color of paper desired.

Chopping-The stem is chopped into small pieces 3-4 inch in sizes using a hammer mill or other
chopping devices. Here the chopped peel must be relatively uniform size, though optimum size
may vary depending on the peel properties.

Digestion- There is several variations of the cooking processes both for the batch and the
continuous digesters. Digesters producing 1,000tonnes of pulp per day and more are common
with the largest producing more than 3,500 tonnes of pulp per day. In a continuous digester the
materials are fed at a rate which allows the pulping reaction to be complete by the time the
materials exit the reactor. Delignification requires several hours at 130 to 180 °C (266 to
356 °F). Under these conditions lignin and hemi cellulose degrade to give fragments that are
soluble in the strongly basic liquid (Lakhan S. and Tarunkanti B., 2013).
Grinding: in this process the cooked banana peel is grinded with starch.

Filtering and Washing: -The softened material is filtered usually by Vacuum filtration
equipment for fiber recovery and washed with water to remove the black liquor. The purpose of
pulp washing is to obtain pulp that is free of unwanted soluble (Lakhan S. and Tarunkanti B.,
2013).
Bleaching: -Bleaching increase brightness as well as reduces the viscosity and yield of bleached
pulp. Bleaching sequences consist of several stages which deal with various chemicals. Today‟s
bleaching stages operate mostly under medium consistency conditions, between 10% and 12%
consistency and is done to change the brown color into white using different kinds of chemicals.
Some chemicals used for bleaching are applied in gaseous form (Smook, 1992).

Commonly Applied Chemical treatments

 Chlorination-reaction with elemental chlorine in acidic medium

 Alkaline Extraction-Dissolution of reaction products with Sodium Hydroxide.
 Chlorine Dioxide- reaction with Chlorine Dioxide in acidic medium.
 Oxygen- reaction with molecular oxygen at high pressure in alkaline medium
 Hypochlorite- reaction with Hypochlorite in alkaline medium

17
  Reaction With Calcium oxide
 Reaction with Activated Carbon
 Peroxide- reaction with peroxide in alkaline medium
 Ozone- reaction with ozone in acidic medium.
Molding: The produced pulp is molded based the desired size of pulp and paper.
Drying: The pulp produced need to be dried based on the acceptable moisture content of pulp.
Storage:-After Filtering and washing the pulp will be molded then the desired pulp is produced
molded pulp or molded fiber, is a packaging material usually used, typically made from recycled
paperboard and/or newsprint. It is used for protective packaging or for food service trays and
beverage carriers. Other typical uses are end caps, trays, plates, bowls and clamshell containers.
For many applications, molded pulp is less expensive than expanded polystyrene (EPS),
vacuumed formed PVC, corrugation, and foams.
Molded pulp products can be made waterproof with a spray or dip coating of wax, which is then
stored in a storage tank (Wiley V. and Co A.2006).
Banana Peel Water

Removal of Drying Chopping Boiling

Unwanted
parts

Water CaO

Molding Bleaching Grinding

Drying
.

Starch
Packaging Storage

Figure 2.7: Block diagram of pulp processing from banana peel waste (Porvika B. and Prashanthi
B., 2014).

18
2.9 Pulp Properties and Applications

2.9.1 Basic Pulp Properties

Moisture Content of Market Pulp: is important from storage, transportation and handling point
of view. Most of the market pulp are sold, stored, transported and used as air dry. The useable
part of pulp is dry fiber only, so the tendency is to minimize the moisture content of pulp
Viscosity of Pulp: Solution viscosity of a pulp gives an estimation of the average degree of
polymerization of the cellulose fiber. So the viscosity indicates the relative degradation of
cellulose fiber during pulping /bleaching process.
Bursting strength: Is a property of pulp or paper that measures its resistance to rupturing,
defined as the hydrostatic pressure needed to burst a pulp sample when it is applied uniformly
across its side. Bursting strength is a function of various processes performed in the pulp making
process. The increased use of longer fibers and surface sizing increases a pulp's bursting
strength. It is measured using a Burst tester.
Tensile Strength of Pulp: Is the maximum strength of randomly oriented pulp fiber when
formed in a sheet. This tensile strength gives an indication of the maximum possible strength of
pulp beaten under ideal condition. This again an indication of what level of tensile strength can
be achieved in real paper making environment
Table 2.5: Basic Pulp Properties (Wiley V. and Co A. 2006).
No Parameters Standard(ASTM)
1. Burst strength 1.9 kg/cm2
2. Breaking length 4326 m
3. Tear factor 108
4. Moisture content 7-9%
5. Viscosity 250-330 CP
6. Consistency >15

19
2.9.2 Pulp application

Pulp represents the major raw material basis for two main applications: (a) for paper and board
production, where the pulp fibers are mechanically modified to give a coherent sheet; and (b) for
chemical conversion to products such as regenerated fibers and cellulose derivatives. The former is
denoted as paper grade, the latter as dissolving grade pulp. Paper-grade pulp is by far the most
dominant field of pulp production (Wiley V. and Co A. 2006).

2.10 Types of Pulping

Pulp for papermaking can be produced from virgin fiber or from re-pulping of recycled paper. To
produce virgin pulp, wood logs are first debarked and chipped. Then water and heat are added
and by mechanical or chemical means the wood is separated into individual fibers (Healy and
Schumacher, 2011).

2.10.1 Mechanical pulping

Mechanical pulping is primarily used in integrated pulp and paper mills. In this process fibers are
separated through the use of mechanical energy. With a raw material conversion efficiency1 of
45%, mechanical pulping is considered both a simple and efficient process, but with the
disadvantage that wood fibers are often damaged (Healy and Schumacher, 2011). Therefore,
mechanical pulping is mainly used for weaker paper such as newspaper, printing paper,
towelling, tissue, or paperboard. Sometimes chemical pulp is mixed for additional strength.
Electricity is the main energy input for mechanical pulping. Mechanical pulp production yields
substantial amounts of heat as side product, which can be used for district heating (Worrell,
2007).

2.10.2 Chemical pulp

Chemical pulp is used both in integrated and non-integrated pulp and paper mills. Through
chemical pulping, non-cellulose wood components are removed leaving the cellulose fibers
intact (Bajpai, 2012). Hence, chemical pulp is better suited for high quality paper. There are
mainly two different processes for chemical pulping: Kraft (sulphate) and Sulphite. Black liquor
is an energy rich by-product of chemical pulping, which is burned in recovery boilers to produce
combined heat and power (Healy and Schumacher, 2011).

20
2.10.2.1 Kraft’s process

The Kraft process (also known as Kraft pulping or sulphate process) is a process for conversion
of wood into wood pulp consisting of almost pure cellulose fibers. It entails treatment of wood
chips with a mixture of Sodium hydroxide (NaOH), Sodium carbonate(Na2CO3) and sodium
sulphate (Na2SO4),known as white liquor, which breaks the bonds that link lignin to the
cellulose. The Kraft process is the dominant chemical pulping method. It involves following
operations (Jaya B. et al., 2014).

Impregnation
Common wood chips used in pulp production are 12-25 millimeters (0.47–0.98 in) long and 2–
10 millimeters (0.079–0.39 in) thick.
The chips normally first enter the pre-steaming where they are wetted and preheated with steam.
Cavities inside fresh wood chips are partly filled with liquid and partly with air. The steam
treatment causes the air to expand and about 25% of the air to be expelled from the chips. The
next step is to impregnate the chips with black and white liquor. Air remaining in chips at the
beginning of liquor impregnation is trapped within the chips. The impregnation can be done
before or after the chips enters the digester and is normally done below 100 °C (212 °F). The
cooking liquors consist of a mixture of white liquor, water in chips, condensed steam
and weak black liquor. In the impregnation, cooking liquor penetrates into the capillary
structure of the chips and low temperature chemical reactions with the wood begin. A good
impregnation is important to get a homogeneous cook and low rejects. About 40–60 % of all
alkali consumption in the continuous process occurs in the impregnation zone (Jaya B. et al.,
2014).

Screening

Screening of the pulp is done to remove oversized and unwanted particles from good
papermaking fibers so that the screened pulp is more suitable for the paper or board product in
which it will be used (Biermann, 1996b). The biggest oversized particles in pulp are knots. Knots
can be defined as uncooked wood particles. The knots are removed before washing and fine
screening. In low-yield pulps they are broken down in refiners and/or fiberizers; they are also
removed in special coarse screens called “knotters.”

21
Washing

The objective of brown stock washing is to remove the maximum amount of liquor dissolved
solids from the pulp while using as little wash water as possible. The dissolved solids left in the
pulp after washing will interfere with later bleaching and papermaking and will increase costs of
these processes. The loss of liquor solids due to solids left in the pulp means that less heat can be
recovered in the recovery furnace. Also, makeup chemicals must be added to the liquor system to
account for lost chemicals (Gullichsen, 2000).
Normally a pulp mill has 3-5 washing stages in series. Washing stages are also placed after
oxygen delignification and between the bleaching stages as well. Pulp washers use counter
current flow between the stages such that the pulp moves in the opposite direction to the flow of
washing waters. Several processes are involved: thickening / dilution, displacement and
diffusion. The dilution factor is the measure of the amount of water used in washing
compared with the theoretical amount required to displace the liquor from the thickened pulp.
Lower dilution factor reduces energy consumption, while higher dilution factor normally gives
cleaner pulp. Thorough washing of the pulp reduces the chemical oxygen demand (COD).
Several types of washing equipment are in use (Jaya B. et al., 2014).
 Pressure diffusers
 Atmospheric diffusers
 Vacuum drum washers
 Drum displacers
 Wash presses
Bleaching

Bleaching is often undertaken, primarily for two purposes: first, to increase brightness; second,
to remove residual lignin. In a modern mill, brown stock (cellulose fibers containing
approximately 5% residual lignin) produced by the pulping is first washed to remove some of the
dissolved organic material and then further De-lignified by a variety of bleaching stages. The
pulp produced up to this point is still in brown color due in the process can be bleached to
produce a white paper product. Bleaching decreases the mass of pulp produced by about 5%,
decreases the strength of the fibers and adds to the cost of manufacture (Smook, 1992).
Bleaching of pulp is also done to increase the brightness of the pulp so that it can be used in

22
paper products such as printing grades and tissue papers. For chemical pulps, an important
benefit is the reduction of fiber bundles and shives as well as the removal of bark fragments. This
improves the cleanliness of the pulp. Bleaching also eliminates the problem of yellowing of
paper in light, as it removes the residual lignin in the unbleached pulp. (Farr et al., 1992;
Fredette, 1996; McDonough, 1992; Reeve, 1989, 1996a)

Figure 2.8: Bleaching of pulp (Smook, 1992).

2.10.2.2 Semi chemical pulping

Semi chemical pulping processes are characterized by a mild chemical treatment preceded by a
mechanical refining step (Biermann, 1996b). Semi chemical pulps, which apply to the category
of chemical pulps, are obtained predominantly from hardwoods in yields of between 65 % and
85 % (∼75 %). The most important semi chemical process is the neutral sulfite semi chemical
process, in which chips undergo partial chemical pulping using a buffered sodium sulfite
solution, and are then treated in disk refiners to complete the fiber separation.

23
Table 2.6: General Classification of pulping processes (Smook, 1992).

Mechanical Hybrid Chemical

Pulping by mechanical Pulping with combination of Pulping with chemicals

energy chemical and mechanical and heat (little or no
treatments mechanical energy)
(small of chemicals and heat)

High yield (85-95%)

Intermediate yield (55-85%) Low yield (40-55%)
Short, impure fibers
“intermediate” pulp properties
Long, strong fibers
 Weak (some unique properties)
 Unstable  Strong
Good print quality  Stable
Examples : Poor print quality
Examples:
 Neutral sulphite semi Examples:
 Stone ground wood
chemical
 Refinery mechanical  Kraft
 High-yield kraft
pulp  Sulphite
 High-yield sulphite
 Thermo mechanical  soda
pulp

Sulphite process

In the sulfite process, a mixture of sulfurous acid (H2SO3) bisulfite ion (HSO3-) is used to attack
and solubilizes the lignin. The sulphites combine with the lignin to form salts of lingo sulfonic
acid which are soluble in the cooking liquor, and the chemical structure of the lignin is left
largely intact. The chemical base for the bisulfite can be ionic calcium, magnesium sodium or

24
ammonium. Black liquor is an energy rich by-product of chemical pulping, which is burned in
recovery boilers to produce combined heat and power (Healy and Schumacher, 2011).

Soda pulping

This pulping process is entirely similar to Kraft‟s process. The only difference exists is the
cooking chemicals used. In soda pulping process only NaOH or combination of NaOH and
Na2CO3 are used. The quality of pulp obtained in soda pulping is low and used for
manufacturing low grade paper. The amount of bleaching agent required is large as the lignin
content is not effectively removed (Jaya B.et al., 2014).

Pulp from banana Peel using surface sizing Starch

Pulp and paper can be produced from banana peel using starch.in this process surface sizing
starch is used. Starch is used as a flocculants and retention aid, as a bonding agent, as a surface size, as a
binder for coatings, and as an adhesives (PorvikaB. and Prashanthi B., 2014).

2.11 Bio-pulping and Bio-bleaching

The pulp and paper industries have been categorized as one of the major sectors that pose a
threat to the environment. Economic conditions and environmental pressures have hit the pulp
and paper industry hard, and it has been under tremendous pressure to improve the performance
related to release of pollutants. Increased environmental concerns are creating pressure for
adoption of new, eco-friendly technologies (Dhiman et al., 2009). An integrated pulp and paper
process includes the general steps of pulping, bleaching, and paper production.

Deployment of bio pulping in pulping is the first processing step in the paper industry. The aim
of the procedure is to eliminate lignin and avoid the cellulose decomposition. Pulping methods
use chemicals to dissolve wood fibers and grind mechanically the fibers to form pulp. Pulp and
paper industries exploit biological raw materials, which are synthesized, modified and degraded
in nature by a number of microbes using a vast array of enzymes. Bio bleaching of pulps is
performed with either hemicellulolytic enzymes, in particular xylanases or lignin-degrading
fungi or their enzymes. The fungal pretreatment of wood chips to reduce consumption of
chemical in pulping, is Bio pulping (Eriksson et.al. 1976,, 1983). Bio pulping use biological
methods as promising alternative to the alkali and chemical bleaches (Keller et al., 2003). Use of

25
fungal inoculums prior to pulping offers an attractive opportunity for mechanical pulp facilities.
This technology could save energy in the refining of mechanical pulp (Shukla et al., 2004).
Likewise, in the case of chemical pulping a biological method can be used to facilitate the
removal of lignin from wood/non-wood by modifying lignin for easier extraction in subsequent
chemical pulping processes (Castillo et al., 2000; Field,, 2000).

The use of suitable lignin-degrading fungi can result in saving of electrical energy during
mechanical pulping, and paper strength properties also can be improved (Kondo et al., 1996).
Bio pulping using cellulases and allied enzymes reduces the energy required to achieve the
desired strength and freeness of the pulp hence it‟s a better alternative to mechanical pulping
(Karmakar and Ray, 2011). About 75% of the pulp in the world is produced by the kraft pulping
process. This process produces paper with very high strength. However, the process has the
disadvantages of being capital and energy intensive, low in yield of production, producing
troublesome waste products, and producing byproducts that are of relatively low value (Kondo et
al. 1994). Biotechnology offers a potential solution to many of these problems (Battan et al.
2007). Bio pulping, in the form of pretreatment of wood chips prior to conventional pulping,
offers the potential for both economic and environmental benefits. Bleaching of pulp is necessary
for whitening of paper and is based on removal of residual lignin from the cellulose fibers but
should have no adverse effect on cellulose fiber quality (Unal and Kolankaya, 2001). However,
use of chlorine-based bleaching process tends to generate toxic and highly persistent chlorinated
organic by-products, which eventually pollute water bodies (Singh et al., 2008). Also, the use of
such chemically-treated pulp/paper in the manufacturing of direct-body-contact consumables like
baby diapers and food packaging is of major concern as it is associated with chlorinated
compounds including the animal carcinogen, dioxin (Shoham et al., 1992). Due to increasing
health awareness and growing public sensitivity towards the negative environmental impact of
chlorinated pulp, there is an ever growing demand for use of chlorine-free paper products all
over the world. In this regard, the world is focusing research on the development of newer
healthy and environmentally friendly technologies. Among these, bio bleaching with enzymes
has shown immense potential in minimizing use of bleaching chemicals containing chlorine
(Singh et al., 2008).

26
2.11.1 Effect of pH on growth of cellulolytic bacteria

Hethener et al. (1992) reported isolation of Clostridium termitidis sp. nov., a cellulolytic
bacterium from the Gut of the Wood-feeding termite. All the bacterial isolates were allowed to
grow in the nutrient broth at various pH concentrations (5.0 to 8.2). It was found that, the
optimum pH for growth of the bacteria was 7.5. Balamurugan et al. (2011) isolated cellulose
degrading bacteria from tea garden soil. Their optimum growth profile was determined at various
pH (6.0, 7.0, 8.0 and 9.0). Results showed that the maximum growth of cellulose degradation
bacteria (CDB) was recorded at pH 7.0 and that even all the strains grew at pH 4.0 to 8.0. An
experiment was conducted for isolation of ten cellulose degrading bacteria from decayed
sawdust. For determination of their growth at optimum pH, they were grown at various pH and
results clearly indicated that isolates such as Bacillus spp1, Bacillus spp2,Micrococcusspp,
Pseudomonas spp1, and Acinetobacter spp. were able to grow in the neutral (pH 7) conditions
better than acidic conditions (pH 6 and pH 5) (Maruthmalai et al. 2011). Isolation of cellulose
degrading bacteria Pseudomonas aeruginosa from termite gut microflora was reported by
Bholay et al. (2014). Optimum pH for the growth of bacteria was determined by incubating test
cultures at different pH concentrations (pH 6.0, pH 7.0, pH 8.0 and pH 9.0) Maximum growth of
Pseudomonasaeruginosa was observed at pH 7.0.

2.11.2 Effect of temperature on growth of cellulolytic bacteria

A novel species Clostridium termitidis sp. nov., a cellulolytic bacterium was isolated from the
Gut of the Wood-feeding termite. All the bacterial isolates were allowed to grow in the nutrient
broth at various incubation temperatures and the optimum growth of the bacteria was observed at
37°C and growth occurred in a temperature range of 20 to 48 °C (Hethner et al. 1992).
Balamurugan et al. (2011) isolated cellulose degrading bacteria from tea garden soil. To
determine their optimum growth profile, these were inoculated with test cultures and were
incubated at different temperatures (25, 30, 35, 40 and 45 °C). The maximum growth of bacteria
was observed at 35 °C. An experiment was conducted for isolation of ten cellulose degrading
bacteria from decayed sawdust. For determination of their optimum growth temperature,
bacterial isolates were incubated at various temperatures (28 °C, 37 °C and 45 °C). The bacterial
isolates such as Bacillus spp.1, Bacillus spp.2, Micrococcus spp, Pseudomonas spp.,
Acinetobacter spp. and Klebsiella spp. Showed better growth performance at 37°C

27
(Muruthamalai et al. 2014).Isolation of cellulose degrading bacteria Pseudomonas aeruginosa
from termite gut microflora was reported by Bholay et al. (2014). Optimum temperature for the
growth of bacteria was determined by incubating test cultures at different temperatures (25°C,
30°C, 35°C, 40°C and 45°C). Maximum growth of Pseudomonas aeruginosa was observed at
30˚C.

2.12 Physico-chemical composition of banana peel

i. Moisture content
Different methods are used to remove all the water but chemically bound water: heating in an
oven, use of microwaves or infrared radiation accordingly direct measurement.

% moisture content = …………………………………………………. (2.1)

Here, Minitial and Mdried are the mass of the sample before and after drying, respectively. The
basic principle of this technique is that water has a lower boiling point than the other major
components within foods, e.g., lipids, proteins, carbohydrates and minerals. Sometimes a related
parameter, known as the total solids, is reported as a measure of the moisture content. The total
solids content is a measure of the amount of material remaining after all the water has been
evaporated (APHA 2540 B, 1999).
ii. Total solid
To calculate TS of sample, heating dishes and watch glasses at 105 OC for 1 hour in an oven is
the first step. Then cooling and storing of the dried equipment in a desiccator follows. A 10 to 50
g aliquot of the pulverized sample on a prepared evaporating dish is needed. If the sample is to
be analyzed in duplicate, the mass of the two aliquots may not differ by more than 10%. Each
sample with a watch glass is covered, and weighied (record weight as “Wsample”). Each sample is
spreaded, so that it is evenly distributed over the evaporating dish (APHA 2540 B, 1999).
Finally, the samples is dried at 105 OC for 12 hours, minimum, cool to balance temperature in an
individual desiccator containing fresh desiccant, and weighied.
The residue heated for 1 hour and cooled it to balance temperature in a desiccator, and weighed.
Record the final weight as “Wtotal.”

28
Thus % of total solid= *100

Or

= *1,000,000…………………………………………………… (2.2)

Where: Wdish = Weight of dish (mg),Wsample = Weight of wet sample and dish (mg) ,Wtotal =
Weight of dried residue and dish (mg).
Debabandhya et al., (2010) reported that Total Solids (TS) of the banana peel was 16.5%.in other
ways Divyabharathi, R.et al., (2017) determined Total solids content of banana peel 19%.
iii. Fixed solid/Ash content and volatile solid/ total organic carbon
Fixed solid/Ash content
Fixed solid is calculated by transferring the evaporating dishes containing the dried residues to a
cool muffle furnace. The furnace is heated to 550 OC and ignited for 2 hours (APHA, 1998). The
terminology fixed solid called as also Ash content.

Then % of fixed solid=

Or

……………………………………………… (2.3)

Where:
Wdish=Weight of dish (mg),Wtotal=Weight of dried residue and dish (mg) , and Wvolatile=Weight of
residue and dish after ignition (mg)
Bilba et al., (2007) determined the ash content of banana fruit bunch to be about 20.6 %.
Volatile solid/total organic carbon
The terminology volatile solid called as also Total organic carbon.

Whereas, % of volatile solid= ……………………………………… (2.4)

Where: Wdish = Weight of dish (mg),Wtotal = Weight of dried residue and dish (mg) Wvolatile =
Weight of residue and dish after ignition (mg) and Dish and watch glass prior to use (record
combined weight as “Wdish”)
VS of the banana peel were about 87% (Tumutegyereize et al., 2011).
iv. Total nitrogen content
Available Nitrogen was estimated in the samples by micro Kjeldahl method. To 1 mL of sample,
2-3 mL of 25 per cent KMnO4 solution was added followed by few drops of Conc. H 2SO4. To

29
this 10-15 mL of diacid (H2SO4& HClO3 in the ratio 5:2) was added and digestion was carried
out in a Kjel plus digestion unit. 5 mL each of the digested samples was distilled with 20 to 50
ml of 40 per cent NaOH and the distillate titrated against 0.05 N H2SO4.

V. C/N ratio
Carbon to nitrogen ratio (C/N) is defined as the relative amounts of elemental carbon and
nitrogen present in the substrate. The C: N ratio was found by separately estimating the Total
Organic Carbon (TOC) and Total Kjeldhal Nitrogen (TKN).
vi. Cellulose and lignin content
Cellulose and lignin content of banana peel are constituents of peel those determine fiber
strength of the peel. Banana peels contain up to 15% of cellulose.

30
CHAPTERTHREE

3. MATERIALS AND METHODS

3.1. Study Site

The study area is Addis Ababa city, where consumption of banana is high and which is located
9.0054012 latitude and 38.763611 longitudes and the gps coordinates of 900‟ 19.4436”N and 380
45‟ 48.9996‟‟E.and elevation 2356 meters above sea level. The experiment was conducted in
Ethiopian pulp and paper factory laboratory and Biotechnology and Environmental Engineering
Laboratory of College of Biological and Chemical Engineering at Addis Ababa Science and
Technology University which is located at a latitude of 9°1‟48‟‟ N, longitude of 38°44‟24‟‟E
and an altitude of 2,355 m.a.s.l (Google earth,2019).

3.2 Samples collection

Giant types of banana peels used for this study were randomly collected from market areas,
where solid wastes are collected, around Kality in Addis Ababa. The peels were originated from
Arbaminch zone of the Southern Nations Nationalities and Peoples‟ Regional State (SNNPRS)
of Ethiopia and were characterized for assessing its suitability for pulp production. About 13kgs
of banana peels were collected for this purpose.

3.3 Experimental set up

In this project, seven experiments were conducted by varying the water content (i. e proportion
of peel to water), duration of drying of banana peel and stage of ripeness of banana peel.
Bananas may be divided into seven different degrees of ripeness in accordance with their
external color. The ripening stage was defined in terms of eel color changes from green to
yellow.
Green (color at time of loading), Green with faint hint of yellow (color at time of unloading
More), green than yellow (incipient discoloration of skin), More yellow than green (correct
degree of ripeness), Yellow with green tip (best condition for retail sale), Completely yellow
(fruit appears at its best and is very tasty) and Yellow with brown spots (small brown spots
indicate that the fruit is fully ripe).

31
Parameter characterizations were done in each experiment as per the experimental set up
described below in diagram:

Raw Material preparation

Collection, Washing, Chopping &

drying

Cooking (Digesting)

Filtering and Washing

Bleaching Pulp

Product Characterization

Figure 3.1: Laboratory experimental set up

3.4 Physico-chemical Composition of sample

Laboratory analysis was done in triplicates for physico-chemical parameters following standard
based on the methods described by (APHA 1998); Undestander et al., 1993). The parameters
analyzed were: moisture content, total solids, volatile solids, ash content, total Kjeldahl nitrogen,
cellulose and lignin content.

32
i. Moisture content
To determine percentage of moisture in the samples, 10 g of fresh substrate was dried in an oven
o
at 105 C for 1 hr and reweighed. The moisture content was then calculated according the
formula stated in equation 2.1.

ii. Total solid

First a clean evaporating dish was oven-dried (at 105 OC for 1 hour) in a muffle furnace, cooled in
desiccators and weighed immediately before use. 10g of banana peel was placed on the evaporating
dish and put in an oven (at 105 OC) using a crucible to evaporate for 24 hours. After 24 hours the
crucible was taken out from the oven, cooled in desiccators and weighed. Each of the samples was
done in triplicates. Then, the percentage of TS was calculated using the following formula (APHA
2540 B, 1999). The residue was dried for 1 hour, cooled to balance temperature in a desiccator,
and weighed. Heating, cooling, desiccating, and weighing procedure was repeated until the
weight change is less than 4 % or 50 mg, whichever is less. The final weight was recorded as
“Wtotal”. Total solid value of banana peel was calculated based the formula stated in equation 2.2.
iii. Fixed solid/Total organic carbon and volatile solid/Ash content
Fixed solid/Ash content
Once the TS were determined, the oven dried substrate was ignited at 550 °C in a muffle furnace
for 3 hours and weighed the weight of crucible. The following formula was employed to
calculate the percentage of volatile solids content of the TS (APHA 2540 E, 1999).
Volatile solid/Total organic carbon
The terminology volatile solid called as also Total organic carbon. Finally, fixed solid and
volatile solid content of banana peel sample was determined according to the formula stated in
equation 2.3 and 2.4 respectively.
iv) Determination of Total nitrogen, C/N ratio, Lignin and Cellulose content of sample
Total Kjeldahl Nitrogen (TKN) measures the concentration total of organic nitrogen and
ammonia.it was determined by Kjeldahl method.
In this experiment, the Total Nitrogen content, was determined at Bless Agro industry whereas
,Lignin and Cellulose content of sample was determined at Ethiopian Environmental and Forest
research Institute Laboratory (Addis Ababa).Moisture content, total solid and volatile solid/total

33
organic carbon was conducted at Addis Ababa Science and Technology University
Environmental and Chemical Engineering laboratory. C/N was the ratio of VS to TKN.

3.5 Preparation of raw materials

Initially banana peels were taken and washed several times with water to remove dust and soil
particles before starting the experiment. The peels were cut into 3-4 inches long pieces by using
a chopper. Then the chopped sample was directly dried on the sun for different durations: 48 hrs,
24 hrs, 12 hrs and the final test was done without drying the peel on the sun. The purpose of
drying was to reduce the water content. Seven different experiments were done based on the
variation of the drying time, proportion of water, and ripeness of the banana peel as briefly
described below.

3.5.1 Effect of drying time and peel to water ratio

In the first experiment, 600 g chopped banana peels was dried in the sun for 48 hrs. The banana
peel was set to hot plate then using the proportion of peel to water as 1:3. The ratio of peel to
water was taken based on w/v. because the banana peel was in a solid state and solvent was
liquid. Distilled water was used in each experiment. The sample was cooked for 4 hours and 30
minutes at 90 0C with intermittent stirring. In the process of digestion the strong basic cooking
liquor and the action of heat combine and help breaking the bonds in lignin molecules. The
duration of drying of peel in the sun and temperature range was taken based on the degree of
ripeness of banana peel. The broken lignin molecules dissolved in cooking liquor there by
turning it into dark brown color called as Black liquor and cellulose remaining unaltered was
present in the cooking medium as brown stock along with the traces of lignin. In this experiment,
unripe banana was used and three experiments were done with this ratio. The second experiment
was done using 600 g banana peel which was dried in the sun for 48 hours but the ratio of peel to
water was 1:2. The sample was cooked for 4 hours and 30 minutes at 90 0C with intermittent
stirring of the sample by using hot a plate. In this experiment unripe banana peel was used and
three experiments were done with this ratio. The third experiment was conducted using 600 g
banana peel which was dried in the sun for 24 hours while the ratio of peel to water was as 1:2
and the sample was cooked for 4 hours and 30 minutes at 90 0C with intermittent stirring of the
sample by using hot plate. In this experiment, unripe banana peel was used and the experiments

34
were done in triplicates. The fourth experiment was conducted using 600 g banana peel which
was dried in the sun for 15 hours with the ratio of peel to water as 1:1.5 and the sample was
cooked for 4 hours and 30 minutes at 100 0C with intermittent stirring of the sample by using hot
plate. In this experiment, ripened banana peel was used and the experiments were done in
triplicates.

3.5.2 Effect of surface sizing starch and bleaching chemical

The fifth experiment was conducted using 600 g banana peel which was dried in the sun for 12
hours with the ratio of peel to water as 1:1.5 and the sample was cooked for 4 hours and 30
minutes at 100 0C with intermittent stirring of the sample by using hot plate. In this experiment,
ripened banana peel was used and three experiments were done with this ratio. The sixth
experiment was conducted using 500 g banana peel which was dried in the sun for 6 hours with
the ratio of peel to water as 1:1 and the sample was cooked for 4 hours and 30 minutes at 100 0C
with intermittent stirring of the sample by using hot plate. In this experiment ripened banana peel
was used and three experiments were done with this ratio. The drying of peel on the sun was
used for reducing the water content of banana peel. The water added during digestion was used
for keeping the sample from burning by continuous heat supply. The seventh experiment was
conducted using 400 g banana peel without drying the peel in the sun with the ratio of peel to
water as 1:0.5 and the sample was cooked for 4 hours and 30 minutes at 100 0C with intermittent
stirring of the sample by using hot plate. In this experiment, ripened banana peel was used and
three experiments were done with this ratio. Fifty grams (50g) of starch was used for the
experiments Five, Six and Seven for surface sizing. The samples were meshed with the starch.

3.6 Procedures for parameter characterization

The results of experiment seven (i.e. the experiment where banana peel was not dried on the sun
and peel to water ratio was 1:0.5) were milled to further reduce the size of the fiber and 400 g of
grist was mixed with 23 L of water using a beating machine. The mixture was put in a measuring
cylinder up to 1200 mL and 800 mL of additional water was added. The mixture was inserted in
a disintegrator to further reduce its size. The solution was put in a pot and an additional of 4000
mL of water was added and it was a total of 6000 mL. Using sheet molding machine the fiber
was separated from water. Here a sample of 400 mL,500 mL were taken from the above solution

35
and inserted it in the sheet molding machine to get the filtered fiber. Then the filtered fiber was
o
put in a Sheet press to remove water. Finally, it was put in oven at 100 c for 1 hour to remove
the water content. Parameter characterization was done based on TAPPI and ASTM testing
method (TAPPI, 1996).
Each experiment was triplicated with the same ratio of peel to pulp, duration of drying in the sun
and ripeness of banana peel and the results were recorded.
3.7 Bio-bleaching and smoothing experiment
Procedure for inoculum preparation

Four bacterial isolates which were originally isolated at AASTU Biotechnology laboratories,
from termite gut taken from different area of our country (Harosurupa termite gut (H), Dida
Yabello termite gut (D),Surupa4 termite gut (S4),and Surupa3 termite gut (S3) for their cellulase
enzyme potential (Personal communication and project undergoing).The isolated bacterial strains
were refreshed at 35 0C for 24 hours and then tested on 2 % of CMC containing nutrient agar for
their cellulase enzyme production growing them at 35 0C for 48 hours.(Balamurgan et
al.,2011).Congo red solution (0.1%) was overplayed for 15-30 minutes with intermittent shaking
to detect cellulose production by the bacterial isolates. About 1M of NaCl solution was used to
destain the Congo red dye and holo zones were detected around bacterial colonies producing
cellulase enzyme.

Secondary screening and production of cellulose enzyme

For preparation of standard innoculum, the isolates which showed a maximum zone of cellulase
production were cultured in 400 mL of nutrient broth medium and incubated at 350C for 24
hours. The nutrient broth contains peptic digest, animal tissue, NaCl, beef extract, yeast extract,
pH 7.4 and distilled water (Wenzel M, et al., 2002).

Preparation of crude enzyme

After incubation, the consortia of three selected cellulase producing bacterial isolate cultures
were centrifuged at 380*10 rpm for 20 minutes and supernatant was used as a source of crude
enzyme. The three identified cellulase producing bacteria were taken in common because; the

36
purpose of the study was using the bacteria for bleaching only. The crude enzyme solution was
used for the determination of enzyme activity.
Partial purification of cellullase

Ammonium sulphate precipitation

Hundred (100) mL of the crude enzyme prepared was brought to 75% saturation with solid
ammonium sulphate. The mixture was left overnight at 4 0C in magnetic stirrer. Then the paper
was inserted in 1000 microlitter of crude enzyme using micropipette which precipitated by
ammonium sulphate for 36-48 hours at room temperature. Finally, the bleaching capacity of the
partially purified crude cellulase enzyme was tested by varying temperature and pH.
Consequently, the selected temperatures value were 40 0C, 50 0C,60 0C, 70 0C,keeping pH at 7.4
and the selected pH values were 5.0,6.0,7.0 and 8.0,keeping temperature at 350C.The pH of the
medium was adjusted with the help of NaOH and HCl (0.2 N). By taking 0.05 mL of inoculum
in each beaker, the samples were incubated and the observation was recorded in 24 hours for
both parameters.

Figure 3.2: pH and Termperature optimization of crude cellulase enzyme for biobleaching
activity

In figure 3.2 (sample preparation for bio bleaching), the innoculum were covered to protect the
sample from contamination. piece of sheet of banana peel pulp was inserted in each beaker
adjusted at different tempreture and pH and the result was observed in 24 hours.

37
CHAPTER FOUR

4. RESULTS AND DISCUSSION

4.1 Physico-Chemical properties of Samples

Laboratory analysis was done in triplicates for physico-chemical parameters of banana peel
sample. Parameters analyzed were: moisture content, total solids, volatile solids, ash content,
total Kjeldahl nitrogen, cellulose,and lignin content as described briefly in table 4.1below
Table 4.1: Physico-chemical characteristics of banana peel sample

Parameters Previous studies This study

Moisture content (%) 6.70±02.22(Anonymous, et al; 2001, 2005 and 9.3±2.63
Ranzani, et al; 1996)
Ash content (%) 8.50±1.52(Anonymous, et al; 2001, 2005 and 10±1.14
Ranzani, et al; 1996)
TS (%) 19 (Divyabharathi, R.et al.,2017) 22
VS (%) 77.6 (Divyabharathi, R.et al.,2017) 88.6
TKN (%) 1.20 ± 0.09(Undestanderet.al., 1993) 1.94±0.02

C/N ratio 29.03 (Divyabharathi, R.et al.,2017) 45.6

Lignin content (%) 13.97 ± 0.02(Undestanderet.al.,1993) 10.45
Cellulose content (%) 13.09 ± 0.09(Undestanderet.al. 1993) 9.072

(TS = Total Solids; VS= Volatile solids; C:N ratio=Carbon to Nitrogen ratio; TKN= Total
Kjeldahl Nitrogen)
As it can be seen from table 4.1, Total Solids (TS) of the banana peel was about 22% which was
higher than the value reported by (Divyabharathi, R.et al.,2017) of about 19%. On the other
hand, the banana peel waste was also lignocellulosic comprising; cellulose 9.072 %, lignin
10.45% which was lower than the value estimated by (Undestanderet.al.,1993).The C: N ratio
was found by separately estimating the Total Organic Carbon (TOC) and Total Kjeldhal
Nitrogen (TKN).

38
4.2 Effect of drying time and temperature

In experiment one, the extracted pulp was found to be too stiff, not stapled, and out of the
expected standard quality. Since the fiber was not strong, the pulp was not used for any purpose
and the color was black. Similarly, in experiment two, it was observed as useless product. Here,
the color of the pulp was black. Thus, in both experiment one and two the pulp attempted to be
produced were almost of the same quality with low standards. In both, the cellulose of unripe
banana peel was low content. The average moisture content of pulp of experiment one and two
were 13.29 % and 13.0 % respectively. The cooking temperature was 90 0C. Thus, the pulp
extracted in both experiments was not good enough to further characterize the basic pulp
properties.

In experiment three, the pulp was cracked, thin and color was found to be slightly improved.
Since it is too fragile and cracked, it was not used for any purposes. Here, unripe banana peel
was used with cooking temperature of 90 0C. Consequently, it was impossible to characterize
basic pulp properties of experiment three as that of experiment one and two. The ash content of
each of the three experiments was greater than 10%, which indicates that the less the cellulose
content, the more the ash content it has.

In experiment four, the extracted pulp was found to be thicker. It was cracked towards the
middle. The pulp was still not usable under these conditions. However, the moisture content and
the ash content of the pulp in this experiment were found to be better compared to the previous
results in experiments 1 to 3. The cooking temperature was 100 OC. Although experiment four
showed decreased moisture and ash contents compared to the previous results, the pulp of
experiments one to four, in general, were not good enough to be selected for use as a pulp.
Because only the banana peel and water was used as a raw material in these experiments, there
were other ingredients should be considered that strengthen fibers of the peels and bleaching
agents to remove black color of the pulp.

39
 (a) (b)

Figure 4.1: Representative results of experiment one and three when peel to water ratio varies as
a) 1:3 and b) 1:2

4.3 Effect of surface sizing starch and bleaching chemical

In these experiments, surface sizing starch and bleaching chemical was used. The pulp of
experiment five and six was better compared to the previous results. The fiber of pulp was
interconnected since the starch was used and also the color of the pulp was better because CaO
was used as a bleaching agent. 20 g of CaO and 50 g of surface sizing starch was used for
experiment five. Ripened banana peel was used and the cooking temperature was 100 OC. since
the peel to water ratio was reduced, the moisture content was better and the ash content of the
pulp was better than the previous results. Similarly the pulp of experiment six was better.it was
attractive. The fiber of both results was better, since surface sizing starch was used. 25 g of CaO
and 50 g of surface sizing starch was used. Here it was possible to characterize basic pulp
properties. The more the peel to water ratio increased, the less the moisture content the pulp has.

40
 (a) (b)

Figure 4.2: Result of experiment when peel to water ratio was a) 1:1.5 and b) 1:1

The pulp of the experiments seven were better than all laboratory test tried so far. The lignin (the
source of black color to the pulp) was removed and the pulp was bright and attractive. The
cooking temperature was 100 OC and ripened banana peel was used. Unlike the previous tests,
the peel was not dried in the sun. This made the pulp whiter. The peel to water ratio was so
reduced that the moisture content of the product was better. The ratio of peel to water was 1:0.5.
Ash content was 8.0 % and 50 g of starch and 25 g of CaO was used.

Generally, the experiment where starch was used was better. Because the parameter
characterization was done in those experiments, indicating that starch was very essential in
interconnecting the fiber of a pulp and smoothness of the product as well. Tensile strength, tear
factor, burst strength and breaking length were the essential characteristics determining the
quality of pulp.

41
Figure 4.3: Result of experiment when peel to water ratio was 1:0.5

42
4.4 Effects of the parameters interaction

Table 4.2: Pulp production yield from different ratio of water to peel, duration of drying and
ripeness of banana peel

Run Factor 1 Factor 2 Factor 3 Facto Facto Factor 6 Product Parameter Characterization
r4 r5
A=peel B=duratio C=Ripe D=Te E= F= Responses
to water n of ness of mper Starc Calcium
drying Banana ature h Oxide
peel
Fiber Ash Tens Tear Burst
Freene cont ile facto
ss ent stren r
gth

O
Ratio Hour Month C Gra Gram(g mL % Kg/ - Kg/cm
2
m(g) ) Nm
100
1:1.5 12 ripened 50 20 24±0. 7±0. 1.7± 103 1.8±0.
8 6 0.0 ±3 1
100
1:1 6 ripened 50 25 27±0. 6±0. 1.8± 106 1.8±0.
04 3 0.0 ±0.0 01
5
100
1:0.5 0 ripened 50 25 28.2± 6±0. 1.8± 108 1.9±0.
0.03 01 0.02 ±0.1 02

From table 4.2, parameter characterization was performed in experiments where bleaching
chemical (CaO) and surface sizing starch was used. Tensile strength, tear factor and burst
strength were analyzed after the pulp was changed into sheet.

43
The results of product characterization using ANOVA design expert are described below

i. Ash content

DESIGN-EXPERT Plot

Ash content
X = A: Peel :water ratio
Y = B: Duration of drying

9.7

9.375

9.05

8.725
Ash content

8.4

12.00
2.00
9.00
1.67
6.00
1.34
B: Duration of drying
3.00 1.00
A: Peel :water ratio
0.00 0.67

Figure 4.4: 3D plot of duration of drying of banana peel interaction with peel to water ratio on
ash content of the product.

From figure 4.4 it was shown that, as the peel to water ratio increased, the moisture content
decreased. The decrease in moisture content results in decrease in ash content. The fiber strength
increased and the ash content decreased from 10 % to 8.5%. The ash content constituent of
typical pulp is 6-8 % (ASTM).

44
ii. Tear factor

Tear strength (internal tearing resistance) is a measure of the toughness of a paper, the ability to
withstand physical shock. This testing method is not particularly useful as a measure of chemical
stability. However, it does give a better indication of brittleness.

DESIGN-EXPERT Plot

Tear factor
X = A: Peel :w ater ratio
Y = B: Duration of drying

108

105.587

103.173

100.76
Tear factor

98.347

12.00
2.00
9.00
1.67
6.00
1.34
B: Duration of drying
3.00 1.00
A: Peel :w ater ratio
0.00 0.67

Figure 4.5: 3D plot of duration of drying of banana peel interaction with peel to water ratio on
tearing resistance of the product.

45
From figure 4.5 above, it was shown that as duration of banana peel drying in the sun decreased
from 12 hour to no drying time, the fiber strength increased with decreasing of moisture content
and resulted in increasing of tear factor of the pulp to the maximum (i.e.108).

The maximum tear factor value was recorded when the peel was not dried in the sun which was
equal with the pulp standard value of tearing resistance of pulp fiber.

iii. Burst strength

Bursting strength is the maximum pressure that the paper can resist without breaking with
pressure applied perpendicular to the plane of the test piece. The burst strength is influenced by
many factors in papermaking, such as the fiber type, degree of refining, presence of strength
additives (e.g. Starch), sheet formation, and moisture content.

DESIGN-EXPERT Plot

Burst
X = A: Peel :water ratio
Y = B: Duration of drying

1.9

1.83875

1.7775

1.71625
Burst

1.655

12.00
2.00
9.00
1.67
6.00
1.34
B: Duration of drying
3.00 1.00
A: Peel :water ratio
0.00 0.67

Figure 4.6: 3D plot of duration of drying of banana peel interaction with peel to water ratio on
burst strength of the product.

46
From figure 4.6, we can conclude that as the peel to water ratio decreased (i.e. from 1:1 to 1:0.5),
the fiber of the pulp is more concentrated and interrelated that the breaking length of the pulp
increased. Strength properties of hand sheets were tested according to TAPPI test methods.

iv. Tensile strength

Tensile strength, the maximum tensile force developed in a test specimen before rupture on a
tensile test carried to rupture under prescribed conditions. Tensile strength (as used here) is the
force per unit width of test specimen.

DESIGN-EXPERT Plot

Tensile strength
X = A: Peel :w ater ratio
Y = B: Duration of drying

1.82

1.7826

1.7452

1.7078
Tensile strength

1.6704

12.00
2.00
9.00
1.67
6.00
1.34
B: Duration of drying
3.00 1.00
A: Peel :w ater ratio
0.00 0.67

Figure 4.7: 3D plot of duration of drying of banana peel interaction with peel to water ratio on
tensile strength of the product.

From figure 4.7 as the moisture content decreased, the tensile strength of the pulp increased.

47
4.5 Screening the isolates for production of extracellular enzymes

Bacteria were successfully isolated and screened from the gut of termites on the basis of
efficiency to degrade cellulose. Among 4 isolates three of them (H, S3, S4) were more efficient.
They made clean zone i.e. they were detected as cellulose degrading bacteria.

Figure 4.8: Screening of cellulolytic bacteria by covering the petri dishes with Congo red dye. A
zone of clearance surrounding a colony is indicative of carboxymethylcellulose (CMC)
hydrolysis by secreted Cellulasase enzyme.

4.6 Effect of pH and Temperature on the growth of the cellulose degrading

bacteria

4.6.1 Growth at different pH

The various ranges of pH from 5.0 to 8.0 were tested for the removal of lignin from banana peel
pulp using bacterial cellulase enzyme. Consequently, it was found that there were significant
changes in enzyme activity with change in pH. Accordingly, the maximum bleaching capacity of
CDB was recorded at pH 6.0 followed by pH 7.0.The least pH was 5.0 (Figure 4.9). This
indicates that the appropriate condition for the growth of the microorganism was around slightly
acidic media and neutral. However, according to the previous study made by Bajaj et al., (2009),
the isolation and identification of cellulose degrading bacteria from soil sample, the highest
activity was recorded at pH 5.5 suggesting that the enzyme was an acid cellulase. Acid cellulases
act at a pH range of 3.8 and 5.8 (Mosjov, 2012; Balamurgan et al., 2011). Optimum pH values of

48
4.5-8.0 have also been reported for different microbial cellulases (Immanuel et al., 2007; Dutta et
al., 2008) for various purposes. In this study, the cellulase enzyme that was partially extracted
and characterized from termite gut bacteria was potentially used for banana peel - derived pulp
for bleaching purposes.

Figure 4.9: Bleached pulps with different pH ranges

49
4.6.2 Growth at different temperature

Bacteria, as a group of organisms, exist over a wide range of temperatures. However, individual
species can only exist within a narrower spectrum of temperatures as it normally influences the
rate of chemical reactions through its action on cellular enzymes (Cappuccino and Sherman,
2002). The aim of the experiment was to determine the optimum temperature requirements for
growth of the isolates. The temperature ranges between 40 °C, 50 °C, 60 °C and 70 °C were
tested for growth of cellulose degradation bacteria, while the maximum bleaching capacity of
CDB was observed at 40°C and followed by 50°C. As shown in Figures 4.10, there was lower
growth of microorganism at temperatures of 60 °C and 70 °C, which made the bleaching
capacity of the CDB also very low. Further increase in temperature declined the enzyme activity.
This indicates that the cellulose degrading bacteria was thermophilic. As described in procedure
described by (Bajaj et al., 2009), there was a significant change in enzyme activity with change
o
in temperature and optimum temperature for activity was recorded at 60 C during isolation and
characterization of cellulolytic bacteria from soil using Bacillus licheniformis. In this study, the
cellulose degrading bacteria from termite gut displayed maximum growth at 40 °C (Figure 4.10).

50
Figure 4.10: Bleached pulps with different temperature ranges.

In this experiment, the effect of pH and temperature was similar to previous studies on activity of
cellulose (Balamurgan et al., 2011; Chung et al, 2009).Based on the conducted experiment
result, it can be concluded that the optimum temperature and pH condition for bacterial growth
varied depending on the bacterial species. Cellulolytic potential of termite gut bacteria for pulp
bleaching was efficiently explored and could be effectively used in the removal of lignin.
In general, temperature profiles of isolates showed that the temperature ranges from 40 °C to 50
°C could be optimal for the growth of cellulose degrading bacteria and the results clearly
indicated that the optimum pH for the growth of cellulose degradation bacteria (CDB) was 6.0-
7.0.

51
CHAPTER FIVE

5. CONCLUSION AND RECOMMENDATION

5.1. Conclusion

From this thesis work, it can be concluded that banana fiber can be a potential raw material for
making pulp in simple processing steps.

The active ingredients of pulp production from banana peel are starch and bleaching chemicals.
We can get these chemicals easily and the basic pulp characterization can be analyzed in
laboratory. Starch is the basic material for the strength of banana fiber.

The source of the black color of the pulp is its lignin content. The cooking temperature range for
cooking the banana peel was 900C-1000C. It can be concluded that the peel to water ratio and
duration of drying time are factors determining the quality of pulp.

In the process of bio-bleaching, micro-organisms play a fundamental role in protecting the

environment.

Isolation of the bacteria from termite proved the presence these isolates inside gut system and
screening the isolates for cellulolytic and hemi cellulolytic activities confirmed their role in
cellulose digestion. From the functional tests carried to determine the optimal conditions for
cellulolytic activity, the isolate„s enzyme activity (bleaching capacity) was found to be high over
a temperature of 40 °C among the selected temperatures. Similarly, enzyme activity was found to
be high at the pH 6.0 of the selected pH values suggesting that the isolate bears around slightly
acidic and neutral media.

The studies further confirmed the potential of bacteria from termite gut for bleaching of banana
peel pulps by using chemical and biological methods. Based on the results, more bleached pulp
was achieved with chemical methods but, in the environmental protection point of view, bio-
bleaching is preferable.

52
5.2 Recommendations

The ever growing population is exploiting nature‟s resources to the maximum. The green cover
of the earth is declining drastically and suitable substitutes for wood like banana peel will go a
long way in healing the world. So, I recommend further detail research in converting banana
fiber into pulp and paper to produce quality product and environmental protection as well.

The cellulose degrading bacteria used in this thesis work was directly taken from biotechnology
laboratory of Addis Ababa science and Technology University. But it is better if sample
collection is done from the field and different cellulose degrading bacteria from different sources
should be identified.

Banana peel properties and parameters used for pulp quality should be further studied.

The investigation of bio bleaching was done at temperature ranges of (40 - 70oC) but it should be
investigated at other temperature ranges (for example Cryophilic (< 20 0C) to investigate more
about the optimum temperature for growth of cellulose degrading bacteria.
The cellulose degrading bacteria used in this thesis displayed highest growth at pH 6.0 from the
tested pH ranges of (pH 5.0 -8.0). Based on this result, I recommend investigation of more pH
ranges to further investigation of the bacterial optimum condition.

Bio-bleaching was done in this thesis by using cellulose degrading bacteria from termite gut.
Based on the result; I recommend pulp bleaching by using enzymes to protect the environment
from pollution.

53
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APPENDICES
Appendix A: ANOVA design result

Response Response Response Response

Factor 1 Factor 2 1 2 Response 3 4 5
A:Peel :water B:Duration of Fiber Ash Tensile Tear
Block ratio drying Freeness content strength factor Burst
2 2
Numeric Hr mL % N/m - kg/cm
Block
1 0.67 0 23.8 9.3 1.82 107.3 1.9
Block
1 0.39 6
Block
1 0.67 12 24.9 9.7 1.72 100 1.8
Block
1 2 0 25.3 8.4 1.8 108 1.9
Block
1 2 12 25 8.8 1.8 108 1.82
Block
1 1.34 -2.49
Block
1 2.28 6
Block
1 1.34 6 26.1 9 1.7 101 1.7
Block
1 1.34 6 26.1 9 1.7 101 1.7
Block
1 1.34 14.49

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Appendix B: Photos during lab works

Raw material (banana peel with different degree of ripennes)

Surface sizing starch

62
Cooking process

Bleaching of pulp

63
Beating machine

64
Sheet making process

65
Product characterization

66