The Pennsylvania State University

The Graduate School

College of Engineering

ETHANOL PRODUCTION FROM WASTE POTATO MASH USING

SACCHAROMYCES CEREVISIAE

A Thesis in

Agricultural and Biological Engineering

by

Gulten Izmirlioglu

2010 Gulten Izmirlioglu

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

August 2010

ii

The thesis of Gulten Izmirlioglu was reviewed and approved* by the following:

Ali Demirci

Professor of Agricultural and Biological Engineering

Thesis Advisor

Virenda M. Puri

Professor of Agricultural and Biological Engineering

Chobi DebRoy

Senior Research Associate

Veterinary and Biomedical Sciences

Paul H. Heinemann

Head and Professor of Agricultural and Biological Engineering

*Signatures are on the file in the Graduate School.

iii

ABSTRACT

Ethanol is one of the bio-energy sources with high efficiency and low

environmental impact. Various raw materials have been used as carbon sources for

ethanol production. In this study, waste potato mash was chosen as a carbon source;

however, a pretreatment process is needed to convert starch of potato to fermentable

carbon sources through liquefaction and saccharification processes. In order to obtain

maximum fermentable sugar conversion, optimum parameters for the liquefaction and

saccharification processes were determined by Box-Behnken Response Surface

Methodology (RSM). The optimum combination of temperature, dose of enzyme (α-

amylase), and amount of potato mash was determined as 95°C, 1 ml of α-amylase

enzyme solution (944 Units/mg protein), and 4.04 g dry-weight potato mash /100 ml DI

water, respectively with a 68.86% loss in dry weight during the liquefaction process. For

the saccharification process, dose of enzyme, temperature, and saccharification time were

also determined by using Box-Behnken RSM. The optimal of amyloglucosidase

combination was 60°C-72 h-0.8 ml (300 Unit/ml) with 34.9 g/L glucose production after

scaling up to increase the glucose level to about 100 g/L. The effect of pH, inoculum size,

and various nitrogen sources to obtain maximum ethanol from waste potato mash was

studied in batch fermentation after. The maximum ethanol concentration and production

rates were 27.7 g/L and 5.47 g/L/h, respectively, at controlled pH 5.5, whereas 22.75 g/L

and 2.22 g/L/h were obtained at uncontrolled pH. Optimum inoculum size was

determined to be 3% for maximum ethanol yield and production rate. Furthermore, five

different nitrogen sources (yeast extract, poultry meal, hull and fines mix, feather meal,

and meat and bone meal) were evaluated to determine an economical alternative of

nitrogen source than yeast extract. In conclusion, this study demonstrated the potential for

utilization of potato waste for ethanol production.

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TABLE OF CONTENTS

LIST OF FIGURES VII

LIST OF TABLE...............................................................................................................IX

ACKNOWLEDGEMENTS............................................................................................X

Chapter I Introduction........................................................................................................1

Chapter II Literature Review ..........................................................................................6

2.1 Introduction………………………………………………..............……..........6

2.2 Environmental Issues Related to Fossil Fuels………………...........................7

2.3 Ecomonic Issues Related to Fossil Fuels……….………..........……..............10

2.4 Bio-energy.......................................................................................................11

2.4.1 Bio-gas...............................…....……..........…………………….................11

2.4.2 Bio-diesel..................….....….……....……..........................................…....12

2.5.Bio-ethanol.......................................................................................................13

2.6. Production of Bio-ethanol...............................................................................15

2.6.1.Producer Microorganims of Ethanol.............................................................16

2.6.2.Feedstocks for Ethanol Fermentation............................................................20

2.6.2.1. Sugars as a Feedstock for Ethanol Fermentation......................................21

2.6.2.2.Lignocellulosic Biomass as a Feedstock for Ethanol Fermentation..........22

2.6.2.3.Starch as a Feedstock for Ethanol Fermentation.......................................24

2.6.3. Fermentation................................................................................................27

2.6.3.1. Batch Process............................................................................................27

2.6.3.2. Fed-batch Process.....................................................................................29

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2.6.3.3. Continuous Process...................................................................................30

2.6.4. Simultaneous Saccharification and Fermentation........................................31

2.7. Potato..............................................................................................................33

2.7.1. Fermentation of Waste Potato Mash.............................................................34

2.8.State -of-the Art...............................................................................................37

Chapter III Goal and Objectives.......................................................................................40

3.1 Goal.................................................................................................................40

3.2 Objectives........................................................................................................40

Chapter IV Methods and Materials...................................................................................42

4.1 Microorganism.................................................................................................42

4.2 Waste Potato Mash..........................................................................................42

4.3 Baseline Fermentation Medium.......................................................................43

4.4 Objective 1: Determination of the Hydrolysis Parameters of Waste

Potato Mash...................................................................................................43

4.4.1 Acid Hydrolysis.............................................................................................43

4.4.2 Enzyme Hydrolysis........................................................................................44

4.5 Ethanol Fermentation in Potato Mash Hydrolyzed Medium

and pH Evaluation (Objective 2).....................................................................48

4.6 Inoculum Size Determination (Objective 3).....................................................49

4.7 Evaluation of Alternative Nitrogen Sources (Objective 4)...............................50

4.8 Analysis.............................................................................................................50

4.8.1 Moisture Analysis..........................................................................................50

4.8.2 Non-Dissolved Solid Analysis.......................................................................50

4.8.3Cell Population................................................................................................51

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4.8.4 Ethanol and Glucose......................................................................................51

4.8.5 Statistical Analysis.........................................................................................51

Chapter V Results and Discussions...................................................................................53

5.1 Hydrolysis of Waste Potato Mash....................................................................53

5.1.1 Acid Hydrolysis............................................................................................54

5.1.2 Enzyme Hydrolysis.......................................................................................56

5.2 Ethanol Fermentation.......................................................................................66

5.2.1. Ethanol Production in Hydrolyzed Waste Potato Mash

Media and Effect of pH...............................................................................67

5.2.2 Effect of Inoculum Size on Ethanol Production...........................................70

5.2.3 Effect of Nitrogen Sources............................................................................73

Chapter VI Conclusions and Suggestions for Future Research......................................80

References..........................................................................................................................83

vii

LIST OF FIGURES

Figure 2.1 Global carbon dioxide emissions from fossil fuels...........................................9

Figure 2.2 Pennsylvania greenhouse gas emissions, 1990 and 1999 ................................10

Figure 2.3 Simplified model of anaerobic fermentation of glucose to ethanol,

glycerol, and polysaccharides in S. cerevisiae..................................................18

Figure 2.4 Ethanol fermentation by Z. mobilis..................................................................19

Figure 2.5 Ethanol production from lignocellulosic materials..........................................23

Figure 2.6 Representative partial structure of amylose and amylopectin.........................24

Figure 2.7 Ethanol production from starchy materials......................................................26

Figure 4.1 Sartorious Biostat B plus bioreactors...............................................................49

Figure 5.1 Glucose release from waste mash potato as a result of acid hydrolysis

at 121°C for 1 h..............................................................................................55

Figure 5.2 Surface plots of liquefaction of waste potato mash..........................................59

Figure 5.3 Surface plots of saccharification of waste potato mash....................................62

Figure 5.4 Correlation of enzyme and generated glucose with enzyme levels

listed in Table 5.3 ............................................................................................64

Figure 5.5 Relationship among enzyme, dry waste potato mash,

and generated glucose with enzyme levels listed in Table 5.4........................66

Figure 5.6 Baseline ethanol fermentation..........................................................................67

Figure 5.7 Glucose and ethanol, and cell populations at pH 5.5 and

uncontrolled pH...............................................................................................69

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Figure 5.8 Ethanol, glucose, and cell population in the fermentation broth with

different inoculum sizes....................................................................................72

Figure 5.9 Ethanol, glucose, and cell populations in the fermentation broth with

different nitrogen sources: Yeast extract, Feather meal, Poultry meal,

Meat bone meal, Hull and fines mix.................................................................77

Figure 6.1 Summary of ethanol production.......................................................................80

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LIST OF TABLES

Table 2.1 Properties of ethanol...................................................................................14

Table 2.2 Ethanol production in the world........................................................................16

Table 2.3 Different feedstocks for bio-ethanol production and their

comparative production potential.....................................................................21

Table 4.1 Box-Behnken Surface response method analysis design for

liquefaction........................................................................................................45

Table 4.2 Box-Behnken Surface response method analysis design for

sachharification................................................................................................. 46

Table 4.3 Increasing of enzyme concentration with a constant amount of

waste potato mash ............................................................................................47

Table 5.1 Box-Behnken design and results of liquefaction...............................................57

Table 5.2 Box-Behnken design and results of saccharification.........................................60

Table 5.3 Relationship between enzyme concentration and glucose ................................63

Table 5.4 Enhancement of glucose concentration.............................................................65

Table 5.5 Comparison of kinetic parameters of controlled pH

vs. uncontrolled pH ..........................................................................................70

Table 5.6 Comparison of kinetic parameters of different inoculum sizes.........................73

Table 5.7 Comparison of kinetic parameters of alternative nitrogen sources....................79

x

ACKNOWLEDGEMENTS

First and foremost I offer my sincerest gratitude to my advisor, Dr. Ali Demirci,

who has supported me throughout my thesis with his patience and knowledge. I attribute

the level of my Masters degree to his encouragement and effort. Without him, this thesis

would not have been completed or written; one simply could not wish for a better or

friendlier supervisor.

I would like to thank my committee members, Dr. Chobi DebRoy and Virenda M.

Puri, who gave me their kind attentions and helpful guidance. It was my pleasure having

a chance to work with them.

I would like to thank my friends and lab mates, Kuan-Chen Cheng, Meltem N.

Keklik, Niharika Mishra, and Manolya Oner, for their support throughout my research. I

also want to thank my closest friend, Aliye Malak, for her patience with me during two

years.

Last but not least, I am sincerely indebted to my parents for their love, guidance,

and support throughout my life. I am also thankful to my siblings for their friendship and

love.

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CHAPTER I

INTRODUCTION

In the 21st century, while the demand for energy for transportation, heating, and

industrial processing is increasing day by day, environmental issues are a point of

concern (Hahn-Hagerdal et al., 2006). According to the Energy Information

Administration (EIA, 2008a), the U.S. is the major consumer of oil, consuming 20.7

millions of barrels per day in 2007. Renewable energy sources receive attention not only

to protect the environment, but also to supply energy needs by reducing dependence on

foreign oil. In recent years, bioenergy sources have become more important as a viable

and economical alternative source.

Ethanol is one of the bioenergy sources with high efficiency and low

environmental impact. Worldwide production of ethanol is approximately 51,000 million

liters. Fuel encompassed 73% of produced ethanol, while beverage and industrial ethanol

constitute 17% and 10%, respectively (Sanchez and Cardona, 2008). As a fuel enhancer,

ethanol has some advantages. Woodson and Jablonowskiy (2008) reported that “As an

additive (ethanol), serves as a fuel volume extender, an oxygenate, and an octane

enhancer. When blended with gasoline, ethanol increases the octane rating and the

oxygen content of the fuel, which results in more complete combustion and reduction in

exhaust emissions such as carbon monoxide and unburned hydrocarbons.” Furthermore,

Reimelt et al., (2002) also listed expected benefits of bio-ethanol as: Lower carbon

dioxide emission, higher octane number, lower dust emission, biodegradable, and

renewable.

2

Most countries have either ethanol blended gasoline or direct ethanol fuel, such as:

Brazil, USA, Canada, Colombia, Spain, and France (Sanchez and Cardona, 2008).

Countries that have added ethanol to fuel are also major ethanol producers. In 2005,

45.42 billion liters of ethanol were produced worldwide (Balat et al., 2008) with Brazil

and the U.S. as the two major producers of ethanol. In 2005, the U.S. and Brazil produced

16.12 billion liters and 16 billion liters of ethanol, respectively (Balat et al., 2008).

However, the U.S. is also the largest petroleum consumer in the world, consuming 20.7

million barrels of petroleum products per day in 2007 (EIA, 2008 a).

Ethanol is an alcohol that is a product of microbial fermentation. Microorganisms

meet their energy demand by converting carbon sources to by-products such as: carbon

dioxide, lactic acid, ethanol, etc. Various feedstock and chemically defined media can be

used for ethanol fermentation. The most commonly used types of feedstock for ethanol

production are corn, sugar cane, and wheat (Balat et al., 2008). Saccharomyces

cerevisiae, Zymomonas mobilis, Kluyveromyces spp., and Schizosaccharomyces pombe

are microorganisms able to convert sugars to ethanol.

Corn is the main raw material for ethanol production in the U.S., accounting for

around 97% of the total ethanol produced. Sugar and other feedstock, can also be a

carbon source for ethanol production by fermentation and are economically viable.

Sugarcane, sugar beets, and molasses are feasible for ethanol fermentation and have been

used; however, these carbon sources are high value products as food sources (Nalley and

Hudson, 2003; USDA, 2006). Less valuable feedstock should be utilized for ethanol

production. In order to meet the low cost requirement, lignocellulosic biomass is another

option for ethanol fermentation. However, lignocellulosic biomass is complex and

3

requires expensive pretreatments. Currently, potatoes are an alternative feedstock for

ethanol production. Potatoes are starchy crops which do not require complex

pretreatments. Although it also is a high value crop, a significant amount of potato (e.g.

18% in the potato chips industry) is lost during processing because of the low quality and

processing. Therefore, waste from the potato industry could be the carbon source for

ethanol fermentation because it is relatively cheaper compared to other feedstock which

are considered food valuable source.

By-products of potato industry are currently, utilized as animal feed, however,

ethanol production could be an alternative for the industry to utilize the waste. At two

plants in Idaho, ethanol is produced from the by-products of French fry processing

whereas the stilliage (unfermented solids) of ethanol production is used for cattle feed

(Mann et al., 2002).

The U.S. produces about 22 million ton of potatoes annually, with approximately

850,500 tons in Pennsylvania, during 2007 fall season (USDA, 2007). In the U.S., the

breakdown of potato use is as follows: 34% frozen potato products, 28% fresh potato,

12% for chips, 10% dehydrated, 15% as potato seed and for consumption, and 1%

canned potato products (NPC, 2008). In the potato chips industry, 18% of company

production is starch waste, which could be a raw material for fermentative alcohol

production (Fadel, 2000). Potato flakes contain 73% available carbohydrate (FFCD,

2008). The mashed potato flake also has vitamins A, D, E, K, C, folate, niacin, riboflavin,

and thiamin. It also contains minerals, sodium, potassium, magnesium, calcium,

phosphorus, zinc, selenium, and iodine. Based on the composition of potato flakes, potato

mash could be a feasible feedstock for microorganisms to produce ethanol.

4

Ethanol production from other sources, such as corn, has been studied extensively.

Studies also indicate that potato can be considered as media for different microbial

products. Oda et al., (2002) studied lactic acid fermentation of potato pulp. The study also

showed that during lactic acid fermentation ethanol was formed by fungus (Oda et al.,

2002). Moreover, Klingspohn et al., (1993) studied utilization of potato pulp from potato

starch processing and reported that potato pulp can be employed to produce ethanol.

Despite this study, ethanol production from waste potato is a relatively new topic and

limited research has been conducted about the utilization of potato waste for ethanol

production. Fadel (2000) and Liimatainen et al. (2004) showed that potato waste can be a

carbon source for yeast during alcohol fermentation from the waste of the potato chips

industry and different potato cultivations, respectively. Yamada et al. (2009) also,

reported that ethanol can be produced from the by-products of potato processing plants.

In the study, the media was liquefied and partially saccharified potato peel and

substandard mash. Because the by-products of the potato industry vary from plant to

plant, further studies are needed to determine the optimal pretreatment of starch and

fermentation conditions (such as: pH, inoculum size, nitrogen sources, etc.). Maximizing

ethanol yield from waste potato fermentation and whether ethanol fermentation can be an

economic alternative from use of the by-products of the potato industry are questions to

be answered.

To reduce the use of fossil fuels, decrease the emission of carbon dioxide, and

reduce dependency on foreign oils, ethanol is an alternative bioenergy source. Since

ethanol is a bioenergy source for the future, there is a need to investigate economical

ethanol production from available raw materials. Ethanol could decrease the dependence

5

on foreign oil and give an opportunity to the potato industry by using their waste product

as a carbon source. However, ethanol fermentation from waste potato still needs to be

studied to optimize the fermentation process.

6

CHAPTER II

LITERATURE REVIEW

2.1 Introduction

During the last decade, demand for energy has been increasing while

environmental issues have become more important. Society has realized that oil fuel is

depleting and is also not an eco-friendly energy source. Therefore, renewable energy

sources attract attention to protect the environment, and to supply our energy needs by

reducing dependence on foreign oil and non-renewable energy sources.

Bioenergy, one renewable energy source, is a potential alternative to petroleum-

derived fuels and has the potential to help meet the increasing demand for energy for

industrial processes, heating, and transportation (Balat et al., 2008). Types of bioenergy

are: Biogas, biodiesel, bio-ethanol, natural gas, etc. Biogas is a digestion of the organic

matter by anaerobic microorganisms under controlled conditions, whereas, biodiesel is a

chemically processed fuel in which fatty acids are transferred to methyl esters and

glycerin by transesterification. Natural gas is a fossil fuel, so while it is clean and safe, it

is not renewable.

Bio–ethanol, a product of fermentation, has been utilized as a bio-fuel, a beverage,

and an industrial alcohol. Most of produced ethanol (73%) is utilized as fuel, while

percentages of beverage and industrial ethanol are 17% and 10%, respectively (Sanchez

and Cardona, 2008). Moreover, bio-ethanol is already being used in pure form or blended

with gasoline for transportation in Brazil and some other countries (de Oliveria et al.,

2005). Bio-ethanol is also one of the components of a fuel called gasohol or E10 (10%

7

ethanol by volume) and is available for transportation use in some states of the U.S.

(Balat et al., 2008). It is recognized that use of bio-ethanol as a fuel may be one of the

solutions to global warming and reducing dependency on foreign oil.

Microorganisms that are ethanol-fermentative, such as Saccharomyces cerevisiae,

can use organic feedstock as a raw material to produce ethanol. Different types of organic

materials, such as starchy materials, lignocellulosic materials, and sucrose- containing

materials, can serve as a raw material for ethanol production. Potato, a starchy material, is

one of these feedstock. The ultimate of potato utility depends on its applicable sugar

content.

This literature review will present background information about environmental

problems due to fossil fuel consumption, bio-ethanol, and ethanol producer

microorganisms, raw materials and methods of ethanol fermentation, and potato as a

medium ingredient for ethanol fermentation.

2.2 Environmental Issues Related to Fossil Fuels

The climate of the Earth has slowly changed since the last ice age. The years

following Industrial Revolution were wrought with human activities that have affected

the climate by changing negatively the composition of the atmosphere (EPA, 2008a).

These activities have caused environmental problems such as: Acid rain, air pollution,

global warming, and ozone depletion. Flooding, starvation, drought, reduction of crop

capacity, and extinction have occurred more often than in the past from these climate

changes.

8

Man-induced global warming is an increase in world temperatures caused by

polluting gases such as carbon dioxide that are collecting in the atmosphere and

preventing some of the heat from escaping into space (Cambridge Online Dictionary,

2008). The Union of Concerned Scientists (UCSUSA) states, “Global warming doesn’t

just mean balmy February days in northern climes, but also means increasingly hot days

in the summer, and a host of negative impacts that are expected to intensify in the coming

decades (UCSUSA, 2008a)”. Heat-related illnesses and deaths, severe flooding, droughts,

and rising sea levels are some of the mentioned negative impacts from global warming.

Greenhouse gases (GHGs) are the chemicals that keep heat from sunlight in the

atmosphere and near the earth's surface, thereby contributing to global warming. GHGs

are like a natural cover on the Earth that do not let radiation back into space thereby

trapping the heat. During the last century, there has been about a 1.8ºC increase in the

temperature of the Earth’s surface from this effect (NSCEP, 2000).

The causes of global warming are: Carbon dioxide (CO2) (from: power plants,

cars, airplanes, buildings), methane, nitrous oxide, deforestation, carbon in the

atmosphere and ocean, and permafrost (Ecobridge,2008). Carbon dioxide occurs in the

atmosphere from the burning of fossil fuels (oil, natural gas, and coal), solid wastes, trees,

wood products, and also as a result of other chemical reactions (e.g., manufacture of

cement). Figure 2.1 shows carbon emissions during the last 200 years.

9

Figure 2.1 Global carbon dioxide emissions from fossil fuels (Moore, 2008).

The greenhouse effect is also a big problem for the U.S. and the Govermenth of

Pennsylvania. Scientists predict that global warming will affect the agricultural

production of Pennsylvania, especially some agricultural products such as Mclntosh or

Granny Smith apples, sweet corn, and Concord grape. Production of these crops will be

affected by higher temperatures (UCSUSA, 2008b). Figure 2.2 shows the gas emissions

in Pennsylvania between 1990 and 1999.

It is possible to slow the greenhouse effect by decreasing emissions of heat-

trapping gases. The use of biofuels and renewable energy sources could decrease GHG

emissions when the demand of energy is met. To decrease use of petroleum derived fuels

is of interest to countries of the world. In the U.S., blending of about 28.5 billion liters of

alternative fuels by 2012 is required by the Energy Policy Act of 2005 (Hahn-Hagerdal

et al., 2006). As an alternative for fossil fuels, biomass, hydro, solar, wind, and bio-

ethanol can be listed as renewable and alternative energy sources (EIA, 2008b).

10

Figure 2.2 Pennsylvania greenhouse gas emissions, 1990 and 1999

(EPA, 2008b). (MMTCE: Million Ton of carbon dioxide)

2.3 Economic Issues Related to Fossil Fuels

Although the U.S. is third in oil production in the world, it counts first in

consumption with 20.7 million barrels of petroleum products consumed per day (MMbd)

during 2007 ( EIA, 2008a). Oil imported goes to the U.S. about 58% of consumed fuel

(EIA, 2008a). Because of the speculations of the oil reserves and economical pressures

on the oil market, the oil price has been increasing day by day (Sanchez and Cardona,

2008). In order to overcome problems due to dependency on foreign oil and

environmental issues, sustainable energy sources should be investigated.

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2.4 Bioenergy

Bioenergy is the production of products including electricity, liquid, solid, and

gaseous fuels, heat, chemicals, and other materials from renewable energy sources.

Bioenergy is clean, pure, non-polluting energy. Bio-ethanol, biogas, biodiesel, and

natural gas are examples of bioenergy.

2.4.1 Biogas

Biogas is a digestion of organic matter by anaerobic microorganisms under

controlled temperature, moisture, and acidity conditions and the main component of

biogas is methane (das Neves et al., 2009). Methane is a colorless, tasteless, and odorless

gas. Biomass can be produced from different raw materials, e.g., animal manure, algae,

landfill, residues of the food industry, etc.

Biogas is produced in four main steps which are: Hydrolysis, acidogenesis,

acetogenesis, and methanogenesis in the study of das Neves et al. (2009). Hydrolysis is

transformation of insoluble compounds to soluble compounds. In the second step,

acidogenic bacteria ferment hydrolyzate to hydrogen, carbon dioxide, and acidic

compounds. The following step is acetogenesis and acetogenic bacteria forming acetic

acid. In methanogenesis, the last step, methanogenic microorganisms convert hydrogen,

carbon dioxide and acetic acid to methane.

Production of biogas can manage solid wastes and supply energy for heating,

transportation, etc in middle-income and developing countries. However, anaerobic

fermentation of biogas is a very slow process which increases the cost of fuel. Another

12

issue, the greenhouse effect of biogas is 21 fold higher than carbon dioxide and must be

stored and managed carefully (das Neves et al., 2009).

2.4.2 Biodiesel

Biodiesel is a renewable, environmentally safe, and energy efficient bioenergy. In

the production of biodiesel, fatty acids are transferred to methyl esters and glycerin by

transesterification, which is catalyzed by an alkali or acid. Although any fatty acid can

be a raw material for the production of biodiesel, waste vegetable oil and animal fats are

preferred due to the fact that they are not food value products (Refaat, 2010).

Production of biodiesel from waste oil starts with pretreatment of raw material,

which includes filtration to remove dirt, food residues, and non-oil materials. After

determining the concentration fatty acid, transesterification is performed to obtain

biodiesel. Tranesterification is a reaction in which fatty acids convert to methyl esters and

glycerin. Korus et al. (1993) reported that the ratio of alcohol to vegetable oil,

temperature, rate of agitation, and amount of water present in the reaction mixture are

essential parameters of the transesterification process. Temperature has no significant

effect on ester conversion except decreasing required conversion time. Because heating

costs are of concern, the transesterification reaction is carried out at room temperature

(Korus et al., 1993). Moreover, a homogenous mixture of alcohol and oil increases

conversion as well as decreasing the time required to reach maximum conversion (Korus

et al., 1993).They also reported that some catalysts can be used to improve

transesterification such as: Potassium hydroxide, sodium hydroxide, sodium methoxide,

or sodium ethoxide. Separation of alcohol and glycerin follows transesterification in the

13

production of biodiesel. Separated alcohol is washed before commercialization to remove

the catalyst from ester.

The limitation of the waste cooking oil is the low market availability of raw

material (Refaat, 2010). Raw material is a very important aspect when the cost of

production of biodiesel is of concern, due to the overall cost of biodiesel which depends

on raw material, chemical technology, and production volume (Refaat, 2010).

2.5 Bio-ethanol

Ethanol, which is known as pure alcohol, ethylalcohol or bio-ethanol, is a

colorless, flammable, volatile liquid with a strong odor. The melting point of ethnaol is –

114.1°C, whereas it boils at 78.5°C. Due to the low freezing point of ethanol, it has been

using in thermometers for temperatures below –40°C, and automobile radiators as

antifreeze (Shakhashiri, 2009). The properties of ethanol are given in Table 2.1. The

chemical formula of ethanol is C2H5OH, contain a –OH group bonded to carbon.

Ethanol can be produced synthetically and naturally by yeasts. Ethanol

fermentation has been used for the production of alcoholic beverages, and for the rising

of bread dough for centuries; recently, it has been produced to use industrially. Since

1908, fuel ethanol has found use for transportation gasoline and today, 73 % of ethanol

production is consumed as fuel worlwide. Bio-ethanol has become an attractive fuel

because it is renewable and oxygenated (Balat et al., 2008). Sanchez and Cardona (2008)

indicate that oxygenated ethanol reduces the emission of carbon dioxide and aromatic

compounds.

14

Table 2.1 Properties of ethanol*

Description Values

Chemical Formula C2H5OH

Molecular weight (g/mol) 46

Density at 20°C (kg/m³) 789

Calorific value (MJ/kg) 26.9

Calorific value of stoichiometric mixture (MJ/m³) 3.85

Heat of evaporation (kJ/kg) 840

Temperature of self-ignition (K) 665

Stoichiometric air/fuel ratio (kg air/kg fuel) 9

Lower flammability (λ1) 2.06

Higher flammability (λh) 0.3

Kinematic viscosity at 40°C (mm²/s) 1.4

Motor octane number /research octane number 89/107

Cetane number 8

Flame temperature (K) 2235

Molecular composition (by mass)

C (%) 52.2

H (%) 13

C (%) 34.8

*Kowalewicz, 2006

Ethanol is also non-toxic, and is a non-contaminant to water sources. Compared to the

other fuel additives such as methyl tertiary butyl ether (MTBE), ethanol’s octane booster

properties are greater (Sanchez and Cardona, 2008). Bio–ethanol is being used purely or

blended with gasoline for transportation in Brazil and in some states of the U.S. (de

Oliveria et al., 2005; Balat et al., 2008). Although bio-ethanol has been introduced as an

alternative to petrolum-derived fuels, corrosiviness, low flame luminosity, low vapor

pressure (compared to gasoline), miscibility with water, and low energy density are some

15

of the disadvantages of bio-ethanol (Balat et al., 2008). Aside from fuel, ethanol has other

applications in various industry branches such as: Personal care products, cleaning agents,

pharmaceuticals, and beverages.

2.6. Production of Bio-ethanol

In 2006, worldwide bio-ethanol production was approximately 51.3 billion liters

(Balat et al., 2008). An increase in fuel ethanol production resulted from the fact that

many countries want to reduce dependency on foreign oil and enhance air quality. Two

leaders of ethanol production in the world are Brazil and the United States as shown in

Table 2.2.

Bio-ethanol can be produced from different feedstock, such as corn, sugar cane,

cellulose, potato, etc. Sugar cane, as a raw material, is used for 60% of global ethanol

production, while 40% of global production of ethanol comes from other crops. Corn

grain is the main raw material of ethanol production in the United States (90% ) whereas

in Brazil, sugar cane is the major source (Balat et al., 2008). Desirable raw materials for

ethanol fermentation should have applicable sugars that can be fermented by

microorganisms. Sucrose containing feedstock, starchy feedstock, and lignocellulose

biomass can be used as raw materials for ethanol production.

Ethanol fermentation is summarized with the chemical equations:

C6H12O6 → 2 CH3COCOO− + 2H

+

CH3COCOO− + H

+ → CH3CHO + CO2

CH3CHO + NADH → C2H5OH + NAD+

16

Table 2.2 Ethanol production in the world *

Ethanol Production (million liters) Country

2005 2006

USA 16,139 18,376

Brazil 15,999 16,998

China 3,800 3,849

India 1,699 1,900

France 908 950

Germany 431 765

Russia 749 647

Canada 231 579

Spain 352 462

* Sanchez and Cardona, 2008.

2.6.1 Producer Microorganisms of Bio-ethanol

Microorganisms meet their energy demand by converting the carbon sources to

by-products such as: carbon dioxide, lactic acid, ethanol, cellulose. Ethanol is one of the

end products of fermentation, which can be performed by either bacteria or yeasts.

Fermentation is an energy generation process with no electron transport mechanism

(Shuler and Kargi, 2008). There are different pathways, which may be different from one

microorganism to another one,such as the Entner-Doudoroff and the Embden-Meyerhof

pathways (Shuler and Kargi, 2008). The Embden-Meyerhof pathway is used by yeast to

17

convert glucose to ethanol under anaerobic conditions during fermentation, whereas the

bacterium Zymomonas mobilis follow the Entner-Doudoroff pathway (Shuler and Kargi,

2008).

Saccharomyces cerevisiae has generally been recognized as safe (GRAS) and is

the most commonly used microorganism in the fermentation industry (Kunz, 2008).

Production of alcoholic beverages and bread dough rising are the two main

responsibilities of S. cerevisiae. Alcohol production occurs by converting sugar to energy,

and simultaneously S. cerevisiae meets its metabolic energy need. Under anaerobic

conditions, yeast ferments glucose, and ethanol and carbon dioxide are the by-products of

the Embden-Meyehof (EM) pathway. Fermentation is carried out in an anaerobic

environment, but S. cerevisiae needs small amounts of oxygen to synthesize fatty acid

and sterols (Sanchez and Cardona, 2008). Although S. cerevisiae is the most common

microorganism in ethanol fermentation, it is not able to break down lignocellulosic and

starchy material. One approach to solve this problem is hydrolysis before the

fermentation process, which converts the unfermentable sugars to glucose by hydrolyzing

enzymes. In pretreatment for hydrolysis, either mixed cultures or genetically modified

microorganisims can be introduced. S. cerevisiae already has some modified strains to

enhance the ethanol yield and assimilate pentoses (Cardona and Sanchez, 2007). Figure

2.5 is an illustration of anaerobic fermentation of glucose to ethanol, glycerol, and

polysaccharides in S. cerevisiae.

18

Figure 2.3 Simplified model of anaerobic fermentation of glucose to ethanol, glycerol,

and polysaccharides in S. cerevisiae (Polisetty et al., 2006). (ATP: Adenosine-

5'-triphosphate, ADP: Adenosine diphosphate, NAD: Nicotinamide adenine

dinucleotide, HK: Hexokinase, GLK:Glucokinase, PFK: Phosphofructokinase,

GADP: Glyceraldehyde-3-phosphate Dehydrogenase)

Zymomonas mobilis, gram negative facultative anaerobe, is also able to

metabolize glucose by the Entner-Doudoroff (ED) pathay (Figure 2.6 shows the pathway

of ethanol fermentation by Z. mobilis). Additionally, Z. mobilis is an ethanol tolerable

microorganism that can tolerate up to 120 g/L ethanol (Lin and Tanaka, 2006).

19

Zymomonas mobilis is also able to ferment just glucose, fructose and sucrose (Classen et

al., 1999). There have been recombinant Z. mobilis strains which have been modified to

transfer not only glucose, but also xylose to ethanol (Cardona and Sanchez, 2007).

Another approach to utilize starchy or lignocellulosic metarials for ethanol production by

Z. mobilis, is various pretreatments.

Figure 2.4. Ethanol fermentation by Z. mobilis (Jeffries, 2005).

After discovering gene transfer from one organims to another, microorganims

have been generated for ethanol fermentation, in which both hydrolysis and fermentation

20

can be done simultaneously. Escherichia coli and Klebsiella oxytoca were modified as

ethanol producers by transfering genes from Z. mobilis and E. coli KO11, yielding 0.49 g

EtOH/g sugar (Hahn-Hagerdal et al., 2006). Eksteen et al. (2003) reported that

recombinant S. cerevisae strains were able to utilize 80% of starch after transfering α-

amylase and amyloglucosidase genes from Lipomyces kononenkoae and

Saccharomycopsis fibuligera. This engineered yeast also produced 6.1 g/L ethanol at the

end of six days of fermentation.

Although Z. mobilis, Kluyveromyces spp., Schizosaccharomyces pombe, and

some recombinant bacteria and yeast can ferment sugars to ethanol, S. cerevisiae is still

the standard microorganism in the industry (Kunz, 2008; Lin and Tanaka, 2006).

2.6.2. Feedstock for Ethanol Fermentation

Bioethanol can be produced from different feedstock including sugar containing

feedstock, starchy feedstock, and lignocellulosic feedstock. For ethanol fermentation, raw

material plays an important role in production costs (Cardona and Sanchez, 2007). Since

30% of medium costs affect the cost of product, composition of media is very important

(Lee et al., 1998). By decreasing the cost of medium, cheap ethanol can be produced

without sacrificing ethanol yield and biomass. The plant design and the process of

fermentation is directly related to the type of raw material. Sugars can be transferred to

ethanol without any pretreatment, however starchy and lignocellulosic materials need

pretreatment prior to the fermentation process. The pretreatment of starch involves

hydrolysis, whereas lignocellulosic metarials reqiure more complicated treatments.

21

Table 2.3 Different feedstock for bio-ethanol production and their comparative

production potential *

Raw Metarial Potential of bio-ethanol production (L/ton)

Sugar cane 70

Sugar beet 110

Sweet potato 125

Potato 110

Cassava 180

Maize 360

Rice 430

Barley 250

Wheat 340

Sweet Sorghum 60

Bagasse and other cellulose biomass 280

*Balat et al., 2008.

2.6.2.1. Sugars as a Feedstock for Ethanol Fermentation

Sugars, hexo and pento carbons, do not require pretreatment, such as hydrolysis,

prior to being fermented. Thus bio-ethanol fermentation is easier, compared to starchy

materials or lignocellulosic feedstock, when the raw material is already in the form of

sugar. However, the limitation of sugars is their high cost, because they are already

valuable as a food source. In addition, availibity and transportation costs of sugar

containing raw metarials increase the cost of ethanol production (Cardona and Sanchez,

2007).

Sugarcane is the major sugar-containing feedstock for ethanol production in

Brazil (Sanchez and Cardona, 2008). In Brazil, sugarcane juice is used to produce

22

approximetly 79% of total ethanol production, and 21% of ethanol is produced from cane

molasses (Wilkie, 2000). In India, however, sugarcane molasses is the main raw material

for ethanol production (Ghosh and Ghose, 2003). The concentration of the sugars and

salts in the medium of cane molasses increases the osmolatily, which is a disadvantage

for the fermentation of ethanol (Sanchez and Cardona, 2008). The juice of sweet sorghum

is another sugar containing feedstock for ethanol due to its high sucrose content (Cardona

and Sanchez, 2007).

2.6.2.2. Lignocellulosic Biomass as a Feedstock for Ethanol Fermentation

This group of bio-ethanol feedstock consists of agricultural residues, wood, and

energy crops (fast growing and low cost agricultural production). Rice straw is also

another lignocellulosic waste material (Balat et al., 2008). Lignocellulosic materials need

to undergo very complex pretreatments prior to the fermentation process. Five main steps

have been used to produce ethanol from lignocellulosic biomass: biomass pretreatment,

cellulose hydrolysis, fermentation of hexoses, separation and effluent treatment (Cardona

and Sanchez, 2007). Figure 2.5 is a flowchart showing the basic steps of ethanol

fermentation from lignocellulosic biomass. Preteatment of lignocellulosic materials can

be performed by physical methods (chipping, grinding, and milling), physical-chemical

methods (thermohydrolysis, ammonia fiber explosion, etc.), chemical methods

(applications of ozone, acids, alkalis, peroxides, and organic solvents), and biological

methods (microbiological applications) (Sanchez and Cardona, 2008). Simultaneous

saccharification and fermentation (SSF), consolidated bioprocessing (CBP), and separate

23

hydrolysis and fermentation are some of the processes studied to improve ethanol

fermentation from lignocelllosic materials.

South et al. (1993) reported that 20 g/L ethanol concentration was obtained in the

application of continuous SSF to pretreated hardwood by using S. cerevisiae and

commercial cellulase supplemented with β-glucosidase. Moreover, F. oxysporum yielded

0.35 g/g cellulose with a productivity of 0.044 g/L/h when batch consolidated

bioprocessing was applied (Cardona and Snachez, 2007).

Figure 2.5 Ethanol production from lignocellulosic materials

(Cardona and Sanchez, 2007).

Cellulosic

Feedstock Pretreatment

Cellulose Hydrolysis

Detoxification

Hexose Fermentation Pentose

Productio

n of

cellulases

Conventional Distillation

Ethanol Dehydration

Effluent

Treatment Waste

Streams

Anhydrous

EtOH

EtOH EtOH

24

Lignocellulosic biomass has a huge potential for bio-ethanol production; however,

the cost of production of bio-ethanol is high due to the expense of a pretreatment process

using current technologies (Balat et al., 2008).

2.6.2.3 Starch as a Feedstock for Ethanol Fermentation

Starch is a polysaccharide composed of amylose and amylopectin, both of which

are glucose units. Amylopectin, which is highly branched by short chains, is 70-80% of

starch by composition. Amylose, a linear polysaccharide formed by α-1, 4-linked glucose

residues is the minor component of starch (20-30%) (Eksteen et al., 2003). Figure 2.6 a

and b illustrate the structure of amylose and amylopectin.

(a) (b)

Figure 2.6 Representative partial structure of amylose (a) and amylopectin (b) (Chaplin,

2008).

25

Hydrolysis is a process of breaking down amylopectin and amylose linkages into

fermentable sugars and is needed before the fermentation of starch materials. Hydrolysis

is carried out at high temperature (90-110ºC0. At low temperatures, hydrolyzing of starch

is possible and can contribute to energy savings (Sanchez and Cardona, 2008). To

convert starch into fermentable sugars, either acid hydrolysis or enzyme addition should

be done. Both hydrolysis methods have disadvantages and advantages. The limitations of

acid hydrolysis include the by-products inhibition on growth of yeast (such as 5-

hydroxymethylfurfural (5-HMF)), neutralization before fermentation, and expensive

constructional material (Tasic et al., 2009). On the other hand, high prices of enzymes

play a crucial role when feasibility is of concern for enzyme hydrolysis. Enzyme

hydrolysis is chosen despite the high cost of enzymes and initial investment (Tasic et al.,

2009) because of the high conversion yield of glucose. Moreover, starch has extended

storage and a low transportation cost with the pretreatment cost of starch still competitive

with pre treatment of lignocellulosic raw materials (Abouzied and Reddy, 1986).

Corn is the main feedstock for ethanol production in the U.S. Corn ethanol is

obtained from corn syrup produced enzymatically after the milling process (Sanchez and

Cardona, 2008). The last step of ethanol production from corn is fermentation at 30-32 ºC

and is accomplished by adding nitrogen sources (ammonium sulfate or urea) to medium

(Sanchez and Cardona, 2008). However, costs of raw metarial, energy costs of wet

milling, and transportation expenses are the limitations of using corn crops. In addition,

Nalley and Hudson (2003) state that “For each gallon of corn ethanol produced, about

160 gallons of waste water are produced”.

26

Figure 2.7 Ethanol production from starchy materials (Cardona and Sanchez,

2007).

Wheat is another starch material that can be used for ethanol fermentation already

used in some countries like France (Sanchez and Cardona, 2008). To enhance ethanol

fermentation from wheat, some reseach such as determing the optimal fermentation

temperature has been completed (Wang et al., 1999;Sanchez and Cardona, 2008). Longer

fermentation times and incomplete fermentations are the difficulties of this feedstock

(Barber et al., 2002). However, Montesinos and Navarro (2000) reported that a mix of S.

Starch Hydrolysis

Fermentation

Production of

amylases

Yeast

Propagation

Conventional

Distillation

Liquefaction Starch

Effluent

Treatment

Ethanol

Dehydration

EtOH

Waste Streams

Anhydrous EtOH

27

cerevisiae and Aspergillus niger and glucoamylase produced 67 g/L ethanol after a pre-

liquefaction with α-amylase from raw wheat flour.

Cassava, a tropical plant, is another starchy raw material that is used for ethanol

fementation. Altough cassava is a good glucose source with high starch content (85-90%

dry matter) (Sanchez and Cardona, 2008), reqiured the tropical climate is a limitation for

cassava production. Jansson et al. (2009) presented that cassava has a 150 conversion

rate (L/ tonne) to ethanol. Other starchy materials that may serve as the source for bio-

ethanol include rye, barley,tricilate, sorghum, and potato (Zhan et al., 2003).

2.6.3. Fermentation

Fermentation is a metabolic process of microorganisms to obtain energy by

breaking down organic compounds. While microorganisms derive their energy, some by-

products are: lactic acid, butane, carbon dioxide, ethanol, cellulose, nisin. In ethanol

fermentation, derivation of energy from sugars by either yeast or bacteria, produce carbon

dioxide and ethanol are produced. Because yeasts produce their energy without the need

for oxygen, ethanol fermentation is a facultative anaerobic process.

Fermentation methods are other important aspects of ethanol fermentation. Batch,

semi-continuous, and continuous processes have been applied in the ethanol industry.

There are also some other fermentation types, such as immobilized cultures.

2.6.3.1. Batch Processes

Batch fermentation is carried out in a cultured vessel with an initial amount of

medium and during the fermentation no medium addition or removal occurs (Shuler and

28

Kargi, 2008). Ethanol fermentation is performed after sterilization of the media and

adjustment of pH by either acid or alkali. After inoculation of yeast or bacteria,

production of ethanol takes place by controlling temperature, pH, agitation, and aeration

depending on the characteristics of the cultured microorganism. Because no medium

addition occurs, the growth of microorganism follows four main phases of the growth

curve which include: the lag phase, the log phase, the stationary phase, and the death

phase. The growth of the microorganism is slow in the lag phase, because this is an

adaptation time for the cells to a new environment and cells may have new metabolic

path ways or synthesize enzymes (Shuler and Kargi, 2008). Logarithmic growth is where

microbial growth performs and reaches its maximum rate. Stationary phase refers to the

period where microbial growth rate equals the death rate. In this phase, cells are still able

to produce secondary products however (Shuler and Kargi, 2008). The last period of

batch fermentation is the death phase in which death rate is higher than the growth rate

due to lack of nutrients or accumulation of inhibitor primary or secondary metabolites,

etc.

In ethanol fermentation, the batch process has a wide application. Although, the

batch system can be conducted directly for fermentation when the raw material is sugar

containing materials, it also can be combined with pretreatment processes for ethanol

fermentation from starchy and lignocellulosic materials. Hydrolysis of starch (mostly

saccharification) and fermentation can be performed simultaneously. Montesinos and

Navarro (2000) studied ethanol fermentation from raw wheat flour by applying batch and

saccharification and fermentation simultaneously. They reported that 67 g/L ethanol

concentration was obtained at the end of 31h fermentation. Mamma et al. (1996) studied

29

ethanol fermentation from sweet sorghum stalks by using mix culture in batch

fermentation and reported 35-49 g/L ethanol concentration.

In applications of batch fermentation, inhibitor effects of primary or secondary

products are a limitation of the process. Furthermore, to reuse the yeast from previous

batch, a centrifugation process is needed.

2.6.3.2. Fed-Batch Processes

Fed-batch process can decrease the disadvantages of batch fermentation. In this

process, fresh sterile medium is added to a reactor continuously, while fermentation broth

is either removed semi-continuously or not removed. By addition of medium, two

benefits can be obtained; microbial growth will not be affected due to lack of nutrients

and substrate inhibition will be overcome, if it is a limitation for the process. Ethanol is a

metabolic inhibitor for yeast and a point of concern in fermentation, which will be

eliminated in the use of fed-batch process. However, to enhance productivity and ethanol

yield, optimization of feeding should be done properly (Sanchez and Cardona, 2008.)

Fed-batch culture is the most common technology in the ethanol industry in Brazil

(Sanchez and Cardona, 2008) by application of cell recycling.

Fed-batch fermentation has been studied to overcome lack of nutrients during

ethanol production by S.cerevisiae by Alfenore et al. (2002). Fermentation medium was

fed by vitamins. By addition of vitamins exponentially, ethanol production increased

from 126 g/L to 147 g/L with a maximum productivity of 9.5 g/L/h. Ozmihci and Kargi

(2007) also studied fed-batch ethanol fermentation. However, in this case medium was

30

fed and 63g/L ethanol and 5.3 g/L/ h productivity were obtained with a 125 g/L feed

sugar.

2.6.3.3. Continuous Process

Continuous process, which is also known as chemostat, continuous-flow, or

stirred-tank fermentation, involves fresh sterile media fed into a reactor continuously. In

addition to feeding the reactor with fresh nutrients, the effluent is removed from the

reactor and the volume of the reactor always is constant. The rates of feeding and

removing are also equal. To avoid wash-out, which means taking away all the cells from

the reactor, growth rate of the microorganism is chosen as a rate of removing cells

(Shuler and Kargi, 2008).

Advantages of continuous process over batch fermentation are low construction

costs of bioreactors, lower maintenance and operational requirements, higher yield, and a

better control of the process (Sanchez and Cardona, 2008). Stability of culture, however,

is an issue for continuous fermentation. Even small changes in any of paramaters, such as:

temperature, dilution rate, substrate concentration of feed,etc., can decrease yield.

Different methods could be used to overcome drawbacks of continuous fermentation. For

instance, Sanchez and Cardona (2008) offer utilization of immobilized cell technology

in which cells are captured in the reactor by biofilms, calcium alginate, chrysolite, etc., to

enhance yield.

Continuous simultanoues saccharification and fermentation was studied to

produce ethanol from grains by S. cerevisiae and yielded 2.75 gal/bushel (Madson and

Monceaux, 1995). Kobayashi and Nakamura (2004) studied continuous ethanol

31

fermentation from starch contaning medium by using recombinant S. cerevisiea and

reported 7.2 g/L ethanol and 0.23 g/L/h maximum ethanol productivity at 0.026 h−1

of the

dilution rate in the free cell culture, however, ethanol productivity increased

approximately 1.5 fold 1 in the immobilized cell culture and reached 3.5 g/L/h

productivity at 0.4 h−1

dilution rate. Cells of recombinant S. cerevisiae were

immobilized in calcium alginate and yeast was able to convert starch to ethnaol by

expressing amyloglucosidase.

2.6.4 Simultaneous saccharification and fermentation (SSF)

There is no process which can give the best productivity, yield, and economic

feasibility at the same time. To attain the maximum ethanol yield and reduce the cost and

time, integration of processes is very effective. By integration of processes, several

operations combine and perform at the same unit. Since the pretreatments play a crucial

role in production of ethanol, most of the process involve integrated hydrolysis and

fermentation.

Simultaneous saccharification and fermentation (SSF) is one of the common

processes. SSF found application to ethanol production in the starch-processing industry

in the 1970’s (Madson and Monceaux, 1995). After liquefaction, saccharification and

ethanol fermentation are carried out simultaneously. The benefit of this process is

elimination of substrate inhibition. Because the glucose is transferred into ethanol right

after its conversion from polysaccharides, no accumulation of glucose occurs in the

media. In addition, the hydrolysis reactor is not needed (Cardona and Sanchez, 2007).

The drawback of SSF is that both saccharification and fermentation have different

32

optimal conditionals to obtain maximum yield, and it is diffucult to optimize parameters

for both hydrolysis and fermentation. Optimizing temperature is especially an issue,

because hydrolysis of starch requires a high temperature, whereas high temperature is an

inhibitor for ethanol production. Costs of enzyme is of concern because more enzyme is

needed for a high yield (Cardona and Sanchez, 2007).

Co-fermentation is another promising application to improve ethanol

fermenatation. In this process, two or more microorganisms are inoculated to enhance

the ethanol yield. While one of the microroganisms converts polysaccharides to glucose,

the second microorganism produces the ethanol. Although, this process prevents substrate

and/or metabolite inhibition, growth parameters of microroganisms are a problem.

Temperature, pH range, requirment of oxygen, and agitation need to be optimized for

both of the microorganisms (Cardona and Sanchez, 2007) . Genetically modified

microorganisms are promising to solve these problems. By transferring the genes which

allow yeast to hydrolyze the starch to yeast, one microorganim can be obtained for a

whole process. S. cerevisiae and Z. mobilis are the two microorganism that are mostly

modified for this purpose, however E. coli and K. oxytoca are designed as ethanologic

bacteria (Cardona and Sanchez, 2007).

Simultaneous saccharification, yeast propagation and fermentation (SSYPF) is an

application which is performed in the starch industry to produce ethanol (Cardona and

Sanchez, 2007). In this process, active dry yeast incorporated in the fermentor during

initial saccharification and cell growth is inhibited by lack of glucose because converted

glucose transfers to ethanol rapidly (Cardona and Sanchez, 2007). Madson and

Monceaux (1995) reported that 2.75-2.8 gal/bushel ethanol was yielded by application of

33

continuous SSYPF. In this study, S. cerevisiae and microbial amylases were inoculated

and this technology has been applied in some plants for corn, wheat, and a veriety of

sorghum.

2.7 Potato

The potato is a starchy, tuberous crop vegetable from the perennial Solanum tuberosum

of the Solanaceae family. Potato is composed of 80% moisture and 18% starch Potato is

a high value crop as a food source, and currently, 34% is frozen, 28% is fresh, 12% is

chip, 10% is dehydrated, 15% is potato seed and on farm consumption, and 1% is canned

in the US (NPC, 2008).The U.S. produced about 22 million tones of potatoes, with

approximately 850,500 ton of potatoes produced in Pennsylvania during fall 2007

(USDA, 2007).

Because of the high starch content, potato can be a feedstock for ethanol

production. Srichuwong et al., (2009) states that “ According to available amount of

fermentable sugars (solubale sugars and starch), 1 kg of fresh tuber (potato) would yield

approximately 126 g or 160 ml of ethanol, if complete conversion of fermentable sugars

to ethanol was accomplised.”. Although potato is a high-value product, the cost of bio-

ethanol production can be decreased by using wastes from the potato industry. Waste

from processing of potatoes, an agro-industrial residue, can be an important feedstock for

ethanol fermentation (Abouzied and Reddy, 1986). Four to five million liters of ethanol

could be produced from 44,000 ton of processing waste potato according to a study in

Canada’s potato-growing province of New Brunswick ( IYP, 2008).

34

Five to twenty percent of crops that are waste potato by-products from potato

cultivation could be utilized for bio-ethanol production (Liimatainen et al., 2004). In the

potato chips industry, 18% of company production is starch waste of potato which could

be a raw material for fermentative alcohol production (Fadel, 2000). According to the

study of Oda et al., (2002) 10% waste potato pulp is produced during the process by

which approximately one million harvested potatoes are processed.

Currently, waste potato is utilized as animal feed after a drying process, which is

an energy demanding process. Without a drying process, waste potato could be used in

the production of ethanol. Waste potato is a promising feedstock for ethanol fermentation

and can provide growth of yeast. 73% available carbohydrate, vitamins (A,D,K,C, foliate,

niacin, riboflavin, and thiamin) and minerals such as sodium, potassium, magnesium,

calcium, zinc, and iodine are included in potato flakes (FFCD, 2008).

2.7.1 Fermentation of Waste Potato

Utilization of waste potato for high yield ethanol production is the main purpose

of the fermentation of potato medium. Because potato is a starchy material the

fermentation process of potato mash starts with hydrolysis. Fadel (2000) used acid

hydrolysis (application of 0.5 N H2SO4) at 121 ºC) in his research of alcohol production

from potato industry starchy waste. However, inhibitory affects on yeast growth of by-

products of acid hydrolysis is reported by Tasic et al., (2009). In their study,

neutralization of hydrolyzates and expensive constructional equipment are also indicated

as disadvantages of acid hydrolysis in industry. Liimatainen et al. (2004) applied an

35

enzyme to carry out the liquefaction and sachharification steps of hydrolysis. After

hydrolyzation of the starch, fermentation can be performed to produce ethanol.

For fermentation, fed-batch, continuous or semi-continuous processes can be

chosen. Ethanol is collected in the medium in the fed-batch culture, and fed-batch allows

reuse of culture (Sanchez and Cardona, 2008). Continuous processes are more cost

effective. In addition, Sanchez and Cardona (2008) indicate other advantages of a

continuous process over a conventional batch process. These advantages include: lower

cost of the bioreactors, higher productivities, better process control, and lower

maintenance and operation requirements. For more effective ethanol fermentation from

waste potato, two or more fermentation processes can be integrated. Simultaneous

saccharification and fermentation of very high gravity potato mash has already been

investigated by Srichuwong et al. (2009) who reported that 16.61% yield of ethanol was

obtained. However, in this study one of the drawbacks was the viscosity of the potato

mash.

Literature for alcohol production from industrial potato waste has been reported

by Fadel (2000), who carried out alcohol fermentation using various S. cerevisiae strains

and reported effects of initial pH value, nitrogen source and level, inoculum size, and

agitation. According to the study of Fadel (2000), the maximum alcohol production

(9.42%) was at pH 5.5, whereas inoculation of 10% culture of S. cerevisiae yielded

12.9% riches of alcohol in fermentation broth. To determine the effect of the nitrogen

source, six different inorganic nitrogen sources (diammonium phosphate, ammonium

hydrogen phosphate, ammonium sulfate, diammonium phosphate:urea (1:2),

urea+KH2PO4, and urea+H3PO4) were studied and it was reported that diammonium

36

phosphate resulted in higher ethanol production (9.42% riches of alcohol) among studied

nitrogen sources. It was also found that ammonium sulfate yielded the lowest riches of

alcohol (8.30%). However, overall results revealed that different nitrogen sources are

suitable for ethanol fermentation.

Liimatainen et al. (2004) studied the effect of potato cultivar. Liimatainen et al.

(2000) reports that “5-20% of potato crops are by-products in potato cultivation.”.

Properties of ten different waste potato cultivars and ethanol yield of these different

potato cultivars were compared after fermentations and the effect of potato cultivar was

reported (Liimatainen et al., 2004). Their study showed that ethanol yield varies among

cultivars. The highest ethanol yield among ten different cultivars was 9.5 g ethanol/100

g potato and the lowest one was 6.5 g ethanol/100 g potato.

By-products (potato peel and substandard mash) of a local plant in Japan were

studied for ethanol production by Yamada et al. (2009). In this study after liquefaction

and partial saccharification, hydrolysis of sugars by glucoamylase and fermentation

were carried out simultaneously. A reasonable conversion of sugars to ethanol was

obtained with a value of 42.5%. Fresh potato peel, 12% (w/w) yielded 20 g/L ethanol,

however, a mixture of substandard mash and potato peel (1:1) yielded 50 g/L ethanol.

Even though waste potato is considered as a medium for ethanol production,

addition of the nitrogen source is still required to attain high ethanol and biomass yield.

Graf-Sirakaya (2004) studied the effect of nitrogen sources for ethanol fermentation

with S. cerevisiae in a chemically defined media to evaluate seven different animal by-

products as a nitrogen source instead of yeast extract. For this study, yeast extract

compared with poultry meal, hulls and fines mix, beef stock, blood meal, feather meal,

37

pork stock and meat and bone meal (Graf –Sirakaya, 2004). Five of seven animal by-

products could be alternative nitrogen sources for ethanol production instead of yeast

extract although all had lower production rate than yeast extract which yielded 44.66

g/L ethanol. Among seven nitrogen sources studied: poultry meal, feather meal, pork

stock, meat and bone meal, and hulls and fine mix, the yields were: 40.88 g/L, 42.16

g/L, 43.26 g/L, 43.06 g/L, and 37.14 g/L ethanol, respectively. To evaluate the various

nitrogen sources, ethanol fermentation using carob (a Mediterranean fruit) extract by S.

cerevisiae was performed by Turhan et al. (2008). It was reported that meat and bone

meal, feather meal, poultry meal, and hulls and fines mix were investigated as an

alternative to yeast extract. Although, maximum production was determined with yeast

extract addition (42.9 g/L), the other four nitrogen sources were still capable to be

alternatives to yeast extract and yielded ethanol in a range of 39.88 – 41.90 g/L (Turhan

et al., 2008).

2.8 State-of-the-Art

Bio-ethanol, one of the renewable energy sources to reduce carbon dioxide

emissions and dependency on foreign oil, has already been used in pure and/or blended

gasoline. About 51.3 billion liters of ethanol was produced worldwide in 2006 and it is

probable that the production of ethanol will increase in the future.

A variety of raw material is used for ethanol production however all of these

materials have advantages and disadvantages. Corn is the major source for ethanol

production in the U.S. (97%), but this production of crops causes more soil erosion and

requires more nitrogen fertilizer than other crops. In Brazil, sugar cane production also

38

has the same environmental limitations (Balat et al., 2008). Cellulosic feedstock needs

expensive pretreatments to be effective for ethanol production. Because of the cost of

ethanol, inexpensive raw material is important to produce cheap ethanol (Sanchez and

Cardona, 2008). The main goals of most of the studies in bio-ethanol production are to

produce cheap and competitive ethanol compared to gasoline (Sanchez and Cardona,

2008). Because waste potato is neither a food source nor requires expensive

pretreatments compared to lignocellulosic raw materials, the waste of the potato industry

could be utilized as a raw material for the ethanol industry to produce cheap and efficient

bio-ethanol. However, the literature does not provide enough information about

utilization of waste potato mash of the potato industry since many types of waste exist for

the potato industry. Limatainen et al. carried out a mashing process before enzyme

hydrolysis, whereas Fadel (2000) used just acid hydrolysis as a pretreatment. Oda et al.

(2009) reported the ethanol production of a mix of potato peel and substandard mash. In

the present study, waste potato mash was evaluated for ethanol production to decrease the

total cost of ethanol by avoiding pretreatment of dry potato waste. Waste potato mash

was utilized as a fermentation medium after enzyme hydrolysis without any other

pretreatment application.

Ethanol production from waste potato mash would decrease the cost of ethanol

production and could offer an alternative to animal feed which is the current use

involving a costly drying process. Therefore, the aim of this study is to produce ethanol

from waste potato mash while decreasing fermentation costs. Properties of waste potato

mash, effect of pH for a high ethanol yield, effect of inoculum size for high ethanol and

biomass yield, effect of nitrogen sources to obtain competitive ethanol yield to yeast

39

extract by using cheap nitrogen sources was studied to determine advantages and

feasibility of waste potato mash fermentation to produce ethanol.

40

CHAPTER III

GOAL and OBJECTIVES

The energy demand of the world has been increasing due to an increasing

population. Bioenergy is an alternative renewable energy. The production of ethanol, a

type of bioenergy, has already occurred all over the world. However, the conventional

raw materials are high value products, such as corn, wheat, cellulose, potato, and

sugarcane. Feedstock, which have already found, use as food. Low value or waste by-

products should be utilized for ethanol production to meet the energy demands. Therefore,

the main goal of this research is to utilize potato mash waste as a carbon source for

Saccharomyces cerevisiae for the fermentation of ethanol.

3.1 Goal

The main goal of this research is to produce ethanol from waste potato mash by

evaluating starch hydrolysis parameters and fermentation conditions such as pH, inoculum

sizes, and four different nitrogen sources.

3.2 Objectives

1. Determine the hydrolysis parameters of waste potato mash to obtain fermentable sugars

from starchy materials.

2. Select the pH in a bench-scale bioreactor to improve ethanol production from waste

potato mash.

3. Optimize the ethanol production in terms of inoculum size.

41

4. Determine the effect of alternative nitrogen sources to obtain high ethanol yield from

potato mash waste fermentation.

42

CHAPTER IV

MATERIALS and METHODS

The purpose of this section is to describe the materials used, experimental designs,

and the procedure of analysis to optimize ethanol production from waste potato mash

by Saccharomyces cerevisiae.

4.1. Microorganism

Saccharomyces cerevisiae (ATCC 36858) was obtained from the American Type

Culture Collection (Manassas, VA). For inoculum preparation, S. cerevisiae was grown

in medium composed of 20 g/L of glucose (Domino Sugar, Domino Foods Inc, Yankers,

NY), 6 g/L of yeast extract (Difco, MD, USA), 0.3 g/L of CaCl2.2H2O, 4 g/L of

(NH4)2SO2, 1 g/L of MgSO4.7H2O, and 1.5 g/L of KH2PO4 at 30°C for 24 h. In order to

maintain viability, the culture was stored at 4°C and sub-cultured biweekly, whereas

stock cultures were kept in 20% glycerol at -80°C for long-term storage.

4.2. Waste Potato Mash

Waste potato mash was obtained from Keystone Potato Products (Hegins, PA),

which produces potato flakes. There were a variety of potatoes used throughout the study,

including Frito-lay FL 1833, Atlantis, and Russet Burbank. After determining moisture

content, waste potato mash was stored in the freezer until use.

43

4.3 Baseline Fermentation Medium

The base-line fermentation medium used in this study as control contained 50

g of glucose (Domino Sugar, Domino Foods Inc, Yankers, NY), 6 g of yeast extract

(Difco, MD, USA), 0.3 g of CaCl2.2H2O, 4 g of (NH4)2SO2, 1 g of MgSO4.7H2O, and

1.5 g of KH2PO4 per liter of deionized water (Demirci et al., 1997). For ethanol

fermentation with hydrolyzed waste potato mash, the potato mash was used as the

base medium supplemented with all of the other ingredients of baseline fermentation

medium except glucose (glucose was replaced with hydrolyzed starch as the carbon

source).

4.4 Determination of the Hydrolysis Parameters of Waste Potato Mash (Objective 1)

Before performing hydrolysis prior to fermentation, acid hydrolysis was

compared to enzyme hydrolysis to determine the best method for maximum glucose

yield.

4.4.1 Acid Hydrolysis

Four different concentrations of waste potato mash were prepared in deionized

water as 5, 10, 15, and 20% (w/v). Since the pHs of mixtures were in a range of

3.8 – 4.2, no acidification was required. Prepared mixtures were then autoclaved

at 121°C, 21 psi for 60 min. Finally, the samples were cooled down and analyzed for

glucose concentration.

44

4.4.2 Enzyme Hydrolysis

α-Amylase (EC 3.2.1.1) for liquefaction and amyloglucosidase (EC 3.2.1.3) for

saccharification were used. These enzymes were manufactured by Novozyme

Corporation and distributed by Sigma-Aldrich (Saint Louis, MO). Their activity or

concentration was reported as 18.8 mg protein/ml for α-amylase and 300 Unit/ml for

amyloglucosidase by the manufacturer.

To minimize the number of trials while gathering useful information, Box-

Behnken response surface method (RSM) was used to determine optimum combinations

of temperature, enzyme concentration and solid waste potato content for liquefaction.

Temperature, enzyme concentration, and time were the evaluated parameters for the

saccharification step. According to full factorial design, the number of data points

required was 27 (3 factors x 3 levels x 3 replicates). However, Box-Behnken RSM

reduced the numbers of data points to 15 (12 edges for 3 factors with 3 levels and 3

replicates for center point) with useful data still generated (Box and Behnken, 1960).

Minitab Software (version 13.3; Minitab Inc., State College, PA) was used to design

RSM tables and to evaluate the data.

For liquefaction, the pH of the slurry was adjusted to 6.5±0.3 by 1 N NaOH, and

α-amylase solution ranged from 0.2 to 1 ml (Table 4.1). The mixture was agitated at 120

rpm agitation in a shaker water bath for 3 hours at 50, 72.5 or 95ºC. Also, based on RSM

design, 0.2, 0.6, and 1 ml of α-amylase, and 1, 5.5, and 10 g dry weight of waste potato

mash / 100 ml of DI water in 250 ml flasks were evaluated to determine the optimum

combination. Initial and final samples were taken and analyzed for residual sugar

45

contents and non-dissolved solid in the mixture to determine optimum condition to yield

the maximum loss in dry weight and non-dissolved solid ratio.

Table 4.1 Box-Behnken Surface response method analysis design for liquefaction.

Temperature (°C) Enzyme (ml)* Dry Weight (g WPM/100 ml DIW)

50.0 0.2 5.5

95.0 0.6 1.0

95.0 0.6 10.0

72.5 0.2 10.0

72.5 1.0 1.0

72.5 0.6 5.5

72.5 0.2 1.0

95.0 1.0 5.5

50.0 1.0 5.5

50.0 0.6 1.0

72.5 0.6 5.5

95.0 0.2 5.5

72.5 1.0 10.0

50.0 0.6 10.0

72.5 0.6 5.5

*α-amylase: 18.8 mg protein/ml

After the liquefaction step was optimized, the sachharification step was carried

out. For saccharification, amyloglucosidase solution ranged 0.2 – 1.0 ml, temperature

ranged 30-60ºC and incubation time ranged 24-72 h. Based on these ranges, the Box-

Behnken RSM design was obtained (Table 4.2) and samples were analyzed for glucose

46

concentration to determine optimum temperature, time, and enzyme concentration

combination.

Table 4.2 Box-Behnken Surface response method analysis design for sachharification.

* Amyloglucosidase: 300 Unit/ml

After the results of acid and enzyme hydrolysis were compared to determine

maximum yield of glucose conversion, hydrolysis of starch was selected for the

remaining of study. However, the glucose concentration was still not high enough for

typical ethanol fermentation; a further procedure was applied to increase the amount of

Temp (°C) Enzyme (ml)* Time (h)

60 0.6 24

60 1.0 48

60 0.2 48

60 0.6 72

45 0.2 24

45 1.0 72

45 1.0 24

45 0.6 48

45 0.6 48

45 0.6 48

45 0.2 72

30 1.0 48

30 0.2 48

30 0.6 72

30 0.6 24

47

converted glucose by increasing amount of dry weight of waste potato mash and enzyme

concentration with the constant ratio. To obtain the desired level of glucose, the

relationship between glucose concentration and the amount of waste potato mash, and

glucose concentration and the amount of enzyme were investigated.

Firstly, the amount of waste potato mash was held constant and amounts of

enzymes were increased to determine the maximum glucose that could be converted

(Table 4.3). Because the increase in enzyme concentration alone did not provide the

desired glucose level, the concentration of waste potato mash was also increased with a

same ratio (Table 4.4). After the desired glucose concentration was obtained, the

hydrolysis of fermentation medium was performed directly in the bioreactor vessel.

Table 4.3 Increasing of enzyme concentration with a constant amount of waste potato

mash.

.

Dry-weight (g/100ml)

WPM (g/100ml)

α-amylase (ml)

Amyloglucosidase (ml)

4.04 16.8 1 0.8

4.04 16.8 4 1.6

4.04 16.8 6 3.2

4.04 16.8 8 4.8

48

4.5 Ethanol Fermentation in Potato Mash Hydrolyzed Medium and pH Evaluation

(Objective 2)

Sartorious Biostat B Plus Bioreactors (Allentown, PA) with 2.5 L vessel (working

volume of 1.5 L) equipped with pH, temperature, and agitation controls were used

(Figure 4.1). Prior to fermentation, liquefaction and saccharification processes were

performed in the bioreactor vessel. Because liquefaction requires a high temperature

(95°C), liquefaction was carried out by autoclaving at the liquefaction temperature. After

slurry preparation, autoclaving was performed at about 95°C for 3 h. Saccharification

followed liquefaction and was carried out after the vessel was connected to the bioreactor

for 72 h at 60°C. Because of the high viscosity of slurry, agitation was maintained at 400

rpm in the bioreactor during saccharification and fermentation. During fermentation,

temperature was maintained at 30°C. Inoculum was grown for 24 h at 30ºC. To evaluate

the pH effect, fermentation at pH 5.5 and uncontrolled pH were performed. pH was kept

constant by adding sterilized 4 N NaOH or H2SO4 at controlled pH 5.5. After inoculation,

48 h fermentation was carried out and samples were taken every one or two hours for the

first 12 hours of fermentation; whereas, every 6 h during the remaining fermentation time.

Samples were analyzed for ethanol and glucose concentrations and cell populations.

49

Figure 4.1 Sartorious Biostat B plus bioreactors.

4.6 Inoculum Size Determination (Objective 3)

Based on the pH evaluation study, pH controlled at 5.5 was selected for the rest of

the fermentations. Temperature and agitation were maintained at 30°C and 400 rpm,

respectively, in the bioreactor vessel as described earlier. Inoculum was grown for 24 h at

30ºC. Three levels of inoculum sizes (1, 3, and 5% (v/v)) were evaluated. After

inoculation, 48 h fermentation was carried out and samples were taken every one or two

hours for the first 12 h of fermentation whereas every 6 h during the remaining

fermentation time. Samples were analyzed for ethanol and glucose concentrations and

cell populations.

50

4.7 Evaluation of Alternative Nitrogen Sources (Objective 4)

Liquefaction and saccharification were carried out in the bioreactor vessel before

fermentation as described earlier. Based on inoculum size study, 3% inoculum size was

selected for the remaining fermentations. Temperature, pH, and agitation were

maintained at 30°C, 5.5, and 400 rpm, respectively. Inoculum was grown for 24 h at 30ºC

and 3% inoculum was used. Poultry meal, hull and fines mix, feather meal, and meat and

bone meal obtained from Griffin Industries, Inc. (Butler, KY) as dry powders were used

as alternative nitrogen sources to replace yeast extract in the medium. After inoculation,

48 h fermentation was carried out and samples were taken every one or two hours during

the first 12 hours of fermentation, and every 6 h during the remaining fermentation time.

Samples were analyzed for ethanol and glucose concentrations and cell population.

4.8 Analysis

4.8.1 Moisture Analysis

To determine moisture of waste potato mash, samples were weighed and the

drying process was carried out at 105ºC in an oven for 48 h until the weight of samples

stabilized.

4.8.2 Non-Dissolved Solid Analysis

The liquefaction slurry was centrifuged at 4,000 rpm for 30 min. After the

supernatant was discarded, distilled water was used to wash the pellet (particles of

centrifuged slurry) and the washing process was repeated two times. Then the mixture

51

was dried in an oven at 105ºC. The pellet was weighed and the percentage was

calculated with respect to initial slurry as the non-dissolved solid.

4.8.3 Cell Population

The spiral plating method was used to determine cell population by using a spiral

auto-plater (Model 4000, Spiral Biotech, Norwood, MA) and Q-count software (Version

2.1, Spiral Biotech, Norwood, MA). Collected samples were serially diluted 0.1%

peptone water and spiral plated on potato dextrose agar (Difco, MD, USA). After plating,

24 h incubation was carried out at 30°C. Q-count software (Version 2.1, Spiral Biotech,

Norwood, MA) was used for enumeration. Results were indicated as log10 CFU/ ml.

4.8.4 Ethanol and Glucose

Samples were analyzed for glucose and ethanol by using YSI 2700 Analyzer

(Yellow Springs, OH). YSI 2700 system included specific membranes for each of the

components as well as system buffers and calibration solutions. The procedure for

ethanol and glucose was as follows: 1 ml of samples was diluted 20 fold to hold the

concentration of either ethanol or glucose in the range provided by manufacturer, then

analyzed by YSI 2700.

4.8.5 Statistical Analysis

Statistical analysis was conducted to test significant differences between each

treatment. In particular, the two-sample t test, ANOVA, and Dunnett tests were used to

test for significant differences between the mean of production rate and growth rate

values of each treatment. Since controlled and uncontrolled ph were compared, the two

52

sample t test was chosen, whereas ANOVA was chosen for inoculum sizes and nitrogen

sources due to number of parameters. The Dunnett test was used to determine whether

any of nitrogen sources yields statistically different than yeast extract. The level of

significance was set at 0.05. Statistical analysis was performed using statistical software

Minitab (State College, PA).

53

CHAPTER V

RESULTS and DISCUSSION

Ethanol was produced by Saccharomyces cerevisiae from waste potato mash.

This study was designed to evaluate waste potato mash as a medium for ethanol

production, but the hydrolysis of waste potato mash and fermentation parameters were

investigated as well. Acid hydrolysis was compared to enzyme hydrolysis to obtain

maximum glucose conversion, whereas effect of pH, inoculum sizes and nitrogen sources

were evaluated to optimize fermentation of ethanol. Because few studies where

conducted on waste potato mash (Chapter 2), this study provides information about the

fermentation conditions of ethanol from waste potato mash by S. cerevisiae.

5.1 Hydrolysis of Waste Potato Mash

Hydrolysis is a process of breaking down amylopectin and amylose linkages into

fermentable sugars and is needed before the fermentation of starchy materials. Because

potato is a starchy material, potato mash needs to be hydrolyzed before fermentation.

Acid hydrolysis and enzyme hydrolysis were evaluated and compared in production of

maximum glucose concentration, which can be utilized later for ethanol fermentation.

54

5.1.1 Acid Hydrolysis

Altough pH of potato solutions was adjusted at pH 5 in the study reported by

Fadel (2000), no acid adjustment was needed in this study, because the waste potato mash

(WPM) mixtures were already at acidic pH, in a range of 3.8 – 4.2. After one hour of

autoclaving at 121°C, glucose concentrations were measured as 0.011 g/L, 0.016g/L,

0.021 g/L, and 0.029 g/L glucose generated from 5, 10, 15, and 20% (w/v) mixtures,

respectively (Figure 5.1). Even though a linear increase occurred, the highest glucose

concentration was still not able to provide enough glucose for fermentation. Increasing

the amount of waste potato mash was not desirable due to an increase in viscosity of

slurry which made the agitation more diffucult (i.e., more energy intensive). Furthermore,

4.04 g dry weight of WPM/100 ml deionized water (DIW) mixture was determined to be

the best combination for enzyme hydrolysis (see section 5.1.2), which was evaluated also

for acid hydrolysis. For this case, 16.8 g WPM was dissolved by 100 ml of deionized

water, and evaulated for glucose concentration after acid hydrolysis. The average of three

replications was 0.0286 g/L glucose which is a low concentration for any fermentation.

55

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0% 5% 10% 15% 20% 25%

% of Solutions (w/v)

Glu

co

se C

on

cen

trati

on

s (

g/L

)

Initial Glucose Conc. (g/L) Final Glucose Conc. (g/L)

Figure 5.1 Glucose release from waste potato mash as a result of acid hydrolysis

at 121°C for 1 h.

Fadel (2000) reported that in the presence of 25% (w/v) sugar in the fermentation

medium, the maximum ethanol yield was with a 90% conversion of sugar. In this study,

the glucose concentration obtained by acid hydrolysis was very low for ethanol

fermentation. Also, it was reported that the success of acid hydrolysis depends on

concentration and type of acid and ratio of potato to acid solution (Tasic et al., 2009).

They also cocluded that the highest dextrose equivalent of 94% was achived by using 1M

HCl at the ratio of plant material to acid solution of 1:2 (w/v). However, in this study no

acid was added to the waste potato mash mixtures due to the fact that waste potato

mixtures were already at acidic pH. However, other chemicals, such as 5-

hydroxymethylfurfural (5-HMF), inhibits ethanol fermentation when acid hydrolysis

applies and 5-HMF should be decreased (Tasic et al., 2009). In their study, no research

56

was carried out to investigate whether 5-HMF was present or not in the waste potato

mash mixtures.

5.1.2 Enzyme Hydrolysis

Box-Behnken response surface design was created to determine optimum

combinations of temperature, enzyme concentration, and dry waste potato mash

concentrations for liquefaction. Results of liquefaction were summarized in terms of %

loss (Table 5.1) because liquefaction is a process which is not a complete conversion of

starch to glucose. Liquefaction is just a partial breakdown of starch and makes insoluble

components of starch soluble. The lowest loss in non-dissolved solids of waste potato

mash was observed at the combination of 50°C, 0.6 ml of α-amylase, and 10 g dry waste

potato mash (43.7% loss). At this combination, the temperature was lower than the

optimum working temperature of α-amylase which might decrease loss in non-dissolved

solids. Moreover, the amount of enzyme might not be enough for 10 g dry waste potato

mash. The maximum loss in non-dissolved solids of waste potato mash was observed at

the combination of 95°C, 0.6 ml of α-amylase, and 1 g dry waste potato mash (79% loss).

In this case, the temperature was high enough to allow α-amylase work however the

amount of enzyme was high for 1 g of dry waste potato mash. When 95°C 0.6 ml of

enzyme1 g dry waste potato mash combination compared to 50°C 0.6 ml of enzyme1 g

dry waste potato mash combination, it was shown that at 95°C temperature, % loss in

non-dissolved solids of waste potato mash obtains, 79% and 61.8% loss, respectively.

57

Table 5.1 Box-Behnken design and results of liquefaction.

Temp (°C)

Enzyme (ml)

Dry Weight (g WPM/100 ml DIW) % loss in Dry Solid

50.0 0.2 5.5 52.1

95.0 0.6 1.0 79.0

95.0 0.6 10.0 52.1

72.5 0.2 10.0 58.1

72.5 1.0 1.0 57.7

72.5 0.6 5.5 60.1

72.5 0.2 1.0 72.4

95.0 1.0 5.5 65.1

50.0 1.0 5.5 50.0

50.0 0.6 1.0 61.8

72.5 0.6 5.5 60.1

95.0 0.2 5.5 65.6

72.5 1.0 10.0 54.4

50.0 0.6 10.0 43.7

72.5 0.6 5.5 52.3

Also, when %loss of 95°C 0.6 ml of enzyme 1 g dry waste potato mash combination and

95°C 0.6 ml of enzyme 10 g dry waste potato mash combination are compared (79 and

52.1%, respectively), the importance of optimum enzyme for substrate can be seen.

By using the measured values of the Box-Behnken design, the Box-Behnken

optimizer was used to determine optimum conditions of liquefaction, the optimum

parameter combination determined was: 95°C, 1 ml of α-amylase, and 4.04 g dry weight

of waste potato mash per 100 ml of deionized water, which yielded 68.8% loss in dry

weight. Verification for this optimum combination was performed with three replications

58

and 74% of loss in non-dissolved components was obtained, which is very close to the

estimated value by the optimizer.

Figure 5.2 shows the surface plots of liquefaction. In this case, effects of enzyme

concentration, dry waste potato mash, and temperature vs % loss were plotted when one

of the factors was held at the center point. From these figures, it can be seen that higher

dry waste potato mash require higher levels of enzymes. However, higher temperature

enhanced % solid loss for dry waste potato mash when enzyme was held constant at 0.6

ml. When dry waste potato mash was kept at 5.5 g/100 ml DI water, higher temperature

(around 95°C) and lower enzyme concentration (around 0.2 ml) resulted higher % loss in

dry waste potato mash. Increasing the temperature (more than 95°C) might have resulted

in higher % loss, but then the enzyme would not be active.

59

Figure 5.2 Surface plots of liquefaction of waste potato mash.

60

After liquefaction was optimized, the sachharification process was carried out.

Following liquefaction, waste potato mash slurries were treated with amyloglucosidase

solution (0.2 - 1 ml) incubated at 30, 45, and 60ºC in a shaker water bath at 120 rpm for

24- 72 h based on the design of Box-Behnken Response Surface Design and to determine

optimum temperature, time, and enzyme concentration combination for the maximum

glucose yield. Results of saccharification were summarized in terms of glucose

concentration (Table 5.2), because saccharification is a complete conversion of starch to

glucose. Minimum glucose (19.2 g/L) was obtained at 30ºC, 0.2 ml of amyloglucosidase,

and 48 h combination. Maximum glucose concentration was obtained at 60ºC, 72 h and

0.6 ml of amyloglucosidase with 30.7 g/L glucose concentration.

Table 5.2 Box-Behnken design and results of saccharification.

Temp (°C)

Enzyme (ml)

Time (h)

Glucose (g/L)

60 0.6 24 21.1

60 1.0 48 28.2

60 0.2 48 27.3

60 0.6 72 30.7

45 0.2 24 20.6

45 1.0 72 20.7

45 1.0 24 21.7

45 0.6 48 24.5

45 0.6 48 22.4

45 0.6 48 22.0

45 0.2 72 26.0

30 1.0 48 24.0

30 0.2 48 19.2

30 0.6 72 20.7

30 0.6 24 22.7

61

Figure 5.3 shows the surface plots of saccharification. In this case, effects of

enzyme concentration, time, and temperature vs. glucose conversion were plotted when

one of the factors was held at the center point. When temperature was kept at 45°C, the

highest glucose conversion was obtained at a low enzyme- high temperature combination.

However, when enzyme was held at 0.6 ml, the highest glucose conversion occurred at

the high temperature (60°C) -long time (72 h) combination. In the case of constant time

(48 h), high temperature resulted in higher glucose conversion regardless of enzyme

concentration but the glucose was not the maximum concentration obtained. Although

longer saccharification might result in higher glucose conversion, this was not feasible

due to energy costs.

62

Figure 5.3 Surface plots of saccharification of waste potato mash.

63

Similar to liquefaction, Box-Behnken optimizer was used to determine optimum

conditions of saccharification. The optimum parameter combination obtained was 60°C,

0.8 ml of amyloglucosidase, and 72 h. Verification for this optimum combination was

performed with three replications and 34.9 g/L glucose was obtained, which is very close

to the estimated value by the optimizer.

Although a higher glucose concentration (34.9 g/L) was generated by enzyme

treatment, glucose concentration was still far from the desired level for typical ethanol

fermentation (about 100 g/L). To reach the ideal high level, the linear relationship

between glucose concentration and amount of waste potato mash and amount of enzyme

was investigated. First, the amount of waste potato mash was held constant and amounts

of enzymes were increased (Table 5.3). The glucose yield was observed to investigate the

maximum obtainable glucose. A linear relationship occurred between the amount of

enzymes and glucose concentration observed with 0.9478 R-sqr and 79.4 g/L as the final

glucose concentration obtained (Figure 5.4) (still less than ideal glucose concentration).

Table 5.3 Relationship between enzyme concentration and glucose

Dry WPM (g/100ml)

WPM (g/100ml)

α-amylase (ml)

Amyloglucosidase (ml)

Glucose (g/L)

4.04a 16.8 1 0.8 37.1

4.04 16.8 4 1.6 61.9

4.04 16.8 6 3.2 64.2

4.04 16.8 8 4.8 79.4

a) First row is the suggested optimum combination from Box-Behnken RSM.

64

Enzyme - Glucose Correlation

R2 = 0.9478

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8 9

Enzyme (ml)

Glu

cose (g/L

Glucose (g/L)

Figure 5.4 Correlation of enzyme and generated glucose with enzyme levels listed in

Table 5.3.

Second, the amounts of waste potato mash and enzyme were increased

simultaneously. The reason behind increasing only the amount of enzymes and waste

potato mash while holding time and temperature constant is because the enzyme and

amount of waste potato mash have a significant effect on glucose conversion yield from

the results of Box-Behnken RSM. To enhance the conversion yield, the amount of

enzyme and waste potato mash were increased together and its effect evaluated (Table

5.4).

65

Table 5.4 Enhancement of glucose concentration

Dry-weight (g/100ml)

WPM (g/100ml)

α-amylase (ml)

Amyloglucosidase (ml)

Glucose (g/L)

4.04a

16.8 1 0.8 37.1

8.08 33.6 2 1.6 54.9

16.16 67.2 4 3.2 93.5

24.24 100.8 6 4.8 137

a) First row is the suggested optimum combination from Box-Behnken RSM.

As seen in Figure 5.5, there is a linear regression between glucose concentration

and amounts of enzyme and dry weight of waste potato mash with a 0.9985 R-sqr. Based

on the average of three replications, 16.16 g dry weight/ 100 ml DIW is the best choice

with a 93.5 g/L glucose yield at the end of saccharification in addition the viscosity of the

mixture allowed for a reasonable agitation rate.

Because the variety of potato which was processed by the supplier was changed

from Fritolay 1833 to Atlantis-Russet Burbank mix, further adjustments were needed for

enzyme hydrolysis before ethanol fermentation was carried out. This would be the case in

the real-world, because plants process various types of potatoes. Therefore, this variation

needs to be a factor in the equation. For this purpose, the dry weight of a new potato

variety, a mix of 50% Atlantis and 50% Russet Burbank, was measured as 19.5 %.

Atlantis and Russet Burbank variety was evaluated by treatment of optimum combination

of dry potato mash and amount of enzyme (16.16 g dry weight/100 ml- 4 ml α-amylase-

3.2 amyloglucosidase) and a slightly lower glucose yield was observed. Even though the

66

initial glucose was lower, due to viscosity problems, the amount of waste potato mash

was kept at 16.16 % dry weight based and the rest of the study was conducted on this

amount.

R2 = 0.9985

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30

Dry Weight (g/100ml)

Glu

co

se

(g

/L)

Glucose (g/L)

Figure 5.5 Relationship among enzyme, dry waste potato mash, and generated glucose

with enzyme levels listed in Table 5.4.

5.2 Ethanol Fermentation

Using baseline medium, ethanol fermentation was evaluated. Temperature and

agitation were maintained at 30°C and 100 rpm, respectively. Inoculum was grown for 24

h at 30ºC. Inoculum size was 1% and pH was held at 5.5. After inoculation, 48 h

fermentation was carried out. Maximum ethanol produced was 55.55 g/L as an average of

two runs. Growth rate was 0.57 g/ml/h, whereas, production rate was 5.81 g/L/h.

67

Baseline Ethanol Fermentation

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40 45 50

Time (h)

Glu

co

se (

g/L

);

Eth

an

ol(

g/L

)

0

1

2

3

4

5

6

7

Bio

mass (

g d

ry w

eig

ht/

L)

Glucose (g/L) Ethanol (g/L) Biomass (g dry weight/L)

Figure 5.6 Baseline ethanol fermentation.

5.2.1. Ethanol Production in Hydrolyzed Waste Potato Mash Media and Effect of

pH

Ethanol fermentation was evaluated at two different pH profiles to determine the

effect of pH: uncontrolled pH and controlled pH at 5.5. Figure 5.7 shows the cell

population and concentrations of glucose and ethanol in the fermentation broth with

controlled pH at 5.5 and uncontrolled pH. For controlled pH, glucose concentration of the

fermentation medium began to increase during the first few hours of fermentation instead

of reduction, which might be because saccharification was still in progress (Figure 5.7A).

This increase of glucose enhances ethanol yield and growth of biomass. Glucose

conversion was continuing simultaneously during the fermentation, and after 24 h of

fermentation, glucose was completely consumed in both cases. After consumption of

glucose, both production of ethanol and growth of biomass slowed as expected.

68

The results clearly indicate that a higher growth rate for biomass was obtained

with the controlled pH at 5.5 (0.496 CFU/ml/h) than uncontrolled pH (0.289 CFU/ml/h)

(Table 5.5). Furthermore, the maximum ethanol concentration and production rates were

27.7 g/L and 5.47 g/L/h, respectively at controlled pH 5.5, whereas 22.75 g/L and 2.22

g/L/h were obtained at uncontrolled pH. Yields of the fermentations (product produced

/substrate consumed) were 46.23% and 44.55% for controlled and uncontrolled pH

profiles, respectively. However, it was diffucult to determine the exact amount of

consumed glucose because saccharification is still going on at the beginning of

fermentation. Therefore, determination of optimum pH was dependent on growth rate,

production rate, and ethanol concentrations, which indicated that controlled pH at 5.5 is

better for ethanol fermentation. However, there is no statistically significant difference in

the growth rate and production rates means (p-value>0.025)

69

Controlled pH at 5.5

(A)

0

10

20

30

40

50

60

0 10 20 30 40 50

Time (h)

Glu

co

se a

nd

Eth

an

ol

(g/L

)

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

Cell P

op

ula

tio

n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

Uncontrolled pH

(B)

0

10

20

30

40

50

60

0 10 20 30 40 50 60

Time (h)

Glu

co

se

an

d E

tha

no

l

(g/L

)

6.0

6.5

7.0

7.5

8.0

8.5

9.0

Cell P

op

ula

tio

n

(lo

gC

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

Figure 5.7 Glucose and ethanol, and cell population at pH 5.5 (A) and

uncontrolled pH (B).

70

Table 5.5 Comparison of kinetic parameters of controlled pH vs. uncontrolled pH.

Kinetic Parameters pH controlled pH Uncontrolled.

Max. Ethanol Produced (g/l) 27.05 22.75

Max. Glucose Consumed (g/l) 58.5 50.5

Growth Rate (log cfu/ml/h) 0.50 0.29

Production Rate (g/l/h) 5.47 2.22

Consumption Rate (g/l/h) -13.1 -11.3

Yield (product/substrate) 46.2% 44.5%

It was reported that high ethanol production was obtained by using initial pH 5.0

to 6.0 (Fadel, 2000) which agreed with the results of this study. It was also shown that no

ethanol production exists lower than pH 4.0 (Graves et al., 2006). During the

uncontrolled pH treatment, however, the minimum observed pH was 4.1. Turhan et al.

(2008) reported that maximum ethanol yield, maximum growth rate, and biomass

concentration were obtained at pH 5.5 on carob as a medium for ethanol production. pH

5.5 was found to be the best pH level and therefore, used for the rest of the study.

5.2.2. Effect of Inoculum Sizes on Ethanol Production

Three different inoculum sizes (1%, 3%, and 5% (v/v)) were investigated to

determine the effect of inoculum size on kinetic parameters of ethanol fermentation from

waste potato mash. Figure 5.8 shows the ethanol production (g/L), glucose consumption

(g/L), and the cell population (log CFU/ml) over 48 h fermentation periods for all cases.

71

1% Inoculum Size

(A)

0

10

20

30

40

50

60

70

0 10 20 30 40 50

Time (h)

Glu

co

se a

nd

Eth

an

ol (g

/L)

4.5

5.5

6.5

7.5

8.5

Cell P

op

ula

tio

n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log CFU/ml)

3% Inoculum Size

(B)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50

Time (h)

Glu

co

se

an

d E

tha

no

l

(g/L

)

5.5

6

6.5

7

7.5

8

8.5

9

Ce

ll P

op

ula

tio

n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log CFU/ml)

72

5% Inoculum Size

(C)

0

10

20

30

40

50

60

70

0 10 20 30 40 50

Time (h)

Glu

co

se

an

d

Eth

an

ol (g

/L)

5.5

6

6.5

7

7.5

8

8.5

9

Ce

ll P

op

ula

tio

n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log CFU/ml)

Figure 5.8 Ethanol, glucose, and cell population in the fermentation broth with

different inoculum sizes.

In these fermentations, glucose conversion was simultaneous, which can be seen

in the graphs. The maximum ethanol productivity (6.48 g/L/h) and maximum growth rate

(0.3 CFU/ml/h) were obtained with 3% inoculation, which produced 30.99 g/L ethanol

(Table 5.6). Although 5% inoculum sizes gave a higher ethanol yield, growth rates,

production rates, and consumption rates were lower than parameters of 3% inoculum size

(Table 5.6). Among 1, 3, and 5 % inoculum sizes, 3% was determined to be the optimum

inoculum by comparing production rate, maximum growth rate and produced ethanol.

The highest production rate, growth rate, and produced ethanol were 6.48 g/l/h, 0.3 log

CFU/ml/h, and 30.99 g/L, respectively, which were produced by 3% inoculum size.

There was no statistically significant difference in mean production rate among the

73

inoculum sizes (p-value>0.05), however, growth rates were statistically different for

inoculum sizes (p<0.05).

Table 5.6 Comparison of kinetic parameters of different inoculum sizes

Inoculum Sizes

Kinetic Parameters 1% 3% 5%

Max. Ethanol Produced (g/l) 27.05 30.99 28.51

Max. Glucose Consumed (g/l) 58.5 70.0 49.6

Growth Rate (log cfu/ml/h) 0.29 0.30 0.17

Production Rate (g/l/h) 5.47 6.48 2.29

Consumption Rate (g/l/h) -13.1 -25.9 -5.3

Yield (product/subs) 46.2% 44.2% 57.4%

The results reveal that there is an increase of ethanol yield up to 3%, however 5%

inoculum causes a decrease of kinetic parameters for ethanol fermentation by S.

cerevisiae. Fadel (2000) reported that ethanol production increases by inoculum up to 4%.

Furthermore, it was reported that 3% inoculum size was the optimum for ethanol

production from carob, which is a fruit grown widely in Mediterranean region (Turhan et

al., 2008). The 3% inoculum size was suggested to be the optimum level for ethanol.

5.3. Effect of Nitrogen Sources

In order to find more economical and efficient alternative nitrogen sources,

ethanol fermentations were performed in hydrolyzed waste potato mash media including

poultry meal, hull and fines mix, feather meal, and meat and bone instead of yeast extract.

74

Because the yeast extract is expensive, an alternative nitrogen source is needed. Figure

5.9 shows ethanol, glucose, and cell populations using four different nitrogen sources in

fermentation medium.

A limitation of using alternative nitrogen sources was that these animal-by-

products were not 100% soluble which caused plugs in the tubes of the reactors.

Although sterilization was done properly, a contamination occurred with meat bone meal

at all replications which might be due to spores.

Based on the results, it can be concluded that all of the animal by-products

investigated in this study supply nitrogen for growth of yeast in ethanol fermentation.

Although the maximum ethanol yield was attained from poultry meal with 35 g/L ethanol,

growth rate, production rate, and consumption rate of poultry meal fermentation (0.211

log CFU/ml/h, 3.2 g/l/h, and -5.35 g/l/h, respectively) were lower compared to yeast

extract (Table 5.7). The second highest ethanol yield (32 g/L ethanol concentration in

final broth) was observed with feather meal which also had very good results for growth

rate (0.28 log CFU/ml), production rate (3.59 g/L/h), and consumption rate (5.06 g/L/h).

Hull and fines mix and meat bone meal were less than yeast extract with 24.59 and 25.54

g/L ethanol, respectively. Moreover, these two nitrogen sources fell behind in growth and

production rates (0.194 log CFU/ml/h and 1.97 g/l/h and 0.13 log CFU/ml/h and 2.36

g/l/h, respectively) (Table 5.7). Overall, the maximum growth rate was observed with

yeast extract and feather meal (both 0.28 log CFU/ml/h). However, maximum production

rate was obtained with yeast extract (3.68 g/L/h) with a 30.8 g/L ethanol production.

75

Yeast Extract

(A)

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50

Time (h)

Glu

co

se

an

d

Eth

an

ol (g

/L)

5.0

5.5

6.0

6.5

7.0

7.5

8.0

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ll P

op

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n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

Feather Meal

(B)

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50

Time (h)

Glu

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Eth

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/L)

5.5

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7

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8

8.5

Cell P

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g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

76

Poultry Meal

(C)

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50

Time (h)

Glu

co

se

an

d

Eth

an

ol (g

/L)

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Ce

ll P

op

ua

ltio

n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

Meat Bone Meal

(D)

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50

Time (h)

Glu

co

se a

nd

Eth

an

ol (g

/L)

4.0

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6.0

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7.5

8.0

Cell P

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(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

77

Hull and Fines

(E)

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40 45 50

Time (h)

Glu

co

se

an

d

Eth

an

ol

(g/L

)

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Ce

ll P

op

ula

tio

n

(lo

g C

FU

/ml)

Glucose (g/L) Ethanol (g/L) Cell Population (log cfu/ml)

Figure 5.9 Ethanol, glucose, and cell populations in the fermentation broth with

different nitrogen sources: Yeast extract (A), Feather meal (B), Poultry meal (C),

Meat bone meal (D), Hull and fines mix (E).

Although poultry meal has the highest ethanol yield, it has lower growth and

production rates compared to yeast extract and feather meal (Table 5.7). This might be

because in poultry meal fermentation, initial glucose concentration was the highest

among the nitrogen sources fermentation. Statistical analysis showed that there is a

significant difference among production rates of different nitrogen sources (p<0.05).

Compared to yeast extract by Dunnett Test and it was concluded that no nitrogen source

produces ethanol less or more than yeast extract at a significance level of 0.05.

A comparison of poultry meal, hull and fines mix, feather meal, and meat bone

meal for ethanol production on carob extract by using S. cerevisiae was reported by

Turhan et al., (2008). In their study, maximum production rate and ethanol yield among

78

four alternative nitrogen sources were determined by addition of meat bone meal,

whereas none of the other evaluated nitrogen sources reached the yield of ethanol and

growth rate when yeast extract was added. Graf-Sirakaya (2004) also studied the effect

of nitrogen sources for ethanol fermentation with S. cerevisiae in a chemically defined

media to evaluate seven different animal by-products as a nitrogen source instead of

yeast extract. For these studies, yeast extract was compared with poultry meal, hulls

and fines mix, beef stock, blood meal, feather meal, pork stock and meat and bone meal

(Graf –Sirakaya, 2004). Pork stock and meat bone meal were reported as giving the

two highest ethanol yields as alternative nitrogen sources, however, the second highest

production rate after yeast extract was obtained with poultry meal (Graf –Sirakaya,

2004). These animal by-products could be alternative nitrogen sources for ethanol

production instead of yeast extract although all of them had lower production rates than

the yeast extract (Graf –Sirakaya, 2004).

Overall, ethanol fermentation from waste potato mash with pH control at 5.5,

inoculum amount of 3%, and yeast extract in the reactor was the best choice. The

maximum production rate was obtained at pH 5.5, 30°C, 400 rpm agitation, and 3%

inoculum size. The addition of alternative nitrogen sources instead of yeast extract into

the fermentation medium resulted in promising ethanol production.

79

Table 5.7 Comparison of kinetic parameters of alternative nitrogen sources.

Nitrogen Sources

Kinetic Parameters YE PM HF FM MBM

Max. Ethanol Produced (g/l) 30.8 35 24.59 32.9 25.54

Max. Glucose Consumed (g/l) 70 73.9 53.2 68.8 53.2

Growth Rate (cfu/ml/h) 0.28 0.21 0.19 0.28 0.13

Production Rate (g/l/h) 3.68 3.20 1.97 3.59 2.36

Consumption Rate (g/l/h) -7.07 -5.35 -4.32 -5.06 -7.25

Yield (product/subs) 43.8% 47.3% 46.2% 47.8% 48.0%

*YE: Yeast extract, PM: Poultry Meal, HF: Hull and Fines Meal, FM: Feather Meal,

MBM: Meat and Bone Meal

80

CHAPTER VI

CONCLUSIONS AND SUGGESTIONS FOR FUTURE RESEARCH

This research successfully demonstrated that waste potato mash can be used for

ethanol production by Saccharomyces cerevisiae. Figure 6.1 shows the overall results for

ethanol at various stages of optimization studies.

0

5

10

15

20

25

30

35

40

pH u

ncont

rolle

d

pH 5

.5

1% In

oculum

3% In

oculu

m

5% In

oculu

m

Hull a

nd Fin

es

Mea

t Bone

Meal

Yeast

Ext

ract

Feath

er Meal

Poultr

y M

eal

Eth

an

ol

(g/L

)

Figure 6.1 Summary of ethanol production

Between acid and enzyme hydrolysis, enzyme hydrolysis turns out to be the better

pretreatment for starch substrates. Acid hydrolysis only reached 0.03 g/L glucose with a

20% (w/v) waste potato mash mixture. Enzyme hydrolysis, on the other hand, after

optimizing liquefaction and saccharification steps, yields 34.9 g/L glucose. After scaling

up, glucose concentration was able to be increased to 93.5 g/L.

81

In terms of the pH strategy for ethanol fermentation from hydrolyzed waste potato

mash, controlling pH at 5.5 is found to be the optimum condition for ethanol fermentation,

which yields 27.05 g/L ethanol. Uncontrolled pH conditions did not improve ethanol

production because the pH level fell down to pH 4 at the end of 48 h. The literature

reveals that low pH levels (under 4.2) do not increase ethanol in fermentation broth

(Fadel 2000) which is consistent with results of this research.

Among inoculum sizes investigated, 3% (v/v) was determined to be the best

choice with production of 30.99 g/L ethanol. To increase amount of culture did increase

neither ethanol production nor kinetic parameters of fermentation and 5% inoculum was

not able to enhance fermentation of ethanol. According to the literature, increasing the

inoculum size increases production of ethanol up to 4% (Fadel, 2000), which is also in

agreement with this study.

Furthermore, four animal by-products were investigated as a substitute of yeast

extract, and poultry meal and feather meal were determined to be economical alternatives

to yeast extract. By combining optimum conditions, 35 g/L ethanol was produced at pH

5.5 with an inoculum size of 3% when the nitrogen source was poultry meal. However,

kinetic parameters of this fermentation were relatively lower than yeast extract and

feather meal. Feather meal yielded 32.9 g/L ethanol with 0.28 log CFU/ml/h growth rate

and 3.59 g/l/h production rate, which are comparable kinetic parameters to yeast extract.

From this work it is clearly indicated that waste potato mash can be an effective

fermentation medium for production of ethanol under conditions of controlled pH at 5.5,

inoculum size of 3% and supplement of the nitrogen source. In order to improve starch

hydrolysis and fermentation conditions and decrease the cost of ethanol, further research

82

is still needed. Co-cultures or genetically modified microorganisms, which might be an

alternative to enzyme or acid hydrolysis, might be evaluated for ethanol fermentation of

waste potato mash. Furthermore, simultaneous saccharification and fermentation might

be applied to have economically feasible production of ethanol from waste potato mash.

83

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