PRODUCTION OF ETHANOL FROM FRUITS WASTE

A Process Engineering Report

Presented to the

Department of Chemical Engineering

Faculty of Mechanical and Chemical Engineering

College of Engineering

Kwame Nkrumah University of Science and Technology, Kumasi

By

AGYAPONG PEREGRINO ABU

AMEHOHO JOSBERT

MENSAH DERRICK

OKAIJA-WELBECK CALEB

In Partial Fulfillment of the Requirements

 for the course

Process Engineering Project

December, 2019

©
 ACKNOWLEDGEMENT

To the Almighty God giver of life, wisdom and understanding be all the glory and

praise.

We sincerely acknowledge the able supervision of our project supervisor, Dr. M.Y

Mensah and assisted by Mr. Ato Fanyin-Martin whose counsel and supervision helped

us through this semester’s project work

Our additional thanks also goes to all the Lab Technicians in the department and any

other person who contributed to the success of this project work; not forgetting the

departmental authorities who brought this whole idea of third year project. We say

thank you all.

A special thanks goes to the lab manager, Mr. Emmanuel Awarikabey and his

assistants for providing us with the laboratory equipments needed for this project.
 Table of Contents

ACKNOWLEDGEMENT................................................................................................ii

LIST OF TABLES..........................................................................................................vii

CHAPTER ONE..............................................................................................................10

INTRODUCTION...........................................................................................................10

Objective..........................................................................................................................15

CHAPTER TWO.............................................................................................................17

2.0. REVIEW OF LITERATURE...................................................................................17

2.1 Brief history of ethanol production........................................................................17

2.1.1 Ethanol and its characteristics.........................................................................18

2.2 Worldwide status of bioethanol production...........................................................18

2.2.1 Ethanol Production in the World.....................................................................18

2.2.1 Ethanol Production in Ghana..........................................................................19

2.3 Bioethanol feedstocks................................................................................................20

2.3.1 First generation feedstocks..............................................................................21

2.3.2 Second generation feedstocks.........................................................................23

2.3.3 Third generation feedstocks............................................................................25

2.3.4 Fourth generation feedstocks..........................................................................26

2.4 Lignocelluloses materials......................................................................................27

Table 2.1 Polymer composition of lignocellulosic biomass........................................28

 2.4.1 Cellulose.............................................................................................................28

2.4.2 Hemicellulose.....................................................................................................30

2.4.3 Lignin..................................................................................................................31

2.5 Fruits and Fruit Wastes..........................................................................................31

2.5.1 Fruits...............................................................................................................31

Composition of Mango................................................................................................36

PINEAPPLE................................................................................................................36

Characterization of Pineapple......................................................................................37

2.5.2 Fruit composition............................................................................................39

2.5.3 Fruit waste.......................................................................................................39

2.5.4 Fruit wastes as a source of bioethanol.............................................................39

2.6 Process in Ethanol Production...............................................................................41

2.6.1 Pretreatment....................................................................................................42

2.6.2 Hydrolysis...........................................................................................................43

2.6.2.2 Enzymatic Hydrolysis..................................................................................44

2.6.2.4 Advantages of Enzymatic Hydrolysis over Acidic Hydrolysis....................48

2.6.2.5 Factors that influence Enzymatic Hydrolysis..............................................48

2.6.3 Fermentation Process......................................................................................49

2.6.4 Factors affecting bioethanol production..........................................................54

2.6.5 Distillation and Purification............................................................................56

CHAPTER THREE.........................................................................................................57

3.0 METHODOLOGY....................................................................................................57
 3.1 Chemicals and Equipment.....................................................................................57

3.1.1 Chemicals........................................................................................................57

3.1.2 Materials used.....................................................................................................57

3.1.3 Equipment.......................................................................................................58

3.2 Sampling and Sample preparation.........................................................................59

3.2.1 Substrate Preparation......................................................................................60

3.3 Proximate and Ultimate Analysis..............................................................................61

3.3.1 Determination of moisture content..................................................................61

Convection oven method.............................................................................................62

3.3.2 Determination of crude fibre...........................................................................64

3.3.5 Analytical techniques......................................................................................64

3.4 Enzymatic hydrolysis of lignocellulosic substrate.................................................65

3.4.1 Reducing Sugars Determination using PAHBAH..............................................66

Reducing Sugars Determination using HPLC.............................................................67

3.5 Fermentation..............................................................................................................68

Apparatus and Materials..............................................................................................68

Reagent........................................................................................................................70

Procedure.....................................................................................................................70

REFERENCE..................................................................................................................73

APPENDIX.....................................................................................................................74

APPENDIX A..............................................................................................................74

Abbreviations...........................................................................................................74
 Meaning of some terms............................................................................................75

APPENDIX B..............................................................................................................77

MEASURED PARAMETERS................................................................................77
 LIST OF TABLES

Table 2.1 Polymer composition of lignocellulosic biomass............................................28

Table 2.2 Varieties of Mangoes Grown in Ghana and Percentage Area Cultivated.......34

Table 3.1; materials used.................................................................................................57

Table 4.1 Composition of fruit waste blend based on waste generation from Blue

Skies..............................................................Error! Bookmark not defined.

Table 4.2 Moisture content Table and Calculation..........Error! Bookmark not defined.

Table 4.3 Hydrolysis conditions for fruit waste..............Error! Bookmark not defined.

Table 4.4 Masses of Different Substrate Concentrations Error! Bookmark not defined.

Table 4.5 Results for PAHBAH Test For Reducing Sugar..........Error! Bookmark not

defined.

Table 4.6 Sugars Detected in HPLC................................Error! Bookmark not defined.

Table 4.7 Total sugar table.............................................Error! Bookmark not defined.

LIST OF FIGURES

Figure 1 Ethanol producing plant in Brazil.....................Error! Bookmark not defined.

Figure 4. Caltech Cassava Processing Plants for Ethanol Production............................20

Figure 5 Bioethanol from first generation feedstocks.....................................................23

Figure 6 General outline of the lignocellulose to bioethanol production process...........25

Figure 7 Structure of Cellulose molecule........................................................................29

Figure 8 Repeating units of hemicelluloses (Source: Scheller and Ulvskov, 2010).......30

Figure 9 Mango fruits.....................................................................................................33

Figure 11 Smooth Cayenne pineapple.............................................................................37

Figure 12 Sugar Loaf Pineapple......................................................................................37

Figure 13 Images of some varieties of pawpaw..............................................................38

Figure 14 A flowchart of Sugarcane conversion to Ethanol...........................................42

Figure 15 Procedural mechanistic action of all three cellulases on the cellulose

polymer.............................................................................................................47

Figure 16 blended sample of pawpaw, mango and pineapple peels in a zip-lock bag....61

Figure 17 some samples of blend in incubated shaker during hydrolysis.......................65

Figure 18 samples to be analysed in UV/VIS spectrophotometer...................................67

Figure 19 samples in vial and racks to be analyse by HPLC device...............................68

Figure 20 HPLC device to analyse fermentation products..............................................71

Figure 21 Plot of total sugars of different substrate concentration with time..........Error!

Bookmark not defined.

Figure 22A graph of hydrolysed sugars using HPLC at 24-hours of hydrolysis.....Error!

Bookmark not defined.

Figure 23 Plot of fermentation products after hydrolysisError! Bookmark not defined.

CHAPTER ONE

INTRODUCTION

Biofuels are renewable energy sources made from organic matter or waste that

can play a valuable role in reducing carbon dioxide (CO2) emissions. Biofuels are one of

the largest sources of renewable energy in use today. In the transport sector they are

blended with existing fuels such as petrol and diesel. In the future, they can be

particularly important to help decarbonize the aviation, marine, heavy duty road

transport sectors. The two main types of biofuels are bioethanol and biodiesel. Biodiesel

is a fuel comprised of the mono-alkyl esters of long chain fatty acids derived from

vegetable oils or animal fats, designated B100, and meeting the requirement of ASTMD

6751.

Since 2000, there has been a rapid growth in demand for fuel ethanol. The rising

oil prices, environmental and climate warming concerns, interests in energy diversity

and security has made fuel ethanol an attractive alternative, particularly in industrial

countries. In developing countries, the focus is more on rural development, job creation,

savings on foreign currency and improving access to commercial energy. In addition,

the agricultural sector which plays a key role in the early development of ethanol, brings

significant benefits to farmers and would be a way to reduce costs and market

distortions of the existing farm support policies. The shift from petroleum- to biomass-

derived materials appears to be the plausible long-term pathway to ensure sustainable

supply of the carbon feedstock for energy and chemical industry (Bairamzadeh et al.,

2018). The use of biomass as a sustainable feedstock, however, does not assure a
successful transition without adapting processes designed with green chemistry and

cost-competitive manufacturing processes. In this regard, the concept of integrated bio

refineries encompassing green chemistry principles for production, enhanced energy

and material efficiency, reduced waste generation and toxicity, and the increased

reusability of the products at the end of their lives, has drawn particular interests and

discussion (Clark et al., 2009; De Bhowmick et al., 2017).

Bioethanol is the product obtained by fermentation of biomass from agricultural

crops, rich in carbohydrates. . Bioethanol is produced from any fodder crop containing

simple sugar or their polymers in abundance (Hughes et al., 2009). The polymers in the

form of starch and cellulose are broken down into simple sugars through chemical or

enzymatic hydrolysis (saccharification), and then fermented to produce ethanol and

carbon dioxide (Jamai et al., 2007). In saccharification, starch is converted into simple

sugar (monosaccharide) using microorganism or enzymes such as glucoamylase and α-

amylase (Shapouri et al., 2004). The yeast, Saccharomyces cerevisiae, produces ethanol

by fermentation of glucose but it is unable to ferment pentose sugars. Ethanol

production for biofuel utilizes the feedstock that are rich in cellulose. The alternative

feedstock available in agriculture is the waste fruits and vegetables that are rich in

carbohydrates including sugars. These raw materials are the conventional and low cost

feed stock that are bio converted through microbial processing to ethanol.

Increased production of biomass for energy, besides being a promising

renewable energy source, has several potential benefits in offsetting substantial use of

fossil fuels, heightening energy security in regions without abundant fossil fuel reserves,

increasing supplies of liquid transportation fuels, and decreasing net emissions of

carbon into the atmosphere per unit of energy delivered (Field et al., 2008). However,

the over-exploitation of biomass, especially consumed by animals, as alternative energy

sources also could threaten food security and generate environmental problems, such as
introducing invasive species by the plantation of foreign energy crops. In this context,

energy conversion from lignocellulosic biomass as feedstock is particularly attractive

because they do not compete with food crops and are abundant in native vegetation.

Lignocellulosic feedstock can be derived from discarded agricultural residues and even

municipal sludge comprising high organic contents.

Lignocellulosic raw materials which include fruit and vegetable waste, forestry waste,

agro- residues, MSW (Municipal Solid Waste) etc. can be used to produce ethanol. The

lignocellulosic biomass provides an alternative attractive fuel source for biofuel

production which can replace conventional fossil fuels (Wan and Li, 2011).

Lignocellulosic material eliminates the utilization of crop land and because of its larger

availability; it provides the domestic source of energy production. This raw material is

less expensive and can produce with lower input of energy, fertilizer and pesticides.

Moreover they reduce the effect of climate change as they generate low greenhouse

gases. Fruit wastes are a rich source of natural sugars. Huge amount of fruits are

consumed world over as health supplements and even as functional foods. Worldwide,

more than 675 million metric tons of fruits are produced each year. The greatest annual

fruit harvest in the world occurs in Asia. China alone produces some 275 million of

fruits annually. Fruit wastes are rich in cellulose and hemicellulose and have low lignin

contents hence very useful for bioethanol production.

Lignocellulosic raw materials are considered renewable sources of energy and their use

for bioethanol production may also help in CO2 mitigation. According to FAO out of the

global food waste, 40-50% arises from fruits, root crops and vegetables. Every year,

there is a loss of about 35-40% of fruits and vegetables as wastes (FAO.org). Even after

consumption, fruit storage and industrial processing, plenty of fruit waste is generated

and its management is also a problem. The cellulose, hemicellulose and lignin contents
of these waste implies that producing bioethanol from these could be a useful process

(Zheng et al., 2009, Del Campo et al., 2006).

This study is aimed at producing ethanol from three types of fruit waste, namely

mango, pawpaw and pineapple. Agricultural wastes are a largely untapped resources in

Ghana, West Africa and Africa as a whole. Fruit waste is one of these Agricultural

feedstock which is in abundance and also contains useful materials other than starches

or sugars. Greater percentage of these agricultural residues are ploughed back into the

soil to be used as manure, some into the sea, rivers or lakes and at times burned or

disposed of in landfills. It has been noted that mango, pawpaw and pineapple are

produced in very large quantities in the country. Ghana’s pineapple production is

estimated between 120000-150000 tonnes annually.

Lignocellulosic raw materials are considered renewable sources of energy and their use

for bioethanol production may also help in CO2 mitigation. According to FAO out of the

global food waste, 40-50% arises from fruits, root crops and vegetables. Every year,

there is a loss of about 35-40% of fruits and vegetables as wastes (FAO.org). Even after

consumption, fruit storage and industrial processing, plenty of fruit waste is generated

and its management is also a problem. The cellulose, hemicellulose and lignin contents

of these waste implies that producing bioethanol from these could be a useful process

(Zheng et al., 2009, Del Campo et al., 2006).

This study is aimed at producing ethanol from three types of fruit waste, namely

mango, pawpaw and pineapple. Agricultural wastes are a largely untapped resources in

Ghana, West Africa and Africa as a whole. Fruit waste is one of these Agricultural

feedstock which is in abundance and also contains useful materials other than starches

or sugars. Greater percentage of these agricultural residues are ploughed back into the

soil to be used as manure, some into the sea, rivers or lakes and at times burned or

disposed of in landfills. It has been noted that mango, pawpaw and pineapple are
produced in very large quantities in the country. Ghana’s pineapple production is

estimated between 120000-150000 tonnes annually.

Blue Skies is a fruit juice processing company found in Ghana and other parts of the

world. 70 tonnes of fruit waste (pineapple, mango and oranges) is generated weekly

from Blue Skies Products (Ghana) Ltd. Dealing with this waste poses a challenge to the

company and thus becomes a nuisance to the environment.

Ghana is said to import about 90 million litres of ethanol yearly. This shows that

Ethanol is one of the most useful fluids in the country. Almost all the alcoholic

beverage companies, petrochemical industries and the health sector imports ethanol in

very large quantities. Wine is an alcoholic beverage made from the fermentation of fruit

juice. The natural chemical balance of fruit is such that they can ferment with or without

the addition of sugars, acids, enzymes or other nutrients. The alcoholic industry is one

of the most lucrative industries in the world today. For example, local beverage

manufacturer, Kasapreko Company Limited imports 25 million litres of ethanol per year

at a cost of GHC 63 million. (GhanaWeb, Local beverage manufacturers battle unfair

taxes, July 2014).

This project has become necessary because of the cost of importing ethanol into Ghana

as a semi-raw material for most process industries in Ghana. Ghana is blessed with so

many raw materials for producing its own ethanol. Some of these materials come in the

form of waste. Though Caltech Bioethanol Distillery and Carbon Dioxide Plant,

produces ethanol from cassava the amount produced is less than 2% of the alcohol

demand of the country. Utilizing the waste cellulosic materials like fruits waste to

produce ethanol is one profitable way to reduce the problem of waste accumulation in

the country and in addition, minimize the cost of importing ethanol into the country.

Thus this project is focused on the production of ethanol from mango peels, pineapple

peels and pawpaw peels

Since large quantities of fruit and vegetable wastes and agro wastes are

available from fruit plantations, vegetable growing fields and wastages

during transportation, their disposal is a problem. Therefore, an attempt

is made to process the fruits and vegetable waste into alcohol. The

present research work was taken up with the following objectives.

1. Isolation of microorganisms from fruit and vegetable wastes and

characterization for saccharifying activity.

2. Optimization of growth conditions for the fermentation process.

3. Selection of efficient bacterial and yeast strains for ethanol production.

Objective

The main objective of this work is to produce bioethanol from fruit wastes.

Specific Objectives:

The specific objectives include to:

• Assess the effect of process parameters such as substrate concentration

and enzyme loading (dosage)

• Determine the effect of hydrolysis and fermentation time on bioethanol

yield
 CHAPTER TWO

2.0. REVIEW OF LITERATURE

2.1 Brief history of ethanol production

Ethanol is an alcohol made through the fermentation of plant sugars from agricultural

crops and biomass resources (NEVC, 1998). With rapid depletion of the world reserves

of petroleum, ethanol in recent years has emerged as one of the alternative liquid fuel

and has generated immense activities of research in the production of ethanol and its

environmental impact. Production of alcoholic beverages is in fact as old as human

civilization. The production of pure ethanol apparently begins in the 12-14th century

along with improvement of distillation. During the middle ages, alcohol was used

mainly for production of medical drugs but also for the manufacture of painting

pigments. The knowledge of using starchy materials for ethanol production was first

employed in the 12th century in typical beer countries like Ireland. Ethanol was one of

the most popular lamp illuminants used in 1850s and approximately 90 million gallons

ethanol was produced in the United States. But due to the tax imposition on ethanol to

assist in financing the civil war and the cheaper price of kerosene, it quickly replaced

ethanol as the premier illuminant in 1861 (Morris, 1993). It was only in the 19th

century that this trade became an industry with enormous production figures due to the

economic improvements of the distilling process. It was at the beginning of the 20th

century that it had become known that alcohol might be used as fuel for various

combustion engines, especially for automobiles. In the 1970‟s, the interest in fuel

ethanol was renewed due to the oil crisis. Nearly 25 federal agencies administered

various ethanol programs and the National Alcohol Fuels Commission was established

to study the potential for alcohol based fuels (Lansing, 1983). Ethanol gained further

support in 1980 when Chrysler, Ford and General Motors released statements that
ethanol with blends of up to 10% would be covered in their vehicle warranties (RFA,

1998). It‟s market grew from less than a billion litres in 1975 to more than 39 billion

litres in 2006 and is expected to reach 100 billion litres in 2015 (Licht, 2006). Interest

in the use of biofuels worldwide has grown strongly in recent years due to the limited

oil reserves, concerns about climate change from greenhouse gas emissions and the

desire to promote domestic rural economies.

2.1.1 Ethanol and its characteristics

Bioethanol or fuel alcohol refers to ethyl alcohol produced by microbial fermentation

(as opposed to petrochemically-derived alcohol) that is used as a transportation biofuel.

It is produced through distillation of the ethanolic wash emanating from fermentation of

biomass derived sugars and can be utilized as a liquid fuel in internal combustion

engines, either neat or in petrol blends (Walker, 2011).

2.2 Worldwide status of bioethanol production

2.2.1 Ethanol Production in the World

Bioethanol production worldwide has increased considerably since the oil crisis in

1970 (Campbell and Laherrere, 1998). Bio-energy ranks second (to hydropower) in

renewable U.S. primary energy production and accounts for 3% of the U.S. primary

energy production (James and Barry, 2007). The United States is the world’s largest

producer of bioethanol fuel, accounting for nearly 47% of global bioethanol production

in 2005 and 2006 (Balat and Balat, 2009).

In 2007, the U.S. president signed the Energy Independence and Security Act of 2007

(EISA, 2007), which required 34 billion litres of biofuels (mainly bioethanol) in 2008

causing a steady increase to 57.5 billion litres in 2012 and expected to reach 136 billion

litres in 2022. The potential demand for bioethanol as a transportation fuel in the EU is

estimated at about 12.6 billion litres in 2010 (Zarzyycki and Polska, 2007).

Brazil is the world's largest exporter of bioethanol and second largest producer after the

United States. With regard to bioethanol, the share of the US in the global production is

50% and Brazil provides 39% of the total global supply, while the share of OECD-

Europe is 5% (Gnansounou, 2010).

Since Brazil is one of the most developed nations in ethanol production, almost all the

Brazilian vehicles use either pure ethanol or the blend of gasoline and ethanol (75:25)

(Mussatto et al., 2010; RFA, 2010). The high percentage in which ethanol is added to

gasoline in Brazil is also an effort on part of the government to reduce the imports of

oil S(Prasad et al., 2007). Production has been expected to rise from 15.4 billion litres

in 2004 to 26.0 billion litres by 2010. Ethanol from sugarcane provides 40% of

automobile fuel in Brazil and approximately 20% is exported to the US, EU and other

markets (Greenergy, 2007).

2.2.1 Ethanol Production in Ghana

Ghana is said to import about 90 million litres of ethanol yearly. Between 1997 and

2016, Ghana fuel ethanol production remained stable at around 0 thousand barrels a

day. Until the year 2016 when Caltech Bioethanol Distillery and Carbon Dioxide Plant,

Ghana started operating at Ho in the Volta Region. Ghana’s first ethanol plant at

Hodzo in the Ho Municipality has produced ethanol from Cassava starting from June

2016. Caltech has the only installed capacity for bioethanol in the nation (300000 litres/

year). Caltech is a Limited liability company incorporated in 2006. The head office is

situated atSpintex in Accra.

The plant is still working and producing ethanol for usage in the country which is in a

small quantity. The company in addition to ethanol produces bio-diesel, starch and

cassava flour.

Figure1. Caltech Cassava Processing Plants for Ethanol Production

2.3 Bioethanol feedstocks

There is a growing interest worldwide to find new and cheap carbohydrate sources for

production of bioethanol (Mohanty et al., 2009). For a given production line, the

comparison of the feedstocks includes several issues (Gnansounou et al., 2005); they

are:

(1) chemical composition of the biomass

(2) cultivation practices

(3) availability of land and land use practices

(4) use of resources and energy balance

(5) emission of greenhouse gases, acidifying gases and ozone depletion gases

(6) absorption of minerals to water and soil, injection of pesticides and soil erosion

(7) contribution to biodiversity and landscape value losses, farm-gate price of the

biomass

(8) logistic cost (transport and storage of the biomass

(9) direct economic value of the feedstocks taking into account the co-products

(10) water requirements and water availability.

Bioethanol feedstocks can be divided into four major groups: (1) First generation

feedstocks (2) Second generation feedstocks and (3) Third generation feedstocks and

(4) Fourth generation feedstock.

2.3.1 First generation feedstocks

First generation bioethanol feedstocks come from agricultural cereal and sugar crops

that are also sources of human (and animal) food. The bioethanol produced by

fermentation of sugars such as sugarcane (Macedo et al., 2008; Leite et al., 2009), sugar

beet (Ogbonna et al., 2001; Icoz et al., 2009), sorghum, wheat (Nigam, 2001), root

crops such as cassava are commonly known as first generation bioethanol.

Sugar crops need only a milling process for the extraction of sugars to fermentation (not

requiring any step of hydrolysis), becoming a relatively simple process of sugar

transformation into ethanol. In this process, ethanol can be fermented directly from cane

juice or beet juice or from molasses generally obtained as a by-product after the

extraction of sugar (Icoz et al., 2009).

In processes that use starch from grains like corn, saccharification is necessary before

fermentation. In this step, starch is gelatinized by cooking and submitted to enzymatic

hydrolysis to form glucose monomers, which can be fermented by microorganisms

(Mussatto et al., 2010). First generation bioethanol has played an important role in

establishing the infrastructure and policy drivers, required to support renewable

transport fuels in the international market place (EIA, 2008).

However, first generation biofuels result in a number of associated problems. There is

much debate over their actually benefit in reducing greenhouse gas and CO2 emissions

due to the fact that biofuels can produce negative Net energy gains, releasing carbon in
their production than their feedstock’s capture in their growth. However, the most

contentious issue with first generation bioethanol is ‘fuel vs food’. As the majority of

biofuels are produced directly from food crops the rise in demand for biofuels has led to

an increase in the amount of crops being diverted away from the global food markets.

This has been blamed for the global increase in food prices over some couple of years

now.

First generation biofuels are the most widely used of the three. They are produced from

mainly starch, oil and sugar-based feedstock (Demirbas, 2011). However, first

generation biofuels have some limitations due to the competition with food production

changes in the land use, and high water requirements (Podkuiko et al., 2014).

Figure 2 Bioethanol from first generation feedstocks.

2.3.2 Second generation feedstocks

Exploitation of first generation feedstocks for future bio-fuel production is ultimately

unsustainable due to food security and land-use issues. Second generation biofuels use

lignocellulosic biomass from forest, agriculture, fishery, and municipal wastes. It

includes non food crops, straw, grass, sawdust and wood chips (Sun & Cheng, 2002;

Nigam & Singh, 2011).

Second-generation bioethanol refers to fuel alcohol produced from non-food biomass

sources, such as lignocellulose, the most abundant form of carbon on the earth. They

account for nearly 50% of world biomass with an estimated annual production of 10 to

50 billion tons, making lignocellulose arguably the most abundant and renewable

organic component of the biosphere (Claassen et al., 1999).

Second Generation biofuels are also aimed at being more cost competitive in relation to

existing fossil fuels. Life cycle assessments of second-generation biofuels have also

indicated that they will increase ‘net energy gains’ over coming another of the main

limitations of first generation biofuels.

Second-generation biofuels are expected to reduce net carbon emission, increase

energy efficiency and reduce energy dependency, potentially overcoming the

limitations of first-generation biofuels (Antizar-Ladislao and Turrion-Gomez, 2008).

The other major benefits of switching to cellulosic ethanol are its renewable nature,

long term sustainability, low net carbon emission, high energy efficiency, low energy

dependency, increase in national security and diversifying rural economies (IEA,

2008b). Polysaccharides present in lignocellulosic materials including cellulose and

hemicellulose are of great interest as feedstocks for second generation ethanol

production.

A schematic for the conversion of biomass to fuel is shown in Fig. 2.2.

 Lignocellulosic biomass

Pretreatment

Hydrolysis Fermentation

Distillation/ Separation

Bioethanol

Figure 3 General outline of the lignocellulose to bioethanol production process

2.3.3 Third generation feedstocks

Third-generation biofuels are produce from algal biomass, which has a very distinctive

growth yield as compared with classical lignocellulosic biomass (Brennan and

Owende, 2010). Microalgae have broad bioenergy potential as they can be used to

produce liquid transportation and heating fuels, such as biodiesel and bioethanol.

The Third Generation of biofuels is based on improvements in the production of

biomass. It takes advantage of specially engineered energy crops such as algae as its

energy source. The algae are cultured to act as a low-cost, high-energy and entirely

renewable feedstock. It is predicted that algae will have the potential to produce more

energy per acre than conventional crops. Algae can also be grown using land and water

unsuitable for food production, therefore reducing the strain on already depleted water

sources. A further benefit of algae based biofuels is that the fuel can be manufactured

into a wide range of fuels such as diesel, petrol and jet fuel.

Microalgae provide carbohydrates (in the form of glucose, starch and other

polysaccharides), proteins and lipids for the production of biofuels. They are
recognised as one of the oldest living organisms, are thallophytes i.e. lacking roots,

stems and leaves have chlorophyll a as their primary photosynthetic pigment and lack a

sterile covering of cells around the reproductive cells (Brennan and Owende, 2010).

Algae structures are primarily for energy conversion without any development beyond

cells and their simple development allows them to adapt to prevailing environmental

conditions and prosper in the long term.

2.3.4 Fourth generation feedstocks

Four Generation Bio-fuels are aimed at not only producing sustainable energy but also a

way of capturing and storing CO2. Biomass materials, which have absorbed CO2 while

growing, are converted into fuel using the same processes as second generation biofuels.

This process differs from second and third generation production as at all stages of

production the carbon dioxide is captured using processes such as oxy-fuel combustion.

The carbon dioxide can then be geosequestered by storing it in old oil and gas fields or

saline aquifers. This carbon capture makes fourth generation biofuel production carbon

negative rather than simply carbon neutral, as it is ‘locks’ away more carbon than it

produces. This system not only captures and stores carbon dioxide from the atmosphere

but it also reduces CO2 emissions by replacing fossil fuel.

2.4 Lignocelluloses materials

Lignocellulosic plant biomass is an important renewable carbon resource for the

biorefinery industry and is thus considered a sustainable and environment friendly

alternative to the current petroleum platform (Wongwilaiwalina et al., 2010).

Lignocellulose refers to plant dry matter (biomass), so called lignocellulosic biomass. It

is the most abundantly available raw material on the Earth for the production of

biofuels, mainly bio-ethanol. It is composed of carbohydrate polymers (cellulose,

hemicellulose), and an aromatic polymer (lignin). These carbohydrate polymers contain

different sugar monomers (six and five carbon sugars) and they are tightly bound to

lignin.

Lignocellulosic feedstocks are cheap, renewable, abundant and do not compete with the

food production. Lignocellulosic biomass such as agricultural residues and herbaceous

energy crops, consists mainly of three different types of biopolymers i.e. cellulose,

hemicellulose, lignin and pectin (Ragauskas et al., 2006; van Maris et al., 2006) (Table

2.1). Removing the lignin and breaking up the crystalline structure of cellulose for

enhancing enzymes accessibility to the cellulose during the hydrolysis is the key task of

the pretreatment (Mosier et al., 2005b).

Table 2.1 Polymer composition of lignocellulosic biomass.

Polymers Content in Major monomers

lignocellulose (%)
Cellulose 33-51 Glucose
Hemicellulose 19-34 Xylose, Glucose, Mannose,
Lignin 20-30 Arabinose, Rhamnose, Galactose
Pectins (when present) 2-20 Aromatic alcohols
Galacturonic acid and Rhamnose
Source: (van Maris et al., 2006)

Lignocellulosic biomass is made of a complex mixture of cellulose, hemicellulose and

lignin regardless of the type of plant it may come from.

2.4.1 Cellulose

Cellulose is a polysaccharide made up of linear glucan chains which are linked by β1,4-

glycosidic bonds and with cellobiose residues as the repeating unit at different degrees

of polymerization depending on the resources and packed into micro fibrils which are

held to close by intramolecular hydrogen bonds as well as in van der Waals

intermolecular forces (Zhao et al., 2011). The chemical formulae of cellulose are

(C6H10O5) n and an example of the structure of one chain of the polymer is presented in

the Fig. 5

Figure 4 Structure of Cellulose molecule

Cellulose the most abundant organic polymer existing on the earth, is a homopolymer of

B-1,4- linked glucose units and composing of the major portion of the plant cell wall in

growing cells (Kumar, 2013). Most of the cellulose is presented as a crystalline form

and only a small amount of the non-organized cellulose chains forms the amorphous

cellulose. Cellulose has high degree of polymerization (DP) from 100-20,000 which is
totally or partially water insoluble and recalcitrant to hydrolysis into its individual

glucose subunit group because of closely packed, highly crystalline structure with

straight, stable supra-molecular fibres of big tensile strength and small accessibility in

its polymer form (Demain et al., 2005).

About 33% of all plant matter is composed of cellulose. Cellulose does not melt with

temperature, but its decomposition starts at 1800C. Cellulose fiber is surrounded by intra

and intermolecular hydrogen bonds (Zhbankov, 1992) which makes cellulose insoluble

in water and most organic solvents available.

The cellulose component can be hydrolyzed by enzymatic treatment or by the action of

microorganisms (Kumar, 2013) once exposed. Although several microorganism

produce cellulases and utilize cellulose directly but pretreatment methods are required

for commercial application on large scale to make the process efficient and

economically viable.

2.4.2 Hemicellulose

Hemicelluloses are a class of heteropolymers made up of various hexoses (D-glucose,

D-galactose and D-mannose) and pentoses (L-arabinose and D-xylose). It is mainly

consisted of xylose-linking compounds like arabinose, glucose, mannose, and other

sugars through an acetyl chain (Chadel et al., 2010). They decrease pore size creating

constraints for the enzymes to act. Xyloses the second most abundant sugar in nature

after D-glucose.

Hemicellulose is insoluble in water at low temperature. However, its hydrolysis starts at

a temperature lower than that of cellulose, which renders it soluble at elevated

temperatures. The presence of acid highly improves the solubility of hemicellulose in

water.
However, there are only a few known microorganisms which act on pentoses for

fermentation. For the process to be economically viable, both the hexoses and pentoses

should be considerably fermented

Figure 5 Repeating units of hemicelluloses (Source: Scheller and Ulvskov, 2010)

2.4.3 Lignin

Lignin is a highly branched polyphenolic, amorphous polymer with wide range of

functional groups consisting of phenyl propanoid monomers of coniferyl, sinapyl and p-

coumaryl alcohols (Fig. 2.6) (Vivekanand et al., 2008). Lignin create matrix holding

together the cellulose and hemicellulose. They are complex, amorphous, three-

dimensional polymers having a phenyl propane structure (Rubin, 2008) and are a

polymer of three phenolic alcohols (p-coumaryl, sinapryl and coniferyl alcohols). They

can be separated from the main biomass but cannot be easily degraded by

microorganisms.

2.5 Fruits and Fruit Wastes

2.5.1 Fruits

A) MANGO

Mango (Mangifera indica L.) may be a case for this purpose because it is one of the

most popular and abundant tropical fruits in Africa and is often in oversupply after each
harvest. This fruit is mainly grown in 85 countries. Asia and Eastern countries produce

about 80 percent of total world production. There are a great number of varieties, which

differ from each other with respect to crop, the color of the skin, pulp, the flavor and

aroma of the fruit, among other features.

Mexico is the fifth producer of mango around the world. The main producers are India,

China, Thailand, Pakistan and Mexico, followed by Indonesia. Together, these six

nations generate three of every four tons of the fruit produced worldwide. The area of

cultivation of mango in Mexico amounts to 186,505 hectors. Mexico has a production

of 1.481 million tons according to the data reported by the SIAP in 2012 [3]. On the

other hand, according to the latest report of SAGARPA, there is a history of losses

amounting to 230 thousand tons per year. According to a report published by Reddy and

Reddy in 2005, Mango contains a high concentration of sugar (16–18% w/v) and acids

with organoleptic properties, and also contains antioxidants. Sucrose, glucose and

fructose are the principal sugars in ripe mango, with small amounts of cellulose,

hemicellulose and pectin [4]

Mango has been identified as one of the main traditional fruits that has demand as an

export commodity and is therefore being promoted in Ghana, to become a major

potential foreign exchange earner in the next 5-10 years. Locally there is a high demand

for mangoes in the food processing industries. It is used for jams, dried fruits, flavours

and juice. The following are some varieties of mangoes and place of origin

Varieties Place of Origin

1. Haden Mexico, Peru, Ecuador

2. Tommy/ Atkins Mexico, brazil

3. Keitt Mexico

4. Kent Mexico

5. Francine Haiti
 6. Ataulfo Mexico

Figure 1 Mango fruits

Table 2.2 Varieties of Mangoes Grown in Ghana and Percentage Area Cultivated

VARIETIES PERCENTAGE AREA CULTIVATED

Dog mouth 0.02

Druelin 0.01

Erwin 0.54

Haden 1.20

Palmer 3.45

Keitt 81.67

Kent 11.33

Tommy Atkins 0.29

Sunset 0.02

Springfield 0.07

Jaffna 0.07

Vorlet 0.02

Zill 0.03

Julie 0.42

Local (Dodowa) 0.15

Njala 0.72

Composition of Mango

Mango is an excellent source of pro vitamin A and vitamin C which varies between a

low of 5mg and as high as 142mg 100g-1 FM (fresh mass).

PINEAPPLE

Pineapple (Ananas comosus) is a tropical plant with a multiple fruit consisting of

coalesced berries, also called pineapples, and the most economically significant plant in
the family Bromeliaceae or Bromalid. Pineapples may be cultivated from the offset

produced at the top of the fruit, possibly flowering in five to ten months and fruiting in

the following six months. Pineapples do not ripen significantly after harvest. In 2016,

Costa Rica, Brazil and the Philippines accounted for nearly one-third of the world’s

production of pineapples.

The most widely grown varieties in Ghana are;

1. Smooth Cayenne (the most spread variety)

2. Sugarloaf

3. MD2

Pineapples do not sweeten after harvest although the acid level may decline. They do

well in regions that have temperature up to 29oC and do not tolerate frost. The plant is

herbaceous or shrubby and classified as epiphytic or terrestrial. The fruit is a syncarp of

fused fruitlets from inferior ovaries.

Pineapples as grown for food, leaf fibre and ornamentals. Pineapple bran (residue) is

used as cattle feed.

Characterization of Pineapple

Pineapple is good source of vitamin C, some vitamin A, Calcium, Phosphorus, Iron,

Potassium and Thiamine. It is low in Sodium. Bromelian, the proteolytic enzyme found

in pineapple plants (particularly stems) has medicinal applications.

Figure 8 Extra MD2 Figure 7 Smooth

Figure 6 Sugar Loaf
pineapple Cayenne pineapple
Pineapple
B) PAWPAW

Botanical name: Carica Papaya, it is one of the 22 accepted species in the genus

Carica of the family Caricae. It belongs to the fruit and vegetable class. Its origin is

in the tropics of the Americas, perhaps from southern Mexico and neighbouring

Central America. It is highly abundant and commonly known as Pawpaw.

The two most common varieties of pawpaw in Ghana are: Solo sunrise and Golden

Caliman.

Nutritionally, the major components of pawpaw fruit pulp dry matter are

carbohydrates. The total dietary fibre content of ripe fruit varies from 11.9 to

21.5g/100g /dry matter. (Puwastien et.al, 2000, Saxholt et al., 2008 and USDA,

2009). There are two major types of carbohydrates in pawpaw fruits, the cell walls

polysaccharides and soluble sugars. At the early stage early stage of growth, glucose

is the main sugar but the sucrose content increases during ripening and can reach up

to 80% of the total sugars (Paul, 1993, and OECD, 2010).

Figure 9 Images of some varieties of pawpaw

2.5.2 Fruit composition

Fruits compose of different components. Both physical and chemical composition. Each

fruit have a moisture content, total solids, total sugars (reducing and non-reducing

sugars), protein, Vitamins and fibre (Organic carbon) and other minute components.

Fruits like pineapple, pawpaw and mango have more than 50 percent of it being

moisture.

2.5.3 Fruit waste

More than 50 percent of fruits and other agricultural foods grown in Ghana are rendered

as waste in one form or the other. Ghana exports fruits to other countries in the world.

These fruits come in the form of mangoes, pineapples, pawpaw, oranges, cocoa and

others. Not all fruits harvested are eaten or exported. Some are left on the farms without

harvesting them. This is because not all farms have good roads for the fruits to be

transported to towns and cities for consumption.

Another factor, the fruits processing industries in the country are small in number thus

only a very little fraction of the fruits are processed into juice and drinks. Blue Skies is

one of the companies that produces natural fruit juice from some fruits like passion,

mango, pineapple and pawpaw. They produce tonnes of fruits waste daily.

2.5.4 Fruit wastes as a source of bioethanol

Itelima et al. estimated bioethanol production by simultaneous saccharification and

fermentation from banana, plantain and pineapple peels by through co-culture of A.

niger and S. cerevisiae .Singh et al. have studied the simultaneous saccharification and

fermentation (SSF) of banana peelsat different temperature (20°C to50°C) to obtain

bioethanol by using co-cultures of A. niger and S.cerevisiae at different pH (4 to 7) for

seven days fermentation for bioethanol . In this study it was observed that the optimum

pH and temperature for the fermentation of banana peels was 6 and 30°C respectively.
With these optimum conditions of pH and temperature,different yeast concentrations of

3% to 12% were used for performing fermentation and it was found that the time

required for the accomplishment of fermentation reduced dramatically.

Grohmann et al. have studied the use of cellulase enzyme for hydrolysis of cellulose of

banana peels and observed that the maximum saccharification was achieved with a

cellulase enzyme from Trichoderma reesei QM 9414 .

Mishra et al. investigated the production of bioethanol from fruit peels of orange, sweet

lime and pineapple. Pineapple was found to produce the maximum sugar amongst these

fruit wastes.

Reddy et al. have reported that mango peels contain larger amounts of reducing sugars

i.e., up to 40% (w/v). Direct fermentation of mango peels yielded very low content of

bioethanol of about 5.4%(v/v). It was reported in this study that this can be enhanced up

to 7.14% (w/v) by using nutrient supplementation such as yeast extract, bran extract,

peptone and wheat.

Arumugam and Manikandan have also reported the production of bioethanol from

banana and mango fruit waste. In this study dilute acid pretreatment of fruit waste was

followed by enzymatic hydrolysis and maximum sugar was produced from the mixed

fruit pulps, this was followed by that from the banana fruit pulp and then followed by

banana peels.

Нe reported that hydrolysate from enzymatic hydrolysis showed fermentation efficiency

of about 70.3% after 48 hours incubation) as compared to 27.1% observed for the

hydrolysate from acid hydrolysis. The authors have reported that production of

bioethanol using S. cerevisiae was far better from enzyme hydrolysate than that from

acid hydrolysate. Several other workers have reported on the use of fruit wastes for the

production of bioethanol.
Presently, studies have been extended on the production of bioethanol from fruit wastes

through enzymatic hydrolysis using commercial cellulase and xylanase enzymes aіer

milder pretreatment with water-steam. A comparison of enzymatic hydrolysis of fruit

wastes with their acidic hydrolysis has also been made.

2.6 Process in Ethanol Production

The process of ethanol production depends on the types of feedstocks used. Generally,

there are three major steps in ethanol production:

(1) obtaining solution that contains fermentable sugars: hydrolysis

(2) converting sugars to ethanol by fermentation

(3) ethanol separation and purification. Feedstocks are thus pretreated in order to reduce

its size and also facilitate subsequent processes. The hemicellulose and cellulose will be

hydrolyzed to fermentable sugars for the production of ethanol. Yeasts are given the

responsibility to ferment these sugars into ethanol for a period of time. Separation

technologies are then used in order to recover ethanol before it can be used as fuel or a

raw material for the beverage industries (C.E. Wyman, et al.,1996).

Figure10 A flowchart of Sugarcane conversion to Ethanol

2.6.1 Pretreatment

Pretreatment is the process through which the cellulose components are exposed to and

made more susceptible to enzymatic hydrolysis. The pretreatment techniques may

involve a synergism between the heat action, the suitability of medium pH and the time

exposure under processing conditions.

The aim of pretreatment is to get rid of lignin and hemicellulose from the material. This

results in a decrease in cellulose crystallinity, and increase in the porosity of cellulose,

thus consequently, making it more susceptible to the action of cellulase. (Lynd et al.,

2002).

Pretreatment has an important effect on the overall process which makes the hydrolysis

easier and leads to the production of higher amount of fermentable sugars. It also

influences the ethanol yield and cost involved in the production. (Srichuwong et
al.,2009). The various methods that are currently used for pretreatments includes:

physical, chemical, biological and physicochemical. Physical-pretreatment uses

mechanical milling to reduce the substrate size by grinding. Ozonolysis is one of the

common chemical pretreatment, others include; acid hydrolysis, alkaline hydrolysis and

organosoly based processes. Different fungal species are involved in biological

pretreatment, moreover physicochemical pretreatment includes ammonia fibre explosion

and steam action (Kurian et al., 2013).

Dehydration of hexose and pentose during the pretreatment process releases furan

compounds like 5-hydroxymethyl-2-furaldehyde (HMF), 2-furaldehyde. These furan

derivatives induce the inhibition of cell growth and reduce ethanol yield and

productivity. Yeasts fermentation is ususally inhibited by the weak acid stress induced

from lignocellulosic materials in the sample. However, the low concentration of weak

acids can increase ethanol production by cellular division and it was reported that the

presence of weak acids improves glucose utilization, ethanol production yield and also

tolerance to HMF and furfural in S. cerevisiae (D. Greetham et al.,2016).

2.6.2 Hydrolysis

Hydrolysis is carried out after pretreatment if any necessary. The process takes place

after pretreatment to break down the feedstocks or lignocellulosic materials into

fermentable sugar for the production of ethanol. The two most commonly used

hydrolysis methods are acidic and enzymatic.

2.6.2.1 Acid hydrolysis

Acid hydrolysis is considered as the oldest and also the most commonly used method.

Acidic hydrolysis is divided into two types namely dilute and concentrated acid

hydrolysis. Dilute acid hydrolysis is done at higher temperature using low acid

concentrations while concentrated acid hydrolysis is performed at lower temperature

using high acid concentrations. Dilute acid hydrolysis is the most commonly used

process in acid hydrolysis. However, it results in the generation of large amount of

inhibitors compared to concentrated acid hydrolysis. Acid hydrolysis of lignocellulosic

biomass is conducted in two-stage process as the pentose sugars degrade more rapidly

compared to hexose sugars.

Hemicellulose is hydrolysed in the first stage using dilute acid while cellulose is

hydrolysed in the second stage using concentrated acid. Concentrated acid process

generates high sugar recovery (90%) in shorter period of time [64].

The disadvantages of acid hydrolysis are the difficulty of performing acid recovery and

recycling process which increases the production cost.

2.6.2.2 Enzymatic Hydrolysis

Enzymatic hydrolysis of cellulose is carried out by the cellulose-hydrolysing enzyme

cellulases, a mixture of several enzymes that hydrolyze crystalline/amorphous cellulose

to fermentable sugars (Duff and Murray, 1996). Enzymatic hydrolysis requires

enzymes to hydrolyse the feedstocks into fermentable sugars. The hydrolysis of

cellulose by cellulolytic enzymes has been investigated intensively since the early

1970s, with the objective of developing a process for the production of ethanol.

Moreover, it does not cause corrosion problem in the reactors which can result in high

sugar yields. Enzymatic saccharification of cellulose can be enhanced by using

surfactants which function to block lignin. The efficiency of cellulose hydrolysis can be

improved by adding Polyethylene glycol (PEG) or Tween 20 to increase enzymatic

saccharification and reduce the adsorption of cellulase on lignin. The limitation of using

enzymes in hydrolysis is because they are too expensive for the economical production

of ethanol from biomass. Three types of enzymes that are commonly used for cellulose

breakdown such as endo-β—1,4-glucanases, cellobio-hydrolases and β-glucosidases.

The products of the hydrolysis are usually reducing sugars majorly glucose. The

utility cost of enzymatic hydrolysis is low compared to acid or alkaline hydrolysis

because enzyme hydrolysis is usually conducted at mild conditions (pH 4-6 and

temperature 45-500C) and does not have a corrosion problem (Kuhad et al., 2010,

2011b). Both bacteria and fungi can produce cellulases for the hydrolysis of

lignocellulosic materials.

These microorganisms can be aerobic or anaerobic, mesophilic or thermophilic.

Bacteria belonging to Clostridium, Cellulomonas, Bacillus, Thermomonospora,

Ruminococcus, Bacteriodes, Erwinia, Acetovibrio, Microbispora and Streptomyces can

produce cellulases and among them Cellulomonas fimi and Thermomonospora fusca

have been studied extensively (Bisaria, 1991; Duff and Murray, 1996; Sun and Cheng,

2002).

Commercial cellulases are mainly obtained from aerobic cultivations of Trichoderma

reesei and to a lesser extent Aspergillus niger (Prasad et al., 2007; Sanchez and

Cardona, 2008). Other fungi that have been reported to produce cellulases include

species of Sclerotium rolfsii, P. chrysosporium and species of Trichoderma,

Aspergillus, Schizophyllum, Fusarium and Penicillium (Sternberg, 1976; Duff and

Murray, 1996; Kuhad et al., 1999; Sun and Cheng, 2002). Of all these fungal genera,

Trichoderma has been most extensively studied for cellulase production.

2.6.2.3 The Major Components of Cellulase

The cellulase system contains of three major enzyme components: β-endoglucanase

(EC 3.2.1.4), β-exoglucanase (EC 3.2.1.91) and β-D-glucosidase (EC 3.2.1.21) (Bhat

and Bhat, 1997; Lynd et al., 2002) (Fig. 2.10).

The exoglucanase act on the ends of the cellulose chain and release β-glucoside as the

end product; endoglucanase randomly attack the internal O-glycosidic bonds, resulting
in glucan chains of different lengths and the β-glycosidases act specifically on the β-

cellobiose disaccharides and produce glucose (Beguin and Aubert, 1994; Kuhad et al.,

1999; Kuhad et al., 2011b).

β-glucosidase catalyzes cleavage of cellobiose, which plays a significant role in the

hydrolysis process, since cellobiose is an end-product inhibitor of many cellulases

including both exo- and endo-glucanases (Lee, 1997; Galbe and Zacchi, 2002;

Rabinovich et al., 2002; Sun and Cheng, 2002). β-glucosidase, in turn is inhibited by

glucose and therefore, enzymatic hydrolysis is sensitive to the substrate concentration

(Philippidis et al., 1993).

In addition to substrate concentration, pretreatment of cellulosic materials and

hydrolyzing conditions such as temperature and pH are among factors influencing the

effectiveness of enzymatic hydrolysis. (Duff and Murray, 1996; Galbe and Zacchi,

2002).

Figure 11 Procedural mechanistic action of all three cellulases on the cellulose polymer.

Hydrolysis of the individual cellulose fibres to break it into smaller sugars by

exocellulase, breakage of the non-covalent interactions presents in the crystalline

Structurally, cellulases typically have two separate domains: a catalytic domain (CD)
and a cellulose binding module (CBM), which is linked by a flexible linker region. The

CBM is comprised of approximately 35 amino acids and the linker region is rich in

serine and threonine (Divne et al., 1998). The nature of the lignocellulosic substrate

changes during the time course of enzymatic hydrolysis (Wang et al., 2006).

2.6.2.4 Advantages of Enzymatic Hydrolysis over Acidic Hydrolysis

Enzymatic hydrolysis methods have shown distinct advantages over acid based

hydrolysis methods; the very mild process conditions give potentially higher yields, the

utility -`cost is low (no corrosion problems), therefore this is the method of choice for

future wood-to-ethanol process (Duff and Murray, 1996). Many experts see enzymatic

hydrolysis as key to cost-effective ethanol production in the long run. Although acid

processes are technically more mature, enzymatic processes have comparable projected

costs and the potential of cost reductions as technology improves.

2.6.2.5 Factors that influence Enzymatic Hydrolysis

Several factors can influence the enzymatic hydrolysis of cellulose. A low substrate

concentration would result in a low overall glucose yield (Hamelinck et al., 2005).

The activity of cellulase enzyme is influenced by the concentration and source of the

enzyme. Cellulose will be degraded into reducing sugars under mild reaction

conditions (pH: 4.8–5.0, temperature: 45–50 °C). The efficiency of enzymatic

hydrolysis is influenced by optimized conditions such temperature, time, pH, enzyme

loading and substrate concentration [65]. The amount of fermentable sugar obtained

increases as the enzyme load increases while cellulose load decreases.

An increase in the substrate concentration would lead to an increased glucose yield

as well as an increased rate of reaction. However, a high substrate concentration can

cause substrate inhibition which would substantially decrease the rate of the hydrolysis

and the extent of substrate inhibition depends on the ratio of total substrate to total
enzyme. A high cellulase dosage would also significantly raise process costs (Prasad et

al., 2007). The susceptibility of cellulosic substrates to enzymatic hydrolysis depends

on the structural feature of the substrate, including cellulose crystallanity, degree of

polymerization, surface area and lignin content (Sun and Cheng, 2002; Taherzadeh and

Karimi, 2008).

Lignin interferes with hydrolysis by acting as a shield, preventing access of cellulases

to cellulose and hemicellulose thereby resulting in extended reaction times to achieve

high conversions. On top of that, lignin irreversibly adsorbs a large portion of the

cellulase rendering it unavailable for further hydrolysis of cellulose (Qing et al., 2010).

Therefore, removal of lignin during pretreatment is essential to dramatically increase

the hydrolysis rate (McMillan, 1994; Prasad et al., 2007). Also, removal of

hemicellulose increases the mean pore size of the substrate, thereby increasing

cellulase accessibility to cellulose (Hendriks and Zeeman, 2009).

2.6.2.6 How to Reduce the Cost of Enzymes in Ethanol Production

To reduce the enzyme cost in the production of ethanol from lignocellulosic biomass,

two aspects are widely addressed: optimization of the cellulases production and

development of a more efficient cellulase-based catalysis system. Recycling and reuse

of the enzymes is also an attractive methodology to reduce enzymatic hydrolysis costs

(Singh et al., 1991; Ramos et al., 1993; Lee et al., 1995; Gregg et al., 1998; Sun and

Cheng, 2002; Mosier et al., 2005)

2.6.3 Fermentation Process

Fermentation is used to convert glucose into ethanol (Raud et al., 2016c). Simultaneous

saccharification and fermentation (SSF) and separated hydrolysis and fermentation

(SHF) are the two most common processes used in the fermentation of lignocellulosic

hydrolysate (Gupta & Verma, 2015). After enzymatic hydrolysis, the lignocellulosic

substrates are converted to monosaccharides, which are further fermented to ethanol by

microorganisms. Approximately 80% of the ethanol produced in the world is still

obtained from the fermentation and the rest comes largely by synthesis from the

petroleum product, ethylene (Lin and Tanaka, 2006).

Ethanol fermentation is a biological process in which sugars are fermented by

microorganisms to produce ethanol and CO2. As compared to starch and molasses, the

fermentation of plant biomass (lignocellulosic) hydrolysate is a complex process.

In SSF, enzymes hydrolyze cellulose into sugars and ferment the hexoses into ethanol,

at the same time (Kamzon et al., 2016). This process has several known advantages.

It has low capital costs, low enzyme requirements, high hydrolysis efficiency and

ethanol yields, reduced process time, low risk of inhibition and contamination, and it

does not require reactors with large volumes. However, it has some limitations

regarding the compatibility of the temperature of the hydrolysis and fermentation, and

inhibition of enzymes (Sun & Cheng, 2002; Chen & Fu, 2016).

In case of SHF on the other hand, hydrolysis and fermentation can proceed at their

optimum conditions, but in separate vessels. Although, it has some problems due to the

inhibition and the possibility of contamination since it is a long process (Kamzon et al.,

2016; Chen & Fu, 2016).

Several microorganisms have been pointed out for fermentation of sugars but, the ideal

microorganism (capable of fermenting efficiently both pentoses and hexoses) has not

been found (Talebnia et al., 2010).

The yeast Saccharomyces and the bacteria Escherichia coli are the most common

microorganisms used to convert the sugars into ethanol (Tong et al., 2012). The yeast

Saccharomyces can produce ethanol from glucose with almost 90% of theoretical yield

(Gupta & Verma, 2015). Nonetheless, it cannot ferment the C5 sugars, so these are
converted into furfural, which is toxic to the yeast itself and affects the ethanol yields

(Saxena et al., 2009; Farias et al., 2017).

Regarding fermentation systems for lignocelluloses to ethanol operations, the

following approaches can be employed depending on the nature of the feedstock

(a) Separate Hydrolysis and Fermentation (SHF) involves four discrete process steps

(b) Simultaneous Saccharification and Fermentation (SiSF) which consolidates

hydrolysis and fermentation of cellulose hydrolysis products into one process step

(c) Simultaneous Saccharification and Co-fermentation (SSCF) involves two process

steps: cellulase production and a second step in which cellulose hydrolysis and

fermentation of both cellulose and hemicellulose hydrolysis products occurs

(d) Consolidated Bioprocessing (CBP) also known as Direct Microbial Conversion

(DMC), cellulase production, hydrolysis and fermentation of products of both cellulose

and hemicellulose hydrolysis are accomplished in a single process step.

All these processes require the hydrolysis of pre-treated biomass (with cellulase and

hemicellulase enzymes or microbes); and fermentation of resultant hexose (Glucose,

Mannose, Galactose) and pentose (Xylose, Arabinose) sugars

There are four processes that are commonly used in bioethanol production which are;

separate hydrolysis and fermentation (SHF), simultaneous saccharification and

fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF).

There is also the fourth method called: Consolidated Bioprocessing (CBP) also known

as Direct Microbial Conversion (DMC).

2.6.3.1 Separate Hydrolysis and Fermentation (SHF)

Enzymatic hydrolysis performed separately from fermentation step is known as separate

hydrolysis and fermentation (SHF) (Wingren et al., 2003). In this the pretreated biomass

first undergoes enzymatic hydrolysis (saccharification) followed by ethanolic

fermentation (Sanchez and Cardona, 2008) (. A major advantage of SHF is that

hydrolysis and fermentation can be performed at their optimum operating conditions.

The enzymes are however, end-product inhibited when cellobiose and glucose

accumulate (Sun and Cheng, 2002; Hahn-Hagerdal et al., 2006).

In SHF, hydrolysis of lignocellulosic materials is separated from ethanol fermentation.

The separation of enzymatic hydrolysis and fermentation allows enzyme to be operated

at high temperature for better performance while fermentation organisms can be

operated at moderate temperature for optimizing sugar utilization.

2.6.3.2 Simultaneous Saccharification and Fermentation (SiSF)

The idea of performing the enzymatic hydrolysis and fermentation simultaneously was

put forward by Gauss and co-workers in a patent from 1976 (Gauss et al., 1976).

In SiSF, hydrolysis and fermentation are performed in a single process unit allowing

reducing sugars produced to be immediately consumed by the fermenting organism.

Thus, the effect of end-product inhibition by sugars is neutralized SiSF also seems to

decrease the inhibition of enzymes by toxic byproducts present in pre-hydrolysate after

pretreatment (Tengborg et al., 2001). This improves the overall ethanol yield and

productivity.

SiSF compared to the two-stage SHF process has several other advantages that

include;

(i) a lower enzyme requirement

(ii) a reduced risk of contamination, since glucose is removed immediately and

ethanol is produced and a shorter process time

(iii) less reactor volume because a single reactor is used and lower capital costs (Sun

and Cheng, 2002).

However, there are some drawbacks of the SiSF process, one of which is the difficulty

encountered with yeast recirculation due to the presence of lignin residues in the

hydrolysate (Ohgren et al., 2007). A major disadvantage of SiSF is that the optimum

temperature condition for enzyme hydrolysis (45-500C) is much higher than what is

required for fermentation (e.g., 300C for S. cerevisiae). Therefore, a compromise

temperature of around 380C is employed meaning hydrolysis is usually the rate limiting

process in SiSF (Philippidis and Smith, 1995; Sun and Cheng, 2002).

2.6.3.3 Simultaneous Saccharification and Co-fermentation (SSCF)

An improvement of the SSF technology called SSCF (Simultaneous Saccharification

and Cofermentation) is targeted at ethanol production from both hexose and pentose

sugars in one step. Thus it basically has the same mechanism as SSF. Cofermentation

refers to the fermentation of both pentoses and hexoses to ethanol.

The hydrolyzed hemicelluloses during pretreatment and the solid cellulose are not

separated after pretreatment allowing the hemicelluloses sugars to be converted to

ethanol together with SSF of the cellulose (Teixeira et al., 2000).

SSF and SSCF have a short overall process as the enzymatic hydrolysis and

fermentation process occur simultaneously to keep the concentration of glucose low.

For SSF, the fermentation of glucose is separated from pentose while SSCF ferment

glucose and pentose in the same reactor. Both SSF and SSCF are preferred over SHF

because the operation can be performed in the same tank. The benefits of both processes

are lower cost, higher ethanol yield and shorter processing time.
SSCF offers increased potential for a more streamlined processing and lower capital

costs. The success of SSCF and co-fermentation of hexose and pentoses in general

requires the construction of genetically engineered microorganisms able to co-ferment

glucose and xylose concurrently with enzymatic hydrolysis of cellulose and

hemicellulose.

In Consolidated Bioprocessing (CBP), ethanol together with all of the required

enzymes is produced in a single bioreactor by a single microorganism’s community.

The process is also known as direct microbial conversion (DMC). It is based on

utilization of mono-co-cultures of microorganisms which ferment cellulose to ethanol.

CBP seems to be an alternative approach with outstanding potential and the logical

endpoint in the evolution of ethanol production from lignocellulosic materials.

Application of CBP entails no operating costs or capital investment for purchasing

enzymes or its production (Hamelinck et al., 2005; Lynd et al., 2005).

2.6.4 Factors affecting bioethanol production

There are several factors which influence the production of bioethanol including

temperature, sugar concentration, pH, fermentation time, agitation rate, and

inoculum size [86]. The growth rate of the microorganisms is directly affected by the

temperature. High temperature which is unfavourable for cells growth becomes a stress

factor for microorganisms [88]. The ideal temperature range for fermentation is between

20 and 35 °C. Free cells of S. cerevisiae have an optimum temperature near 30 °C

whereas immobilized cells have slightly higher optimum temperature due to its ability

to transfer heat from particle surface to inside the cells [89]. Moreover, enzymes which

regulate microbial activity and fermentation process are sensitive to high temperature

which can denature its tertiary structure and inactivates the enzymes [90].

Thus, temperature is carefully regulated throughout the fermentation process. The

increase in sugar concentration up to a certain level caused fermentation rate to increase.

However, the use of excessive sugar concentration will cause steady fermentation rate.

This is because the concentration of sugar use is beyond the uptake capacity of the

microbial cells.

Generally, the maximum rate of ethanol production is achieved when using sugars at the

concentration of 150 g/L. The initial sugar concentration also has been considered as an

important factor in ethanol production. High ethanol productivity and yield in batch

fermentation can be obtained by using higher initial sugar concentration.

In fermentation for ethanol production, the optimum pH range of S. cerevisiae is 4.0–

5.0. When the pH was below than 4.0, a longer incubation period is required but the

ethanol concentration was not reduced significantly. However, when then pH was above

5.0, the concentration of ethanol reduced substantially.

Fermentation time affects the growth of microorganisms. Shorter fermentation time

causes inefficient fermentation due to inadequate growth of microorganisms. On the

other hand, longer fermentation time gives toxic effect on microbial growth especially

in batch mode due to the high concentration of ethanol in the fermented broth.

Complete fermentation can be achieved at lower temperature by using longer

fermentation time which results in lowest ethanol yield. Agitation rate controls the

permeability of nutrients from the fermentation broth to inside the cells and removal of

ethanol from the cell to the fermentation broth. The greater the agitation rate, the higher

the amount of ethanol produced. Besides, it increases the amount of sugar consumption

and reduces the inhibition of ethanol on cells.

The common agitation rate for fermentation by yeast cells is 150–200 rpm. Excess

agitation rate is not suitable for smooth ethanol production as it causes limitation to the

metabolic activities of the cells.

Most of fermentation process using S. cerevisiae was carried out the at 30 °C whereas

fermentation using K. marxianus was performed at 42 °C.

The ideal temperature for bioethanol production depends on the ideal temperature of the

yeasts. Most of the fermenting medium used for bioethanol production has pH in the

range of 4.5–5.5 with various sugar concentrations. Fermentation and hydrolysis

processes are commonly carried out 72h and 24h with rotation at 150 and 120 rpm

respectively.

2.6.5 Distillation and Purification

The recovery of ethanol from fermentation broth is done by distillation. Ethanol

purification is critical for any kind of purpose. In the industry, purification is done by

mainly distillation. Although distillation is a strong separation technique, it has several

advantages, mainly its separation capacity of volatile compounds and cost.

Different separation methods can be used, and include ordinary distillation, azeotropic

distillation, extractive distillation, liquid extraction fermentation hybrid, absorption and

membrane separation (Adekunle et al., 2016).

The ordinary distillation can give an ethanol recovery of 95% however, to achieve

99.9% of ethanol recovery (anhydrous ethanol), further dehydration is needed (Zabed et

al., 2016). Nevertheless, this purification technique has high-energy requirements,

limited capacity for the separation of volatile compounds (since they tend to lodge more

in ethanol), and high costs. The costs of the distillation depend on the efficiency of the

enzymatic hydrolysis and on the fermentation, and increase with low ethanol

concentrations (Onuki et al., 2008; Saini et al., 2015; Farias et al., 2017).
 CHAPTER THREE

3.0 METHODOLOGY

3.1 Chemicals and Equipment

3.1.1 Chemicals

1. NaOH (Sodium Hydroxide) pellets

2. Citric acid pellets

3. Absolute Ethanol

4. Deionized water

5. Buffer solution of Citric Acid and Sodium Hydroxide

3.1.2 Materials used

Table 3.1; materials used

SN Material Quantity Uses

1. Evaporating dish 5 To ash
2. Beaker 4 Holding and transferring liquid
3. Volumetric flask 5 Solution holder and measuring
solution
4. Masking Tape 2 Labelling
5. Test tubes 20 Holding solution
6. 100ml pyrex glass 30 Hydrolysis/Fermentation
7. Conical flask 2 Preparing and holding solution
8. Plastic container 20 Keeping samples
9. Filter paper 5 Filtration
10. Stirrer 5 Agitation
11. Desiccator 1 Prevent moisture from entering
sample
12. Aluminium foil 5 Covering content
13. Erlenmeyer flask with spout 2 Preparation of solution
14. Pipette 5 Picking solution
15. Spatula 1 Picking small matter

3.1.3 Equipment

1. Oven: for drying

2. Blender: for blending the substrate

3. Refrigerator: for keeping the substrate in other to avoid spoilage

 4. Refractometer: test for sugar

5. Autoclave: for sterilizing the materials

6. HPLC Equipment: for solid or liquid analyte phase separation

7. Thermo-Shaker: for mixing and temperature control of sample

8. UV/VIS Spectrophotometer: for quantitative determination of different

analytes

9. Water bath: for incubation of samples in water at a constant temperature

10. Incubator: a device used to grow and maintain cell cultures

11. Analytical balance: for accurate measurement of mass of sample

12. Centrifuge : for separation of fluids based on their densities

13. Fume hood : it provides protection against toxic fumes

14. System controller: determines the slope and level values by setting up

fractionation parameters graphically

3.2 Sampling and Sample preparation

Fruit wastes from Blue Skies, pineapple, pawpaw and mango were collected and used

in the research. The selection of fruits waste was done due to their availability. The

waste includes peels of pineapple, peels of mango, peels of pawpaw and pineapple core.

The substrates (samples) were washed with deionized water. It was later kept in the

freezer for some time. The sample was then taken from the refrigerator to defrosts, after

which it was weighed (3000g in total) and then blended using an industrial blender and

aliquots made in ziploc bags and stored in a freezer at -20 oC. A 1.3Litre volume of

deionized water was added during the blending. The pH was measured and recorded.

Substrate Acquisition

The substrate used was fruit waste obtained from Blue Skies Company Limited, Ghana.

• The fruit waste comprised of:

 • Pineapple core (Ananas sp)

• Pineapple peels (Ananas sp)

• Pawpaw peels (Asimina sp)

• Mango peels (Mangifera sp)

3.2.1 Substrate Preparation

• The Fruit waste was macerated with an industrial blender and aliquots made and

stored in a freezer at -20 oC.

• 2.5% ,5%, 7.5 % and 10% substrate concentrations were used for the analysis.

• Enzyme dosage of 15u/g.DM was used for the hydrolysis.

Figure 2 blended sample of pawpaw, mango and pineapple peels in a zip-lock bag

3.3 Proximate and Ultimate Analysis

3.3.1 Determination of moisture content

About 4g of the blend of pineapple, pawpaw and mango peels sample was weighed out

in the evaporating dish, before then the evaporating dish was dried in the oven at a

temperature of 105 ± 2oC to take of all the moisture on the dish and then weighed. After

which the sample on the evaporating dish has been heated and weighed. This was done

in three different evaporating dishes. The final reading was taken when a constant mass
was attained. NREL protocol for determination of moisture content was used as a guide.

The method is as follows:

Convection oven method

Three evaporating dishes weighed and pre-dry by placing them in a 103ºC drying oven

for a minimum of four hours. The dishes were then cool in a desiccator. The pre-dried

dishes were then weighed to the nearest 0.1 mg. These weights were recorded.

The blended sample was then weigh out an appropriate amount to the nearest 0.1 mg,

into the weighing dish. The weight of the sample plus weighing dishes were recorded.

The samples were then placed into the convection oven at 103oC for 4 hours and. They

were then removed from the oven and allow to cool to room temperature in a desiccator.

Each of the three dishes containing the oven-dried sample was weighed to the nearest

0.1mg. They weight was then recorded

The samples were placed back into the convection oven at 103oC and dry to constant

weight. Constant weight is defined as ± 0.1% change in the weight percent solids upon

one hour of re-heating the sample.

The total solids were calculated using the formula below.

(Weight dry dish plus dry sample - Weight dry dish)

%Total Solids = ×100

weight sample as received

The moisture content was calculated using the formula below.

% moisture content =

Weight of sample before drying−Weight of sample after drying

×100
Weight of sample beforedrying
3.3.2 Determination of crude fibre

Material and reagent

Conical flask, glass, stirring rod, funnel, filter paper are the materials used while the

reagents used include distilled water, ethanol, boiling water, acetic acid, trichloroacetic

acid.

Procedure

5g of sample was digested into trichloroacetic acid by refluxing for 40 minutes and then

filtered. The residue was washed with boiling distilled water and then with acetone. The

washed residue was dry-heated at 103oC in oven and the dried residue was scraped into

porcelain crucible, weighed and then placed in furnace for ashing at 550 0C for 2 hours,

after which it was removed and cooled in desiccator and weighed.

weight of crucible+residue −weight of crucible+ ash

The fibre content = ×100
Initial weight of sample

3.3.5 Analytical techniques

Chemical composition of pineapple, banana and pawpaw: The cellulose, hemicellulose

and lignin were estimated using Proximate and Ultimate analysis

3.4 Enzymatic hydrolysis of lignocellulosic substrate

Enzymatic hydrolysis of substrate (blend of pineapple, mango and pawpaw) was carried

out in 0.05 M citrate acid monohydrate buffer of 750ml (pH 5.0). A buffer of pH 5.0

was prepared. For each substrate concentration (2.5%, 5%, 7.5%, 10%) of the blended

sample was weighed in a sterilised air-tight bottles(flask) on an analytical balance.

Labels were put on each flask according to the substrate concentrations.

A calculated amount of the prepared buffer solution is added to each of the weighed

samples of different concentrations. This was done using a pipette which has also been
sterilized. Thereafter, commercial enzyme (containing 15 U per g of substrate) was

added. The flask or bottles were then placed into the thermos-shaker (an incubator) with

the following conditions:500 C and 150 rpm.

Smaller quantities from the nine different samples were picked at different time

intervals of 12hours (5 days), centrifuged at 150 rpm and the supernatant was analysed

for total reducing sugars released. The samples were then removed on the fifth day after

hydrolysis. The test tubes were labelled accordingly and placed in a freezer. This was

done in other to stop the enzymatic reaction.

Figure 3 some samples of blend in incubated shaker during hydrolysis

3.4.1 Reducing Sugars Determination using PAHBAH

Reducing sugars are carbohydrates with an aldehyde group that act as reducing agent

for some chemicals. Examples of reducing sugars are: glucose, galacturic acid,

galactose, fructose, mannose, xylose, rhamnose, fucose and arabinose are reducing

sugars. The concentration of the reducing sugars is quantitatively correlated to the

intensity of bright yellow colouration by the reaction with the chromogenic reagent, 4-

hydroxybenzoic acid hydrazide (PAHBAH)

Assay Procedure

0.5% w/v of PAHBAH solution was prepared in 0.5M NaOH. Glucose standards with

concentrations (0, 0.4, 0.8, 1.5, 3.0, and 5Mm) to determine the standard curve.
0.1ml of standards was added to 2.9ml of the PAHBAH solution in falcon tubes. It was

mixed well and retained in boiling water for 5minutes.

The samples in the tubes were allowed to cool to room temperature (about 20-30mins)

and the absorbance was measured at 410nm in a spectrophotometer using PAHPAH

solution as the blank.

Figure 4 samples to be analysed in UV/VIS spectrophotometer

Reducing Sugars Determination using HPLC

The determination of reducing sugars by the HPLC device was done by Schimadzu 10-

20 A with an Aminex HPX -87 column. Calibration standards were prepared and run in

the column with a retention time of 20 minutes. About 0.5ml of each sample was

collected from test tubes, then collected using a 1.0ml syringe (BD Plastipak). It was

then filtered through 0.2µm filter into an autosampler vial cronus. Each vial was sealed

and labelled. The vials were put on a rack which was then loaded into the HPLC

containing a series of calibration standards. The samples were analysed by using a

Biorad Aminex HPX-87H column. The calibration standards used were glucose,

cellobiose, fructose, fucose.

The HPLC was run at the following conditions for hydrolysis analysis :

Column/ Detector temperature: 550 C , Oven temperature:600 C

Flowrate: 0.6ml/min, Run time: 20 minutes, Inlection volume: 10µl

 Figure 5 samples in vial and racks to be analyse by HPLC device

3.5 Fermentation

Organism used for Fermentation

Fermentation was carried in a 100ml Durham flask at 30 oC and 120 RPM for 24 hours

using an engineered Saccharomyces cerevisiae (Fast Turbo Yeast) obtained from Still

Spirits in Denmark

Also, Brewer’s yeast was used for fermentation. Selection of given yeast was done due

to their ability to produce high yield of alcohol by fermenting sugars.

Standard methods involved in fermentation

Apparatus and Materials

1. Balance

2. Pipette

3. Fermentation tubes

4. pH equipment
Reagent

1. Buffer Solution:

To prepare stock Citric Acid Monohydrate buffer solution, dissolve 7.9280g of citirc

acid monohydrate in 500 mL de-ionised water, adjust to pH 4.8 there about with 0.5M

sodium hydroxide (NaOH), and dilute to 750ml.

Procedure

For each substrate concentration (2.5%, 5%, 7.5%, 10%) of the blended sample was

weighed in a sterilised air-tight bottles(flask) on an analytical balance. Labels were put

on each flask according to the substrate concentrations. A calculated amount of the

prepared buffer solution is added to each of the weighed samples of different

concentrations. This was done using a pipette which has also been sterilized. The flask

or bottles were then placed into the thermos-shaker (an incubator) with the following

conditions:500 C and 150 rpm. The samples were then removed on the fifth day after

hydrolysis

Brewer’s yeast was then added, and stored in the shaker for 24 hours for the

fermentation. It was done at 300 C and 120 rpm.

Method for analysing the fermented sugar. (HPLC DEVICE)

The determination of reducing sugars by the HPLC device was done by Schimadzu 10-

20 A with an Aminex HPX -87 column. Calibration standards were prepared and run in

the column with a retention time of 20 minutes. About 0.5ml of each sample was

collected from test tubes, then collected using a 1.0ml syringe (BD Plastipak). It was

then filtered through 0.2µm filter into an autosampler vial cronus. Each vial was sealed

and labelled. The vials were put on a rack which was then loaded into the HPLC

containing a series of calibration standards. The samples were analysed by using a

Biorad Aminex HPX-87H column.

The HPLC was run at the following conditions for fermentation analysis:

Column/ Detector temperature: 55

Oven temperature: 60

Flowrate: 0.6ml/min

Run time: 45 minutes

Inlection volume: 10µl

Figure 6 HPLC device to analyse fermentation products

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APPENDIX

APPENDIX A

Abbreviations

HC- Hydrolysis control sample

HY- Hydrolysis sample

F- Fermentation sample

FC- Fermentation control sample

SC- Substrate concentration

g/l- gram per litre (unit for molar concentration)

Meaning of some terms

1. Ethanol: It is a clear, colorless, flammable solvent; also known as ethyl alcohol,

grain spirits, or neat alcohol (anhydrous). Unlike other alcohols of similar

molecular weight, ethanol is considered non-toxic or a drinking alcohol.

2. Flash point (The flash point of gasoline is -45°F; the flash point of ethanol is

-5°F): The lowest temperature at which a flammable liquid can form an ignitable

mixture in air near the surface of the liquid; the lower the value is, the easier it is

to ignite. This is the minimum temperature at which a liquid gives off vapor in

sufficient concentrations to allow the substance to ignite.

3. Auto-ignition temperature: The minimum temperature required to ignite a gas or

vapor to spontaneously combust in air without a spark or flame being present.

4. Specific gravity: The ratio of the density of a substance to the density of water.

5. Vapor density: Relative weight of a gas or vapor in comparison to air.

6. Boiling point: The temperature at which the vapor pressure of a liquid equals the

environmental pressure surrounding the liquid.

7. Polar Solvent: A compound such as alcohol, acid, or ammonia with a separation

of charge in the chemical bonds. These have an affinity for water and will

readily go into solution.

8. Hydrocarbon: A compound composed of only carbon and hydrogen and

commonly obtained through the refining of crude oil; these are the primary

constituent parts of both gasoline and diesel fuel.

9. Flammable liquid: Any liquid with a flash point under 100°F; referred to as

Class I liquids; examples are gasoline and ethanol.

10. Combustible liquid: Any liquid with a flash point above 100°F but below 200°F;

examples include diesel fuel and kerosene.

APPENDIX B

MEASURED PARAMETERS

• Total Reducing Sugars – Schimadzu 10-20 A with an Aminex HPX -87

column Carbohydrates – PABA Assay

• Hydrolysates- Schimadzu 10-20 A with an Aminex HPX -87 column

• Fermentation products - Schimadzu 10-20 A with an Aminex HPX -87 column

• Dry Matter – Gravimetric Method