See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/284174732

Optimized ethanol production from banana peels

Thesis June 2014

DOI: 10.13140/RG.2.1.1421.7200

CITATIONS READS

0 1,135

2 authors, including:

Mebrahtom Gebresemati
Gachon University
4 PUBLICATIONS 1 CITATION

SEE PROFILE

All content following this page was uploaded by Mebrahtom Gebresemati on 27 July 2016.

The user has requested enhancement of the downloaded file.

 MEKELLE UNIVERSITY

MEKELLE INSTITUTE OF TECHNOLOGY

DEPARTMENT OF BIOLOGICAL AND CHEMICAL ENGINEERING

Submitted to Mekelle University - Mekelle Institute of Technology in partial fulfillment of the Bachelor
of Science Degree in Biological and Chemical Engineering

By:

Alula Gebregergs

Mebrahtom Gebresemati

Advisors:

Mr. Abadi G/yesus (MSc)

Mr. Hailekiros Tadesse (MSc)
Mr. Kibrom G/egzabher (MSc)
June, 2014

Mekelle, Ethiopia
ACKNOWLEDGEMENT
We would like to acknowledge our advisors Mr. Abadi Gebreyesus (MSc), Mr. Hailekiros Tadesse (MSc)
and Kibrom Gebreegzabher (MSc) for their efforts, comments, fruit full advice, proper supervision. Their
knowledge and hands on experience in biological and chemical engineering discipline has added greatly to
the depth of information provided by this research paper.
We would like to express our gratitude to the laboratory of organic chemistry in Mekelle university for they
have granted us full access to laboratory equipments. Moreover, our deepest gratitude goes to the laboratory
technicians of the microbiology department at the college of veterinary medicine, Mekelle University, in
general and to Mr. Melesew in particular for his time and help during our laboratory work.

Finally, we would like to thank Desta Alcohol and Liquor Factory (DALF) in general and to Mr.
Gebremeskel Gebrewahd in particular for his help during experimental work.

II
CONTENTS
ACKNOWLEDGEMENT ........................................................................................................................... II
LIST OF FIGURES ..................................................................................................................................... V
LIST OF TABLES ...................................................................................................................................... VI
LIST OF ACRONYMS AND ABBREVIATIONS .................................................................................. VII
LIST OF APPENDICES .......................................................................................................................... VIII
ABSTRACT ................................................................................................................................................ IX
1. INTRODUCTION ................................................................................................................................. 1
1.1. BACKGROUND .......................................................................................................................... 1
1.2. STATEMENT OF THE PROBLEM ............................................................................................ 2
1.3. OBJECTIVES ............................................................................................................................... 3
1.3.1. GENERAL OBJECTIVE ...................................................................................................... 3
1.3.2. SPECIFIC OBJECTIVES ..................................................................................................... 3
1.4. SIGNIFICANCE OF THE STUDY .............................................................................................. 3
2. LITERATURE REVIEW ...................................................................................................................... 4
2.1. FEEDSTOCKS FOR ETHANOL PRODUCTION ...................................................................... 4
2.1.1. ETHANOL FROM SUGARS ............................................................................................... 5
2.1.2. ETHANOL FROM STARCH ............................................................................................... 5
2.1.3. ETHANOL FROM LIGNOCELLULOSIC BIOMASS ....................................................... 7
2.1.4. BANANA PEELS AS FEED STOCK FOR ETHANOL PRODUCTION .......................... 9
2.1.5. OVERVIEW OF ETHANOL PRODUCTION PROCESS ................................................ 10
2.2. GLOBAL TREND IN ETHANOL PRODUCTION AND USE ................................................ 14
2.3. CURRENT ETHANOL PRODUCTION IN ETHIOPIA ........................................................... 16
2.4. ECONOMIC AND ENVIRONMENTAL IMPACTS OF USING ETHANOL ......................... 17
2.4.1. ECONOMIC IMPACTS ..................................................................................................... 17
2.4.2. ENVIRONMENTAL IMPACTS ........................................................................................ 17
3. MATERIALS AND METHODS ........................................................................................................ 19
3.1. MATERIALS .............................................................................................................................. 19
3.1.1. CHEMICALS ..................................................................................................................... 19
3.1.2. EQUIPMENTS ................................................................................................................... 19
3.2. PROCEDURES........................................................................................................................... 20
3.2.1. SAMPLE COLLECTION AND PREPARATION ............................................................. 20
3.2.2. PRETREATMENT ............................................................................................................. 21
3.2.3. ACID HYDROLYSIS......................................................................................................... 21
3.2.4. MEASURING SUGAR CONTENT ................................................................................... 23

III
 3.2.5. FERMENTATION.............................................................................................................. 23
3.2.6. DISTILLATION ................................................................................................................. 24
3.2.7. DENSITY MEASUREMENT ............................................................................................ 24
4. RESULT AND DISCUSSION ............................................................................................................ 26
4.1. EXPERIMENTAL RESULTS .................................................................................................... 26
4.2. STATISTICAL ANALYSIS....................................................................................................... 27
4.3. EFFECTS OF EXPERIMENTAL VARIABLES ON HYDROLYSIS ...................................... 33
4.4. OPTIMIZATIONS ...................................................................................................................... 40
4.5. EFFECT OF BENZATHINE PENICILLIN G ON ETHANOL PRODUCTION ...................... 43
5. PLANT DESIGN AND COST ANALYSIS ....................................................................................... 44
5.1. PLANT DESIGN ........................................................................................................................ 44
5.1.1. FEEDSTOCK REPRESENTATION .................................................................................. 44
5.1.2. PROCESS DESCRIPTION ................................................................................................ 45
5.2. COST ANALYSIS...................................................................................................................... 47
5.2.1. EXECUTIVE SUMMARY (2014 Prices) .......................................................................... 47
5.2.2. ANNUAL OPERATING COST (2014 Prices)................................................................... 47
5.2.3. MATERIALS COST (2014 Prices) .................................................................................... 48
5.2.4. UTILITIES COST (2014 Prices) ........................................................................................ 48
6. CONCLUSIONS AND RECOMMENDATIONS .............................................................................. 50
6.1. CONCLUSION ........................................................................................................................... 50
6.2. RECOMMENDATION .............................................................................................................. 50
BIBLIOGRAPHY ....................................................................................................................................... 51
APPENDICES ............................................................................................................................................ 54

IV
LIST OF FIGURES
Figure 2-1: Structure of single cellulose molecule ....................................................................................... 8
Figure 2-2: A schematic representation of the hemicellulose backbone of arborescent plants. ................... 9
Figure 2-3: Dominant building blocks of the three-dimensional polymer lignin ......................................... 9
Figure 2-5: Schematic of lignocellulose pretreatment ................................................................................ 11
Figure 3-1: (a) Chopped sample, (b) Dried sample, (c) Grinder machine, and (d) Proportioned sample ... 20
Figure 3-2: Samples being hydrolyzing ...................................................................................................... 23
Figure 3-3: Media prepared ........................................................................................................................ 24
Figure 4-1: Sugar content and parameters condition .................................................................................. 27
Figure 4-2: Normal plots of residuals ......................................................................................................... 32
Figure 4-3: Residual versus predicted values ............................................................................................. 33
Figure 4-4: Effect of acid concentration and time on the yield of ethanol when temperature was at the
center point (a, b & c) ................................................................................................................................. 35
Figure 4-5: Effect of temperature and time on yield of ethanol when acid concentration was at the center
point (a, b & C) ........................................................................................................................................... 37
Figure 4-6: Effect of temperature and acid concentration on the yield of ethanol when time was at the
center point (a, b & c) ................................................................................................................................. 39
Figure 4-7: (a), (b) and (c) optimization of contours plot in sugar yield .................................................... 42
Figure 5-1: Process flow sheet for ethanol production from banana peel (SuperPro Designer) ................. 46

V
LIST OF TABLES
Table 2-1: Top ten banana producing nations in 2012 ................................................................................ 10
Table 2-2: Pretreatment methods of lignocellulosic biomass for ethanol production ................................. 12
Table 2-3: World Fuel Ethanol Production by Country or Region (Million Gallons) ................................ 15
Table 3-1: Experimental design formulation for hydrolysis stage .............................................................. 22
Table 4-1: Mass percentage of sugars ......................................................................................................... 26
Table 4-2: Specific gravity measurement results at 25oC ........................................................................... 27
Table 4-3: Design Summary of designs ...................................................................................................... 28
Table 4-4: Analysis of variance (ANOVA) for the quadratic model .......................................................... 29
Table 4-5: Model adequacy measures ......................................................................................................... 30
Table 4-6: Regression coefficients and the corresponding 95% CI High and Low .................................... 30
Table 4-7: Optimization criteria for optimum yield of soluble sugar content ............................................ 40
Table 4-8: Optimum possible solutions (10 solutions found) ..................................................................... 41
Table 5-1: Executive summary of Economic Evaluation Report................................................................ 47
Table 5-2: Annual operating cost ................................................................................................................ 48
Table 5-3: Material costs ............................................................................................................................ 48
Table 5-4: Utilities costs ............................................................................................................................. 48

VI
LIST OF ACRONYMS AND ABBREVIATIONS

AFEX Ammonia Fiber Explosion

ANOVA Analysis of Variance
CI Confidence Interval)
CO Carbon monoxide
CO2 Carbon dioxide
EIA Energy Information Administration
ESDA Ethiopia Sugar Development Agency
E5 Ethanol mixture that contain 5% ethanol and 95% unleaded gasoline
GHG Green House Gas
GTP Growth and Transformation Plan
H2SO4 Sulfuric acid
LHW Liquid hot water
IEA International Energy Agency
MgSO4. 7H2O Magnesium sulfate
MoWE Ministry of Water and Energy
MSW Municipal solid wastes
MTBE Methyl tertiary butyl ether
NaOH Sodium hydroxide
NRBP Northeast Regional Biomass Program
PAN Peroxyacetylnitrate
pH Power of Hydrogen
POM Poly-cyclic organic matter
SSF Simultaneous Saccharification and Fermentaion
USA United States of America

VII
LIST OF APPENDICES
Appendix A: Experimental results of mass percentage of sugars ............................................................... 54
Appendix B: Diagnostics Case Statistics: actual versus model Predicted of ethanol yield ........................ 54
Appendix C: Properties of ethanol .............................................................................................................. 55
Appendix D: Density of ethanol ................................................................................................................. 55
Appendix E: Materials overall balances ..................................................................................................... 56

VIII
ABSTRACT
Energy consumption has increased steadily over the last two decades as the population has grown and more
countries have become industrialized. On the other hand waste disposal has become the major concern of
developing cities. This study aims at utilizing banana peels for the production of bioethanol by using the
yeast Saccharomyces cerevisiae. The banana peels were collected and dried. After drying, each of the
samples was milled separately. The mill samples of 10gm were taken and passed through steam
pretreatment and hydrolysis to get maximum amount of soluble fermentable sugars. The effects of factors
in hydrolysis were investigated and the optimum combination of factors (acid concentration, temperature
and time) was set by response surface design software in which each of the three parameters are varied over
3 levels and 17 experimental runs were conducted to produce fermentable sugar. The sugar content of the
hydrolyzed samples were carefully measured and the specific mass percentage of the produced sugar was
determined. Box-Behnken response surface was applied to study the interaction effects, to see the contour
and surface plot of each variables. The significance of the result was set from analysis of variance
(ANOVA). The optimum results were obtained at 1.50 % v/v acid concentration, 91.02 C temperature and
21.66 min retention time. At this optimum condition, fermentation with and without benzyl penicillin was
performed to determine its effect on bioethanol. The specific alcohol content of the produced alcohol was
determined using pycnometer. From the results obtained the addition of benzyl penicillin during
fermentation enhanced the percentage of ethanol yield by 8.97%.

Keywords: Ethanol, Bioethanol, Biofuel, Banana peel, Pretreatment, Hydrolysis, Fermentation, Benzyl
penicillin, Sugar content

IX
1. INTRODUCTION

1.1. BACKGROUND
The overall well-being of the world; industrial competitiveness and the functioning of society are dependent
on safe, sustainable and affordable energy. Energy provides an essential power for almost all human
activities: it provides services for cooking, heating, lighting, health, food production and storage, education,
mineral extraction, industrial production and transportation.

Even though, world primary energy source nowadays is dominated by fossil fuels (coal, oil and natural
gas), the acceleration of technological development has opened door to continuous and rapid worldwide
economic growth and in fact allowed the world to achieve energy sustainability using many different energy
sources called renewable energy sources, comprising mainly biofuel, hydropower, geothermal, wind and
solar energy, currently represent less percentage of the primary energy use (Anonym, 2001).

Energy consumption has increased steadily over the last century as the world population has grown and
more countries have become industrialized. Crude oil has been the major resource to meet the increased
energy demand. Several dierent techniques has been used to estimate the current known crude oil reserves
and concluded that the annual global oil production would decline from the current 25 billion barrels to
approximately 5 billion barrels in 2050. Because the economy in the US and many other nations depends
on oil, the consequences of inadequate oil availability could be severe. Therefore, there is a great interest
in exploring alternative energy sources (Cheng & Sun, 2001).

Ethanol (ethyl alcohol, bioethanol) is the most employed liquid biofuel either as a fuel or as a gasoline
enhancer. Ethanol has some advantages when it is used as an oxygenate. Firstly, it has a higher oxygen
content that implies a less amount of required additive. The increased percentage of oxygen allows a better
oxidation of the gasoline hydrocarbons with the consequent reduction in the emission of CO and aromatic
compounds. Related to MTBE, ethanol has greater octane booster properties, it is not toxic, and does not
contaminate water sources. Nevertheless, ethanol production costs are higher than those of MTBE, gasoline
mixed with alcohol conduces the electricity, and Reid vapor pressure is higher that entails a greater
volatilization, which can contribute to ozone and smog formation. Many countries have implemented or are
implementing programs for addition of ethanol to gasoline (Sanchez & Cardona, 2007).

The fuel ethanol can be obtained from energy crops and lignocellulosic biomass. The complexity of the
production process depends on the feedstock. In this way, the spectrum of designed and implemented

1
technologies goes from the simple conversion of sugars by fermentation, to the multistage conversion of
lignocellulosic biomass into ethanol (Sanchez & Cardona, 2007).

Different biofuels such as ethanol, methanol, bio-diesels, etc. are produced by fermentation of agricultural
wastes, fruit wastes, municipal and industrial wastes using Saccharomyces Cerevisiae (bakers yeast) as
food for the microorganisms. Amongst these biofuels, ethanol has great demand as it is widely accepted
and it is clean burning (Raikar , 2012). A treatment of municipal solid waste goes far back into 18th century
when burying the waste was the best option of management. In course of time, the scarcity of resources and
the soaring pollution level necessitated the need for alternative treatment options. Recently, there are many
treatment options for municipal solid waste among which composting, incineration, land filling and
production of different biofuels are some. The choice among these options must be based on different
technical and economic criteria of the situation in question (Ashenafi, 2009).

In most developing African countries, municipal solid wastes are disposed of in non-engineered landfills
which have always been known to generate greenhouse gases. In developed nations, conversely, municipal
solid wastes are treated through such advanced methods as controlled incineration and production of
biofuels because of both increasing need of using the so called waste as resource and stringent
environmental regulations (Raikar , 2012). In this study the possibility of converting waste fruit peels into
valuable product (bioethanol) with optimum parameters was carried out.

1.2. STATEMENT OF THE PROBLEM

The massive dependence on fossil fuel import increasingly exposed Ethiopia to large international price
volatility that intensified its balance of payment. Today, an overriding emphasis has been given to biofuel
production. The government has set clear plan of increasing biofuel production to 1.8 billion liters by 2015
(Tesfaye & Gebru, 2011), consisting of 195 million liters of ethanol. Even though the four state owned
sugar factories; Fincha, Metahara, Wonji and Tendaho are included as main sources of ethanol in the plan,
according to The Ethiopian Sugar Development Agency (ESDA), by the year 2014/15 the projected total
bioethanol production form these factories is only 137,051,930 liters (Tesfaye & Gebru, 2011). On top of
that the country is also seeking increased percentage of ethanol blending for the future. Therefore, finding
another alternative source of bioethanol would be crucial to fulfill the growing demand.

On the other hand inadequate municipal and industrial solid waste collection and disposal creates a range
of environmental problems in our country. A considerable amount of waste ends up in open dumps or
drainage system, threatening both surface water and ground water quality and causing serious of
environmental and health problems. Open air burning of waste, spontaneous combustion in landfills and
incinerating plants that lack effective treatment for gas emissions are causing air pollution. The adverse

2
effects of inadequate solid waste service creates significant effect on productivity and economic
development. Solid waste such as fruit peels largely obtained as a byproduct from food processing
industries, juice processing houses, hotels and restaurants in our country. These wastes can entail serious
environmental problems unless they change or convert in to some useful products or disposed properly.

1.3. OBJECTIVES
1.3.1. GENERAL OBJECTIVE
The general objective of this project study was determining an optimized way of conversion of banana peel
wastes to ethanol.

1.3.2. SPECIFIC OBJECTIVES

The specific objectives of the study were:

Developing optimized parameters condition (acid concentration, temperature and time) for
hydrolyzing waste banana peels to obtain maximum amount of fermentable sugars by conducting
series of experimental analysis.
Determining the effect of benzyl penicillin in enhancing s. cerevisiae during fermentation.

1.4. SIGNIFICANCE OF THE STUDY

These days, the world is phasing acute shortage of energy. As a result, cost of energy is increasing
alarmingly. In addition to these problems, global warming is increasing at rate which seems out of control.
These are because of the nonrenewable source of energy we are currently depending on, which are also the
main source of greenhouse gases. As a result, the nature itself is calling for the generation to use alternative
renewable sources of energy such as fuel ethanol from biomass (Teshale, 2012).

Even though, efficient utilization of the already existing energy is the best and top rating both to overcome
the shortage of energy, searching renewable energy sources are important to solve the shortage of energy
and to reduce global warming. The world economy is now on much focus on possible ways to produce
renewable energy. As one and most promising solution, production of bio-ethanol from biomass has grown
steadily during the last four decades (Raikar , 2012). Ethiopia is also working strongly on the area of
renewable energy (bio-ethanol) production from molasses.

Ethiopia in its Growth and Transformation Plan (GTP) considers biofuel development as an opportunity
for providing domestic energy security, rapid economic development and creation of wealth. There were
great expectations that biofuel can solve major development challenges the country faces today. It is also
hoped that biofuel would meet substantial proportions of the national energy needs, reduce the need for

3
diminishing supplies of fossil fuels contributes to the solution of global climate change crisis, and create
new business opportunities (Tesfaye & Gebru, 2011).

2. LITERATURE REVIEW
Everything we do is connected to energy in one form or another. Concerns about the greenhouse effect and
global warming, air pollution, and energy security have led to increasing interest and more development in
renewable energy sources such as biofuel, solar, wind, geothermal, and hydrogen. Long term economic and
environmental concerns have resulted in a great amount of research in the past couple of decades on
renewable sources of liquid fuels to replace fossil fuels. Burning fossil fuels such as coal and oil releases
CO2, which is a major cause of global warming. Conversion of abundant lignocellulosic biomass to biofuels
as transportation fuels presents a viable option form proving energy security and reducing greenhouse
emissions (Kumar & Barrett, 2009).

Biofuels are energy carriers that store energy derived from biomass. Many types of biomass can be used to
produce biofuels, including agricultural residues, woods, fiber residues, grasses, as well as food crops. As
specified by different books one can distinguish between primary and secondary biofuels. Primary biofuels
are essentially raw biomass fuels (such as firewood, wood chips or sawdust pellets) that have undergone no
or only minor processing prior to conversion to energy, usually combustion. Secondary biofuels are solid,
liquid or gaseous energy carriers (such as charcoal, ethanol, biogas) that are produced after considerable
processing of raw biomass. Secondary biofuels can be used for a wider range of applications including
transportation and industrial processes (Langeveld & Sanders, 2010).

Lignocellulosic materials such as agricultural residues (e.g., wheat straw, sugarcane bagasse, corn stover),
forest products (hardwood and softwood), and dedicated crops (switchgrass, salix) are renewable sources
of energy. These raw materials are sufficiently abundant and generate very low net greenhouse emissions
(Kumar & Barrett, 2009).

2.1. FEEDSTOCKS FOR ETHANOL PRODUCTION

The complexity of the production fuel ethanol depends on the feedstock. The spectrum of designed and
implemented technologies goes from the simple conversion of sugars by fermentation, to the multi-stage
conversion of lignocellulosic biomass into ethanol. The big diversity of technological alternatives requires
the analysis of the global process along with the design and development of each one of the involved
operations. Among the new research trends in this field, process integration has the key for reducing costs
in ethanol industry and increasing bioethanol competitiveness related to gasoline (Sanchez & Cardona,
2007).

4
Several reviews have been published on the theme of fuel ethanol production especially from
lignocellulosic biomass. The amount of reviews covering ethanol production from other types of feedstocks
like sucrose-based or starchy materials is more reduced. Nevertheless, an analysis of this process from the
viewpoint of the three major types of feedstock has not been the main objective of those works. In addition,
some issues concerning the feedstocks features on a comparative basis have not always been sufficiently
emphasized (Sanchez & Cardona, 2007).

2.1.1. ETHANOL FROM SUGARS

Main feedstock for ethanol production is sugar cane in form of either cane juice or molasses (by-product of
sugar mills). About 79% of ethanol in Brazil is produced from fresh sugar cane juice and the remaining
percentage from cane molasses (Ghosh & Ghose, 2003). Sugar cane molasses is the main feedstock for
ethanol production in India; cane juice is not presently used with this purpose (Ghosh & Ghose, 2003). Beet
molasses are other source of fermentable sugars for ethanologenic fermentation.

The most employed microorganism in the conversion of sugars into ethanol is Saccharomyces cerevisiae
due to its capability to hydrolyze cane sucrose into glucose and fructose, two easily assimilable hexoses.
Other yeasts, as Schizosaccharomyces pombe, present the additional advantage of tolerating high osmotic
pressures (high amounts of salts) and high solids content (Bullock, 2002). In fact, a fermentation process
using a wild strain of this yeast has been patented (Carrascosa, 2006).

Among bacteria, the most promising microorganism is Zymomonas mobilis, which has a low energy
efficiency resulting in a higher ethanol yield (up to 97% of theoretical maximum). However, its range of
fermentable substrates is too narrow (glucose, fructose and sucrose) (Claassen, Van Lier, Lopez, Sijtsma,
& Stams, 1999). Other disadvantage of the use of this bacterium during the fermentation of sugar cane
syrup and other sucrose based media is the formation of the polysaccharide levan (made up of fructose
units), which increases the viscosity of fermentation broth, and of sorbitol, a product of fructose reduction
that decreases the efficiency of the conversion of sucrose into ethanol (Lee & Huang, 2000).

2.1.2. ETHANOL FROM STARCH

Starch is a high yield feedstock for ethanol production, but its hydrolysis is required to produce ethanol by
fermentation. Starch was traditionally hydrolyzed by acids, but the specificity of the enzymes, their inherent
mild reaction conditions and the absence of secondary reactions have made the amylases to be the catalysts
generally used for this process. -amylase obtained from thermoresistant bacteria like Bacillus
licheniformis or from engineered strains of Escherichia coli or Bacillus subtilis is used during the first step

5
of hydrolysis of starch suspensions. For amylases to attack starch, these suspensions should be brought to
high temperatures (90110 C) for the breakdown of starch kernels (Harmsen, Huijgen, Bermudez, &
Bakker, 2010).

Apar & Ozbek (2004) provides information about the effects of operating conditions on the enzymatic
hydrolysis of corn starch using commercial -amylase. The possibility of hydrolyzing starch at low
temperatures for achieving energy savings is being investigated (Burmaster, 2007). The product of this first
step, called liquefaction, is a starch solution containing dextrines and small amounts of glucose. The
liquefied starch is subject to saccharification at lower temperatures (6070 C) through glucoamylase
obtained generally from Aspergillus niger or Rhizopus species (Pandey, et al., 2004).
Ethanol is produced almost exclusively from corn in the USA. Corn is milled for extracting starch, which
is enzymatically treated for obtaining glucose syrup. Then, this syrup is fermented into ethanol. There are
two types of corn milling in the industry: wet and dry. During wet-milling process, corn grain is separated
into its components. Starch is converted into ethanol and the remaining components are sold as co-products
(Harmsen, Huijgen, Bermudez, & Bakker, 2010).
Although in France ethanol is mostly produced from beet molasses, it is also produced from wheat by a
process similar to that of corn. Some efforts have been done for optimizing fermentation conditions. For
example, Dai et al. (2006) have determined the optimal fermentation temperature and specific gravity of
the wheat mash. Barber et al. (2002) have optimized the conditions for starch hydrolysis using -amylase
and glucoamylase obtained by solid-state fermentation of wheat bran.

To enhance fermentation performance, high gravity fermentations have been proposed, particularly for the
case of wheat mashes. In this kind of process, the initial dissolved solids concentration exceeds 200 g/L
implying a higher substrate load. Therefore, higher ethanol concentrations are obtained, lower amounts of
process water are required, and energy costs are decreased. The drawbacks of this technology include longer
fermentation times, and sometimes incomplete fermentations probably caused by product inhibition, high
osmotic pressures and inadequate nutrition (Barber, Henningsson, & Pamment, 2002).
Cassava represents an important alternative source of starch not only for ethanol production, but also for
production of glucose syrups. In fact, cassava is the tuber that has gained most interest due to its availability
in tropical countries being one of the top ten more important tropical crops. Ethanol production from
cassava can be accomplished using either the whole cassava tuber or the starch extracted from it. Starch
extraction can be carried out through a high-yield large-volume industrialized process as the Alfa Laval
extraction method (FAO, 2004), or by a traditional process for small- and mid-scale plants. This process
can be considered as the equivalent of the wet-milling process for ethanol production from corn.

6
The production of cassava with high starch content (8590% dry matter) and less protein and minerals
content is relatively simple. Cassava starch has a lower gelatinization temperature and offers a higher
solubility for amylases in comparison to corn starch. The hydrolysis of cassava flour has been proposed for
the production of glucose in an enzymatic hollow-fiber reactor with 97.3% conversion (Hazell & Pachauri,
2006) considering that cassava flour production is more simple and economic than cassava starch
production. However, it is considered that cassava ethanol would have better economic indicators if the
whole tuber is used as feedstock, especially when small producers are involved.
Fuel ethanol production from whole cassava is equivalent to ethanol production from corn by dry-milling
technology. For this, cassava should be transported as soon as possible from cropping areas considering its
rapid deterioration due to its higher moisture content (about 70%). Hence, this feedstock should be
processed within 34 days after its harvest. One of the solutions to this problem consists in the use of sun-
dried cassava chips. The farmers send the cassava roots to small chipping factories where they are peeled
and chopped into small pieces. The chips are sun-dried during 23 days. The final moisture content is about
14% and the starch content reaches 65% (Raikar , 2012).
Besides corn and wheat, ethanol can be produced from rye, barley, triticale (Dai, Hu, Pu, Li, & Wang,
2006), and sorghum (Zhan, et al., 2003). For these cereals, some pretreatments have proven to be useful.
Dai et al. (2006) have employed the pearling of wheat, barley, rye and triticale grains for increasing starch
content of the feedstock in an average of 12% obtaining a 6.522.5% enhance in ethanol yield during
fermentation.

2.1.3. ETHANOL FROM LIGNOCELLULOSIC BIOMASS

It is evident the importance of lignocellulosic biomass as a feedstock for ethanol production.
Lignocellulosic complex is the most abundant biopolymer in the Earth. It is considered that lignocellulosic
biomass comprises about 50% of world biomass and its annual production was estimated in 1050 billion
ton (Claassen, Van Lier, Lopez, Sijtsma, & Stams, 1999). Many lignocellulosic materials have been tested
for bioethanol production. In general, prospective lignocellulosic materials for fuel ethanol production can
be divided into six main groups: crop residues (cane bagasse, corn stover, wheat straw, rice straw, rice hulls,
barley straw, sweet sorghum bagasse, olive stones and pulp), hardwood (aspen, poplar), softwood (pine,
spruce), cellulose wastes (newsprint, waste office paper, recycled paper sludge), herbaceous biomass
(alfalfa hay, switchgrass, reed canary grass, coastal Bermudagrass, thimothy grass), and municipal solid
wastes (MSW) (Cheng & Sun, 2001).
Lignocellulosic materials represent a promising option as a feedstock for ethanol production considering
their output/input energy ratio, their great availability both in tropical and temperate countries, their low
cost (primarily related to their transport), and their ethanol yields. One of the advantages of the use of
lignocellulosic biomass is that this feedstock is not directly related to food production. This implies the

7
production of bioethanol without the need of employing vast extensions of fertile cultivable land for
cropping cane or corn exclusively dedicated to the bioenergy production. In addition, lignocellulosics is a
resource that can be processed in different ways for production of many other products like synthesis gas,
methanol, hydrogen and electricity (Chung & Nam, 2002).
Lignocelluloses is the primary building block of plant cell walls. It is a renewable organic material
contained by all plants. Fruit peels are the rich source of lignocellulose. Lignocellulosic biomass is
composed of cellulose, hemicellulose and lignin, as well as other minor components. Both the cellulose and
hemicellulose fractions are polymers of sugars, and thereby a potential source of fermentable sugars
(Harmsen, Huijgen, Bermudez, & Bakker, 2010).The composition of these constituents can vary from one
plant species to another. For example, hardwood has greater amounts of cellulose, whereas wheat straw and
leaves have more hemicellulose (Kumar & Barrett, 2009). In addition, the ratios between various
constituents within a single plant vary with age, stage of growth, and other conditions (Kumar & Barrett,
2009).
Lignocellulosic biomass has a complex internal structure. It is comprised of a number of major components
that have, in their turn, also complex structures (Harmsen, Huijgen, Bermudez, & Bakker, 2010). Cellulose
is the -1, 4-polyacetal of cellobiose (4-O--D-glucopyranosyl-D-glucose). Cellulose is more commonly
considered as a polymer of glucose because cellobiose consists of two molecules of glucose. The chemical
formula of cellulose is (C6H10O5)n and the structure of one chain of the polymer is presented in Figure 2-1.

Source: (Harmsen, Huijgen, Bermudez, & Bakker, 2010)

F IGURE 2-1: S TRUCTURE OF SINGLE CELLULOSE MOLECULE

The term hemicellulose is a collective term. It is used to represent a family of polysaccharides such as
arabino-xylans, gluco-mannans, galactans, and others that are found in the plant cell wall and have different
composition and structure depending on their source and the extraction method.

8
Source: (Harmsen, Huijgen, Bermudez, & Bakker, 2010)
F IGURE 2-2: A SCHEMATIC REPRESENTATION OF THE HEMICELLULOSE BACKBONE OF ARBORESCENT PLANTS .

Lignin is the most complex natural polymer. It is an amorphous three-dimensional polymer with
phenylpropane units as the predominant building blocks. More specifically, p-coumaryl alcohol, coniferyl
alcohol and sinapyl alcohol (Figure 2-3) are the ones most commonly encountered.

P-coumaryl-1 , coniferyl-2 and sinapyl alcohol-3

Source: (Harmsen, Huijgen, Bermudez, & Bakker, 2010)
F IGURE 2-3: D OMINANT BUILDING BLOCKS OF THE THREE -DIMENSIONAL POLYMER LIGNIN

Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore
lignocelluloses degradation is essential for maintaining the global carbon cycle. Cellulose-hydrolysis results
in the production of glucose, which is readily fermented with existing organisms in much the same way as
has been done for centuries. Hemicellulose hydrolysis produces both hexose and pentose sugars: mannose,
galactose, xylose and arabinose that are not all fermented with existing strains. Other compounds primarily
acetic acid is also produced during the hydrolysis that can inhibit the ethanol fermentation process. By-
products include mostly lignin. When compared to existing grain ethanol by-products they have much less
food value and are used as fuel (Gaikwad, 2013).

2.1.4. BANANA PEELS AS FEED STOCK FOR ETHANOL PRODUCTION

Banana is a tropical fruit grown in over 122 countries worldwide. Until 2004, the cultivated area of 3.8
million hectares and a total production of 56.4 million metric tons of the fruit were produced ranking it
fourth behind rice, corn and milk (Jamal, Saheed, & Alam, 2012). The top ten world producers of banana
are presented in the below table.

9
T ABLE 2-1: T OP TEN BANANA PRODUCING NATIONS IN 2012

Country Production (million of tones)

1. India 29.7
2. Uganda 11.1
3. China 10.7
4. Philippines 9.2
5. Ecuador 8.0
6. Brazil 7.3
7. Indonesia 6.1
8. Colombia 5.1
9. Cameroon 4.8
10. Tanzania 3.9
Source: Food and Agricultural Organization of the United Nations (FAO, 2012)

The distributions showed that Asian countries (India, Philippines, China, Indonesia and Thailand) had more
productions while Latin America countries (Ecuador, Brazil, Columbia, Costa Rica, Mexico) were
seconding.

In Ethiopia however, banana is the second major fruit crop produced next to citrus. Domestic needs are
largely met from local production which spreads over most parts of the country. Ethiopia lies entirely in the
tropics where vast area are suitable for banana growing with an average annual temperature of 20 oC and a
well distributed annual rainfall of about 1000mm. As source of food many peoples eat banana and threw
banana peels in roads, drains etc. that can create health problems and environmental impacts in many parties
of the nation (Seifu, 2001).

2.1.5. OVERVIEW OF ETHANOL PRODUCTION PROCESS

The production of bioethanol from lignocellulosic material consists of a pretreatment, a hydrolysis and a
fermentation steps. In these processes, lignin is discharged as a by-product and can be used to generate
electricity to supply the process with energy, or to export electricity to the grid (Cheng & Sun, 2001). Lignin
is composed of phenolic components, which are not fermentable under anaerobic conditions. Pretreatment
is necessary to break open the lignocellulosic structures and to facilitate the separation of the main
carbohydrate fractions hemicellulose and cellulose from lignin, in order to make these better accessible for
hydrolysis, the next step in the process. Hydrolysis is the process to convert the carbohydrate polymers
cellulose and hemicellulose into fermentable sugars. Hydrolysis can be performed either chemically in a
process involving the use of concentrated acids, or enzymatically by using enzymes. Most pathways

10
developed today are based on enzymatic hydrolysis, by using cellulases and hemicellulases (Cheng & Sun,
2001).
Fermentation is the main process used to convert fermentable sugars, produced from the previous hydrolysis
step, into ethanol. In principle, the fermentation process is largely similar to that in the current ethanol
production facilities; however, a major fraction of sugars produced from lignocellulosic are pentoses (5-
carbon sugars such as xylose) (Cheng & Sun, 2001), which are difficult to ferment with standard industrial
microorganisms. In some processes, the hydrolysis and fermentation steps are combined into one step, in a
process often referred to as simultaneous saccharification and fermentation (SSF).

a) PRETREATMENT
Pretreatment is applied to remove lignin and hemicellulose, reduce cellulose crystallinity, and increase the
porosity of the materials. Pretreatment must meet the following requirements: improve the formation of
sugars or the ability to subsequently form sugars by enzymatic hydrolysis, avoid the degradation or loss of
carbohydrate, avoid the formation of byproducts inhibitory to the subsequent hydrolysis and fermentation
processes, and be cost-eective. Physical, physicochemical, chemical, and biological processes have been
used for pretreatment of lignocellulosic materials (Sanchez & Cardona, 2007).

Source: (Kumar & Barrett, 2009)

F IGURE 2-4: SCHEMATIC OF LIGNOCELLULOSE PRETREATMENT

11
T ABLE 2-2: P RETREATMENT METHODS OF LIGNOCELLULOSIC BIOMASS FOR ETHANOL PRODUCTION

Methods Procedure/agents Remarks Examples

Physical Mechanical Chipping, grinding, Milling: vibratory ball mill (final Wood and forestry
comminution milling size: 0.2 -2 mm), knife or hammer wastes
mill (final size: 3 6 mm)
o
Pyrolysis T>300 C, then Formation of volatile products Wood, waste cotton,
cooling and corn stover
condensing
Physco- Steam Saturated steam at 80 -100% hemicellulose Poplar, aspen,
chemical explosion 160 290 C, P (0.69
o
hydrolysis, it can handle high eucalyptus
-4.85 MPa) solids loads, size reduction with
lower energy input
Liquid hot Pressurized water, 80 -100% hemicellulose Bagasse, corn, olive
water (LHW) P>5 MPa, T (170 hydrolysis, cellulose pulp
230 oC) depolymerization, partial
solubilization of lignin
Ammonia fiber 1 -2kg ammonia/kg Ammonia recovery required, 0 - Wood chips, wheat
explosion dry biomass, 90 oC, 30 60% hemicellulose hydrolysis straw, rice
(AFEX) min, 1.12 -1.36 MPa
CO2 explosion 4kg CO2/kg fiber, P Cellulose conversion (>75%) Alfalfa, paper
(5.62 MPa)
Chemical Ozonolysis Ozone, room Cellulose conversion (>57%) and Pine, wheat straw,
temperature and lignin degradation cotton straw, peanut
pressure
Dilute acid 0.5 - 5% H2SO4, HCl, pH neutralization required, 80 10 Bagasse, corn
hydrolysis or HNO3, P~1 MPa hemicellulose hydrolysis, lignin stover, wheat straw,
redistributed switchgrass
Concentrated 10-30% H2SO4, 170 Acid recovery required, greater Poplar sawdust
acid hydrolysis 190 oC time residence than dilute acid
Alkaline Dilute NaOH, 24 h, >50% hemicellulose hydrolysis Hardwood
hydrolysis 60 oC, Ca(OH)2, 4h,
120 oC
Biological Fungal Brown-white-and Fungi produce Degrading enzymes Corn stover, wheat
pretreatment soft-rot fungi straw
Source: (Sanchez & Cardona, 2007)

12
 b) HYDROLYSIS
In the hydrolysis reaction, the complex chains of sugars that make up the hemicllulose are broken, releasing
simple sugars. The complex hemi-cellulose sugars are converted to a mix of soluble five-carbon sugars,
xylose and arabinose, and soluble six-carbon sugars, mannose and galactose. The rest of hemicelluloses are
degraded to weak acids, furan derivates, and phenolics. These compounds, however, are potential
fermentation inhibitors (Gupta, 2009). By the action of acids and/or enzymes, the glucose yields of cellulose
hydrolysis often exceed 90%, but hydrolysis without preceding pretreatment yields typically less than 20%
only. The pretreated feedstock can be hydrolyzed by two methods (Gupta, 2009).

c) FERMENTATION
The fermenting of the biomass is conducted under standard fermenting conditions and will utilize all the
major biomass. Yeast is the most commonly used microorganism in fermentation processes. Yeasts are
minute, often unicellular, fungi. The yeasts used are typically brewers' yeasts. Examples of yeast capable
of fermenting the decaying biomass include, but are not limited to, Saccharomyces cerevisiae and
Saccharomyces uvarum. (Sanchez & Cardona, 2007)

Non-Sacharomyces yeasts, also known as non-conventional yeasts, are also used to make a number of
commercial products. Some examples of non-conventional yeasts include Kuyberomyces lactis, Yarrowia
lipolytica, Hansenula polymorpha and Pichia pastoris. Microorganisms other than yeast can also be useful
in making fermentation products. For example, cellulosic ethanol production also utilizes fungi and bacteria
(Cheng & Sun, 2001).

3C5H10O5 yeast 5C2H5OH + 5CO2 H = -74.986 kJ/mol

C6H12O6 yeast 2C2H5OH + 2CO2 H = -84.394 kJ/mol

d) DISTILLATION
Distillation is one of the steps of the purifications. Distillation is the method used to separate two liquid
based on their different boiling points. However, to achieve high purification, several distillations are
required. This is because all materials have intermolecular interactions with each other, and two materials
will co-distill during distillation. This means that proportion between two materials, in this case ethanol and
water can be changed, and still, there are two materials in layers, the liquid and the vapor layers (Teshale,
2012).

Whatever method of preparation is used, the ethanol is initially obtained in a mixture with water. The
ethanol is then extracted from this solution by fractional distillation. Although the boiling point of ethanol,
0 0
78.3 C, is significantly lower than the boiling point of water, 100 C, these materials cannot be separated

13
completely by distillation. Instead, an azeotrope mixture (i.e. a mixture of 95% ethanol and 5% water) is
0
obtained, and the boiling point of the azeotrope is 78.15 C. In a distillation, the most volatile material (i.e.
the material that has the lowest boiling point) is the first material to distill from the distillation flask, and
this material is the azeotrope of 95% ethanol which has the lowest boiling point. If an efficient fractionating
column is used, 95% alcohol could be obtained first and then a small intermediate fraction of lower
concentration, and then water. But no matter how efficient the fractionating column used, 95% alcohol
cannot be further concentrated by distillation because the vapor has exactly the same composition as the
liquid; towards distillation, then, 95% alcohol behaves exactly like a pure compound (Acevedo, Godoy, &
Bolanos, 2003).

e) DEHYDRATION
After distillation, about 5% of water remains in ethanol. Especially, this water is a big problem for fuel
ethanol because the presence of this amount of water enhances the molecular polarity of ethanol when it is
mixed with gasoline. Consequently, they separate into two phases, ethanol phase and gasoline phase. It is
easy to imagine that this inhomogeneous fuel is not acceptable. Thus, dehydration can be another issue. For
the ethanol to be usable as a fuel, water must be removed. Most of the water is removed by distillation, but
the purity is limited to 95-96% due to the formation of a low boiling water-ethanol azeotrope. For blending
with gasoline, purity of 99.5 to 99.9% is required, depending on temperature, to avoid separation. Currently,
the most widely used purification method is a physical absorption process using molecular sieves and
another method is azeotropic distillation (Krishnan, Nghiem, & Davison, 1999).

2.2. GLOBAL TREND IN ETHANOL PRODUCTION AND USE

Fuel ethanol production has increased remarkably because many countries look for reducing oil imports,
boosting rural economies and improving air quality. The world ethyl alcohol production has reached about
51,000 mill liters (Renewable Fuels Association, 2012), being the USA and Brazil the first producers (see
table 2-3). In average, 73% of produced ethanol worldwide corresponds to fuel ethanol, 17% to beverage
ethanol and 10% to industrial ethanol.

14
T ABLE 2-3: W ORLD FUEL E THANOL P RODUCTION BY C OUNTRY OR REGION (M ILLION G ALLONS )

Country 2007 2008 2009 2010 2011 2012

USA 6,521 9,309 10,938 13,298 13,948 13,300

Brazil 5,019 6,472 6,578 6,922 5,573 5,577

Europe 570 734 1,040 1,209 1,168 1,179

China 486 502 542 542 555 555

Canada 211 238 291 357 462 449

Asia (minus China) 132 156 527 244 335 397

South America (minus
Brazil) 75 79 83 200 199 223

Mexico & Central America na na na 364 39 19

Australia 26 26 57 66 87 71

Africa na na na 44 38 42

Other 82 128 247 66 na na

WORLD 13,123 17,644 20,303 23,311 22,356 21,812

Sourced from Renewable Fuels Association, 2012.

The world ethyl alcohol production has reached about 21, 812 million gallons (Renewable Fuels
Association, 2012), being the USA and Brazil the first producers.

15
2.3. CURRENT ETHANOL PRODUCTION IN ETHIOPIA
There are apparent reasons for promoting biofuels in a country like Ethiopia that depends heavily on
imported fossil fuels. The current plan is to produce 1.8 billion liters of liquid biofuels by 2015, consisting
of 195 million liters of ethanol and 1.6 billion liters of biodiesel. Biofuel is considered as an opportunity
for providing domestic energy security, rapid economic development and creation of wealth. There is great
expectations that biofuel could contribute to solve main development challenges the country is facing today
(Tesfaye & Gebru, 2011).
Commercial scale liquid biofuel production in Ethiopia dates back to the 1990s when production of fuel
grade ethanol from sugar cane molasses was initiated at the Fincha sugar factory. Production was limited
to a few million liters and most of the produce was exported to Italy as there was no domestic market for
fuel ethanol. Beginning around 2005 there appeared widespread global interest to introduce liquid biofuels
as substitutes to petroleum fuels for both energy security and greenhouse gas emission reduction reasons
(Ashenafi, 2009).
Since 2009, the country has been provided a 5% ethanol and 95% benzene blended (technically known as
E5) for the market in which a Sudanese owned oil company, Nile Petrol, was a sole agent that was carrying
out the blending process and distributing for all local oil stations. The blending of E10 (10% ethanol blended
gasoline) has been effective in the country since March 2011. Currently, ethanol blending is only for
benzene consuming automobiles. According to MoWE there is another project on the pipeline to produce
biodiesel for diesel consuming motors (Tesfaye & Gebru, 2011).
For bioethanol there are six developers in the country of which four of them are government owned sugar
factories and two private companies. The three state owned sugar factories Fincha, Metahara and Wonji
have been operational for long time. However, the country produces ethanol from byproduct of sugar
(molasses) solely from Fincha which has been so far producing 8 million liters every year since 2009
(Tesfaye & Gebru, 2011). Similarly, Metahara Sugar Factory is currently set to start producing ethanol that
amounts to 10.5 million liters per year as of the current year. The fourth factory which is under construction
is the Tendaho Sugar Factory which has been said to be the biggest and most promising factory expected
to produce huge amount of ethanol upon its completion.
According to MoWE, upon the completion of all sugar factories including the private factories, the country
will be able to produce over 195 million liters of ethanol by the year 2015. However, the total irrigable land
for sugar cane based ethanol production is estimated to be 1,390,000 ha. With this potential Ethiopia has a
total ethanol production potential of 2.2 billion liters per year (Tesfaye & Gebru, 2011).

16
2.4. ECONOMIC AND ENVIRONMENTAL IMPACTS OF USING
ETHANOL
The following section provides a summary of some of the expected impacts from increased ethanol use in
the Northeast. In general, compared with alternatives, the use of ethanol is expected to result in significant
economic development, environmental benefits, and greater energy independence. Unanswered questions
about the fate and transport of ethanol in groundwater will soon be addressed (NRBP, 2001).

2.4.1. ECONOMIC IMPACTS

Development of ethanol represents a long-term economic development strategy for Northeast states.
Expanded production capacity will foster job growth and boost state and local tax revenues.

At present, an estimated 200,000 Americans are directly and indirectly employed by the ethanol industry.
Development of an ethanol industry in the Northeast could potentially bring thousands of additional jobs
into the region (NRBP, 2001). The replacement of MTBE with ethanol in Northeast gasoline would require
749 million gallons per year of ethanol. To meet this demand, approximately fifty ethanol plants, each
supplying 15 million gallons per year of ethanol, would be required. Applying job data from the above
California feasibility study suggests that operation of these plants would result in an estimated 1400
permanent operational jobs, 3150 to 5000 feedstock supply jobs, and 4650 to 6100 indirect jobs in the
Northeast (NRBP, 2001).

In addition to job creation, ethanol production would provide benefits to rural economies, help diversify
the tax base, and result in increased tax revenues. Importantly, many of the benefits of a new ethanol
industry would provide a welcome boost to ailing resource-based industries in Ethiopia, such as pulp and
paper industries and food processing industries (Tesfaye & Gebru, 2011).

2.4.2. ENVIRONMENTAL IMPACTS

Studies suggest that increased use of ethanol would result in important environmental benefits related to air
quality and greenhouse gas emissions.

a) AIR QUALITY
In general, predictive and actual measured research shows that the use of gasoline formulated to meet the
Clean Air Acts reformulated gasoline specifications provides a range of air quality benefits. Ethanol is
one potential oxygenate that can be blended with gasoline to achieve these benefits. Research by the
California Air Resources Board has shown that the use of ethanol as a gasoline additive in reformulated
gasoline helps contribute to reduced carbon monoxide (CO), nitrogen oxide (NOx), and volatile organic
compound (VOC) emissions. In addition, the use of ethanol (as well as other oxygenates) dilutes toxic

17
components such as the aromatic hydrocarbons benzene and toluene, enabling ethanol-blended gasoline to
potentially over comply with toxic content regulations for gasoline. Used as an additive in diesel fuel,
ethanol has been shown to exhibit similar positive air quality impacts (NRBP, 2001).

b) GREENHOUSE GAS EMISSIONS

The use of ethanol, a renewable fuel, is also capable of reducing greenhouse gas emissions relative to the
use of fossil fuels. It is important to note, however, that fossil fuel inputs are generally required to process
the plants and plant wastes into ethanol (and in the case of corn, to grow the plant material in the first place).
Additionally, the magnitude of the emissions reduction depends on the type of ethanol feedstock utilized.
A life cycle analysis conducted by the Argonne National Laboratory found that use of a gallon of corn-
based ethanol reduces full fuel cycle greenhouse gas emissions by 12 to 19 percent, assuming that the
ethanol is blended in gasoline at 10 percent. Meanwhile, for a gallon of cellulosic-based ethanol,
greenhouse gas emissions can be reduced by up to 100 percent (or more10) when compared to conventional
fuel (NRBP, 2001).

c) WATER QUALITY
Reviews of the fate and transport of ethanol in water and groundwater generally show that ethanol, unlike
MTBE, is not likely to accumulate or persist for long in the environment. Regarding surface water
environments, it is commonly accepted by the scientific community that ethanol evaporates quickly in
surface water, and poses little threat to environmental or human health. Ethanol is also expected to be
rapidly degraded in groundwater, and is not expected to move beyond source areas (Anonym, 2001).

However, the behavior of ethanol-blended gasoline in subsurface environments is a subject of ongoing

study. Of particular concern are the BTEX13 compounds all six of which are powerful depressants to the
central nervous system and potentially serious threats to public health. For instance, some laboratory
evidence has suggested that in some cases BTEX biodegradation may be inhibited in the presence of
ethanol, since ethanol will preferentially degrade relative to BTEX compounds. Due to limited laboratory
testing and a paucity of field evidence, overarching conclusions cannot yet be made (Kumar & Barrett,
2009).

18
3. MATERIALS AND METHODS
The experiment were carried out in the Microbiology Department Laboratory in the Veterinary Medicine
College of Mekelle University.

3.1. MATERIALS
3.1.1. CHEMICALS
The following chemicals were used in the experiment.

98% sulfuric acid (H2SO4)

Sodium hydroxide (NaOH)
Yeast extracts agar
Urea
Dextrose sugar
Mg SO4.7H2O
Baker yeast/Yeast (Saccharomyces cerevisiae)
Distilled water
Benzathine penicillin G

3.1.2. EQUIPMENTS
The equipments used in the experiment comprised of the following:

Plastic bags
Knife
Oven
Grinder
Electronic balance
Digital pH meter
Flasks
Graduated cylinders of different volumes
Autoclave
Filter
Shaking incubator
Rotary evaporator
Pycnometer

19
3.2. PROCEDURES
The basic steps for the production of ethanol alcohol from banana peels were; sample collection, size
reduction, pretreatment to make peels agreeable to hydrolysis, hydrolysis to break down cellulose and
hemicelluloses into simple sugars, fermentation and evaporation to produce alcohol.

3.2.1. SAMPLE COLLECTION AND PREPARATION

Banana peels were collected in plastic bags from one juice house, two fruit and vegetable shops near
Kedamay Woyane, Mekelle, Ethiopia and transported to the laboratory of microbiology department at
the College of Veterinary Medicine, Mekelle University for analysis.

The gathered peels were chopped into small pieces about 2-4 cm length using knife. The pieces were then
sun dried under mild sunlight for two days and finally dried at 60oC in oven for one day. Then the samples
were taken out of the drier when they were dried enough to be crushed. The cut pieces were then crushed
in the mixer grinder. Grinding helps in somewhat reduction in the chemical bonds between the celluloses,
hemicelluloses. The ground sample was proportioned into seventeen separate samples of 10gm of ground
banana peels and another two separate samples of 20gm of ground banana peels. For all the samples juice
were prepared, by adding 10:1(v/w) ratio of distilled water to the sample in separate flasks.

(a) (b) (c)

(d)
F IGURE 3-1: ( A) C HOPPED SAMPLE , (B) DRIED SAMPLE , (C) GRINDER MACHINE , AND ( D) P ROPORTIONED SAMPLE

20
3.2.2. PRETREATMENT
The lignocelluloses molecules must be break down to free sugars, before fermentation for alcohol
production. Though hydrolysis is of many types, steam pretreatment followed by dilute acid hydrolysis
was performed in this particular study.

STEAM TREATMENT UNDER HIGH PRESSURE

This pretreatment helped in loosening of lignocellulosic material. Bonding present between cellulose and
hemicelluloses breaks due to steam under high pressure. The separate samples capped with aluminum
foil was autoclaved at 15psi pressure for 30mins. After autoclaving the samples was allowed to cool and
the soluble portion was separated from the nonsoluble by using filtration. Next, the nonsoluble portion
was made to hydrolyze in the next step and amount of sugar produced was measured for each sample.

3.2.3. ACID HYDROLYSIS

The cellulose molecules should be broken down to simple sugar, before fermentation. Even though there
are many types of hydrolysis types, dilute acid hydrolysis, was used for hydrolysis of nonsoluble part.
Oven was used to control temperature of the sample under experiment isothermally at the set point and
hydrolysis was conducted in three variable parameters; acid concentration, temperature, and time.
Experimental design formulation was set all the parameters to vary by three level factors (minimum,
medium, maximum) by using Box-Behnken response surface of Design-Expert 6.0.8 software as follows:

21
T ABLE 3-1: E XPERIMENTAL DESIGN FORMULATION FOR HYDROLYSIS STAGE

Exp. Run Acid conc. (% v/v Hydrolysis Hydrolysis

to dist. water) temp. (oC) time(minutes)
1 2.5 90 30
2 1.5 90 20
3 2.5 70 20
4 1.5 90 20
5 0.5 110 20
6 2.5 90 10
7 1.5 90 20
8 2.5 110 20
9 1.5 90 20
10 1.5 90 20
11 1.5 110 10
12 0.5 70 20
13 0.5 90 30
14 0.5 90 10
15 1.5 70 10
16 1.5 70 30
17 1.5 110 30

PROCEDURES FOR ACID HYDROLYSIS

First 25ml of 0.5% to 2.5% (v/v) diluted sulfuric acid was added to the nonsoluble component from
pretreatment steps. The fruit peels were then hydrolyzed in the reactor between 70 and 110oC for 10 to 30
min as the experimental design order. After hydrolysis, neutralization was carried out with 1M NaOH until
the pH became around 7. Nonsoluble particles were separated from the hydrolyzate by filtration (to remove
the non-fermentable lignin portion). Then the soluble component was added with the previously filtered
solution from the pretreatment step for the next procedure.

22
 F IGURE 3-2: S AMPLES BEING HYDROLYZING

3.2.4. MEASURING SUGAR CONTENT

Brix method was used to measure the sugar contents of each sample. First, the weight of an empty dry
flask equal with the sample flask was weighed. Next, the weight of the flask with distilled water up to
the level of the juice on the sample flask was weighed. The procedure was repeated for each flasks with
the juice. Finally, the maximum sugar content percentage was calculated and the parameters condition
(acid concentration, temperature, and time) that gave rise to it was taken as an optimum condition. Using
this optimum condition, hydrolysis was carried out for the two samples containing 20gm banana peel
powder. Once again the lignin were removed from the soluble portion and the soluble solution found was
added into the previously separated soluble portions. The residue and remaining samples was discarded
into waste basket. Finally, these two samples were used to produce ethanol from banana peels by the
Saccharomyces cerevisiae.

3.2.5. FERMENTATION
MEDIA PREPARATION
Before fermentation process conditioning inoculum was prepared for S. cerevisiae. The following
constituents was used for inoculum preparation for S. cerevisiae growth medium.

Potato Dextrose 10gm

Yeast extract agar 0.4gm
Urea 1gm
MgSO4. 7H2O 1gm
Distilled water 100ml

23
Media was prepared for fermentation process; fermentation with S. cerevisiae only and fermentation with
S. cerevisiae and benzathine penicillin G. 100ml from the inoculum was added to 0.5gm of yeast (5gm/L
yeast), S. cerevisiae, in 250ml flask. The flask was properly covered with aluminum foil to prevent air
entrance (the growth was made anaerobically). The conical flask was then placed in a shaking incubator
for 24 hours at temperature of 30oC and 200 rpm, which was used for the next process of fermentation.

F IGURE 3-3: MEDIA PREPARED

PROCEDURE FOR FERMENTATION

In the fermentation step, the two samples was conditioned to a temperature of 30oC before fermentation
step started. This was the working temperature throughout the whole fermentation process. At this step the
pH was set to 5.0 - 5.5, optimum pH for the activity of S. cerevisiae by the addition of 1 M NaOH. Then
25ml freshly prepared yeast culture was added into each of the flasks (1:4 (v/v) of samples) and the flasks
mouth was raped with aluminum foil. Next, 0.05gm of benzathine penicillin G (0.5gm/L) was added into
the second flask while the first remains as it is. Finally, both of the samples were placed in shaker incubator
at 200 rpm, 30oC, for 3 days. The samples were checked every day by adjusting the pH set between 5.0 and
5.5.

3.2.6. DISTILLATION
In the production of ethanol from the banana peel juice distillation was proposed to be carried out using
distillation unit. However, distillation unit was not available during the experimentation, instead rotary
evaporator was used as an alternative method for the separation and purification of ethanol from the
samples.

3.2.7. DENSITY MEASUREMENT

The final products were evaluated for their ethanol content by measuring the corresponding density using
pycnometer at room temperature. The specific gravity of the produced alcohol was determined and
alcohol concentration was got from the relationship between the specific gravity and the proportion of

24
ethanol in alcohol solution at room temperature. Weigh the pycnometer (specific gravity bottle) with
stopper after cleaning, drying and note the weight as W1. Filled the pycnometer with distilled water and
take the weight of the water and note as W3. Make the pycnometer empty, clean, dry and then filled with
sample (alcohol) of the experimental result. Determine the weight of the sample and note as W2.
Calculate the net weight in grams of the alcoholic liquid in the pycnometer by subtracting the weight of
the empty specific gravity bottle or pycnometer. Calculate specific gravity of sample according to the
formula given.

2 1
Specific gravity of sample = 31

Where: W1 = weight of empty pycnometer

W2 = weight of pycnometer plus sample

W3 = weight of pycnometer filled with distilled water

25
4. RESULT AND DISCUSSION

4.1. EXPERIMENTAL RESULTS

During the experimentation the following results was found after successfully measuring the sugar
contents of each samples (see Appendix A for full table).

T ABLE 4-1: M ASS PERCENTAGE OF SUGARS

Run Percent weight of sugar content

(%w/w to original sample)
1 43.56
2 45.10
3 44.01
4 45.00
5 44.27
6 42.40
7 45.00
8 42.98
9 45.00
10 45.15
11 42.96
12 43.21
13 43.00
14 42.56
15 42.20
16 43.85
17 43.40

After, the optimum conditions of hydrolysis, which give maximum amount of sugar content was
determined from the series of experiments and analyzing the results by using Design Expert Software,
another two separate samples of equal amount was prepared for ethanol production analysis at these
optimum conditions. As a result the following data was found after measuring the specific gravities of
produced ethanol at 25oC.

26
T ABLE 4-2: SPECIFIC GRAVITY MEASUREMENT RESULTS AT 25 O C

W1 Weight of empty pycnometer 25.100gm

W21 Weight of pycnometer plus sample one 48.100gm
W22 Weight of pycnometer plus sample two 48.073gm
W3 Weight of pycnometer filled with distilled water 48.500gm

4.2. STATISTICAL ANALYSIS

From the result shown above (table 4-1) the maximum sugar yields are run number: 10, 2, 4, 7 & 9
descending order. The minimum results were obtained at run number: 15, 6, 14, 8 & 11.

120 110 110 110 110

100 90 90 90 90 90 90 90 90 90

80 70 70 70 70

60
43.56 45.10 44.01 45.00 44.27 42.40 45.00 42.98 45.00 45.15 42.96 43.21 43.00 42.56 42.20 43.85 43.40
40 30 30 30 30
20 20 20 20 20 20 20 20 20 16 17
11 10 12 13 14 15
20 10 8 9 10 10 10
2.5 21.5 32.5 41.5 50.5 62.5 71.5 2.5 1.5 1.5 1.5 0.5 0.5 0.5 1.5 1.5 1.5
1
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Exp.R 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
A conc. 2.5 1.5 2.5 1.5 0.5 2.5 1.5 2.5 1.5 1.5 1.5 0.5 0.5 0.5 1.5 1.5 1.5
Temp. 90 90 70 90 110 90 90 110 90 90 110 70 90 90 70 70 110
Time 30 20 20 20 20 10 20 20 20 20 10 20 30 10 10 30 30
Sugar 43.56 45.10 44.01 45.00 44.27 42.40 45.00 42.98 45.00 45.15 42.96 43.21 43.00 42.56 42.20 43.85 43.40

F IGURE 4-1: S UGAR CONTENT AND PARAMETERS CONDITION

As we see from the above figure 4-1 high sugar content were observed at 1.5% acid concentration, 90 oC
temperature and 20 min time. The maximum sugar content found here, 45.15%, was an impressive result
comparing to 21% (w/w) sugar production, which investigated from the utilization of waste ripe banana,
and peels for bioethanol production using Saccharomyces cerevisiae investigation in India (KUMAR,
MOORTHY, RAJESWARI, & HARIKRISHNAN, 2011).In this study experimental design techniques
were used to determine the effects of the acid concentration, hydrolysis time and temperature on the
efficiency of soluble sugar yield. A total of 17 experiments were carried out for optimization purpose where

27
the effect of each factor was analyzed by using lower and higher values from optimized conditions. The
sugar yields obtained from experiments were used as a response parameter for optimization and table 4-3
show respective yields of each run with the factor.

The resulting data obtained was analyzed using Design-expert 6.0.8 software to decide the effects of
operating parameters; acid concentration, temperature and time. The dependent variable used as a response
parameter was percent of mass of soluble sugar content. All experiments were carried out in a randomized
order to minimize the effect of unexpected variability in the observed response due to extraneous factors.
Design Summary for ethanol production with three levels and three factors. The design model of the
experiments are quadratic polynomial and the center point is zero using Design expert 6.0.8 software.

T ABLE 4-3: DESIGN S UMMARY OF DESIGNS

Design Summary
Study Type Response Surface
Initial Design Box Behnken
Design Model Quadratic
Experiments 17
Blocks No Blocks
Response Name Units Minimum Maximum
Y1 Sugar %w/w 42.2 45.15
Factor Name Units Low Actual High Actual
A Acid conc. %v/v 0.5 2.5
B Temp. deg. C 70 110
C Time min 10 30

To determine whether or not the quadratic model is significant, it was crucial to perform analysis of variance
(ANOVA), table 4-4. The probability (P-values) values were used as a device to check the significance of
each coefficient, which also showed the interaction strength of each parameter. The smaller the P-values
are, the bigger the significance of the corresponding coefficient.

28
T ABLE 4-4: ANALYSIS OF VARIANCE (ANOVA) FOR THE QUADRATIC MODEL

Source Sum of square DF Mean square F-value P-valu >F

Model 16.52939 9 1.836599 80.94563 < 0.0001 (significant)
A 0.001013 1 0.001013 0.044625 0.8387
B 0.01445 1 0.01445 0.636864 0.4511
C 1.702013 1 1.702013 75.01393 < 0.0001
A2 2.883184 1 2.883184 127.0725 < 0.0001
B2 1.541158 1 1.541158 67.92448 < 0.0001
C2 7.588658 1 7.588658 334.46 < 0.0001
AB 1.092025 1 1.092025 48.12955 0.0002
AC 0.1296 1 0.1296 5.711947 0.0482
BC 0.366025 1 0.366025 16.13206 0.0051
Residual 0.158825 7 0.022689
Lack of Fit 0.138825 3 0.046275 9.255 0.0285 (significant)
Pure Error 0.02 4 0.005E-003
Cor Total 16.68821 16

F- Value is a test for comparing model variance with residual (error) variance. If the variances are close to
the same, the ratio will be close to one and it is less likely that any of the factors have a significant effect
on the response. It is calculated by Model Mean Square divided by Residual Mean Square. Here the Model
The Model F-value of 80.95 implies the model is significant. There is only a 0.01% chance that a "Model
F-Value" this large could occur due to noise.

Probability Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case C, A2,
B2, C2, AB, AC, BC are significant model terms. Values greater than 0.1000 indicate the model terms are
not significant. If there are many insignificant model terms, model reduction may improve your model.
The "Lack of Fit F-value" of 9.25 implies the Lack of Fit is significant. There is only a 2.85% chance that
a "Lack of Fit F-value" this large could occur due to noise.

Coefficient of Variation, the standard deviation expressed as a percentage of the mean; Predicted Residual
Error Sum of Squares, which is a measure of how the model fits each point in the design; the R-Squared,
measure of the amount of variation around the mean explained by the model; Adj R-Squared that is a
measure of the amount of variation around the mean explained by the model, Pred R-Squared, a measure

29
of the amount of variation in new data explained by the model, and Adequate Precision, this is a signal to
disturbance ratio due to random error, presented in the table 4-5, below, are used to decide whether the
model can be used or not.

T ABLE 4-5: MODEL ADEQUACY MEASURES

Std. Dev. 0.15063 R-Squared 0.990483

Mean 43.74412 Adj R-Squared 0.978246
C.V.% 0.344343 Pred R-Squared 0.865027
PRESS 2.25245 Adeq Precision 24.43138

The "Pred R-Squared" of 0.8650 is in reasonable agreement with the "Adj R-Squared" of 0.9782."Adeq
Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 24.431
indicates an adequate signal. This model can be used to navigate the design space.

The regression coefficients and the corresponding 95% CI (Confidence Interval) High and Low were
presented in table 4-6 below. If zero was in the range High and Low 95% Confidence Interval, the factors
has no effect. From the 95% CI High and Low values of each model term, it could be concluded that the
regression coefficients of acid concentration and the interaction terms of time and acid concentration have
highly significant effect in ethanol production.

T ABLE 4-6: REGRESSION COEFFICIENTS AND THE CORRESPONDING 95% CI H IGH AND LOW

Factor Coefficient Estimate DF Standard Error 95% CI Low T95% CI High

Intercept 45.05 1 0.067364 44.89071 45.20929
A-Acid
conc. -0.01125 1 0.053256 -0.13718 0.11468
B-Temp. 0.0425 1 0.053256 -0.08343 0.16843
C-Time 0.46125 1 0.053256 0.33532 0.58718
A2 -0.8275 1 0.073408 -1.00108 -0.65392
2
B -0.605 1 0.073408 -0.77858 -0.43142
C2 -1.3425 1 0.073408 -1.51608 -1.16892
AB -0.5225 1 0.075315 -0.70059 -0.34441
AC 0.18 1 0.075315 0.001909 0.358091
BC -0.3025 1 0.075315 -0.48059 -0.12441

30
 Final Equation in Terms of Coded Factors:

Sugar = +45.05
-0.01125 *A
+0.0425 *B
+0.46125 *C
-0.8275 * A2
-0.605 * B2
-1.3425 * C2
-0.5225 *A*B
+0.1800 *A*C
-0.3025 *B*C

Final Equation in Terms of Actual Factors:

Sugar =
+18.761
+4.462 * Acid conc.
+0.344 * Temp.
+0.692 * Time
-0.827 * Acid conc. * Acid conc.
-0.002 * Temp. * Temp.
-0.013 * Time* Time
-0.026 * Acid conc. * Temp.
+0.018 * Acid conc. * Time
-0.002 * Temp. * Time

To see how well the model satisfies the assumptions of the analysis of variance (ANOVA), the plots of
residuals versus predicted (Appendix B) were analyzed.
Normal probability plot of the raw data used to check the assumption of normality when using t-test. In the
analysis of variance, it is usually more effective (and straight forward) to do this with the residuals. This

31
shown below resembles a straight line. In visualizing the straight line, place more emphasis on the central
values of the plot than on the extremes.

F IGURE 4-2: N ORMAL PLOTS OF RESIDUALS

From the plot as shown above, the normal probability plot indicates the residuals following a normal
distribution, in the case of this experiment the points in the plots shows fit to a straight line in the figure,
this shows that the quadratic polynomial model satisfies the assumptions analysis of variance (ANOVA)
i.e. the error distribution is approximately normal.

32
F IGURE 4-3: RESIDUAL VERSUS PREDICTED VALUES

If the model is correct and the assumptions are satisfied, the residuals should be structure less; in particular,
they should be unrelated to any other variable including the predicted response. A simple check is to plot
the residuals versus the fitted (predicted) values. A plot of the residuals versus the rising predicted response
values tests the assumption of constant variance. The plot shows random scatter which justifying no need
for an alteration to minimize personal error.

4.3. EFFECTS OF EXPERIMENTAL VARIABLES ON HYDROLYSIS

Ethanol production can be affected by many parameters starting from sample preparation to distillation, the
hydrolysis steps has a complex connection with independent variables. The best way of showing the effects
of this parameter for the yield of ethanol are to generate response surface plots of the equation. The three
dimensional i.e. interactions, contours and response surfaces effect were plotted in figures (4.4), (4.5) and
(4.6) below as a function of the interactions of any two of the variables by holding the other value of the
variable at middle. For the interaction figures, black and red line indicates low and high level of parameters
respectively.

33
Figure 4.4 (a): The effects of time and acid concentration (fixed) on the yield of ethanol, when the
temperature was at the center point

Figure 4.4 (b): The effects of time (fixed) and acid concentration on the yield of ethanol, when the
temperature was at the center point

34
Figure 4.4 (c): Response surface plots of the effects of acid concentration and time on ethanol yield

F IGURE 4-4: E FFECT OF ACID CONCENTRATION AND TIME ON THE YIELD OF ETHANOL WHEN TEMPERATURE WAS
AT THE CENTER POINT ( A, B & C)

The effects of acid concentration and time on the yield of sugar temperature was selected at the center point,
are shown in figure 4.4 (a) & (b). At the lower and higher levels of acid concentration and time, the
production of ethanol yield level decrease since it has effect of the hydrolysis treatment. At lower acid
concentration and time the cellulose might not hydrolysis to simple glucose and at higher acid concentration
and time the cellulose might convert to other molecules which might not be fermentable. Hence both acid
concentration and time have strong relationship for the yield of ethanol production.

The response surface figure 4.4 (c), obtained from hydrolysis time and acid concentration was conical
shape. Hence from the result, there were well defined optimums operating conditions. As hydrolysis time
increases at lower level of acid concentration and as increase level of acid concentration and lower level of
time gives a positive effect on the yield of ethanol. The response surface suggests that there were dominance
interactions of these two factors.

35
Figure 4.5 (a): The effect of time and temperature (fixed) on the yield of ethanol, when acid concentration
was at the center point

Figure 4.5 (b): The effect of time (fixed) and temperature on the yield of ethanol, when acid concentration
was at the center point

36
Figure 4.5 (c): Response surfaces plot of the effects of temperature and time on the yield ethanol

F IGURE 4-5: E FFECT OF TEMPERATURE AND TIME ON YIELD OF ETHANOL WHEN ACID CONCENTRATION WAS AT
THE CENTER POINT ( A, B & C)

The effects of acid concentration and temperature on the yield of ethanol, acid concentration was selected
at the center point, are shown in figure 4.5 (a) and (b). When the levels of temperature increase hydrolysis
resulted in higher yield of ethanol. However, as you seen from the graph after some increments of
temperature, the yield of ethanol became decreases since the possible formation of other molecules instead
of glucose formation due to high temperature. Similarly, at low and high time, the yield of ethanol decrease.
The response surface figure 4.5 (c), obtained from hydrolysis time and hydrolysis temperature was conical
shape. It suggests that there were well-defined optimum operating conditions. The response optimized value
for the production of ethanol was based on both in hydrolysis time and temperature.

37
Figure 4.6 (a): The effects of temperature and acid concentration (fixed) on the yield of ethanol, when and
time was at the center

Figure 4.6 (b): The effects of temperature (fixed) and acid concentration on the yield of ethanol, when time
was at the center

38
Figure 4.6 (c): Response surface plots of the effects acid concentration and temperature

F IGURE 4-6: E FFECT OF TEMPERATURE AND ACID CONCENTRATION ON THE YIELD OF ETHANOL WHEN TIME WAS
AT THE CENTER POINT ( A, B & C)

At the lower and higher levels of temperature and acid concentration, the production of ethanol yield
decrease since it has effect of the hydrolysis treatment. At lower temperature and acid concentration the
cellulose might not hydrolysis to simple glucose and at higher acid concentration and time the cellulose
might convert to other molecules which might not be fermentable. Hence both temperature and acid
concentration have strong relationship for the yield of ethanol production.

The response surface figure 4.6 (c), obtained from hydrolysis temperature and acid concentration was
conical shape. Hence from the result, there were well defined optimums operating conditions. As hydrolysis
temperature increases at lower level of acid concentration and as increase level of acid concentration and
lower level of temperature gives a positive effect on the yield of ethanol. The response surface suggests that
there were dominance interactions of these two factors.

39
4.4. OPTIMIZATIONS
Soluble sugar production can be optimized numerically, graphically or by point prediction. Numerical
optimization sets optimization goals for each response either with objective to minimize, maximize, set in
range, in target or equal to specific value. Based on the data and goals numerical optimization produces
possible solutions with full report; constraints, number of starting points, ramps, histogram and desirability,
which is also possible graphically.

In graphical optimization goals are set to minimum or maximum limits for each response then create an
overlay graph highlighting an area of operability. Simply enter your desired operating conditions and
discover your predicted response values with confidence intervals in point prediction optimization.

The numerical optimization of hydrolysis criteria for ethanol production from banana peel using dilute acid
treatment are summarized as follows (using Design-Expert 6.0.8):

Constraints
Name Goal Lower Limit Upper Limit
Acid conc. is in range 0.5 2.5
Temp. is in range 70 110
Time is in range 10 30
Sugar maximize 42.2 45.05
T ABLE 4-7: OPTIMIZATION CRITERIA FOR OPTIMUM YIELD OF SOLUBLE SUGAR CONTENT

40
The optimum possible solutions in hydrolysis for different parameters for ethanol production and
corresponding surface plot are presented in table 4-9 and figure 4.7 below.

Number Acid conc. Temp. Time Sugar Desirability

1 1.50 91.02 21.66 45.088
1 (Selected)
2 1.43 87.55 22.05 45.070
1
3 1.60 86.97 23.02 45.064
1
4 1.74 86.8 22.10 45.055
1
5 1.31 89.76 20.93 45.050
1
6 1.57 94.03 22.04 45.051
1
7 1.51 92.51 21.62 45.079
1
8 1.40 93.81 22.29 45.057
1
9 1.31 92.42 21.53 45.059
1
10 1.51 85.73 21.45 45.063
1
T ABLE 4-8: OPTIMUM POSSIBLE SOLUTIONS (10 SOLUTIONS FOUND)

(a)

41
 (b)

(c)

F IGURE 4-7: ( A), (B) AND (C ) OPTIMIZATION OF CONTOURS PLOT IN SUGAR YIELD

42
4.5. EFFECT OF BENZATHINE PENICILLIN G ON ETHANOL
PRODUCTION
From the experimental result table 4-2, the specific gravity of alcohol (ethanol) produced was calculated as
follows;

Specific gravity of sample one = (48.100 25.100)/(48.500 25.100) = 0.98291

Specific gravity of sample two = (48.073 25.100)/(48.500 25.100) = 0.98175

From ethanol density standard table (Appendix D), the % ethanol yield at 25oC was then found by linear
interpolation:

Sample % EtOH yield

Sample one 8.36
Sample two 9.11

As it is indicated above the addition of benzathine penicillin G enhanced the fermentation process by 8.97%.
Even though there is no many related research done before, Brooks (2008) has investigated ethanol
production potential of local yeast strains isolated from ripe banana peels, and he found that Saccharomyces
kluvveri K-6 ferment 40%(v/v) of the glucose at 30 oC to yield 3.6 - 5.8% ethanol (Brooks, 2008). Hence,
the result found here, 8.36% ethanol and 9.11% ethanol is impressive.

43
5. PLANT DESIGN AND COST ANALYSIS

5.1. PLANT DESIGN

For large scale analysis of production of ethanol from banana peel the following plant model was developed
using Intelligen SuperPro Designer v9.0. The model can be readily modified to represent conversion of
other types of lignocellulosic biomass, such as sugar cane bagasse, wheat straw, rice straw, and softwood,
switch grass, etc. The plant scale for the base case has a feedstock flowrate of 550 kg/h (0.55MT/h) of dry
mass of banana peel by assuming 345 working days annually. The plant generates 3.02 kg/h of fuel-grade
ethanol.

5.1.1. FEEDSTOCK REPRESENTATION

A mixture, called banana peel, was created in SuperPro to represent the dry mass of banana peel. The table
below displays its composition.

Bananapeel Composition

Component % w/w

Cellulose 9.00

Crude fat 6.00

Dietary fiber 19.00

Glucose 2.00

Hemicellulose 8.00

Lignin 9.00

Other solids 6.00

Pectin 11.00

Proteins 6.00

Starch 3.00

Water 20.00

Xylose 1.00

44
A 20% moisture level was assumed for feedstock banana peels. Hence, the feedstock stream of the flow
sheet contains 80% of dry peel and 20% moisture level. The impact of different moisture levels can be
investigated by simply changing the water content of the Feedstock stream.

5.1.2. PROCESS DESCRIPTION

The overall flowsheet is divided into four sections: Pretreatment, Hydrolysis, Fermentation, and
Distillation. A flowsheet section is a set of associated unit procedures. The unit procedure icons of each
section are displayed in a unique color (BLACK for Pretreatment, RED for Hydrolysis, BLUE for
Fermentation and GREEN for Distillation) (see Figure below).

45
F IGURE 5-1: P ROCESS FLOW SHEET FOR ETHANOL PRODUCTION FROM BANANA PEEL (S UPER P RO DESIGNER )

46
5.2. COST ANALYSIS
The table below displays the key results of cost analysis for a plant capacity. It was assumed that a new
facility will be built with a project lifetime of 14 years. The total capital investment for such a plant is
around 3 billion ETB. The net annual operating cost is around 61.2 million ETB leading to a unit production
cost of around 3.1 million ETB/MT MP of product ethanol.

5.2.1. EXECUTIVE SUMMARY (2014 Prices)

T ABLE 5-1: E XECUTIVE SUMMARY OF ECONOMIC E VALUATION R EPORT

Total Capital Investment 3,080,063,000 ETB

Capital Investment Charged to This Project 3,080,063,000 ETB
Operating Cost 61,204,000 ETB/yr
Main Revenue 376,000 ETB/yr
Other Revenues 309,520,768 ETB/yr
Total Revenues 309,896,000 ETB/yr
Cost Basis Annual Rate 20.02 MT/MP/yr
Unit Production Cost 3,056,604.70 ETB/MT MP
Unit Production Revenue 15,476,506.42 ETB/MT MP
Gross Margin 8.25 %
Return On Investment 13.88 %
Payback Time 7.21 years
IRR (After Taxes) 8.83 %
NPV (at 7.0% Interest) 283,438,000 ETB
MP = Total Flow of compenent Ethyl Achol in Stream Product

The above table was copied from the Economic Evaluation Report (ERR) generated by SuperPro Designer.
This table provides a summary of the overall economics of the process. The information above (as well as
additional information) is also displayed in the Executive Summary dialog of the application. The rest of
the EER provides detailed information on capital and operating costs.

5.2.2. ANNUAL OPERATING COST (2014 Prices)

The table below, which was also copied from the EER, provides a summary of the annual operating cost.
The cost of Utilities is the most important cost item (76.19% of total) followed by the Transportation and
Raw Materials cost, which mainly accounts for the long distance collection and transportation of raw
materials.

47
T ABLE 5-2: ANNUAL OPERATING COST

Cost Item ETB %

Raw Materials 457,000 0.75
Labor-Dependent 6,834,000 11.17
Consumables 13,000 0.02
Waste Treatment/Disposal 0 0
Utilities 46,630,000 76.19
Transportation 7,271,000 11.88
Miscellaneous 0 0.00
Advertising/Selling 0 0.00
Running Royalties 0 0.00
Failed Product Disposal 0 0.00
TOTAL 61,204,000 100.00

5.2.3. MATERIALS COST (2014 Prices)

The table below displays the breakdown of the materials cost, which is the main cost item of this process.
The cost of the feedstock followed by water and sulfuric acid cost was estimated to be higher in the process.

T ABLE 5-3: M ATERIAL COSTS

Bulk Material Unit Cost Annual Unit Annual Cost %

(ETB) Amount (ETB)
Banana peel 0.050 4,554,000 kg 227,700 49.84
Water 0.005 40,673,690 L(STP) 203,368 44.52
HP Steam 0.006 251,140 kg 1,507 0.33
Air 0.00 228,144 kg 0.00 0.00
Yeast 50.00 33 MT 1,656 0.36
Sulfuric acid 3.60 6,278 kg 22,603 4.95
TOTAL 456,834 100.00

5.2.4. UTILITIES COST (2014 Prices)

The table below, which was copied from the EER, provides detailed information on utilities costs.

T ABLE 5-4: U TILITIES COSTS

Utility Annual Amount Reference Units Annual Cost (ETB) %

Std Power 0.00 52,121,444 kW/h 10,424 0.02

Steam 14.00 106,083 MT 1,485,166 3.19
Cooling Water 10.00 117,743 MT 1,177,430 2.53
Chilled Water 8.00 5,494,597 MT 43,956,778 94.27
TOTAL 46,629,799 100.00

48
The SuperPro Designer also generates two other cost-related reports, and many other useful reports. The
Cash Flow Analysis (CFR) and the Item Cost report (ICR) are important cost related reports, and overall
material balances (see Appendix E), major equipment specification and equipment summary are also
generated by the software.

49
6. CONCLUSIONS AND RECOMMENDATIONS

6.1. CONCLUSION
Hydrolysis of banana peel waste was carried out with dilute sulfuric acid with the objective to determine
the optimum operating conditions that yield maximum sugar concentration. The experiment was designed
by Box-Behnken response surface method. The optimum conditions (acid concentration, temperature and
time) was examined by plotting the interaction, contour and 3D plots. Those all three factors were
significant variables for the yield of optimum fermentable sugars. Very high and low acid concentration,
temperature and retention time have negative effect on the yield of sugars. In addition to this the effect of
benzathine penicillin G on fermentation process was examined by measuring the specific gravity of the
produced ethanol. The presence of benzathine penicillin G had enhanced the final ethanol production.

6.2. RECOMMENDATION
1. Further researches have to be done to improve the production of high quality and quantity of fruit peel
ethanol.
2. Alternative extraction methods of ethanol such as enzymatic extraction have to be done in order to
investigate the variation that could be arise on the quality and quantity of the ethanol yield as a result
of using different extraction methods.
3. Most of the solid wastes including fruit peel waste in our country have no or very low conversion to
different usable products and as such among the major problems of health especially for our cities.
Hence, it recommended that government or other investors to recover this very valuable product as
well as to contribute to the country in reducing the highly rising quantity of wastes.
4. To conclude the recommendation, there is an urgent need for proper collection, documentation and
assessment of fruit peel yields and their potential as source of renewable energy in our country.

50
BIBLIOGRAPHY
Abd-Aziz, S. (2002). Sago starch and its utilization. Journal of Bioscience and Bioengineering 94 (6),
526529.
Acevedo, A., Godoy, R., & Bolanos, G. (2003). Increase in ethanol production during fermentation of
molasses using the enzymatic complex Rhyzozyme. Congreso Colombiano de Ingenieria
Quimica Bucaramanga, Colombia (in Spanish).
Agrocadenas, M. (2007). Segundo informe de coyuntura maiz 2006. Observatorio Agrocadenas
Colombia. Retrieved from <http://www.agrocadenas.gov.co/home.htm>
Anonym. (2001). An Ethanol Production Guidebook for Northeast States. Washington: Northeast
Regional Biomass Program.
Apar, K., & Ozbek, B. (2004). r-Amylase inactivation during corn starch hydrolysis process. Process
Biochemistry 39 (12), 18771892.
Ashenafi, T. (2009). CONVERSION OF SELECTED ADDIS ABABA MUNICIPAL SOLID WASTE TO
ETHANOL (Case study of Yeka sub city). ADDIS ABABA: ADDIS ABABA UNIVERSITY.
Barber, R., Henningsson, M., & Pamment, B. (2002). Acceleration of high gravity yeast fermentations by
acetaldehyde addition. Biotechnology Letters 24, 891895.
Bayrock, P., & Ingledew, M. (2001). Application of multistage continuous fermentation for production of
fuel alcohol by very-high-gravity fermentation technology. Journal of Industrial Microbiology
and Biotechnology 27, 8793.
Bothast, J., & Schlicher, A. (2005). Biotechnological processes for conversion of corn into ethanol.
Applied Microbiology and Biotechnology 67, 1925.
Brooks, A. A. (2008). Ethanol production potential of local yeast strains isolated from ripe banana peels.
Calabar, Nigeria: Department of Microbiology, University of Calabar.
Bullock, E. (2002). Ethanol from Sugarcane. Sugar Research Institute, Australia.
Burmaster, B. (2007). Improved ethanol fermentation using oxidation reduction potential. PHB
WO2007064545.
Carrascosa, V. (2006). Production of ethanol under high osmotic pressure conditions comprises a
microorganism for fermentation of molasses must. PLD Publisher.
Cheng, J., & Sun, Y. (2001). Hydrolysis of lignocellulosic materials for ethanol production: a review.
Raleigh, USA, NC 27695-7625, USA: North Carolina State University.
Chung, H., & Nam, G. (2002). Process for producing high concentration of ethanol using food wastes by
ermentation. Bright plc.
Claassen, M., Van Lier, B., Lopez, C. V., Sijtsma, L., & Stams, M. (1999). Utilisation of biomass for the
supply of energy carriers. Applied Microbiology and Biotechnology 52, 741755.
Dai, D., Hu, Z., Pu, G., Li, H., & Wang, C. (2006). Energy efficiency and potentials of cassava fuel
ethanol in Guangxi region of China. China: Energy Conversion and Management.

51
EIA. (2007). EIA. US Energy Information Administration World Outlook. DC, USA: EIA: DOE/EIA-
0484.
Ergun, M., Mutlu, F., & Gurel, O. (1997). Improved ethanol production by Saccharomyces cerevisiae
with EDTA, ferrocyanide and zeolite X addition to sugar beet molasses. Journal of Chemical
Technology and Biotechnology.
Espinal, F., Martinez, J., & Acevedo, X. (2005). The chain of cereals, balanced feed, aviculture, and pig
farming in Colombia. An overview of its structure and dynamics 19912005. Spanish:
Observatorio Agrocadenas Colombia, Ministry of Agriculture and Rural Development.
FAO. (2004). Global cassava market study. Business opportunities for the use of cassava. Proceedings of
the Validation Forum on the Global Cassava Development Strategy, vol. 6. Rome: Food and
Agriculture Organization of the United Nations (FAO).
Fearnside, P. (2002). Soybean Cultivation as a Threat to the Environment in Brazil, Environmental
Conservation. Brazil.
Fulton, L., Howes, T., & J, H. (2004). Biofuels for Transport: An International Perspective. Paris:
International Energy Agency.
Gaikwad, H. (2013). PRODUCTION OF BIOETHANOL FROM BANANA WASTE. Lovely Professional
University.
Ghosh, P., & Ghose, K. (2003). Bioethanol in India: recent past and emerging future. New Delhi:
Advances in Biochemical Engineering/Biotechnology.
Gupta, N. (2009). HYDROLYSIS OF LIGNOCELLULOSIC MATERIALS FOR ETHANOL
PRODUCTION. Rourkela: National Institute of Technology.
Harmsen, P., Huijgen, W., Bermudez, L., & Bakker, R. (2010). Literature review of physical and
chemical pretreatment processes for lignocellulosic biomass. Energy Research Centre of the
Netherlands, 3-31.
Hazell, P., & Pachauri, K. (2006). Bioenergy and Agriculture: Promises and Challenges. International
Food Policy Research Institute 2020 Focus No. 14.
IEA. (2004). Biofuels for Transport: An International Perspective. International Energy Agency (IEA).
Jamal, P., Saheed, O. K., & Alam, Z. (2012). Bio-Valorization Potential of Banana Peels (Musa
sapientum): An Overview. Asian Journal of Biotechnology, 4: 1-14. Retrieved from URL:
http://scialert.net/abstract/?doi=ajbkr.2012.1.14
Kammen, M. (2006). Bioenergy in Developing Countries: Experiences and Prospects, in Bioenergy
and Agriculture: Promises and Challenges, International Food Policy. Research Institute 2020
Focus No. 14.
Krishnan, S., Nghiem, P., & Davison, H. (1999). Ethanol production from corn starch in a fluidized-bed
bioreactor. Applied Biochemistry and Biotechnology.
Kumar, P., & Barrett, D. M. (2009). Methods for Pretreatment of Lignocellulosic Biomass for Efficient
Hydrolysis and Biofuel Production. Industrial and Engineering Chemistry Research, 2-7.

52
KUMAR, S. R., MOORTHY, G. I., RAJESWARI, R., & HARIKRISHNAN, H. (2011). Utilization of
waste ripe Banana, and peels for Bio ethanol production using Saccharomyces cerevisiae. Tamil
Nadu, India: Kamaraj College of Engineering & Technology.
Langeveld, H., & Sanders, J. (2010). The Biobased Economy: Biofuels, Materials and Chemicals in the
Post-oil Era. London, Sterling, VA: Earthscan.
Lee, C., & Huang, T. (2000). Modeling of ethanol fermentation using Zymomonas mobilis ATCC 10988
grown on the media containing glucose and fructose. Biochemical Engineering Journal.
Maye, P. (2006). Use of hop acids in fuel ethanol production. DC: BLP: plc: US2006263484.
Mohapatra, D., & Mishra, S. S. (2010). Banana and its by product utilisation: an overview. Journal of
Scientific and Industrial Research Vol. 69, 2-5.
Msangi, T., Sulser, R., Valmonte, S., & Ringler, C. (2006). Global Scenarios for Biofuels: Impacts and
Implications. International Food Policy Research Institute (IFPRI).
NRBP. (2001). An Ethanol Production Guidebook for Northeast States. Washington, DC 20001:
Northeast Regional Biomass Program.
Pandey, A., Nigam, P., Soccol, R., Soccol, T., Singh, D., & Mohan, R. (2004). Advances in microbial
amylases. Biotechnology and Applied Biochemistry.
Poitrat, E. (1999). The potential of liquid biofuels in France. Renewable Energy.
Raikar , R. V. (2012). Enhanced production of Ethanol from grape waste . INTERNATIONAL JOURNAL
OF ENVIRONMENTAL SCIENCES Volume, 1-5.
Sanchez, O. J., & Cardona, C. A. (2007). Trends in biotechnological production of fuel ethanol from
different feedstocks: a review. Manizales: National University of Colombia.
Seifu, G. (2001). Banana Production and Utilization in Ethiopia. Addis Ababa: Ethiopian Agricultural
Research Organization.
Singh, B. (2010). Industrial Crops and Uses. Chippenham, UK: CPI Antony Rowe.
Tesfaye, M., & Gebru, Y. (2011). Assessment of biofuel development activities in Ethiopia. Addis Ababa:
Forum for Environment.
Teshale, F. (2012). Design and Optimization of Molasses Treatment to Reduce Scale Formation in
Ethanol Production (The Case of Metahara Sugar Factory Ethanol Plant). Addis Ababa: Addis
Ababa University.
Tiffany, G., & Eidman, R. (2003). Factors associated with success of fuel ethanol producers. Staff Paper
Series. USA: University of Minnesota.
Watson, T., Zinyowera, R., & Moss, H. (1996). Technologies, Policies and Measures for Mitigating
Climate Change. Technical Paper Series No. 1. Geneva: Geneva.
Zhan, X., Wang, D., Tuinstra, R., Bean, S., Seib, A., & Sun, S. (2003). Ethanol and lactic acid
production as affected by sorghum genotype and location. Industrial Crops and Products.

53
APPENDICES

APPENDIX A: EXPERIMENTAL RESULTS OF MASS PERCENTAGE OF SUGARS

Run Acid Hydrolysis Hydrolysis Total Weight Weight Weight Mass %w/w
o
conc. temp. ( C) time (min) weight of of of dist. sugar of
(%v/v) of empty sample water found sugar
samples flask (gm) (gm) (gm) to
(gm) (gm) original
1 2.5 90 30 143.62 98.33 45.29 40.93 4.356 43.56
2 1.5 90 20 145.24 98.33 46.91 42.40 4.510 45.10
3 2.5 70 20 144.72 98.33 46.39 41.99 4.401 44.01
4 1.5 90 20 144.95 98.33 46.62 42.12 4.500 45.00
5 0.5 110 20 143.91 98.33 45.58 41.15 4.427 44.27
6 2.5 90 10 143.41 98.33 45.08 40.84 4.240 42.40
7 1.5 90 20 145.01 98.33 46.68 42.18 4.500 45.00
8 2.5 110 20 142.75 98.33 44.42 40.12 4.298 42.98
9 1.5 90 20 144.89 98.33 46.56 42.06 4.500 45.00
10 1.5 90 20 145.31 98.33 46.98 42.47 4.515 45.15
11 1.5 110 10 144.67 98.33 46.34 42.04 4.296 42.96
12 0.5 70 20 142.56 98.33 44.23 39.91 4.321 43.21
13 0.5 90 30 143.27 98.33 44.94 40.64 4.300 43.00
14 0.5 90 10 143.09 98.33 44.76 40.50 4.256 42.56
15 1.5 70 10 143.18 98.33 44.85 40.63 4.220 42.20
16 1.5 70 30 144.35 98.33 46.02 41.64 4.385 43.85
17 1.5 110 30 142.97 98.33 44.64 40.30 4.340 43.40

A PPENDIX B: D IAGNOSTICS C ASE S TATISTICS : ACTUAL VERSUS MODEL P REDICTED OF ETHANOL YIELD

54
 Standard Actual Predicted Residual Leverage Student Cook's Outlier t Run
Value Value Residual Distance Order
1 42.980 43.126 -0.146 0.75 -1.942 1.131 -2.647 8
2 42.400 42.228 0.172 0.75 2.290 1.574 4.236 * 6
3 42.960 42.986 -0.026 0.75 -0.349 0.036 -0.326 11
4 42.560 42.610 -0.050 0.75 -0.664 0.132 -0.635 14
5 45.000 45.050 -0.050 0.20 -0.371 0.003 -0.347 9
6 43.210 43.064 0.146 0.75 1.942 1.131 2.647 12
7 43.560 43.510 0.050 0.75 0.664 0.132 0.635 1
8 45.150 45.050 0.100 0.20 0.742 0.014 0.716 10
9 45.100 45.050 0.050 0.20 0.371 0.003 0.347 2
10 45.000 45.050 -0.050 0.20 -0.371 0.003 -0.347 7
11 42.200 42.296 -0.096 0.75 -1.278 0.490 -1.351 15
12 44.010 44.086 -0.076 0.75 -1.012 0.307 -1.015 3
13 43.400 43.304 0.096 0.75 1.278 0.490 1.351 17
14 43.000 43.173 -0.173 0.75 -2.290 1.574 -4.236 * 13
15 45.000 45.050 -0.050 0.20 -0.371 0.003 -0.347 4
16 44.270 44.194 0.076 0.75 1.012 0.307 1.015 5
17 43.850 43.824 0.026 0.75 0.349 0.036 0.326 16

A PPENDIX C: P ROPERTIES OF ETHANOL

Acidity (Pka) 15.9 (H plus from OH group)

Appearance Colorless
Boiling point 78.4oC (351.6k)
Density 0.789 g/cm3
Dipole moment 1.69D (gas)
Melting point -114.3 oC (158.8k)
Molecular formula C2H5OH
Phase liquid
Solubility in water Fully miscible
Viscosity 1.200 cp at 20 oC
A PPENDIX D: DENSITY OF ETHANOL

55
APPENDIX E: M ATERIALS OVERALL BALANCES

56
COMPONENT IN OUT OUT-IN

Carb. Dioxide 0.00 3.45 3.45

Cellulose 49.50 49.41 - 0.09

Crude fat 33.00 33.00 - 0.00

Dietary fiber 104.50 104.50 - 0.00

Ethyl Alcohol 0.00 2.44 2.44

Glucose 11.00 5.73 - 5.27

Hemicellulose 44.00 43.92 - 0.08

Lignin 49.50 49.50 0.00

Nitrogen 21.12 21.12 0.00

Other solids 33.00 33.00 - 0.00

Oxygen 6.41 6.41 0.00

Pectine 60.50 60.50 - 0.00

Proteins 33.00 33.00 - 0.00

Starch 16.50 16.50 - 0.00

Sulfuric Acid 0.76 0.76 - 0.00

Water 5068.69 5069.36 0.66

Xylose 5.50 0.96 - 4.54

Yeast 0.00 2.94 2.94

TOTAL 5536.99 5536.50 - 0.49

57
 Mekelle University
Mekelle Institute of Technology
Department of Biological and Chemical Engineering

Project presentation
By:
Alula Gebregergs
Mebrahtom Gebresemati
Advisors:
Abadi G/yesus (MSc)
H/kiros Tadesse (MSc)
Kibrom G/egzabher (MSc)
June 24, 2014
Mekelle, Ethiopia

1
 Problem Statement

Introduction Objectives

Significance of the Study

Contents

Materials and Methods

Plant Design and Cost Analysis

Result and Discussion

Conclusion and Recommendation

2
Introduction
Overall wellbeing of Primary Energy Renewable Energy
the world Sources Sources
Fossil fuels Hydropower
Safe
Coal Geothermal
Oil
Sustainable Natural gas
Wind
Solar
Affordable energy
Biofuel
3
Statement of the Problem

Massive dependence on fossil Daily waste generation and An overriding emphasis

fuel waste management
biofuel
exposed Ethiopia to large international balanced 2009 (E5)
price volatility
2011 (E10)

4
Objectives
General

determining Specific
an optimized
way of Determining the Promoting
Developing optimized
conversion parameters condition effect of Benzyl national energy Decreasing
of banana (acid conc., temp. &
time)
penicillin in demand health and
enhancing S. fulfillment by environmental
peels to to obtain maximum cerevisiae generating impacts of
ethanol amount of fermentable during energy from banana peels
sugars
fermentation wastes
5
Significance of the Study
Successful completion of this study
Global warming
will show the production of renewable energy from
banana peels
will help as technical input for ethanol producers from
lignocellulosic biomass

Shortage of Energy

6
Materials and Methods >> Materials
H2SO4
NaOH
Plastic bags Knife Oven Grinder
Yeast extracts agar

Equipments
Chemicals

Urea
Dextrose sugar
Mg SO4.7H2O Balance pH meter Flasks Autoclave

Baker yeast
Distilled water
Shaking Rotary
Benzathine penicillin G Filter
incubator evaporator
Pycnometer
7
Materials and Methods >> Sample Collection and Preparation
Chopping: Knife (2-4 cm)
Sample Collection
Drying: Sunlight
(48hrs) & Oven (60oC,
24hrs)
Fruit shops
Sample
Preparation Grinding: Grinder
(<3mm)
Juice houses sample Preparation 19: 17
(10gm) + 2(20gm) with 1:10
%v/v dist. water
8
Materials and Methods >> Pretreatment

Cooling and Separating

Steam under high pressure nonsoluble from soluble portion
9
Materials and Methods >> Hydrolysis
Experimental design formulation for hydrolysis
Exp. Run Acid conc. (% v/v Hydrolysis Hydrolysis
to dist. water) temp. (oC) time(minutes)
1 2.5 90 30
2 1.5 90 20
3 2.5 70 20
4 1.5 90 20
5 0.5 110 20
6 2.5 90 10
7 1.5 90 20
8 2.5 110 20
9 1.5 90 20
10 1.5 90 20
11 1.5 110 10
12 0.5 70 20
13 0.5 90 30
14 0.5 90 10 Hydrolysis with dilute sulfuric acid in oven
15 1.5 70 10
16 1.5 70 30
17 1.5 110 30 10
Materials and Methods >> Measuring Sugar

Hydrolyzates
sample

Soluble sugars
Brix method (weight measuring)

11
Materials and Methods >> Fermentation
Sample
conditioned: 20 oC
Media (working
temperature)
Dextrose (10gm)
Yeast extract sugar
(0.4gm)
Inoculum Urea (1gm)
Addition of yeast
culture (1:4 v/v)
MgSO4.7H2O (1gm)
Dist. water (100ml)
Benzathine
penicillin G
addition (0.5gm/L)
(Sample Two)

Incubating in
S. cerevisiae shaking incubator
at 30 oC, 200 rpm,
3 days

pH adjusting
between 5.0 5.5

12
Materials and Methods >> Alcohol Measurement
Filtration Measuring W1 Empty Pycnometer
Specific W2 P + Sample
W3 P + Dist. water
Gravity
Distillation
SG = (W2-W1)/(W3-W1)

Rotary evaporator
EtOH Density
Pycnometer Table
13
 Result and Discussion >> Experimental Results
Table 1: Sugar Mass Percentage
Percent weight of sugar content (%w/w to original
Run
sample)
1 43.56
2 45.10
3 44.01 Table 2: Alcohol Specific Gravities
4 45.00 W1 Weight of empty pycnometer 25.10gm
5 44.27
6 42.40 W21 Weight of pycnometer plus sample one 48.100gm
7 45.00 W22 Weight of pycnometer plus sample two 48.070gm
8 42.98
9 45.00 W3 Weight of pycnometer filled with dist. water 48.50gm
10 45.15
11 42.96
12 43.21
13 43.00
14 42.56
15 42.20
16 43.85
17 43.40 14
Result and Discussion >> Statistical Analysis
120
110 110 110 110

100
90 90 90 90 90 90 90 90 90

80
70 70 70 70

60
43.56 45.10 44.01 45.00 44.27 42.40 45.00 42.98 45.00 45.15 42.96 43.21 43.00 42.56 43.85 43.40
42.20
40
30 30 30 30
20 20 20 20 20 20 20 20 20
15 16 17
20 13 14
10 8 9 10 11 10 12 10 10
5 6 7
32.5 41.5
12.5 21.5 0.5 2.5 1.5 2.5 1.5 1.5 1.5 0.5 0.5 0.5 1.5 1.5 1.5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Exp.R 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
A conc. 2.5 1.5 2.5 1.5 0.5 2.5 1.5 2.5 1.5 1.5 1.5 0.5 0.5 0.5 1.5 1.5 1.5
Temp. 90 90 70 90 110 90 90 110 90 90 110 70 90 90 70 70 110
Time 30 20 20 20 20 10 20 20 20 20 10 20 30 10 10 30 30
Sugar 43.56 45.10 44.01 45.00 44.27 42.40 45.00 42.98 45.00 45.15 42.96 43.21 43.00 42.56 42.20 43.85 43.40
15
Effects of Experimental Variables on Sugar Yield

16
 Optimizations
Constraints
N0. Acid conc. Temp. Time Sugar Desirability
Name Goal Lower Limit Upper Limit
1
Acid 1.50 91.02 21.66 45.088 1 (Selected)
conc. is in range 0.5 2.5 2
Temp. is in range 70 110 1.43 87.55 22.05 45.070 1
Time is in range 10 30 3
1.60 86.97 23.02 45.064 1
Sugar maximize 42.2 45.05
4
1.74 86.8 22.10 45.055 1
5
1.31 89.76 20.93 45.050 1
6
1.57 94.03 22.04 45.051 1
7
1.51 92.51 21.62 45.079 1
8
1.40 93.81 22.29 45.057 1
9
1.31 92.42 21.53 45.059 1
10
1.51 85.73 21.45 45.063 1
17
Effects of Benzathine penicillin G on Ethanol Production

Specific gravity calculated Percentage of EtOH

(from data results) obtained from Std table
( . . ) Sample one 8.36%
Sample one = = 0.98921
( . . )
( . . )
Sample two 9.11%
Sample two = = 0.91453
( . . )

Presence of benzathine penicillin G enhanced the fermentation process by 8.97%

18
Plant Design and
Cost Analysis
Feedstock
Component % w/w

Process Flow Sheet

Cellulose 9.00
Crude fat 6.00

Dietary fiber 19.00

Glucose 2.00
Hemicellulose 8.00

Lignin 9.00

Other solids 6.00

Pectin 11.00
Proteins 6.00

Starch 3.00
Water 20.00
Xylose 1.00

19
 Cost Analysis >> Executive Summary
Total Capital Investment 3,080,063,000 ETB
Cost Item ETB %
Capital Investment Charged to This Project 3,080,063,000 ETB
Raw Materials 457,000 0.75
Operating Cost 61,204,000 ETB/yr
Labor-Dependent 6,834,000 11.17
Main Revenue 376,000 ETB/yr
Consumables 13,000 0.02
Other Revenues 309,520,768 ETB/yr
Waste Treatment/Disposal 0 0
Total Revenues 309,896,000 ETB/yr
Utilities 46,630,000 76.19
Cost Basis Annual Rate 20.02 MT/MP/yr
Transportation 7,271,000 11.88
Unit Production Cost 3,056,604.70 ETB/MT MP
Miscellaneous 0 0.00
Unit Production Revenue 15,476,506.42 ETB/MT MP
Advertising/Selling 0 0.00
Return On Investment 13.88 %
Running Royalties 0 0.00
Payback Time 7.21 years
Failed Product Disposal 0 0.00
IRR (After Taxes) 8.83 %
TOTAL 61,204,000 100.00
NPV (at 7.0% Interest) 283,438,000 ETB
Total Capital Investment 3,080,063,000 ETB

20
Conclusion

Effect of benzathine penicillin

Hydrolysis of banana peel
G on fermentation process
To determine the optimum
conditions, which yield max. sugar
By measuring the SG of the
content produced ethanol

Presence of benzathine
All have negative effect at high and
low level penicillin G had enhanced
ethanol production
21
Recommendation

Alternative
Effective waste extraction
Researches
Further study management methods of
have to be done
method ethanol have to
be done

Fruit peel yields and their Conversion of other fruit Enzymatic extraction, To improve the quality
potential as source of wastes to valuable Microbial (Strain and quantity of fruit peel
renewable energy products Improvement) ethanol production

22
Acknowledgment
1. Advisors
Mr. Abadi Gebreyesus (MSc)
Mr. Hailekiros Tadesse (MSc)
Mr. Kibrom Gebreegzabher (MSc)
2. Laboratory of organic chemistry in Mekelle university
3. Microbiology department at the college of veterinary
medicine
Mr. Melesew
4. Desta Alcohol and Liquor Factory (DALF)
Mr. Gebremeskel Gebrewahd
23
 24
View publication stats