Saimaa University of Applied Sciences Faculty of Technology, Imatra

Saimaa University of Applied Sciences Faculty of Technology, Imatra
Saimaa University of Applied Sciences
Faculty of Technology, Imatra
Degree in Paper Technology
Liu Guifang
INFLUENCE OF YEAST AND ENZYME VARIATION
ON BIOETHANOL YIELD
Bachelor’s Thesis 2011
ABSTRACT
Liu Guifang
Influence of Yeast and Enzyme Variation on Bioethanol Yield,
46 pages, 12 appendices
Saimaa University of Applied Sciences, Imatra, Finland
Faculty of Technology, Bachelor’s Degree in Paper Technology
Bachelor’s Thesis 2011
Instructor: Lahdenperä Esko, Saimaa University of Applied Sciences
This is a study concerning the procedures of bio-ethanol production from the
wood based biomass hydrolysates’ fermentation process. Required process
conditions are analyzed and experimental data include raw material properties;
bio-ethanol productivity and its impacts are evaluated to illustrate how the
bio-ethanol production potential relates with the variation of the yeasts types.
Theoretical background and experimental based research are majorly applied to
achieve two targets that the final thesis composition requires: first process
knowledge accumulation which gives the solid foundation for principle
understanding and related experimental operations; and secondly real-life
process data and result analysis of the experiments that provide the scientific
support to the thesis opinion establishment.
The result of this study can be concluded as follow: after the study of the
bio-ethanol production from woody raw material, the related chemical and
biological process as are expected to receive higher productivity and faster
process period by applying the additional usage of specific and appropriate type
of enzyme. According to the final results, we might understand that although the
wood chips are able to be used as the raw material for bio-ethanol production, its
productivity is relatively lower and process is considerably more difficult than the
traditional agriculture based bio-ethanol production, for instance: bio-ethanol
production from using sugar products and starch crops as raw materials.
However, due to the concerns of world`s food security and the trend of the new
bio-energy developments and applications, the usage of forest based raw
materials is still considered as one of the major methods of bio-ethanol
production.
Key Words: Enzyme, Yeast, Bio-ethanol, Fermentation, Hydrolysis, Spruce,
Lignocelluloses.
CONTENTS
1 INTRODUCTION……………………………………….……..….……………...…1
2 BIO-ETHANOL…………………………………………………………….…….….2
2.1 Characteristics of Bio-Ethanol……………….....…….….………...…….…2
2.2 Condition and Applications of Bio-Ethanol……………..……………...…..4
3 WOOD AS RAW MATERIAL………………………………………………………6
3.1 Chemical Structure of Wood…………...……………...………………….…6
3.2 Cellulose……………………...……………………………………………….7
3.3 Hemicelluloses………...…………....………………………………......……8
3.4 Lignin…………………………………………..…………………..……..……9
4 MATERIAL PRETREATMENT AND HYDROLYSIS..................................…11
4.1 Pretreatment……………………..…………………….….….……..…...…11
4.1.1 Physical Pretreatment………………………………………………11
4.1.2 Chemical Pretreatment……………………………………………..13
4.2 Hydrolysis…………………………………………………………..…..……14
4.2.1 Acid Hydrolysis…………….................................................…….14
4.2.2 Enzymatic Hydrolysis…………………….…………………………16
5 FERMENTATION………………………………………………..………………..18
5.1 Fermentation Microorganism….……….......................…………….....…18
5.1.1 Yeast…………………………………...……..……………….…..…18
5.1.2 Bacteria…………………………………………...…………….……19
5.1.3 Fungi………………………………………………………………….20
5.2 Fermentation’s Techniques……………….…………………..…………...20
5.2.1 Batch Process…………………………...……….…………….……21
5.2.2 Fed-batch Process………………………………………...…..……21
5.2.3 Continues Process……………………………………………...…..22
5.3 Distillation………………………………………...………………...………..23
6 EXPERIMENTAL SECTION……………….........................…………………..24
6.1 Materials………………………………………………………………......…24
6.1.1 Raw Lignocellulosic Material………………………………...…….24
6.1.2 Categories of Yeast ………………………………………………...25
6.1.3 Categories of Enzymes…………………………………...……..…27
6.2 Experimental Equipment and Procedures…………………………..……27
6.2.1 Wood Selection…………………………….…………………...…..27
6.2.2 Hydrolyzation………………………………………………………..28
6.2.3 Yeast Cultivation……………………………...…………….......…..30
6.2.4 Fermentation…………....…………………………...……………...31
6.2.5 Distillation…………………………………………………...……….32
6.2.6 Distillate Analyzed by Gas Chromatograph…........................….33
7 RESULTS AND DISCUSSION………………………………………………..…35
7.1 Yield Comparison between Two Yeast……………………………..…….35
7.2 Yield Comparison between Two Enzymes……………………...…….....37
7.3 Discussion……………………………………………………................….38
8 CONCLUSION………………………………………………….................……..39
REFERENCES...........................................................................…....................40
APPENDICES
1 INTRODUCTION
Global warming and climate change are acknowledged as the impacts from
fossil fuel consumption. As the current situation that traditional fossil fuel is
reaching its storage limitation quickly and global energy consumption never
meets a break, it has become more necessary than ever to locate an alternative
solution to reduce the fossil fuel dependence within appropriate resource and
investment implantations. Nowadays, new technology and scientific research
based on the alternative energy production is rapidly growing. Bio-energy,
especially the bio-ethanol as energy source ranks ahead, and its importance can
be noticed as approximately 30% of total biomass is transferred into bio-ethanol
annually [1] and used in different fields.
Forest, or wood to be more specific is considered as one of the most essential
natural resources on this planet; its valuable factors are covering not only on the
aspect of traditional paper and pulp manufacturing, but a new trend of energy
source such as the mentioned bio-ethanol research and production is inspired
as well by the certain wood process technology. As figure 1 indicates below, this
process shows how the bio-ethanol is product from the biomass hydrolysates.
Figure1. General flowchart of bio-ethanol production from biomass [2]
This thesis project is mainly following this process with using different yeasts and
specific enzymes to produce the bio-ethanol, and to clarify ethanol productivity
and other related conditions according to the analytical chemistry testing results.
1
2 BIO-ETHANOL
Bioethanol is the principle fuel used as the petrol substitute for road transport
vehicles. It is mainly produced by the sugar fermentation process. Several
advantages of using ethanol as fuel should be mentioned. It is good for the
environment and it can reduce dependence on oil imports
2.1 Characteristics of Bio-Ethanol
Ethanol, which is also named as ethyl alcohol, pure alcohol, grain alcohol, or
drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive
drug and one of the oldest recreational drugs. It is best known as the type of
alcohol found in alcoholic beverages. Ethanol is a straight-chain alcohol, and
molecular formula is C2H5OH, its chemical structure is given in the figure 2
below:
Figure2. Chemical structure of Ethanol [3]
Its empirical formula is C2H6O. An alternative notation is CH3–CH2–OH, which
indicates that the carbon of a methyl group (CH 3–) is attached to the carbon of a
Methylene group (–CH2–), which is attached to the oxygen of a hydroxyl group
(–OH). It is a constitutional isomer of Dimethyl ether. [3]
Bio-ethanol has the same chemical properties as the regular ethanol that is
2
actually a petroleum product; the difference only exists in their raw material of
production, which regular Petroleum ethanol is made by the hydrolysis of
ethylene, a major petrochemical [4]. Ethanol is used as an industrial feedstock, or
solvent is often made from petrochemical feed stocks, primarily by the
acid-catalyzed hydration of ethylene as the formula 1 indicates below:
C2H4 + H2O → CH3CH2OH
(1)
As the bio-ethanol, its production is connected with the fermentation process of
sugar from living organisms, or named as biomass, which is divided into:
agriculture based sources and forestry based sources.
The agriculture based biomass includes straw of cereals and pulses, stalks and
seed coats of oil seeds, stalks and sticks of fiber crops, pulp and wastes of the
plantation crops, peelings, pulp and stalks of fruits and vegetables and other
wastes like sugarcane trash, rice husk, molasses, coconut shells etc. In another
direction, harvesting and thinning residues, thinning from hazardous fuels
reductions, habitat improvement, and other ecosystem restoration projects,
sawdust from paper mills, trees and woody plants and their other woody parts
are all included as the biomass sources from the forest [5].
The production of bio-ethanol is through the fermentation process, scientifically,
the biomass fermentation is a process where microbes use sugars as food and
produce alcohols (bio-ethanol) as a product of their metabolism. The
fermentation process is usually anaerobic but can also be aerobic; it is
depending on the microbes that are used in the fermentation process. When
proceeding the biomass fermentation process, the basic stages are illustrated as
the figure 3.
3
Figure3. Biomass fermentation procedures briefing
2.2 Conditions and Applications of Bio-Ethanol
Bio-ethanol, as one of the most important and valuable bio-fuels, acts an
essential role in the transportation field. It is mainly used as a fuel additive for
gasoline. World ethanol production for transport fuel was tripled between 2000
and 2007; it was reported from 17 billion to over 52 billion liters. In 2010
worldwide ethanol fuel production reached 86.9 billion liters, its popularity is
easily being seen from this number
[6]
.
Among the numbers of production and consumption of bio-ethanol fuel,
countries of Brazil and the United States, and together both countries were
responsible for nearly 90% of the world's ethanol fuel production in 2010. Table 1
4
indicates their production facts of bio-ethanol as fuel usage between 2007 and
2010. As we can notice, gloally, except for the U.S. and Brazil, many countries
are interested in the bio-ethanol production and applications as well;
Table1. Annual Fuel Ethanol Productions by Country
[7]
However, today’s opinions of bio-ethanol are not as positive as the trend of its
production, because the bio-ethanol is produced from agricultural and forest
based biomass. In developing countries there are certain concerns and worries
of its production and use, related to increased food prices due to the large
amount of arable land required for crops, few the energy and pollution balance of
the ethanol production cycle are being argued and tested.
5
3 WOOD AS RAW MATERIAL
There are numbers of natural materials, for instance: agriculture residues,
forestry residues, food waste, industrial waste can all be categorized as biomass,
but in this specific process, wood is selected as the biomass raw material for
certain mentioned process.
3.1 Chemical Structure of Wood
Wood is defined as a hard, fibrous tissue type of material that exists in trees. It
has been used for hundreds of thousands of years for both paper & pulp
manufacturing, fuel and as a construction material. Wood is a heterogeneous,
hygroscopic, cellular and anisotropic material. Cell is the basic structure unit of
wood. Chemically, wood is composed principally of carbon, hydrogen, and
oxygen, data is given in the table 2 provides the percentage that each major
chemical element holds inside the wood structure.
Table2. Chemical Element Distribution [8]
There are three types of organic polymers that are responsible for the main
function and structure of the wood: cellulose, hemicelluloses and lignin. In
chemical terms, the difference between hardwood and softwood is reflected in
the composition of the constituent lignin. Hardwood lignin is primarily derived
6
from sinapyl alcohol and coniferyl alcohol. Softwood lignin is mainly derived from
coniferyl alcohol
[9]
. However, as the table 3 indicates, due to the difference
between softwood and hardwood, the distributions of mentioned organic
polymers are various within certain scale of range.
Table3. Distributions of major organic polymers in wood [10]
Except for the lignocellulose, wood consists of a number of low molecular weight
organic compounds, such as terpenes, diterpenes, and fatty acids. For example,
rosin is exuded by conifers as protection from insects.
3.2 Cellulose
Cellulose is an organic compound with the formula of (C6H10O5)n. Like the figure
4 illustrates, this compound is a polysaccharide consisting of a linear chain of
hundreds to over ten thousand β(1→4) linked with D-glucose units [11].
Figure4. Chemical Structure of Cellulose
It is a principal chemical constituent of the cell walls of the higher plants, and a
complex carbohydrate as major structure in the form of polymer chains.
7
Cellulose is the key for the biological production of ethanol by proceeding two
main methods: a) Cellulolysis processes which consist of hydrolysis on
pretreated lignocellulosic materials, using enzymes to break complex cellulose
into simple sugars such as glucose and followed by fermentation and distillation.
b) Gasification process that transforms the lignocellulosic raw material into
gaseous
carbon monoxide and hydrogen, those gases can be converted to
ethanol by fermentation or by chemical catalysis [12].
3.3 Hemicellulose
Hemicelluloses are polysaccharides in plant cell walls. These types of
hemicelluloses are found in the cell walls of all terrestrial plants, the detailed
structure of the hemicelluloses and their abundance vary widely between
different species and cell types. The most important function of the
hemicelluloses is to strengthen the cell wall by interaction with cellulose and
lignin.
Glucomannans are the principal hemicelluloses in softwood. The backbone is a
linear or slightly branched chain of β-(14)-linked D-mannopyranose and
D-glucopyranose units like the figure 5 illustrates [13].
Figure5. Chemical Structure of Glucomannans
8
The main hemicellulose compound in the hardwood is a xylan, more specifically
an O-acetyl-4-O-methylglucurono-ß-D-xylan, as the figure 6 shows below.
The backbone consists of β-(14)-linked xylopyranose units. Most of the hydroxyl
groups at C2 and/or C3 of the xylose units are substituted with acetyl groups. [13]
Figure6. Chemical Structure of Xylan
Hemicellulose is constructed by hexoses, pentoses and uronic acids. Comparing
with
cellulose,
hemicellulose
is
easily
hydrolyzed
to
its
constituent
monosaccha-rides [14].
3.4 Lignin
Lignin is defined as a chemical compound that has the cross-linked aromatic
polymer property, which is complex and hydrophobic. The lignin is functioning as
an integral part of the plant cell wall. Several possible monomers can be found in
lignin. This molecule of phenolic character as dehydration product contains three
monomeric alcohols:
Trans-p-coumaryl alcohol, Trans-coniferyl alcohol and
Trans sinapyl alcohol [15]. The structures of mentioned three monomers are given
in the figure 7.
9
Figure7. Chemical Structure of lignin’s monomers[15]
The function of the cellulosic based lignin component biomass is to provide large
extent for the difficulties inherent in cellulose hydrolysis.
The main precursor of lignin in softwoods is trans-coniferyl alcohol In hardwoods,
trans-sinapyl alcohol and trans -p-coumaryl alcohol are also lignin precursors [16].
The composition of lignin is different based on the source of raw material. If
softwood is taken as material, it contains a higher amount of lignin of nearly 30%
and the hardwood has lower lignin content of 20% approximately.
10
4 MATERIAL PRETREATMENT AND HYDROLYSIS
There are two fundamental stages during the lignocellulose materials’
degradation in producing fermentable sugars, which are:
A) Pretreatment (chemical and mechanical)
B) Hydrolysis (chemical and enzymatic)
The pretreatment is required to increase the surface area of the feedstock, which
makes the lignocellulose accessible for hydrolysis process more effectively [17].
4.1 Pretreatment
The goal of pretreatment is to destroy the lignocellulose cell structure in order to
make it more approachable for further treatment. During the pretreatment,
hemicellulose is chemically hydrolyzed into monomer sugars. The sugars are
converted into a mixture of soluble sugars, xylose, arabinose, mannose and
galactose
[18]
. The chemical stability of cellulose is better than that of
hemicellulose. There is only a small part of cellulose that can be converted into
glucose as the result of this step. The pretreatments are performed physically or
chemically. In order to maximize the performance of the pretreatment stage,
normally the process is done in both ways.
4.1.1
Physical Pretreatment
The most popularly used physical pretreatment method is steam explosion
where the lignocellulose is heated by using high-pressure steam with the
pressure between 20 and 50 bars at the temperature from 210 to 290 oC for few
11
minutes. During the steam explosion process, the steam with high pressure and
thermal energy penetrates the structure of lignocellulose, and is released out
from the closed pores of the lignocellulose [19].
The high temperature and high pressure causes the damage of hydrogen bonds
of the cellulose, where new and free hydroxyl appears. As a result, the ordered
structure of cellulose is changing; the adsorption capacity of cellulose is
increasing. The conventional pretreatment methods only can change the
solubility of hemicellulose and the enzymatic conversions rate etc. The steam
explosion pretreatment is to control the temperature; manage time, and change
the cellulose particle size for reaching the purpose of physical and chemical
property changing of the cellulose.
The initial steam explosion was proposed and patented in 1927 by Mason. The
further research which is combined with chemical treatment was done by Mason
later which made the steam explosion technology more effective. The treatment
effect of steam explosion is not only related with the selective chemical reagents,
but also with the granularity of the raw material. By using a larger particle size
(8mm - 12mm), energy can be saved, and the operating conditions can be more
severe. Sugar loss in hemicellulose hydrolysis decreases, and cellulose enzyme
hydrolysis rate can be raised. The advantage of steam explosion method mainly
concerns the low energy consumption and performance effectiveness. However,
disadvantage concerns the loss of xylose, and harmful substances produced in
the fermentation. When the intensity of pre-treatment is getting greater, the
easier enzymatic hydrolysis of cellulose can be done, but the less sugar is
obtained from hemicellulose, and there are more harmful substances from the
fermentation [19].
12
4.1.2
Chemical Pretreatment
Chemical method is the use of acids, alkalis, organic solvents, such as the
method of lignocellulose pretreatment. The method is mainly aimed at cellulose,
hemicellulose and lignin imbibed with the destruction of its crystalline structure.
As similar as the physical pretreatment, there are two methods available for the
chemical pretreatment:
Dilute acid hydrolysis pretreatment has been successfully used in pretreatment.
The dilute sulphuric acid with concentration between 0.5 and1.5% is in use,
treatment temperature should be above 160 Co. It is used as the most favored
pretreatment for industrial application, because it achieves reasonably high
sugar yields from hemi-cellulose: minimum xylose yield is 75–90% [20].
During this treatment, lignin content keeps unchanged, the average degree of
polymerization of cellulose is decreased, and ability to respond is corresponding
increased. As a result, the cellulose contact area is raised, additionally, dilute
acid pretreatment produces fermentation inhibitors, corrosion of metal
equipment, for which certain containesr and devices are required to avoid
damages from happening by using the chemical pretreatment.
Alkaline pre-treatment uses chemicals of sodium hydroxide or calcium hydroxide.
The function of the alkali pretreatment is to remove lignin and to increase the
reactivity of the remaining polysaccharides. All lignin and part of the
hemicellulose are removed, and the treated cellulose performance for later
hydrolysis is sufficiently increased. The effect depends on the characteristics of
raw materials with certain lignin contents and properties. If the lignin content of
lignocellulose raw materials is more than 20%, the alkali treatment can hardly
improve subsequent enzymatic hydrolysis rate. The mechanism of Alkali
13
treatment is to weaken the hydrogen bonds between hemicellulose and lignin
and the saponification of the ester bond between. After the alkali treatment of
wood, fiber becomes more porous, which makes wood more suitable for the
growth of filamentous fungi. NaOH solution for processing of wood cellulose,
which can cause swelling of wood fiber raw material profit, lowers the degree of
polymerization and crystallinity. [21]
However, the use of mentioned treatments is believed to highly raise
environmental concerns and may lead to prohibitive recycling, it also increases
the wastewater treatment and residual handling costs.
4.2 Hydrolysis
The aim of the hydrolysis is to cleave the polymers of celluloses and
hemicelluloses to monomeric sugars which are able to be fermented to ethanol
by microorganisms. The hydrolysis is essential before fermentation to release
the fermentable sugars. The theory difference between cellulose hydrolysis and
hemicellulose hydrolysis is indicated in the formulas 2 and 3.
Cellulose
Hemicellulose
Hydrolysis
Hydrolysis
Glucose
Fermentation
Pentosed &Hexoses
Ethanol
Fermentation
(2)
Ethanol
(3)
In ethanol production, the process of hydrolysis is very sophisticated, depending
on several aspects, for example: properties of substrate, acidity, and
decomposition rate during hydrolysis process
[22]
. The hydrolysis can be made
either chemically or by a combined chemical and enzymatic treatment. Acids are
predominantly applied in chemical hydrolysis and Sulphuric acid is the most
frequently used.
14
4.2.1
Acid Hydrolysis
The solubility of cellulose in acid was detected already in 1815. Concentrated
acid hydrolysis technology began in the 1820s, the first concentrated acid
hydrolysis process was developed by the Department of Agriculture in the U.S.
The required condition, the acid hydrolysis, can be performed by high acid
concentration at a low temperature or that of low concentration at a high
temperature in contrast [23].
The scientific explanation of concentrated acid hydrolysis is described as follows:
the cellulose can be dissolved in the 72% sulfuric acid, 42% hydrochloric acid or
77% and 83% phosphoric acid solution at a lower temperature
[24]
. Then the
cellulose is transformed into monomeric sugars. Within the concentrated
hydrolysis, dimerization reaction will occur in some monosaccharose. The
monomeric sugars start to rejoin and form polysaccharide. This reaction is the
reverse process of cellulose hydrolysis.
The higher the hydrolyzed monomeric sugars contents and acid concentration,
the greater sensitivity is obtained from the dimerization reaction. The monomeric
sugars rejoin to generate the glucose disaccharide or three glycans. The
hydrolytic solution must be diluted and heated in order to prevent hydrolysis
forming
polysaccharide.
The
yield
of
glucose
will
increase
in
the
hydrolysis-operative period [24].
Dilute acid hydrolysis refers to use within 10% acid as a catalyst to hydrolysis of
the cellulose and hemicellulose into monomeric sugars. The reaction condition is
harder to achieve than in concentrated acid hydrolysis. The required reacting
temperature is from 100 ºC to 240 ºC and the pressure is higher than 10
atmospheres in dilute acid hydrolysis process conditions.
15
The sugar degradation happens in high temperature and highly pressurized
environment; the advantages along with some expected problems as
disadvantages of the concentrated acid hydrolysis and dilute acid hydrolysis are
shown in the table 4 [25]:
Table4. Comparison between concentrated and dilute acid hydrolysis
The monosaccharose will break down into formic acid further which results in
lower sugar yield and inhibition of the fermentation. But this problem can be
solved by a two stage process, in which the hemicellulose is mainly hydrolysed
in the initial step at temperature of 150 ºC to 190ºC and the remaining cellulose
subsequently hydrolysed at more severe conditions at minimally 90 to 230ºC [26].
However, the concentrated sulfuric acid hydrolysis is still the most commonly
concentrated acid hydrolysis method although obvious disadvantages exist.
4.2.2
Enzymatic Hydrolysis
The degradation of cellulose to monomer sugars in enzymatic hydrolysis is
catalyzed by specific cellulolytic enzymes which are called cellulases. Cellulases
are produced from both bacteria and fungi, which can decompose cellulosic
material. [27]
16
The enzymatic hydrolysis of cellulose is a complex process. There are three
different chemical reactions which take place at the same time. [28]
1. Residual (not yet solubilized) solid-phase cellulose changes chemically and
physically.
2. Release of soluble intermediates from the surface of reacting cellulose
molecules (primary hydrolysis).
3. Hydrolysis of soluble intermediates to lower molecular weight intermediates
and finally to glucose (secondary hydrolysis).
Generally, degradation of cellulose by enzymatic hydrolysis is characterized by a
rapid initial phase, and then a slow secondary phase follows. Enzymatic
hydrolysis can occur under milder conditions (typically 40-50oC and pH 4.5-5),
which give rise to two advantages of the process; low utility cost since there are
few corrosion problems and low toxicity of the hydrolysates. In addition, it is also
an environmental friendly process [29].
Enzymatic hydrolysis differs from the Acidic hydrolysis. The difference in
functional environment is shown in the table 5.
Table5. Comparison of acid and enzymatic hydrolysis
17
5 FERMENTATION
During fermentation monomeric sugars released in the hydrolysis are converted
into the desired product, by a microorganism, which is required to ferment these
sugars to produce bio-ethanol by different fermentation techniques [30].
The principles of the glucose fermentation can be indicated as the chemical
reaction below:
C6H12O6 → 2C2H5OH + 2CO2
(4)
5.1 Fermentation’s Microorganisms
There are a variety of microorganisms which are able to produce ethanol in
alcoholic fermentation process, including yeasts, bacteria and fungi. Among
them there are several types of bacteria, yeasts and filamentous fungi. The
specific organisms with their advantages and disadvantages will be discussed
below.
5.1.1
Yeast
Yeast is the eukaryotic microorganism in the fungi family. There are many
different strains of yeast. More than one thousand species of yeasts have been
found. The most commonly used yeast is Saccharomyces cerevisiae, which can
convert sugars into carbon dioxide and alcohol. Baking yeast and brewing yeast
are the most important yeasts belonging to Saccharomyces cerevisiae. In
brewing, the yeast is used to ferment alcoholic beverages, the ethanol is final
production. While in baking, the yeast is used to leaven bread, the carbon
18
dioxide raises the bread and the ethanol evaporates
[31]
. Yeasts have recently
been used to produce ethanol for the biofuel industry.
Yeast requires suitable conditions to grow. When water, nutrient, oxygen, and a
proper temperature occur, the life cycle of yeast will become activated. Water is
needed by the yeast in order for it to absorb nutrients. The ammonia and urea
can be used as nutrient for yeast grow. Oxygen enables the yeast to metabolize
nutrients and to multiply. The temperature range of yeast growing best is from
30oC to 40 oC.
The yeast cannot survive when the temperature is higher than
40 oC. But it can survive freezing under certain conditions.
5.1.2
Bacteria
Fermentation bacteria are anaerobic, but use organic molecules as their final
electron acceptor to produce fermentation end-products. Different bacteria
produce different fermentation end products. Streptococcus, Lactobacillus, and
Bacillus produce lactic acid, while Escherichia and Salmonella produce ethanol,
lactic acid, succinic acid, acetic acid, CO2, and H2. Fermenting bacteria have a
characteristic in sugar fermentation that only they can decompose some specific
sugars. For example, Neisseria meningitidis ferments glucose and maltose, but
not sucrose and lactose, while Neisseria gonorrhoea ferments glucose, but not
maltose, sucrose or lactose. This characteristic can be used to identify and
classify bacteria [32].
During the 1860s, the French microbiologist Louis Pasteur studied fermenting
bacteria. He demonstrated that fermenting bacteria could contaminate wine and
beer during manufacturing, turning the alcohol produced by yeast into acetic
acid (vinegar). Pasteur also showed that heating the beer and wine to kill the
bacteria preserved the flavor of these beverages. The process of heating, now
19
called pasteurization in his honor, is still used to kill bacteria in some alcoholic
beverages, as well as milk.
5.1.3
Fungi
Fungi are a group of organisms and microorganisms which are separate from
plants, animals, and bacteria. The fungi include the fleshy fungi, the hyphae, and
the yeast. Fungi are widely distributed and are found wherever moisture is
present. Fungi exist primarily as filamentous hyphae. Like some bacteria, fungi
digest insoluble organic matter by secreting exoenzymes, then absorbing the
soluble nutrients.
The fungi contain a large-scale diversification of classification with varied
ecologies, life cycle strategies. Its biological conformation ranges from
single-celled aquatic chytrids to large mushrooms. Fungi present a significant
role in the decay of organic matter and they have elementary role in nutrient
cycling and exchange. They can be used as a direct source of food, such as
mushrooms and truffles; they can also be used as a leavening agent for bread,
and in fermentation of various food products. [33]
5.2 Fermentation Techniques
The fermentation process can be performed in majorly three types of operations,
depending on different conditions such as properties of microorganisms and
types of lignocellulosic hydrolysates. They are batch process, fed batch process
and continuous process [34].
20
5.2.1
Batch Process
The batch process is a closed fermentation process. In the batch process,
nutrients and the inoculums are added to the reactor only once at the start of the
process. When the maximum amount of product is present in the reactor, the
product is extracted from the solution. Then the reactor is cleaned and used for
other batch processes.
During the batch fermentation process, the microorganism works in high
substrate concentration initially and a high product concentration in the end
[35]
.
The batch process is a multi-vessel process, allows flexible operation and easy
control over the process. Generally batch fermentation is defined as low
productivity with an intensive labor. For batch fermentation, elaborate
preparatory procedures are needed; and because of the discontinuous start up
and shut down operations, high labor costs are incurred. This inherent
disadvantage and the low productivity offered by the batch process have led
many commercial operators to consider the other fermentation methods
5.2.2
[35]
.
Fed batch Process
The fed batch process is based on feeding the reacting solution into the reactor.
This is called controlled feeding process. During the process, feed solution
contains substrate yeast culture, important minerals and vitamins. They are
added at regular intervals after the start
[36]
.The concentration of substrate in the
reacting solution must be kept constant in the reactor while the feeding is made.
Fed batch process is a very popular fermentation process which is mostly used
in ethanol industry. It is a production technique in between batch and continuous
fermentation process. It combines the advantages from them both. No more
21
equipment is needed compared to the batch process requirement. And it
provides better yield than batch process for the production of ethanol under
controlled conditions in the fed-batch process.
5.2.3
Continuous Process
During continuous process, nutrients are continuously supplied to the bioreactor
and metabolites and other wastes are continually removed at the same rate as
the supply, resulting in a constant volume. This method prolongs the exponential
growth phase of microbial growth and promotes continual growth of the
microorganisms [37].
Two control methods are used in continuous culture fermentation, namely,
chemostat and turbidostat. Continuous fermentation can be completed in
different kind of reactors – stirred tank reactors (single or series) or plug flow
reactors [37].
Continuous fermentation often gives a higher productivity than batch
fermentation. Continuous operation offers ease of control and is less labor
intensive than batch operation. The continuous process eliminates much of the
unproductive time associated with cleaning, recharging, adjustment of media
and sterilization.
5.3 Distillation
Distillation is a separation process for a mixture of liquids by taking advantage of
their difference in boiling point temperatures. A distillation step is required after
fermentation to separate the ethanol from the mixed solution. The boiling point of
ethanol is 80oC, and water is 100 oC. Ethanol will preferentially vaporize first
22
during heat the mixed solution to be boiling. The ethanol concentration in the
condensate of the vapor is high. [38]
23
6 EXPERIMENTAL SECTION
Experimental operation is the most essential and direct method to allocate the
performance, effects and appearance of scientific research and study. The way
how experimental devices, tested raw materials and other related factors are
prepared and processed is the key to the accuracy of the mentioned
chemical/biological process.
6.1 Materials
In this process, only spruce wood chips are used lignocellulosic raw material.
Besides, two types of yeast and two types of enzyme are used for the biological
treatment during the bioethanol production process.
6.1.1
Raw Lignocellulosic Material
Wood chips from spruce were used as lignocellulosic raw material in my thesis
work. Spruce is one kind of softwood. Table 6 shows representative values
which are taken from the literature for the composition of spruce. However, the
values can differ quite much due to species and environmental variations for
each material
[39]
.
Table6. Composition of the lignocellulosic material in spruce
(Percentage of dry material)[39]
24
6.1.2
Categories of Yeast
Three different kinds of yeast are used in my thesis work. They are baking yeast
and brewing yeast. The characteristics of dry baking yeast are given in the table
7 below:
Table7. Characteristics of Dry Baking yeast
Name
Dry Baking yeast (Active dry yeast)
Ingredients
Yeast(Saccharomyces cerevisiae),
Rehydrating agent
Properties
Very long standing natural product.
Commonly used as a leavening agent in
baking bread and bakery products.
Dry matter and density
Dry matter range is 92 – 96 %.
Density is about 0.75 – 0.95
Pitching instructions
The yeast is rehydrated to reactivate it in
axenic water at around 38 oC before use.
Fermentation T and pH T is about 30oC. pH is 6-7.
Packaging
1 X 11 g packed in paper bag
Storage
Store at room temperature (24oC) for a year.
Frozen for more than a decade.
Once opened, the yeast is best stored dry
and refrigerated and used as quickly as
possible.
25
For the dry brewing yeast, characteristics are given in the table 8:
Table8. Characteristics of Dry Brewing yeast
Name
Dry Brewing yeast
Ingredients
Yeast(Saccharomyces cerevisiae),
Rehydrating agent
Properties
Very popular general purpose yeast.
Used for the production of a varied range of
top fermented special beers.
Excellent performance in beers with alcohol
contents of up to 7.5% v/v but can ferment up
to 11.5% v/v.
Dosage
50 g/hl to 80 g/hl in primary fermentation
2.5 g/hl to 5.0 g/hl in bottle-conditioning
Pitching instructions
Re-hydrate the dry yeast into water before
utilization. Sprinkle the dry yeast in 10 times
its own weight of sterile water at 27±3oC.
Fermentation T
T is about 135oC. PH is 6-7.
Packaging
1 X 6 g vacuum-packed in paper bag
Storage
Store in cool (< 10oC), dry conditions.
Opened sachets must be sealed and stored
at 4oC and used within 7 days of opening.
26
6.1.3
Categories of Enzymes
Two types of enzymes were used in the hydrolysis process. They are cellulase
and xylanase.
Cellulase is a digestive system enzyme that helps to break down the cellulose
into glucose. Xylanase is a class of enzymes which degrade the hemicellulose
into xylose.
The Biobake TR and Biobake Optum 815 are the commercial products that
contain mentioned enzymes, and thus they were both in use for the experiments.
Biobake TR holds the Cellulase, and the Biobake Optum 815 contains the
Xylanase. The concentration of each enzyme in the product is 10% to 25%. The
working conditions of the enzymes are almost the same:
Active temperature range: 45oC – 60 oC
Active pH range: 4.3 – 6.3
6.2 Experimental Equipment and Procedures
The experimental operation was done under the laboratory scale by using the
laboratory facility at the Saimaa University of Applied Sciences, Imatra, Finland.
All equipment operations and safety precautions are following certain
supervisions from the university regulations.
6.2.1
Wood Selection
The yeast fermentation requires certain experimental equipment to provide
reaction environment and other required conditions for further treatment with
quality. Wood chips are used as the raw material in this experiment, but due to
27
its production process, the difference of wood chip sizes is very significant,
which is not ideal for the further treatment. Under this condition, the selection
process of wood chips becomes is necessarily required. In order to select the
wood chips within proper sizes, Gyratory Screening device is in use as the figure
8 indicates:
Figure8. Gyratory Screening for wood chips selection
In this process, only select-sized wood chips, which are in the range from 5 x 10
x 3 mm to 15 x 20 x 3 mm (length x width x thickness) are able to be gathered.
Approximately 1 kg of the wood chips was collected and stored in an air tight bag
under the room temperature.
6.2.2
Hydrolyzation
As the illustration in the figure 9 shows, the equipment of Ethylene Glycol Bath is
in use to produce hydrolyzed and treated lignocellulosic materials from the wood
chips. The control panel is shown on the left of the figure and working part of
rotating reactor is on the right.
28
Figure9. Ethylene Glycol Bath
During this stage, sulfuric acid with 1.5% concentration is needed; however only
98% concentrated sulfuric acid was available in the laboratory, where a manual
dilution of the concentrated sulfuric acid was applied. Safety operations had to
be taken into account when proceeding: Adding concentrated sulfuric acid into
water.
This step can be divided into two parts. In the first part, 22g of wood chips and
165ml of 1.5% sulfuric acid were added into the small reaction unit inside
Ethylene Glycol Bath for a reaction, which temperature requirement is 130 C o
and duration is 2 hours. After the reaction, through the filtration process, the
solvent is left for further treatment, which contains the pH of 1.9. Then the
hydrolysate can be treated in the next fermentation stage.
In the second part, the mentioned process is considered as the pretreatment.
The enzymatic hydrolyzation is executed right after completion of the
pretreatment. The pH of the solution should be modified through the sodium
hydroxide solution in order to achieve the suitable condition for enzymes working.
100ml of solution is taken into the flask and the enzymes are added directly into
the solution. Flux is sealed by cotton. The solution is kept in the water base for
48 hours at 55 oC. Then the solution is ready to be fermented in the next phase.
29
6.2.3
Yeast Cultivation
Yeast cultivation is critical to ensure the success and quality of the final results of
the experiment; there are five major stages to properly control the cultivation,
which are:
1. Water boiling: this is to provide disinfected water as reaction media and
bacterial-free experimental environment. Water is cooked inside flux for 10
minutes.
2. Water cooling: it is impossible to perform the experiment when water is at
100 oC, so the water has to be cooled. The water is removed from flame and
cooled to be 40 oC, which is the same as the best temperature of the yeast’s
cultivation.
3. Yeast adding: when required temperature of 40 oC is reached, yeast is
added into the flux. The mixture is well mixed.
4. Nutrition adding: 10 minutes after adding the yeasts, necessary nutrition is
added to provide the cultivation fundamentals. To this stage, 2g of
(NH4)2PO4 is added.
5. After building the cultivation fundamentals, the flux is sealed by cotton, in
order to maintain the air exchange (O2 is required during the cultivation) and
keep any external contamination outside.
6. Keeping the yeasts cultivation in the water base for 24 hours. The
temperature is 35 oC.
30
As the figure 10 illustrates below, the preparation stage is ready for yeast
cultivation.
Figure10. Preparation and Environment for Yeasts Cultivation
6.2.4
Fermentation
The batch process is run in this fermentation stage. The pH of the hydrolysate
should be modified through the sodium hydroxide solution in order to achieve the
suitable condition for yeast working. 100ml of hydrolysate is mixed with the
active yeast solution in flask. The flask is closed with a rubber balloon. The
function of the curving conduit is to eliminate the carbon dioxide which is
produced by the fermentation process. One side of the curving conduit is on the
top of the mixed solution in the flask. The other side is immerged to the NaOH
solution in the beaker. The oil layer floats on the solution. The oil is used to
prevent the air from entering into the NaOH solution, and then further come into
the ferment mixed solution. Fermentation is an anaerobic process which does
not need oxygen. The connective structure of the laboratory equipment for the
fermentation process is shown in the next figure.
31
Figure11. Connective structure of the equipments
After building the fermentation fundamentals, it is then put into the oven which
can keep the temperature at 35oC well for 24 hours.
6.2.5
Distillation
The distillation apparatus is set up as figure 12 indicates below.
Figure12. Connective structure of the distillation apparatus
The side of water inlet and outlet must be carefully identified while installing the
equipment. The mixed solution is heated by the heat source. When temperature
achieves a certain degree, the ethanol vapor passes into the condenser. The
32
vapor is cooled and liquefied through the condenser. Then the resulting liquid is
collected in a flask. The heat source should be closed, when the temperature
achieves 100oC.
6.2.6
Distillate Analyzed by Gas Chromatograph
Each distillate was analyzed twice through the Gas Chromatograph in order to
identify the ethanol concentration based on the areas; the reason of the double
analysis is to increase the accuracy of the results. During the GC analysis, the
“Ethanol - 1” method was applied. The device of the GC analysis is presented in
the figure 13 below:
Figure13. Gas Chromatograph analysis device
The figure 14 describes the principle of how the ethanol concentration can be
calculated from the area, based on the “Ethanol-1” analysis method.
33
Figure14. Ethanol concentration calculation principle function
In the figure 14, the calculation principle formula is presented as:
y = 3960.3 * x
Where “y” represents the area which is obtained from the analysis;
“3960.3” is a constant number of this function
“x” is the ethanol concentration that indicates the percentage.
34
(5)
7 RESULTS AND DISCUSSION
7.1 Yield Comparison between Two Yeasts
After the fermentation process by using two different types of yeast without
enzymatic hydrolysis, totally 4 kinds of distillates samples were received from
each mixture which is indicated below:
A) 40ml of Baking Yeast solution
+ 100ml of hydrolysate
B) 60ml of Baking Yeast solution
+ 100ml of hydrolysate
C) 40ml of Brewing Yeast solution + 100ml of hydrolysate
D) 60ml of Brewing Yeast solution + 100ml of hydrolysate
Based on the calculation principle formula 5, the 8 analysis results are given in
the table 9. (The results from the GC analyses are given as the appendix.)
Table9. Analysis results and ethanol concentration (A, B, C, D)
Sample Type
Area (pA*s)
Ethanol Concentration (%)
A1
8638.2
2.18
A2
8852.8
2.23
B1
8770.4
2.21
B2
8829.9
2.23
C1
8788.3
2.22
C2
8215.9
2.07
D1
10142.1
2.56
D2
10023.2
2.53
35
Average C. (%)
2.205
2.220
2.145
2.540
The volumes of all the distillates were 10ml. The yield of the ethanol can be
calculated based on the concentration. So the concentration of the distillate is in
direct proportion to the yield of ethanol. In the other words, the raise of the
ethanol concentration in the distillates reflex the increase of the ethanol yield.
The differences of average concentration values in two groups of A-B samples
and C-D samples were compared. In each group, all conditions in the process
were the same, expect for the amount of the yeast which was used in the
fermentation process. It shows that when the volume of hydrolysate solution is
constant, the demand for the yeast has not reached saturation point in the
fermentation process; with more yeast used, the more ethanol is fermented. On
the other hand, if the yeast had reached saturation point in the fermentation
process, more yeast will be an inhibitor which can restrict the process.
The comparison was also made between the A-C samples and B-D samples
groups; however the regular change did not appear. But if another method was
applied on the comparison, if A and B samples are considered as one unified
object which is named AB sample and the same method was applied to C and D
samples, then under this condition, both AB and CD samples contain 100ml of
yeast solution and 200ml of hydrolysate solution. The total ethanol concentration
from AB samples (Baking yeast) is smaller than the CD sample (Brewing yeast).
This fact proves that the ethanol productivity when using the Brewing yeast is
slightly higher than that of the Baking yeast. However, considering they belong to
the same yeast family, the differences of functions and working conditions are
very similar.
36
7.2 Yield Comparison between Two Enzymes
After the fermentation process by using Baking yeast with enzymatic hydrolysis,
totally 2 kinds of distillates were received from each mixture, which is indicated
below:
E) 40ml of Baking Yeast solution + 100ml of hydrolysate with xylanase
F) 40ml of Baking Yeast solution + 100ml of hydrolysate with cellulase
Based on the same calculation principle of formula 5, the 4 analysis results are
given in the table 10. (The results from the GC analyses are given as the
appendix.)
Table10. Analysis results and ethanol concentration (E, F)
Area (pA*s)
Ethanol Concentration
E1
E2
F1
F2
11605.6
13691.2
13717.2
11584.3
2.93
3.45
3.72
3.14
(%)
Average C. (%)
3.19
3.43
The average ethanol concentration values in E and F samples are obviously
higher than in samples A, B, C, and D. This data describes that adding specific
enzyme can enlarge the ethanol yield.
By comparing the average ethanol concentration values between samples E and
F, the results prove that the ethanol yield from the enzymatic hydrolyzation with
xylanase is lower than the ethanol yield from the enzymatic hydrolyzation with
37
cellulose which concludes that the working efficiency of cellulase is better than
that if the xylanase in this process.
7.3 Discussion
By analyzing the results from the experiments, no solid evidence was found to
prove that large difference in ethanol yield exists between the usage of baking
yeast and the usage of brewing yeast in the fermentation process. Biologically,
the baking yeast and brewing yeast are both categorized as fungus, formally
known as Saccharomyces cerevisiae. Both of them can be used in alcoholic
fermentation. The functions of these two yeasts are almost the same. This is why
no significant difference in the ethanol yield between these two yeasts was
appeared.
The amount of the cellulose is much greater than hemicellulose in wood
structure. The cellulase is active on the cellulose which holds the largest share
of the wood structure. The xylanase is a type of hemicellulase which is active on
the hemicellulose. Based on the biological characteristics of enzymes, specific
type of enzyme must be properly applied to certain raw materials in order to
release its maximum efficiency. An efficient enzymatic hydrolyzation acts an
import role in the ethanol production by using the lignocellulosic materials as
source, since it largely increases the ethanol productivity.
However, this work is done within the laboratory scale; there are still several
factors which can influence the ethanol yield, for example: experimental device
restrictions and the controls of the experimental conditions.
38
8 CONCLUSION
The theoretical study and experimental operations of the particular wood
process have offered the understanding on how wood as raw material is used in
the bio-ethanol production.
During this study, two types of yeasts were in use for the fermentation process,
the variation of the yeast influenced the ethanol production, but the difference is
not significantly large since the biological characteristics of mentioned yeast are
similar. But compared with the yeast, the difference between using and not using
the specific enzyme is obvious; the same appearance can also be found in the
comparison between acidic hydrolyzation and the enzymatic hydrolyzation,
which both comparisons indicate that the usage of enzyme is able to highly
enlarge the productivity. This fact tells that if the condition is available, it is best
to apply a certain enzyme to the chemical/biological process to reach higher
productivity and faster production period.
After analyzing the final results, we might understand that although the wood
chips are able to be used as the raw material for bio-ethanol production, its
productivity is relatively lower and process is considerably more difficult than in
the agricultural bio-ethanol production. For instance: producing bio-ethanol by
using sugar products and starch crops. However, due to the concerns of world`s
food security and the trend of the new bio-energy developments and
applications , the usage of forest based raw materials is still considered as one
of the major methods of bio-ethanol production.
39
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Figures
Figure1. General flowchart of bio-ethanol production from biomass
Figure2. Chemical structure of Ethanol
Figure3. Biomass fermentation procedures briefing
Figure4. Chemical Structure of Cellulose
Figure5. Chemical Structure of Glucomannans
Figure6. Chemical Structure of Xylan
Figure7. Chemical Structure of lignin’s monomers
Figure8. Gyratory Screening for wood chips selection
Figure9. Ethylene Glycol Bath
Figure10. Preparation and Environment for Yeasts Cultivation
45
Figure11. Connective structure of the equipments
Figure12. Connective structure of the distillation apparatus
Figure13. Gas Chromatograph analysis device
Figure14. Ethanol concentration calculation principle function
Tables
Table1. Annual Fuel Ethanol Productions by Country
Table2. Chemical Element Distribution
Table3. Distributions of major organic polymers in wood
Table4. Comparison between concentrated and dilute acid hydrolysis
Table5. Comparison of acid and enzymatic hydrolysis
Table6. Composition of the lignocellulosic material spruce
Table7. Characteristics of Dry Baking yeast
Table8. Characteristics of Dry Brewing yeast
Table9. Analysis results and ethanol concentration (A, B, C, D)
Table10. Analysis results and ethanol concentration (E, F)
Formula
Formula1
C2H4(g) + H2O(g) → CH3CH2OH(l)
Formula2 Cellulose
Hydrolysis
Formula3 Hemicellulose
Glucose
Hydrolysis
Fermentation
Ethanol
Pentosed &Hexoses
Formula4
C6H12O6 → 2C2H5OH + 2CO2
Formula5
y = 3960.3 * x
46
Fermentation
Ethanol
APPENDIX 1
GC analysis report: Sample A1
i
APPENDIX 2
GC analysis report: Sample A2
ii
APPENDIX 3
GC analysis report: Sample B1
iii
APPENDIX 4
GC analysis report: Sample B2
iv
APPENDIX 5
GC analysis report: Sample C1
v
APPENDIX 6
GC analysis report: Sample C2
vi
APPENDIX 7
GC analysis report: Sample D1
vii
APPENDIX 8
GC analysis report: Sample D2
viii
APPENDIX 9
GC analysis report: Sample E1
ix
APPENDIX 10
GC analysis report: Sample E2
x
APPENDIX 11
GC analysis report: Sample F1
xi
APPENDIX 12
GC analysis report: Sample F2
xii
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