Fossil fuels like coal and oil have played a critical role in humanitys recent history,

providing a vast energy source which has fueled much of societys development and
industrialization. These fuels are still the primary source of energy for the worlds developed
nations, and yet it is agreed that these traditional sources of energy cannot continue to power
humanitys growth into the future. The demand for oil production is at an all-time high, and
will only increase as developing nations continue to grow. Furthermore, many experts predict
that the rate of world oil production has already peaked, and that it will only decrease from
now onwards as fewer and fewer oil reserves are discovered. This decreasing supply and
rising demand will drive up the price of oil and other fossil fuels, and will eventually make
them economically unsustainable. The use of fossil fuels poses other problems as well, most
notably that their consumption is environmentally unsustainable. Burning fossil fuels
produces enormous quantities of the greenhouse gas carbon dioxide, which has a negative
impact on the Earths environment by contributing to global warming. For all of these
reasons, there is great incentive to pursue the development of renewable energy sources,
particularly microbial biofuels.

Microbial metabolism is incredibly varied, and can both utilize and produce a wide variety of
useful molecules. Many microbial systems are also well characterized and easy to manipulate
genetically, and scientific advances will only make these systems easier to work with in the
future. Although there is no biofuel option currently available which solves all of the
economic and environmental issues associated with fossil fuels, the potential for both fine-
tuning biofuel-producing microbes, and genetically modifying species to be able to
efficiently make use of otherwise useless materials and byproducts, makes microbial biofuels
an appealing target for research.
Bioethanol is ethanol (alcohol) that is derived exclusively from the fermentation of plant
starches. Though ethanol can be extracted as a by-product from a chemical reaction with
ethylene and other petroleum products, these sources are not considered renewable.

Chemically, bioethanol is identical to ethanol and can be represented by either the formula
C
2
H
6
O or C
2
H
5
OH.
There are two biochemical production routes for bioethanol: (1) First generation technologies
utilizing crops rich in starch, e.g. corn, grain and sugar cane, and (2) second generation
technologies utilizing lignocellulosic biomass, e.g. wood, agricultural residues and energy
crops.



First generation technologies (Starch Ethanol)
Starch is a glucose-containing polymer which can readily be hydrolyzed by industrially
produced enzymes and fermented by e.g. yeast. This is a wellknown process and is presently
used for production of bioethanol in many parts of the world, especially in the USA (corn /
maize) and Brazil (sugar cane). However, the feedstock costs are high and still rising due to
rising demand for both food and fuel, limited area of cultivation and bad harvests. The ethical
question of using food for fuel has become more and more relevant and thus the sustainability
of first generation bioethanol production is questioned.

Second generation technologies (Lignocellulosic Ethanol)
Lignocellulose is a complex molecule consisting of cellulose, hemicellulose and lignin. The
complexity of the lignocellulosic biomass makes it difficult to utilize lignocellulose
compared to starchy biomass. This difficulty is overcome by introducing an additional step to
the production strategy: pretreatment. Most often the pretreatment is a thermo-chemical
treatment of the biomass, making the glucose containing polymer, i.e. cellulose, susceptible
to enzymatic hydrolysis. Afterwards, the sugar released during the pretreatment and the
enzymatic hydrolysis is then fermented to ethanol.


Bioethanol Production

Ethanol can be produced from biomass by the hydrolysis and sugar fermentation processes.
Biomass wastes contain a complex mixture of carbohydrate polymers from the plant cell
walls known as cellulose, hemi cellulose and lignin. In order to produce sugars from the
biomass, the biomass is pre-treated with acids or enzymes in order to reduce the size of the
feedstock and to open up the plant structure. The cellulose and the hemi cellulose portions are
broken down (hydrolysed) by enzymes or dilute acids into sucrose sugar that is then
fermented into ethanol. The lignin which is also present in the biomass is normally used as a
fuel for the ethanol production plants boilers. There are three principle methods of extracting
sugars from biomass. These are concentrated acid hydrolysis, dilute acid hydrolysis and
enzymatic hydrolysis.
Concentrated Acid Hydrolysis Process
The Arkanol process works by adding 70-77% sulphuric acid to the biomass that has been
dried to a 10% moisture content. The acid is added in the ratio of 1.25 acid to 1 biomass and
the temperature is controlled to 50C. Water is then added to dilute the acid to 20-30% and the
mixture is again heated to 100C for 1 hour. The gel produced from this mixture is then
pressed to release an acid sugar mixture and a chromatographic column is used to separate
the acid and sugar mixture.
Dilute Acid Hydrolysis
The dilute acid hydrolysis process is one of the oldest, simplest and most efficient methods of
producing ethanol from biomass. Dilute acid is used to hydrolyse the biomass to sucrose. The
first stage uses 0.7% sulphuric acid at 190C to hydrolyse the hemi cellulose present in the
biomass. The second stage is optimised to yield the more resistant cellulose fraction. This is
achieved by using 0.4% sulphuric acid at 215C.The liquid hydrolates are then neutralised and
recovered from the process.
Enzymatic Hydrolysis
Instead of using acid to hydrolyse the biomass into sucrose, we can use enzymes to break
down the biomass in a similar way. However this process is very expensive and is still in its
early stages of development.
Wet Milling Processes
Corn can be processed into ethanol by either the dry milling or the wet milling process. In the
wet milling process, the corn kernel is steeped in warm water, this helps to break down the
proteins and release the starch present in the corn and helps to soften the kernel for the
milling process. The corn is then milled to produce germ, fibre and starch products. The germ
is extracted to produce corn oil and the starch fraction undergoes centrifugation and
saccharifcation to produce gluten wet cake. The ethanol is then extracted by the distillation
process. The wet milling process is normally used in factories producing several hundred
million gallons of ethanol every Year.
Dry Milling Process
The dry milling process involves cleaning and breaking down the corn kernel into fine
particles using a hammer mill process. This creates a powder with a course flour type
consistency. The powder contains the corn germ, starch and fibre. In order to produce a sugar
solution the mixture is then hydrolysed or broken down into sucrose sugars using enzymes or
a dilute acid. The mixture is then cooled and yeast is added in order to ferment the mixture
into ethanol. The dry milling process is normally used in factories producing less than 50
million gallons of ethanol every Year.
Sugar Fermentation Process
The hydrolysis process breaks down the cellulostic part of the biomass or corn into sugar
solutions that can then be fermented into ethanol. Yeast is added to the solution, which is then
heated. The yeast contains an enzyme called invertase, which acts as a catalyst and helps to
convert the sucrose sugars into glucose and fructose (both C6H12O6).

The chemical reaction is shown below:

The fructose and glucose sugars then react with another enzyme called zymase, which is also
contained in the yeast to produce ethanol and carbon dioxide.

The chemical reaction is shown below:

The fermentation process takes around three days to complete and is carried out at a
temperature of between 250C and 300C.
Fractional Distillation Process
The ethanol, which is produced from the fermentation process, still contains a significant
quantity of water, which must be removed. This is achieved by using the fractional
distillation process. The distillation process works by boiling the water and ethanol mixture.
Since ethanol has a lower boiling point (78.3C) compared to that of water (100C), the ethanol
turns into the vapour state before the water and can be condensed and separated.
Microorganisms Used for Bioethanol Production
Microbial biofuel production is already in use, principally in the form of sugar fermentation
by yeast to produce ethanol. Although many microbes have been used in ethanol production,
the yeast species Saccharomyces cerevisiae is primarily used in industry, using starch and
sugars from plants as the starting material for the process . The most common feedstocks
(carbon source utilized by the microbes) are agricultural products which can easily be
processed to create the simple sugars needed for fermentation. This is primarily corn in the
United States, wheat in the European Union, and sugar cane in Brazil.
A major problem that plagues ethanol fermentation by S. cerevisiae is that the fermentation
of sugars by glycolysis also produces ATP, which is then used in reactions of yeast cell
growth and division. Although the production of growth-inducing ATP is a natural function
of glycolysis, in this industrial setting the creation of undesired biomass is essentially an
unwanted byproduct, and represents wasted carbon and energy resources which could have
gone into increased ethanol production. Although this problem can be somewhat resolved by
immobilizing the yeast cells to retard cell growth, the consequence of slowing growth is that
the intracellular ATP concentration quickly increases, and the accumulation of this product of
glycolysis downregulates the entire glycolytic pathway, thereby decreasing ethanol
production. The use of yeast presents other problems as well, particularly the negative effects
of environmental and intracellular stress which accumulate in the fermentation environment.
The challenges faced in such an environment include high concentrations of ethanol, high
sugar concentrations which increase osmostic pressure on the cells, temperatures as high as
38C, high sodium ion concentrations, and others. These factors will often be a greater source
of stress when acting together, making the conditions for S. cerevisiae even worse. The stress
reduces the yeasts overall viability, thereby inhibiting its ability to produce ethanol.
Overall, S. cerevisiae can make ethanol and carbon dioxide from glucose at an efficiency of
about 90-93% of the theoretical yield.

Some of these efficiency problems could potentially be overcome by using different
organisms to perform the fermentation reaction .The production of fuel-grade ethanol is
primarily done by various species of bacteria.The reason bacteria are used is due to their
ability to ferment carbohydrates that cannot be broken down by other microorganisms such as
yeast.
They most common species of bacteria used are Escherichia coli, Klebsiella oxytoca,
and Zymomonas mobilis. All of these species can naturally ferment various types of sugar.
However, in order to maximize ethanol production and meet industrial demands, these
baterica have been genetically modified.

Where are they found?
> Klebsiella Oxytoca is closely related to Klebsiella Pneumonia, which is commonly found
in the mouth, skin and the intestines.
> E. coli is found in the intestinal tracts (lower Intestines) of some mammals, in raw milk,
unchlorinated or contaminated water, unpasteurized apple juice or cider, and uncooked cut
fruits and vegetables.
> Zymomonas mobilis is found in sugar enriched plant saps.
Scientists have turned to their attention to microbes to see if they can find any that are
capable of converting the cellulose and even hemicellulose in lignocellulose into ethanol. The
remaining lignin by-product can be burned to produce energy. They have looked in the
strangest of places from termites stomachs to the soil surrounding volcanoes. What they
have found is a range of very different microbes that all have one thing in common - they
produce a group of enzymes called cellulase.
An archaeon Sulfolobus solfataricus lives in volcanic pools near Mount Vesuvius in Italy. It
produces cellulase. Researchers are looking at ways of genetically modifying this microbe to
see if they can get it to improve its performance and produce more cellulase. In the future S.
solfataricus may be used to produce biofuel.
Another common wood digester is the fungus Trichoderma reesei. It is found in nearly all
soils and secretes huge quantities of cellulase. The fungus was originally discovered by the
United States army during the Second World War. It was responsible for breaking down the
cellulose in the soldiers canvas tents and uniforms which meant they became very holey. It
was known as jungle rot.
A company in Canada has harnessed the microbes ability to convert straw into glucose. The
company genetically modified the fungus so that it produces even larger quantities of
cellulase. A staggering 75 % of the straw fibre is converted into sugar. The left over woody
matter, lignin, is dried and then pressed into burnable cakes. The glucose is then fermented
with yeast to produce the biofuel ethanol.
Algae also could be used for bioethanol production in near future. Algae carry out
photosynthesis. They use the energy from sunlight to convert carbon dioxide into sugar,
which they then metabolize into lipids. Algae lipids can be processed into biodiesel while
algae carbohydrates can be processed into bioethanol. This works well in the laboratory in
small-scale bioreactors. Scientists are investigating if it is practical to grow algae on a large
enough scale to see if they may be the biofuel producers of the future.
Advantages
Exhaust gases of ethanol are much cleaner, it burns more cleanly (more complete
combustion)
The use of ethanol-blended fuels such as E85 (85% ethanol and 15% gasoline) can reduce the
net emissions of greenhouse gases by as much as 37.1%, which is a significant amount.
Positive energy balance Depending to the type of raw stock it can vary from 1.24 to 8. The
output of energy during the production is more than the input. (controversial)
You can use any plant for production of bioethanol, it only has to contain sugar and starch.
The best choice is sugar cane, but you can also use potatoes, barley, wheat etc.
It is carbon neutral i.e. the carbon dioxide released in the bioethanol production process is
the same amount as the one the crops previously absorbed during photosynthes
Ethanol-blended fuel as E10 (10% ethanol and 90% gasoline) reduces greenhouse gases by
up to 3.9%.
The net effect of ethanol use results in an overall decrease in ozone formation, an important
environmental issue. (The emissions produced by burning ethanol are less reactive with
sunlight than those produced by burning gasoline, which results in a lower potential for
forming the damaging ozone).
Ethanol is considered a renewable energy resource because it is primarily the result of
conversion of the sun's energy into usable energy. Creation of ethanol starts with
photosynthesis, which causes feedstocks, such as sugar cane, to grow. These particular
feedstocks are processed into ethanol.
It benefits energy security as it shifts the need for some foreign-produced oil to
domestically-produced energy sources. Countries that do not have access to crude oil
resources can grow crops for energy use and gain some economic freedom.
It reduces greenhouse gases.
It reduces the amount of high-octane additives.
The fuel spills are more easily biodegraded or diluted to non toxic concentrations.
Disadvantages
Biodiversity A large amount of arable land is required to grow crops. This could see some
natural habitats destroyed including rainforests.
The food V fuel debate There is concern that due to the lucrative prices of bioethanol
some farmers may sacrifice food crops for biofuel production which will increase food prices
around the world.
Carbon emissions There is debate over the neutrality of bioethanol when all elements are
taken into consideration including the cost of changing the land use of an area,
transportation and the burning of the crop.
During the production process of bioethanol a huge amount of carbon dioxide is released
which makes its ecological effectiveness close to zero.
The production of ethanol fills the air with greenhouse gases (GHG) in the amounts
comparable to the emissions of internal-combustion engines
The energy content of the petrol is much higher than the one of
bioethanol. Burning 1 liter of ethanol gives 34% less energy than burning
the same amount of petrol. In other words, Bioethanol is not as efficient as
petroleum. Its energy content is 70% of that of petrol.
pay more for more fuel to do the same work. So the use of bioethanol in public
transportation will lead to increasing the price of the service.
There are also concerns over the fuel systems used. Too many older cars
are currently unequipped to handle even 10% ethanol while there is
concern that using 100% ethanol decreases fuel economy by around 15-
30% compared with 100% petroleum.
The octane number of bioethanol is at around 105. It means that this
substance can be burned in the engines with much higher compression
ratio. The engines made for working on the new energy cannot be used for
their petrol or diesel variants.
It can negatively affect electric fuel pumps by increasing internal wear and
undesirable spark generation.
It is not compatible with capacitance fuel level gauging indicators and may
result in erroneous fuel quantity indications in vehicles that employ that
system.
phosphorous and nitrogen used in the production have negative effect on
the environment
Transportation ethanol is hygroscopic, it means that it absorbs water
from the air and thus has high corrosion aggressiveness. Thats why it is
transported only by auto transport or railroad.
Pure ethanol is also difficult to vaporise which can make starting a car in
cold weather difficult and that is why most fuels retain at least a small
amount of petrol such as E85 cars with 85% ethanol and 15% petroleum.