1.

General view of Biochemical or Distillery

2. Fermentation

 Raw Material
 Molasses
 Definition

A dark brown thicker liquid which is by product of making of sugar. For example sugar cane
molasses, Beet molasses. The thick syrup that’s left behind is molasses. The boiling process can be
repeated multiple times, creating the different varieties of molasses.

Chemical Properties of Molasses

Molasses is a by-product of the sugar manufacturing process, and it has several chemical properties
that give it unique characteristics and uses. Here are some of the chemical properties of molasses:

 High sugar content: Molasses is composed mainly of sugar and water, with sucrose being the
most abundant sugar. It typically contains around 45-55% sucrose.
 High mineral content: Molasses is also rich in minerals like calcium, potassium, and
magnesium. These minerals are necessary for several essential bodily functions.
 Low pH: Molasses has a slightly acidic pH, around 5-6. This low pH makes it useful in
preserving foods, acting as a natural preservative.
 Antimicrobial properties: The high mineral content and low pH of molasses also give it
antimicrobial properties, which makes it useful in preventing the growth of harmful bacteria
in certain applications.
 High viscosity: Molasses has a high viscosity, or thickness, which makes it useful in various
applications such as in the production of animal feed supplements.
 High calorific value: Due to its high sugar content, molasses has a high calorific value, which
makes it a valuable source of energy in animal feed.

Overall, the chemical properties of molasses make it a valuable ingredient in many applications,
ranging from animal feed supplements to food flavorings and syrups.

 Types of molasses
 Blackstrap molasses
 Dark molasses
 Light molasses
Description

The main difference between these types comes down to the maturity of the sugar cane or sugar beet, the
amount sugar extracted throw the boiling process and extraction method. Light molasses gets after the
first boiling process.it is sweetest of all type of molasses because it has most sugar left in it most
commonly used in molasses cookies cakes and bread.

Light Molasses

After the first processing of sugar, you’re left with light molasses: The sweetest and lightest-colored type
in the group. It’s the most popular type of molasses because it has the highest sugar content. It has a mild
flavor and can be used as a substitute for maple syrup on pancakes, sugar in coffee or bake with it to
make your favorite molasses cookie recipe.

Dark Molasses

If the molasses is boiled a second time, you end up with dark molasses. It’s darker and thicker the light
variety, with a deeper, richer flavor that hints at bitterness. It’s not quite as sweet, but it’s also not as bitter
as blackstrap molasses and it makes an incredible gingerbread.

Blackstrap Molasses

The third and last boiling of molasses results in the deepest, darkest, most bitter version of molasses:
blackstrap molasses. It’s sometimes referred to as the healthiest molasses because it contains a ton of
vitamins and minerals, including iron, manganese, copper, calcium and potassium. It also has a lower
glycemic value because most of the sugar was extracted during the triple processing. It’s strong and bitter,
but it’s great for savory cooking like baked beans. The analysis of blackstrap, and particularly of sugars in
it, may vary considerably depending on the variety of sugar cane, soil, climate, period of the crop,
efficiency of the factory operation, system of sugar boiling, type and capacity of crystallizers etc.
 Composition of molasses
 Molasses Composition

Sr. # Composition Parameters Sr. # Composition Parameters

1
Brix spindle 86.0degrees 22 Magnesium 0.27%
2
Weight /Gallon 11.8-12.0lbs 23 Sulfur 0.78%
3
Nitrogen 1.01% 24 Sodium 0.06
4
Crude Protein 6.30% 25 Copper 14ppm
5
Total Sugar 48.30% 26 Iron 130ppm
6
Density (as fed ) 11.8lbs /gal 27 Manganese 5ppm
7
Dry Matter 76.50% 28 Zinc 8ppm
8
Moisture 23.50% 29 Cobalt Negligible
9
Ash 16.00%- 30 iodine Negligible
10
Organic Matter 62.50%. 31 Selenium Negligible
11
Reducing Substance 11.50% 32 Biotin 3ppm
12
as dextrose 33 Folic Acid 0.04ppm
13
Sucrose 35.90% 34 Inositol 6000ppm
14 Calcium
Fructose 5.60% 35 Pantothenic 60ppm
15
Glucose 2.60% 36 Pyridoxine 4ppm
16
pH 4.9-5.4 37 Riboflavin 2.5ppm
17
Calcium 0.80% 38 Thiamine 1.8ppm
18
Phosphorus Negligible 39 Niacin 500ppm
19
Not for Use 40 Choline 700ppm
20
Potassium 4.20% 41 Choline 700ppm
21
Chloride 2.10% 42

 Molasses Content
  Total Sugar
 Total Sugar As Invert
 Reducing Sugar
 Non-Reducing sugar
 Fermentable Sugar
 Non-Fermentable Sugar
 Un Fermentable Sugar

Total Sugar

Total Sugar (Reducing Sugar - Inverted) is a measurement of sucrose and reducing sugars. The most
common reducing sugars are glucose and fructose. Sucrose is a non-reducing sugar. Any sucrose present
in a sample must be broken down (inverted) into its individual component parts, glucose and fructose,
before running Total Sugar analysis.

Total Sugar as Inver

Total sugar as invert refers to the conversion of sucrose into its constituent sugar, glucose, and fructose.

Reducing Sugar

A sugar that serves as a reducing agent due to its free aldehyde or ketone functional groups in its
molecular structure. Examples are glucose, fructose, glyceraldehyde’s, lactose, arabinose and maltose,
except for sucrose.

Is glucose a reducing sugar? Yes.

Is fructose a reducing sugar? Yes.

Is maltose a reducing sugar? Yes

Is lactose a reducing sugar? Yes

All monosaccharaides are reducing sugars. Glucose, fructose, and galactose are monosaccharide’s and are
all reducing sugars. . Maltose (glucose + glucose) and lactose (glucose + glucose) have a free aldehyde
group and thus are reducing sugars.

Non-Reducing sugar
Is sucrose a reducing sugar? No

Sucrose (glucose + fructose) lacks a free aldehyde or ketone group and therefore is non-reducing.

Fermentable Sugar

Fermentable carbohydrates are the sugars that are easily fermented in your digestive system, which
include oligosaccharides, disaccharides, monosaccharaides and polyols.

Non-Fermentable Sugar

Non Fermentable Sugar is fermented by bacteria to form lactic acid, and sucrose, which cannot be broken
down by yeast. Non-fermentable sugars are usually added to beverages to sweeten them and to add body.
Examples of non-fermentable sugars include lactose.

Un-Fermentable Sugar

Un fermentable sugar refers to a type of that cannot be easily or completely converted into alcohol or
carbon dioxide through the process of fermentation, unfermentable sugar are not metabolized by yeast or
bacteria during fermentation.

 Storage of Molasses
• Molasses at over 80 brix is quite stable but reactions an gas evolution do occur.
• Molasses is usually stored in tanks or covered underground pits.
• Storage should be “cool”. Cooled after sugar extraction and not in direct sun. Tanks should be in
shady area.
• Tanks painted a light color.
• Clean up spills.
• Air flow to stop condensation and release gasses Molasses Storage.
• Molasses degrades during storage producing gases and losing fermentable sugars.
• Storage over 60 risks explosion.
• Molasses at over 80 brix has low bacteria loading.
• Leuconostoc can be active creating polymer chains dextran’s also lactic acid and diacetyl.
• Spores may be present but not active. Heat does not kill spores.
• Heat does not move easily through molasses.
• Does not mix well so laying can form.
Molasses Degradation

MOLASSES has sugar content around 50% and, with a Brix above 85, it should not deteriorate during
storage. However, it is not uncommon to observe reduction in sugars during storage. Sometimes, this
deterioration is accelerated and instances of foaming with or without a rise in temperature are observed,
which changes the colour and smell of molasses. There is a rapid reduction in sugars and a rise in acidity.
Microscopic observation revealed that the microbial population is very high, and further microbial
evaluation confirmed the growth of two bacteria growing in consortium as single bacteria which required
a minimum sucrose content of 30% when grown under anaerobic conditions. When this consortium, was
broken in aerobic conditions, none of the isolates could tolerate more than 5% sucrose. Their growth and
thus molasses deterioration was controlled only with the use of Polmax ESR, a special biocide
formulation used with continuous circulation of molasses with cooling. The possible reasons for such
infection and efforts to control or avoid such deterioration in some factories will be presented.

Molasses usually has more than 850 Brix i.e. only 15% moisture. It contains more than 45% fermentable
sugars. If we apply theory, it is impossible to think of any microorganism which could survive in this
environment for too long! We know that fungi, the best known living entities at low moisture, do not
grow when moisture is lower than 18%, and there are very few microbes that can tolerate more than 30%
sugar. It is believed that molasses is stable and does not deteriorate during storage. All reports of
degradation of molasses during storage indicate that it is due to the Millard reaction. This is the
chemical reaction between reducing sugars and nitrogenous compounds / amino acids present in
molasses. This reaction is exothermic, generates tremendous heat and usually is an uncontrollable
reaction. Many molasses tanks are known to have exploded / burned where molasses mass is charred to
black mass. The correct reason for the initiation of Millard reaction is yet to be known and nothing.

Foaming of Molasses

Foaming of molasses revealed that two types of bacteria are involved in the deterioration. These bacteria
find their way to molasses from cane, form a consortium and initiate growth in combination under
anaerobic conditions in molasses. One of them is a Gram +Ve rod and the other one is a Gram – Ve
short rod. It was very difficult to separate them anaerobically, and they required minimum 30% sucrose
concentration to grow. However, on separation, none of them could tolerate even 5% sucrose! They
possibly belong to Lactobacillus and Pseudomonas respectively, and produce acid and gas by
consuming sugars in molasses. This causes molasses to foam and the cell count is often more than 1010
per g! We have controlled this foaming by treating it with continuous addition of Polmax ESR @ 100
ppm dose.
Effect of Temperature:

Fermentation
Yeast
A microscopic fungus consisting of single oval cells that reproduce by budding, and capable of
converting sugar into alcohol and carbon dioxide. A greyish-yellow preparation of the yeast fungus
obtained chiefly from fermented beer, used as a fermenting agent, to raise bread dough, and as a food
supplement.
1. Saccharomyces cerevisiae
2. Zymomonas mobilis
3. Pichia stipites
4. Kluyveromyces marxianus
5. Candida spp

Saccharomyces cerevisiae:

Saccharomyces cerevisiae, commonly known as baker's yeast or brewer's yeast, has undergone various
modifications and improvements over the years through genetic engineering and selective breeding.
These modifications aim to enhance its performance and suitability for specific industrial applications,
including ethanol fermentation. Some of the key modifications made to Saccharomyces cerevisiae yeast
strains include
Following brand of Saccharomyces cerevisiae are used in the Pakistan,
1. Red ethanol
2. Thermosacc
3. Pinnacle
4. Ravi
5. Razavi
6. Saf instant
7. Mauripan

Name of Yeast Brand Temperature Life Span

Red Ethanol High temperature bearable

Angel yeast low temperature bearable

Mauripan low temperature bearable Moderate life span

Saf-Gold Moderate life span

Pinnacle Moderate life span

Thermosac High temperature bearable

Active Yeasts
Active yeast refers to fresh or live yeast that is still active and capable of fermentation. It is typically sold
in moist or semi-moist form, often as compressed yeast cakes or blocks. Active yeast contains a high
moisture content and needs to be refrigerated to maintain its viability. It is used in baking and
fermentation processes where it is added directly to the dough or fermentation mixture. Active yeast
requires proofing or activation in warm water before use to ensure its viability and to quick start the
fermentation process.
Dry Yeasts:
Dry yeast refers to yeast that has been dehydrated to reduce its moisture content. It is available in the
form of small granules or powder. Dry yeast has a longer shelf life and does not require refrigeration,
making it more convenient for storage and transportation. Dry yeast can be added directly to the dough or
fermentation mixture without the need for proofing. It activates and rehydrates during the mixing or
fermentation process, provided there is sufficient moisture and suitable conditions.
Instant Dry Yeasts:
Instant dry yeast is a type of dry yeast that has been further processed to have finer particles and higher
potency. It is also known as rapid-rise yeast or quick-rise yeast. Instant dry yeast dissolves and rehydrates
more quickly than regular dry yeast, allowing for faster fermentation. It can be directly added to the dry
ingredients in baking recipes without the need for proofing or activation in warm water. Instant dry yeast
is commonly used in commercial baking and is convenient for home baking as well, as it speeds up the
rising time of the dough.
Both dry yeast and instant dry yeast have a longer shelf life compared to active yeast and can be stored at
room temperature. They are widely used in baking, including bread-making, where they provide reliable
and consistent results while offering convenience and ease of use

GMO:

GMO (Genetically Modified Organism): An organism, typically a plant or an animal, whose genetic
material has been modified using genetic engineering techniques. This involves the manipulation and
transfer of genes from one organism to another, often unrelated species, to introduce specific desired
traits or characteristics.

GMO Purpose:

GMOs are created for various purposes, including improving ethanol production, enhancing nutritional
content, and increasing resistance to pests, diseases, or environmental conditions.

GMO certified yeast:

These Saccharomyces cerevisiae yeast strains are genetically modified organism (GMO) certified.

 Red ethanol
 Thermosacc
 Pinnacle

Ethanol Tolerance:
Ethanol fermentation generates high concentrations of ethanol, which can be toxic to yeast cells. To
improve ethanol tolerance, yeast strains have been selected or engineered to withstand higher ethanol
concentrations. This allows for more efficient fermentation and higher ethanol yields.
Sugar Utilization:
Yeast strains have been modified to enhance their ability to ferment a broader range of sugars. This
includes the utilization of alternative sugars like xylose, arabinose, and lactose, which are present in
various biomass feed stocks. By expanding the range of fermentable sugars, yeast strains can be used in
diverse ethanol production processes.
Stress Resistance:
Yeast strains have been developed with increased resistance to various stress conditions encountered
during fermentation, such as high temperatures, acidic pH, and osmotic stress. These modifications ensure
that the yeast can perform optimally under challenging fermentation environments.
Nutrient Utilization:
Yeast strains have been engineered to improve their utilization of essential nutrients, such as nitrogen
sources. This helps enhance the overall fermentation efficiency and ethanol production rate.
Coenzyme Preferences:
The coenzymes involved in yeast metabolism, such as NADH and NADPH, have been modified to
optimize their utilization and enhance the efficiency of metabolic pathways related to ethanol production.
This can improve the overall yield and productivity of ethanol fermentation.
Today's yeast strains, compared to earlier strains, exhibit improved fermentation performance, higher
ethanol tolerance, broader substrate utilization, and increased stress resistance. These modifications have
been made through a combination of traditional breeding methods and genetic engineering techniques. As
a result, modern yeast strains are more robust, efficient, and adaptable for various ethanol production
processes. The modifications have played a crucial role in advancing the field of biofuels and industrial
fermentation.
Selective breeding, also known as strain improvement or strain selection, has been a key approach in
improving yeast strains for various industrial applications, including ethanol fermentation. Through
selective breeding, yeast strains with desirable traits are selected and propagated, leading to the
development of improved strains. Some common selective breeding techniques employed to enhance
yeast strains include:
Phenotypic Selection:
This method involves screening yeast populations for specific phenotypic traits. Yeast strains that exhibit
desirable characteristics, such as high ethanol production, ethanol tolerance, or robustness under specific
fermentation conditions, are selected for further breeding.
Mutagenesis:
Mutagenesis involves introducing mutations into the yeast genome to generate genetic diversity. This can
be achieved through exposure to mutagens such as chemicals or radiation. Mutagenized yeast populations
are then screened for desirable traits, and strains with improved fermentation performance or other
desired attributes are selected for further breeding.
Hybridization:
Hybridization involves crossing different yeast strains with complementary traits to obtain offspring with
a combination of desired characteristics. By carefully selecting parent strains with specific traits,
hybridization can lead to the development of new yeast strains with improved properties, such as
enhanced ethanol yield or broader substrate utilization.
Adaptive Laboratory Evolution (ALE): ALE is a method of long-term selection and cultivation of
yeast under specific environmental conditions. Yeast strains are subjected to repeated cycles of growth
and selection under specific fermentation conditions, such as high ethanol concentrations. Over time,
yeast cells adapt and evolve to better tolerate and thrive in the given conditions, leading to the
development of strains with improved performance.
Marker-Assisted Selection:
Marker-assisted selection involves the use of genetic markers to identify and select yeast strains with
specific traits. Genetic markers linked to desired traits, such as ethanol tolerance or sugar utilization, are
identified, and strains carrying these markers are selected for further breeding.
Selective breeding techniques aim to optimize the combination of traits in yeast strains, such as high
ethanol production, ethanol tolerance, stress resistance, and substrate utilization. These techniques have
been crucial in developing yeast strains that are more efficient, robust, and well-suited for industrial-scale
ethanol fermentation and other biotechnological applications. Genetic engineering techniques have played
a significant role in improving yeast strains for various industrial applications, including ethanol
fermentation. Some common genetic engineering approaches used to enhance yeast strains include: Gene
Overexpression:
By introducing extra copies of specific genes, genetic engineers can enhance the production of key
enzymes involved in ethanol fermentation. For example, genes encoding enzymes like pyruvate
decarboxylase and alcohol dehydrogenase, which are responsible for converting pyruvate to ethanol, can
be overexpressed to increase ethanol production.
Gene Knockout:
Genetic engineers can selectively delete or "knock out" specific genes to eliminate or modify metabolic
pathways that compete with ethanol production. This can redirect metabolic flux towards ethanol
synthesis, resulting in higher ethanol yields.
For example, knocking out genes involved in byproduct formation or alternative metabolic pathways can
enhance ethanol production.
Metabolic Pathway Engineering: Genetic engineering techniques can be used to introduce or optimize
metabolic pathways in yeast cells to enhance ethanol production. This involves introducing genes from
other organisms or modifying existing genes to enable the efficient utilization of alternative sugars or to
enhance the efficiency of ethanol synthesis pathways.
Stress Tolerance Enhancement: Genetic engineering can be employed to enhance yeast strains'
tolerance to stress conditions encountered during fermentation, such as high temperatures, ethanol
toxicity, or osmotic stress. This can involve introducing genes encoding stress response proteins,
chaperones, or transporters that improve cellular resilience and viability under challenging fermentation
conditions.
Genome Editing:
Advanced genome editing techniques, such as CRISPR-Cas9, allow for precise modifications of the yeast
genome. Genetic engineers can use these tools to introduce specific genetic changes or modifications to
optimize yeast strains for ethanol fermentation. This includes precise gene deletions, insertions, or
modifications to improve metabolic pathways or other desired traits.
Synthetic Biology Approaches:
Synthetic biology combines genetic engineering with principles from engineering and computer science
to design and construct novel biological systems. By applying synthetic biology approaches, researchers
can engineer yeast strains with customized features, such as enhanced ethanol production, improved
substrate utilization, or tunable metabolic pathways. These genetic engineering techniques enable
researchers to tailor yeast strains to meet specific industrial requirements, such as increased ethanol
yields, improved fermentation efficiency, or utilization of diverse feed stocks. They have significantly
contributed to the development of advanced yeast strains for ethanol production and other
biotechnological applications.