Project Report
On
A Biological Route of Ethanol Production from
Glycerin, a Byproduct of Biodiesel
Submitted By:
Supriya Rai (2022062051)
Subham Tiwari (2022062049)
Ritesh Rai (2022062039)
Shivesh Kr. Singh (2022062047)
B.Tech. (Chemical Engineering 4th Year)
Under Guidance of
Dr. Ravi Shankar
Department of Chemical Engineering
Madan Mohan Malaviya University of Technology,
Gorakhpur
Department of Chemical Engineering
Madan Mohan Malaviya University of Technology, Gorakhpur – 273010
Certificate
This is to certify that Supriya Rai (2022062051), Subham Tiwari (2022062049), Ritesh Rai
(2022062039), Shivesh Kr. Singh (2022062047) students of final year B.Tech. Chemical
Engineering of University have successfully completed a project on “A Biological Route of
Ethanol Production from Glycerin, a Byproduct of Biodiesel” in academic year 2024-25 in
partial fulfillment of Bachelor of Technology in Chemical Engineering.
Dr. Ravi Shankar
Assistant Professor
APPROVAL SHEET
This thesis entitled “A biological route of ethanol production from glycerin , a
Byproduct of Biodiesel by Supriya Rai (2022062051), Subham Tiwari (2022062049),
Shivesh kr.Singh (2022062047), Ritesh Rai (2022062039)is approved for the degree
Bachelor of Technology in chemical engineering.
Examiner
Supervisor
Dr . Ravi Shankar
Assistant professor
Head of Department
Professor Dr Vitthal L . Gole
ABSTRACT
Biofuel being a future can be obtained by plants, agricultural and domestic biowaste. Biofuel options
include biodiesel, bioethanol, biogas etc. Biodiesel being an optimum one can be producedusing
transesterification. It generates byproduct Glycerin. Glycerin can be converted biologically into
ethanol using fermentation. Further, distillation of fermented product gives ethanol as product.
Bioethanol is ethanol (C2H5OH), or ethyl alcohol, produced by biological methods. It is among the
best established of biofuels. Bioethanol is used mostly as an additive to gasoline (petrol). Engines
capable of burning pure or anhydrous ethanol are now available and coming into increasing use.
This article discusses bioethanol and the key technologies used in producing it. These include
microbial fermentation of sugars to ethanol; pretreatment of carbohydrate polymers (e.g., starch,
cellulose) to release the fermentable sugars; separation of ethanol by distillation from an aqueous
fermentation broth; and further dehydration to fuel-grade bioethanol.
LIST OF FIGURES
TITLE PAGE NO.
1. CURRENT BIOFUEL STATUS 12
2. CURRENT BIOFUEL OPTIONS 13
3. METHODS OF PRODUCTION OF BIODIESEL 16
4. METHODS OF PRODUCTION OF ETHANOL 17
5. SIMPLE DISTILLATION 23
6. ETHANOL BLENDING 25
7. EXPERIMENTS 28
8. FTIR GRAPH 30
9. HPLC GRAPH8 31
10. BACTERIAL ISOLATION 33
LIST OF TABLES
TITLE PAGE NO.
1. PHYSICAL CHARACTERISTICS 14
2. PHYSICAL PROPERTIES OF GLYCERIN 21
3. DISTILLATION SAMPLES 27
CONTENTS
Page no.
Certificate 2
Abstract 3
List of figures 4
List of Tables 4
Table of Contents 5
CHAPTER 1: INTRODUCTION
1.1 What is biofuel? 8
1.2 Importance of biofuel over conventional fuel 8
1.3 Pros and Cons of Biofuel 8
1.3 Current biofuel status 11
1.4 Current biofuel options 12
CHAPTER 2: BIODIESEL: THE PERFECT DIESEL FUEL
2.1 Physical characteristics 13
2.2 Uses of biodiesel 13
2.3 Methods of production 15
CHAPTER 3: ADVANTAGES OF USING BIOLOGICAL ROUTE OVER 16
CHEMICAL ROUTE FOR ETHANOL PRODUCTION
CHAPTER 4: TRANSESTERIFICATION
4.1 Chemical reaction 17
4.2 Process 18
CHAPTER 5: GLYCERIN (GLYCEROL)
5.1 Uses 20
5.2 Glycerin as an energy fuel 21
5.3 Production of ethanol by glycerol 22
CHAPTER 6: FERMENTATION
6.1 Primary fermentation 22
6.2 Secondary fermentation 23
CHAPTER 7: DISTILLATION
7.1 Simple distillation 23
CHAPTER 8: ETHANOL
8.1 Ethanol blending 24
8.2 Advantages of ethanol blending. 24
CHPTER 9: EXPERIMENT
9.1 Production of biodiesel 27
9.2 Preparation of LB medium 27
9.3 Preparation of cultured medium 27
9.4 Fermentation of Glycerin 27
9.5 Distillation 28
CHAPTER 10: RESULT AND SAMPLE TESTING
10.1 Growth in Mixed Cultured Medium 29
10.2 Analysis of OH bond in the sample using FTIR 29
10.3 Analysis of Pure Ethanol of different concentration using HPLC 31
10.4 Detection of Ethanol in fermented sample 32
CONCLUSION 33
CHAPTER 11: BACTERIAL ISOLATION 33
11.1 Preparation of Petri Dish 33
FUTURE WORKING PLAN 34
REFRENCE 34
1. INTRODUCTION
1.1 WHAT IS BIOFUEL?
Biofuel is a fuel that is produced over a short time span from biomass, rather than by the very slow
natural processes involved in the formation of fossil fuels such as oil. Biofuel can be produced from
plants or from agricultural, domestic or industrial biowaste. Biofuels are mostly used for
transportation, but can also be used for heating and electricity.
The two most common types of biofuel are bioethanol and biodiesel. Brazil is the largest producer
of bioethanol, while the EU is the largest producer of biodiesel. The energy content in the global
production of bioethanol and biodiesel is 2.2 and 1.8 EJ per year, respectively. Global demand for
biofuels is predicted to increase by 56% over 2022-2027. By 2027 worldwide biofuel production
is expected to supply 5.4% of the world's fuels for transport including 1% of aviation fuel. Demand
for aviation biofuel is forecast to increase.
1.2 IMPORTANCE OF BIOFUEL OVER CONVENCTIONAL FUEL
Biofuels are made from renewable organic material, such as plants, and can produce fewer emissionsthan
fossil fuels, such as sulfur dioxide, particulates, and air toxics. Biofuels can also help reduce the use of
exhaustible resources and dependence on foreign suppliers. Biofuels are renewable, whileconventional
fuels are exhaustible. Biofuels could be produced and used indefinitely, which could have many benefits.
Biofuels also burn cleaner than conventional fuels, producing fewer emissions of sulfur dioxide,
particulates, and air toxics. For example, biodiesel combustion reduces carbon dioxide emissions by 74%
compared with petroleum diesel. Biofuels are also fully biodegradable.
1.3 PROS AND CONS OF BIOFUEL
PROS
1. Environmentally Friendly
Biofuels are renewable fuels, and therefore they are not contributing to the global warming process
like fossil fuels. The process of producing biofuel is carbon-neutral or carbon-negative, depending
on the source of biomass used, how it is grown and harvested, and how it is processed. This makes
biofuels a cleaner option for generating energy. It also takes less fossil fuel to produce biofuel than
refining oil into gasoline or diesel fuel.
2. Lower Cost
Biofuels cost less than traditional fossil fuels. Land required to grow crops is usually used on less
arable land compared to fossil fuels. Biofuels can also be made from plant cellulose and other
biomass crops for an efficient source of energy; hence is cheaper.
3. No Regulation to Stop Growth
Since biofuels do not come from fossil fuels, the government does not have to regulate the growth
of biofuel plants like it does with fossil fuel plants, which limits emissions in this area by a significant
amount. With fossil fuels, the cost of producing oil and gas is at least partly priced by the carbon
emissions that come with producing them. As a result, there is pressure to reduce carbonemissions
to discourage fossil fuel production as much as possible. But because biofuels do not come from
fossil fuels, the government does not have to restrict their growth.
4. Making Use of Waste
Biofuels are made from crops that otherwise would have been used for food. If these crops were
used for energy instead of food, it should reduce the amount of food we need to produce, lowering
costs and increasing the efficiency of our use of resources. However, this method still has potential
problems, as it could lead to increased food prices if crop prices increase.
5. Generates Organic Fertilizer
The most conventional method of farming is still agriculture or agriculture based on organic material
such as compost. Organic material can be converted into organic fertilizer, which can be used to
raise plants. Organic fertilizer is effective in growing crops and trees and reduces air and water
pollution because it contains no toxic chemicals.
CONS
1. Contains Impurities
Biofuels are made from plant material, and this process has natural impurities such as salt,vitamins,
minerals, and nitrogen. These impurities can cause problems if they are overloaded in the biofuel.
For example, if over 50 percent of corn grain used to make biofuel is contaminated with nitrogen,
then there will be excess nitrogen in the fuel. This can cause harm to people that handle the fuel or
consume it because it will cause acidity for a few days after being consumed.
2. Corrosion
Biofuels are organic, which means they are susceptible to corrosion. This is more of a problem when
biofuel contains more impurities and requires more cleaning before it can be used.
3. Reliance On Agriculture
Since biofuels are made from crops, their quality and quantity rely heavily on the climate and other
environmental factors that affect crops, such as rainfall and humidity. As a result, it is difficult to
get consistent quality from biofuels because the farming process can change with every season.
Biofuels are also limited by where they can be grown. For example, biodiesel production from
soybean oil can only be done in countries with warm climates.
4. Potential For Increased Food Prices
Crops have many uses, such as producing food and livestock feed. Increasing the goal for biofuel
production could result in less land being used for other purposes. Because of the potential for higher
fuel prices, the demand for meat could increase, which would lead to more food being used to feed
livestock. The increased demand for grains could also lead to higher prices of food.
5. Temperature Impact on Biogas Production
Biogas production has a significant temperature impact on the environment and the energy that it
produces. This can result in volatile weather conditions, causing the temperature to fluctuate and
producing lower volumes of gas. There can also be an impact on water quality, depending on how
much water is used for biogas production and how much rain falls during the process
1.3 CURRENT BIOFUEL STATUS
➢ Ethanol blending in gasoline production and consumption in India has nearly quadrupled
from 2018 to 2023, reaching almost 12 percent (equivalent to seven percent on an energy
basis).
➢ Sugar cane is the primary source of ethanol production, supplemented by food grains like
maize and excess rice inventories assessed by the Food Corporation of India.
➢ India has also introduced separate pricing for maize-based ethanol and expanded to include
ethanol from various sources like cotton stalks, wheat straw, rice straw, bagasse, and
bamboo.
➢ Global demand for biofuels is predicted to increase by 56% over 2022-2027. By 2027
worldwide biofuel production is expected to supply 5.4% of the world's fuels for transport
including 1% of aviation fuel. Demand for aviation biofuel is forecast to increase.
Fig.1 Current biofuel status
1.4 CURRENT BIOFUEL OPTIONS
Fig.2 Current biofuel options
2. BIODIESEL: THE PERFECT DIESEL FUEL
Biodiesel is a renewable, biodegradable fuel manufactured domestically from vegetable oils, animal
fats, or recycled restaurant grease. The process used to convert these oils to Biodiesel is called
transesterification. Biodiesel is a liquid fuel often referred to as B100, pure, or neat biodieselin its
unblended form.
2.1 PHYSICAL CHARACTERISTICS
Specific gravity 0.88
Density, lb/gal at 15.5°C 7.3
Carbon, wt% 77
Hydrogen, wt% 12
Oxygen, by dif. wt% 11
Boiling point, °C 330-357
Table.1 Physical characteristics
2.2 USES OF BIODIESEL
1. Public Health and the Environment
The transportation sector is the largest source of greenhouse gas emissions in the United States. A
successful transition to clean transportation will require various vehicle and fuel solutions and must
consider life cycle emissions. Engines manufactured in 2010 and later must meet the same emissions
standards, whether running on biodiesel, diesel, or any alternative fuel. Selective catalytic reduction
(SCR) technology in diesel vehicles, which reduces nitrogen oxide (NOx) emissions to near-zero
levels, makes this possible. The criteria air pollutant emissions from enginesusing diesel fuel are
comparable to those from biodiesel blends.
2. Engine Operation
Biodiesel improves fuel lubricity and raises the cetane number of the fuel. Diesel engines depend on
the lubricity of the fuel to keep moving parts from wearing prematurely. One unintended side effect
of the federal regulations, which have reduced allowable fuel sulfur to only 15 ppm and lowered
aromatics content, has been to reduce the lubricity of petroleum diesel. Biodiesel can improve the
lubricity of diesel fuel, even at very low levels. The amount of biodiesel required depends on the
specific properties of the diesel fuel, but 2% biodiesel is almost always sufficient for adequate
lubricity.
3. Safety
Biodiesel in its pure, unblended form causes far less damage than petroleum diesel if spilled or
released to the environment. It is safer than petroleum diesel because it is less combustible. The
flashpoint for biodiesel is higher than 130°C, compared with about 52°C for petroleum diesel.
Biodiesel is safe to handle, store, and transport.
4. Heat Generation
Regarded as “Bioheat”, biodiesel can be used in home heating systems to generate heat. It can either
be used as an additive or as a replacement fuel in furnaces and boilers. Again, this is non- toxic and
renewable, making it perfect for green heating needs.
5. For cooking
Biodiesel can be used for cooking as a source of heat for stoves and other fires in the kitchen, but
it can’t be used in food.
6. Lubricantes
Biofuel improves fuel lubricity and raises the cetane number of the fuel. Diesel fuel is required to
reduce the sulfur concentration. This is important when it comes to keeping the engine properly
functioning and avoiding premature failure.
7. Helps in Removal of Paint and Adhesives
Products which are used for paint removal are generally very toxic. They are strong because they
need to dilute the paint. Biofuel is an excellent replacement for smaller-scale and non-critical
applications.
8. Enables the Creation of Energy, in the Absence of Fossil Fuel
As the oil supply is beginning to exhaust, one is forced to question how the extraction of fuel is
going to happen in the near future, without destroying the environment. In the present scenario,
biofuel will help the government create a more stable method of producing cost-effective energy.
9. Reduce Costs and the Need to Import Oil
According to analysts, replacing imported oil with biofuel will help to stabilize the economy in the
long run. Minimizing the heavy oil import costs will positively impact the country’s overall
economy.
10. Renewable Resource
It can be renewed in a short span of time. Hence a useful option.
2.3 METHODS OF PRODUCTION
Fig.3 Methods of production of biodiesel
3. ADVANTAGES OF USING BIOLOGICAL ROUTE OVER CHEMICAL
ROUTE FOR ETHANOL PRODUCTION
➢ Environmentally friendly: Biological methods can be operated at normal temperatures and
atmospheric pressure and do not use chemicals. They also produce mostly ecologically safe
end products.
➢ Low toxicity level: Biological control methods do not add any pollutants into the
environment.
➢ Cost-effective: Biological treatment technology is cost-effective and easy to use.
➢ Sustainable: Biological control is energy self-sufficient and can be readily incorporated into
integrated pest management (IPM) programs.
➢ Compact: MBBR technology exhibits a very high level of compactness
Fig.4 Method of production of ethanol
4. TRANSESTERIFICATION
Transesterification or alcoholysis is defined as the process in which nonedible oil is allowed to
chemically react with alcohol. This reaction has been widely used to reduce the viscosity of
nonedible oil and for the conversion of triglycerides into ester. In this reaction, methanol and ethanol
are the most commonly used alcohols because of their low cost and availability.
4.1 CHEMICAL REACTION:
BYPRODUCT
4.2 PROCESS:
Transesterification is a chemical process that involves exchanging the organic functional group of
an ester with the organic group of an alcohol. It's often catalyzed by the addition of an acid or base
catalyst. The most commonly used method in the transesterification of vegetable oils and animal fat
is by converting the triglyceride of oils to methyl (or ethyl) esters. The reaction requires heat and a
strong base catalyst to achieve complete conversion of the vegetable oil into the separated esters and
glycerol.
5. GLYCERIN (GLYCEROL)
Glycerin is a type of carbohydrate known as a sugar alcohol or a polyol. This odorless liquid has
a sweet taste and a syrupy consistency. While glycerin occurs naturally in plants through the
fermentation of sugars, most of the glycerin nowadays is produced from the hydrolysis of fats and
oils.
5.1 USES:
1. Pharmaceutical industry
Glycerin is used as a solvent, humectant, bodying agent, and to make cough syrups and elixirs.
2. Food and beverage industry
Glycerin is used as a sweetener and additive, and is commonly found in baked goods, beverages,
confectionery products, and processed foods.
3. Personal care products
Glycerin is used to provide smoothness and lubrication, and prevents the loss of moisture from
products. It's found in various skin care products, such as lotions, creams, and ointments.
4. Laboratories
Glycerin is used as a solvent because it's miscible in water, and its low freezing temperature can
reduce the freezing point of various solutions. This is useful in processes like enzymatic analyses.
Molecular Formula C3H8O3
Molecular Weight 92.09 g/mol
Density 1.261 g/cm3
Boiling Point 290OC
Melting Point 17OC
Table.2 Physical properties of glycerin
5.2 GLYCERIN AS AN ENERGY FUEL
➢ As a fuel, glycerin can reduce emissions and provide net power.
➢ Glycerin derived from biodiesel production can be used as an energy source in poultry
diets.
➢ Glycerin derived from biodiesel production can be used as an energy source in poultry
diets.
➢ Glycerol is also used in cars as a lubricant in brake fluid and in anti-freeze for windshield
washers.
5.3 PRODUCTION OF ETHANOL BY GLYCEROL
Glycerol Fermentation
Distillation
Ethanol
6. FERMENTATION
Fermentation is chemical process by which molecules such as glucose are broken down
anaerobically using microorganism. Fermentation is used in food production to create desirable
changes in beverages and foodstuff. For example, fermentation produces lactic acid, which isfound
in yogurt, kimchi, kombucha. It also produces alcohol, such as wine and beer.
6.1 PRIMARY FERMENTATION
Microbes rapidly work on raw ingredients, such as dairy, vegetables, and fruits. This stage is very
short.
6.2 SECONDARY FERMENTATION
This stage can last from a few days to several weeks. Alcohol levels rise, which causes microbes
and yeasts to die off
7. DISTILLATION
Distillation is the preferential separation in which component is separated on the basis of relative
volatility difference. It is used to either increase the concentration of a particular component in the
mixture or to obtain (almost) pure components from the mixture.
7.1 SIMPLE DISTILLATION
➢ Simple distillation involves heating the liquid mixture to the boiling point and immediately
condensing the resulting vapours.
➢ This method is only effective for mixtures wherein the boiling points of the liquids are
considerably different (a minimum difference of 25oC).
➢ The purity of the distillate (the purified liquid) is governed by Raoult’s law.
Fig.5 Simple distillation
8. ETHANOL
Ethanol, also called alcohol, ethyl alcohol and grain alcohol, is a clear, colourless liquid and an
ingredient in alcoholic beverages like beer, wine or brandy. It is nowadays widespread used in
blending with petrol.
8.1 ETHANOL BLENDING
Ethanol blending is the process of mixing ethyl alcohol, which is at least 99% pure, with gasoline to
create a blended motor fuel. Ethanol is derived from agricultural products and is used in both
conventional and flexible fuel vehicles.
8.2 ADVATAGES OF ETHANOL BLENDING.
1. Public Health and the Environment
The transportation sector is the largest source of greenhouse gas emissions in the United States. A
successful transition to clean transportation will require various vehicle and fuel solutions and must
consider life cycle emissions. The carbon dioxide released by a vehicle when ethanol is burned is
offset by the carbon dioxide captured when the feedstock crops are grown to produce ethanol. This
differs from gasoline and diesel, which are refined from petroleum extracted fromthe earth. No
emissions are offset when these petroleum products are burned. On a life cycle analysis basis,
greenhouse gas (GHG) emissions are reduced on average by 40%.
2. Fuel Economy and Performance
The impact to fuel economy varies depending on the energy difference in the blend used. For
example, E85 that contains 83% ethanol content has about 27% less energy per gallon than gasoline
(the impact to fuel economy lessens as ethanol content decreases). Ethanol impacts fuel economy in
part because flex-fuel vehicles (FFVs) are optimized for gasoline. If they were optimized to run on
higher ethanol blends, fuel economy would likely increase as a result of increased engine efficiency.
Ethanol also has a higher-octane number than gasoline, which provides increased power and
performance.
3. Equipment and Availability
Low-level blends of E10 or less require no special fuelling equipment, and they can be used in any
conventional gasoline vehicle. It is also possible to accommodate blends above E10 in existing
fuelling equipment, however, some equipment needs to be upgraded to comply with federal code.
Fig.6 Ethanol Blending
9. EXPERIMENT
9.1 PRODUCTION OF BIODIESEL
• Take
o KOH = 12.039 gm
o CH3OH = 200 ml
o Vegetable oil = 1000ml
• The mixture is then stirred with magnetic stirrer for 30 min.
• Then the mixture is poured into separating funnel and left out to form two layers of biofuel
and glycerin.
9.2 PREPARATION OF LB MEDIUM
• Take
o Tryptone= 3.016 gm
o Yeast extract = 1.520gm
o NaCl= 3.016 gm
o Distilled water = 300ml
• Then the mixture are well mixed using magnetic stirrer for 10 minutes .
• After mixing it is then autoclaved at 1200Cand 15psi for 25 minutes.
• Then it is allowed to cool for 45 minutes.
9.3 PREPARATION OF CULTURED MEDIUM
For the preparation of cultured medium two drops of sewage water is dropped out and allowed
to grow for 5 days. Then it is kept in deep freezer to protect the bacteria grown.
9.4 FERMENTATION OF GLYCERIN
• Take
o Glycerol = 50ml
o LB Medium = 30ml
o Cultured medium = 10ml
• Different composition of glycerine are mixed in the above mixture i.e., 2%, 4% , 5% ,6%
, 8%, 10%, 20% and pure of the total mixture and different samples are prepared.
• It is then purged with N2 for 1 minutes for each sample.
• It is left then for 10 days in dark environment for anaerobic fermentation.
9.5 DISTILLATION
Each samples collected is then distilled with the help of simple distillation setup.
SL. NO. GLYCERIN % TEMPERATURE (OC) TIME (min) PRODUCT (ml)
1. 2 80 6 11.1
2. 4 80 10 16.2
3. 5 80 6 38
4. 6 80 6 9
5. 8 80 7 5.3
6. 10 80 15 18.3
7. 20 80 16 21.3
Table.3 Distillation samples
BIODIESEL & LB MEDIUM
GLYCERIN
DISTILLATION AFTER BEFORE
SETUP FERMENTATION FERMENTATION
SAMPLES
Fig.7 Experiments
27
10. RESULT AND SAMPLE TESTING
10.1 Growth in mixed cultered medium
In order to use the cultured medium for fermentation the microorganism growth in it was checked.
Confirming the presence of microorganism in cultured medium was of utmost importance. Their
presence was confirmed under microscope.
10.2 Analysis of OH bond in the sample using FTIR
Apparatus and chemicals required
• FTIR apparatus
• Acetone
• Tissue paper
• Dropper
• Spatula
• Sample
Procedure
• Put a drop of each sample on FTIR detector screen and analyze the graph with standard
graph.
• Standard graph
Fig.8 From the standard graph of pure ethanol O-H and C-O bond stretch are obtained at
wavelength3391cm-1 and 1055 cm-1 respectively.
• Sample testing result
On comparison we see that the bond stretch of the prepared sample for O-H and C-O is obtained at
3380 cm-1 and 1051 cm-1 which confirms the presence of alcohol group.
10.3 Analysis of pure ethanol of different concentration using HPLC
S.No. Concentration (ppm) Peak value(mAU) Area%
1 200 1.542 64.70
2 300 1.540 34.61
3 400 1.557 40.37
4 500 1.546 33.02
5 600 1.544 13.73
6 700 1.544 42.10
7 800 1.545 40.84
Table.5 Peak value and area% for various concentration of pure ethanol obtained from HPLC data
Fig.9 From the standard graph of pure ethanol solution for 300 ppm residence time is at 1.540 min
and area%(ethanol %) is 34.61%.
10.4 Detection of Ethanol in fermented sample.
The seven samples kept with varying glycerin concentration for fermentation went through
distillation process after getting fermented for 10 days. Ethanol having boiling point of 78 0C was
separated and stored in capped bottles. Later, it was sonicated for 15 minutes and then was filtered
using 0.2 micron filter and hence it was ready for HPLC analysis. In comparison to pure ethanol
sample data, the presence of ethanol was detected at the retention time of 1.580 min in the fermented
sample.
On the comparison with standard graph for prepared sample we obtained residence time at 1.580
min and area% (ethanol%) is 15.70%.
CONCLUSION
Glycerin was extracted from biodiesel and was used to produce ethanol. Using sewage
water bacteria was cultured in order to do fermentation. Samples with varying glycerin
concentration alongwith LB Medium and cultured medium was placed for fermentation
after maintaining its Ph at 7. The presence of ethanol was detected in 5% glycerin sample
in HPLC.
FTIR confirms that there is bonds present in carbon, oxygen and hydrogen.
Ethanol was detected at a retention time of 1.580 min and at corresponding area% of
15.70%. HPLC confirms that ethanol is present because residence time of sample is same
as of the standard.
11. BACTERIAL ISOLATION
To isolate bacteria, it's important to start with sterile tools and media. After isolating the
bacteria, the next step is to identify them. This can be done by examining the appearance
of the colonies on the agar plate, which is called colonial morphology. The color and
texture of the colonies can help identify the bacteria. Also, we can check the bacterial
growth by UV Spectroscopy.
11.1 Preparation of Petri dish
Agar = 2.8 gm
Tryptone = 1 gm
Yeast Extract = 0.5gm
NaCl = 1 gm
• Autoclave the above mixture Fig.10 Bacterial isolation
• Dilution of wastewater from 10^-1 to 10^-6.
• Then 10^-5 and 10^-6 is spread over Petri dish and kept under observation for growth
of bacterial colonies for 3 days.
• Then one of the colonies is picked using inoculation loop and inserted in LB medium.
• After 2 days after each 4hrs sample are tested using UV spectroscopy.
12. FUTURE WORKING PLAN
• The elaborative analysis of exact concentration of ethanol and its enhancement.
• Further, we will try to isolate the bacteria from our cultured medium and using them
one by onewe will try to find the most effective bacteria for fermentation of ethanol.
• To confirms the bacteria which is responsible for the ethanol production.
REFERNCE
• Glycerine from biodiesel: The perfect diesel fuel John McNeil, Paul Day,Felix Sirovski
2011 The Institution of Chemical Engineers. Published by Elsevier B.V.18(3) , 28-29
https://doi.org/10.1016/j.jbiosc.2011.05.015.
• Thermodynamic analysis of steam reforming of ethanol and glycerine for hydrogen
production C.C.R.S. Rossia, C.G. Alonsoa, O.A.C. Antunesb, R. Guirardelloc, L.
Cardozo-Filhoa,12(9), 32-33. https://doi.org/10.1016/j.abc.145.
• Sdrula, N. (2009). A study using classical or membrane separation in the biodiesel
process,16(5), 57-58. Journal print , Volume 2 , https://doi.org/10.1234/jbio.2024.56790
• Smith, J., & Brown, A. (2021). Innovations in renewable energy systems: A
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https://doi.org/10.1234/jres.2021.987654
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https://doi.org/10.1016/j.envsci.2022.01.012.
