ABSTRACT
In this work, the bioconversion of Citrus sinensis peels for the production of ethanol using Sacchromyces cerevisae underwent fermentation process for eleven days. The foliowing parameters such as Total solids, pH, Ttratables acidity, Temperature, Specific gravity, Biomass, and Alcohol were observed. The values of the following parameters were 3.77 pH , 1.473%, titratable acidity, 0.72% sugar, 331.5oC temperature, 410.6mg/l Biomass, 1.011 Specific gravity and 9.32 % v/v alcohol. The parameters were checked during fermentation period and different changes also occurred starting from the day zero. This work shows that Citrus sinensis peels are very useful wastes used for biotechnological application for maximum yield of ethanol. The result of the experiment conducted shows that Citrus sinensis is a potential substrate which can be exploited in industries tor ethanol production in a commercial scale as they are cheap and importantly renewable.
TABLE OF CONTENTS
Tile
page i
Certification ii
Dedication iii
Acknowledgement
iv
Table
of Contents v
List
of Tables vii
List
of Figures viii
Abstract
ix
CHAPTER ONE
1.0 INTRODUCTION
1.1 FERMENTATION 1
1.2 Types of Fermentation 2
1.2.1 Lactic acid fermentation 2
1.2.2 Alcoholic fermentation 3
1.2.3 Solid State Fermentation 3
1.2.4 Ethanol fermentation 4
1.2.5 Propionate fermentation 4
1.2.6 Homoacetate fermentation 5
1.2.7 Mixed acid and butanediol fermentation 6
1.3 Aim and Objectives 8
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Citrus fruits 9
2.2 Citrus
sinesis 9
2.2.1 Citrus
sinesis peels 10
2.3 Yeast 11
2.4 Bioethanol 13
2.4.1 Ethanol production technologies 14
2.4.2 Production of bioethanol 15
2.4.3 Microorganisms for ethanol production 16
2.5 Ethanol as a Fuel 16
2.6 Environmental Impact of Bioethanol
Productions 17
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1
Sources of materials 19
3.2
Sample preparation 19
3.3
Preparation of Fungi Inoculums
Aspergillus niger 19
3.4 Preparation of yeast moculum (palm wine
yeast) 20
3.5 Media preparation
20
3.6 Enzymatic hydrolysis of orange peels 21
3.7
Optimization of the hydrolysate 21
3.8.
Saccharification for bioconversion to
produce ethanol 22
3.9.
Method of analysis 22
3.9.1
Determination of pH 22
3.9.2
Determination of titrable acidity 22
3.9.3
Determination of total solid 23
3.9.4
Determination of sugar content 23
3.9.5
Determination of temperature 24
3.9.6 Determination of specific gravity 25
3.9.7
Determination of ethanol content 25
3.9.8
Determination of biomass
26
3.9.1 Statistical Analysis 27
CHAPTER FOUR
RESULT 28
CHAPTER FIVE
5.0
DISCUSSION 38
5.1
CONCLUSION 38
REFERENCES
LIST OF TABLE
Table
Title Page
4.1: Changes
of properties of fermented Citrus sinensis peels 29
LIST OF FIGURE
Figures
Title Page
1 Generalized pathway for the production
of some fermentation
products
from glucose to various organisms represented by letter A to G 7
2 Changes
in pH of Ethanol 30
3 Changes
in Titratable acidity 31
4 Changes in Total solid in Ethanol 32
5
Changes in Specific gravity of
Ethanol 33
6
Changes in % Sugar of Ethanol 34
7
Changes in %alcohol (v/v) in
Ethanol 35
8
Changes in Biomass mg/L 36
9 Changes in Temperature (OC)
37
CHAPTER ONE
1.0 INTRODUCTION
1.1 FERMENTATION
Fermentation is the process of anaerobic or partial
anaerobic oxidation of carbohydrates to produce intermediate substrate (organic
acid, ethanol etc) with the release of carbondioxide and water (Prescott et al., 2005).
The fermentation process occurs naturally in many
foods and humans have naturally used it sense ancient time to improve both the
preservation and organoleptic properties of food (Invi et al., 2010). In developed and less technically developed
countries; fermentation is one of the important techniques employed to extend
the shell life of raw food materials while in the technically advanced
countries, it is used more to develop and add flavor to variety of diet
(Abd Elmoneom et al., 2005, Achi, 2005and Isabel et al., 2005).
Fermentation is also an anaerobic redox process, in
which the oxidation of the substrate is coupled to the reduction of another
substrate or an intermediate derived from the oxidation with the difference in
redox potential of the substrate and the end product providing energy for ATP
synthesis.
In fermentation, the substrate is only partly oxidized
and a small amount of energy stored in the substrate is condensed. In most
fermentation organisms, ATP is produced only by substrate level
phosphorylation, but there are also a few example of an additional
ion-gradient-driven phosphorylation, the ion gradient is either a proton or a
sodium ion gradient and is generated by electron transport (Dimroth, 1997).
Patril and Dayanand (2006) reported that the period of
fermentation depends upon the nature of medium; fermenting organisms, concentration
of nutrient and the process physiological conditions.
Fermentation is also used in a broader sense for the
intentional use of microorganisms such as bacteria, yeast and fungi to make
products useful to humans (biomass, enzymes, primary and secondary metabolites,
recombinants products of biotransformation on an industrial scale.
1.2 Types of Fermentation
There are different types
of fermentation which include the following.
1.2.1 Lactic acid fermentation
In lactic acid fermentation, lactate is a common end
product of fermentation and the large amount of lactation as lactic acid
bacteria. The lactic acid bacteria are nutritionally very versatile and grow
not only on glucose but also on other substrate such as fructose, galatose,
certain variations of fermentation pathways occur. For example, pentoses are
fermented by facultative homo fermentative organisms through the
phosphaketolase pathelectron. Citrate is an ingredient of milk, is converted to
diacetyl, the typical flavor of butter (Kandler, 1983). Lactic acid is mostly
used in food and pharmaceutical processes is produced by homofermentative
lactic acid bacteria such as Lacto
bacillus dehbruckii strains.
Lactic acid bacteria are sub-divided according to
their fermentative products. The homo fermentative species produce a single end
product, lactic acid according to the equation below.
glucose ®2
lactate
Whereas, the hetero fermentative species produce other
compounds mostly ethanol and carbondioxide along with lactate in the equation
below.
glucose ®CO2
+ lactate + ethanol
1.2.2 Alcoholic fermentation
This is generally produced by yeast of kluyureromyces and
saccharomyces families which are respectively spire farming and non-spore
formily. It is produced at the expense of the hexoses and hexobises, all of
which are transferred into glucose – 6 – phosphate to enter the cycle of
anaerobic glycosis.
C6H12O6 ®2CH3
– CH2OH + 2CO2
(Alais and Linden., 1999)
Alcoholic fermentation by Zymomonas species is not through glycosis but the Enter-Doudoroff
pathway which leads to 2 mol pyruvate per mol glucose.
1.2.3 Solid State Fermentation
This technique are developed in the Eastern countries
which it has been used for centuries for the production of traditional foods
such as Soy Science, Koji, Mise or Sake using different substrate and
microorganism. Solid state fermentation is used for systems where organisms are
cultured on the surface of a concentrated water insoluble substrate (usually
containing polysaccharide as a carbon and energy source with a low level of
free water (Bellon-Murel et al., 2003).
1.2.4 Ethanol fermentation
Ethanol is the major end product of the anaerobic
metabolism of yeast but also of Zymomoas species in both ethanol is fermented
according to
Glucose®
2CO2 + 2 ethanol
Ethanol fermentation by yeast is an ancient process
used by humans to produce alcohol beverages. Yeast ferment glucose by way of
glycosis to pyruvate, which is decarboxylated to acetyaldehyde and
carbondioxide.
This reaction is catalysed by pyruvate decarboxylase
key enzyme of alcohol fermentation by yeast (Bock and Sawers, 1996).
Ethanol is used as a raw material in the chemical
industry for various purposes and as an additive to feel. Therefore, for
ethanol production on an industrial scale yeast strains have been selected for
features such as higher yield and glucose.
1.2.5 Propionate fermentation
Propionate is a major end product of various
fermentation and many bacteria convert glucose to a mixture of propionate,
acetate and carbondioxide. However, most propionic acid bacteria are also able
to fervent the end product of lactic acid fermentation, lactate to propionate.
There are two pathways for propionate format from lactate, both of which have
the same fermentative equation.
3 lactate ®
2 propionate + acetate + CO2
The acrylate pathway is the first pathway carried out
by Clostridum propionicum consist of
an oxidative and a reductive branch, 1 and lactate is oxidized to acetate this
giving rise to CO2, 1 mol ATA and four reducing equivalent . The electrons are
fed into the reductive branch in which lactate is activated by a COA transfer
the lacty-COA is dehydrated to acryloyl – COA and then reduced to a propionyl –
COA (Ljungdahl, 1999). The second pathway is methylmalonyl-COA which is carried
out by the propionic acid bacteria. Again, 1 mol lactate is oxidized to acetate
giving rise to ATP but also to reducing equivalent. The electrons are fed into
the reductive branch which is very interesting from a biochemical point of view
since it contains a number of unusual enzyme such as COA trnaferases, a
transcarboxylase and a B12-containig enzyme (Ljungdahl, 1994).
1.2.6 Homoacetate fermentation
Acetate is an end product of many fermentation but
only a few microorganisms such as Moorella
thermoacetica ferment organic compounds exclusively to acetate according to
Glucose 
3 acetate
Hexose conversion is by way of glycolysis to pyruvate
which is then converted to acetyl-CoA, carbondioxide and reduced ferredoxin by
pyruvate. The carbondioxide formed is then reduced through the acetyl-CoA or
Wood-Ljungdahl pathway. The monoxide is derived from the reduction of the
second mole of carbondioxide catalysed by carbon monoxide dehydrogenase
activity of the acetyl-CoA synthase. The net production of ATP by substrate
level phosphorylation is only two. However, in addition to substrate level
phosphorylation the acetyl-CoA pathway is coupled to ion-gradient-driven
phosphorylation and with respect to their energy metabolism homoacetogens can
abe divided into two groups; the proton and sodium ion organisms. In M. thermoacetica, a proton motive force
is established most probably by electron transport to methylene-THF
(tetrahydrofolate) (Ljungdahl,., 1994).
1.2.7 Mixed acid and butanediol fermentation
Mixed acid and butanediol fermentation is carried out
by the facultative anaerobic Enterobacteria (Bock and Sewers, 1996). Members of
the genera such as Salmonella,
Escherichia, Citrobacter, Shigella and
Proteus ferment glucose to a mixture of acids (acetic, lactic and formic
acids), carbondioxide and some ethanol. In the mixed acid fermentation, glucose
is converted by way of glycolysis. The fate of pyruvate is a reduction to
lactate by the action of lactate dehydrogenase, a reduction of succinate after
carboxylation to oxaloacetate and a cleavage to acetyl-CoA and formate by
pyruvate.
During butanediol fermentation, fewer acids are formed
by pyruvate. Instead two molecules of pyruvate are condensed under
decarboxylation to α-acetolactate, this reaction is catalysed by α-acetolactate
synthase. Since butanediol formation is coupled to two decarboxylation
reactions, butanediol fermenters produce much more gas than do mixed acid
fermenters.
Fig. 1: Generalized pathway for the production of some
fermentation products from glucose to various organisms represented by letter A
to G
Source: Alais and Lindem, (1999)
A – Homofermentation lactic
B – Heterofermentation lactic
C and D – propionibacteria
E – Saccharomyces
spp
F – Acetobacter
spp
G – Acetobacter
spp
1.3 Aim and Objectives
1. To
produce ethanol from orange peels
2. To
produce biomass from orange peels using S.
cerevisae
3. To
monitor the rate at which S. cerevisae utility
the available sugar present in orange peels to produce alcohol.
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