Alcohol Fermenttion by Immobilised Yeast Cells

“ALCOHOL FERMENTATION BY IMMOBILIZED YEAST CELLS”
Thesis submitted to the
Institute of Biosciences and Technology,
Shri Ramswaroop Memorial University,
Lucknow Deva Road, Uttar Pradesh
For
The partial fulfillment of the requirement for the degree of
MASTER OF SCIENCE
In
BIOTECHNOLOGY
Submitted by
Ms. VANISTHA SHUKLA
Under guidance and supervision of
Dr. SANTOSH KUMAR
ASSISTANT PROFESSER OF
BIOCHEMISTRY DEPARTMENT
NATIONAL SUGAR INSTITUTE
Ministry of Consumer Affairs, Food & Public Distribution,
Department of Food & Public Distribution ,Government of India, Kanpur-208017
2018
CERTIFICATE
This is to certify that the thesis entitled ―ALCOHOL FERMENTATION BY

IMMOBILIZED YEAST” submitted in partial fulfillment of the requirements for the degree of
Master of Science in M.sc in Biotechnology of the Institute of Bioscience and Technology, Shri
Ramswaroop Memorial University, is a record of bonafide research carried out by
Ms.Vanistha shukla Roll No. 201610901010002.
The assistance and help received during the course of this investigation have been
acknowledged.
Place: Barabanki Signature of Director
Date:
ACKNOWLEDGEMENT
First and foremost I bow my head with humanity before almighty GOD who is prevailing upon
all the living and dead and is the cause behind each effect and his abundant love and blessing
provide me the wisdom and strength to complete this dissertation in this manner.
I would exert my greatest thanks to Shree Narendra Mohan, Director, National Sugar
Institute (NSI), Kalyanpur , Kanpur , for allowing me to carry out the work at institute.
I would like to express my grandiose thanks and sincere gratitude to Dr. Santosh Kumar
Assistant professor of Biochemistry, National Sugar Institute, Kanpur, for his fruitful
supervision, valuable suggestion which help me to go in a right direction.
A plethora of thanks also goes to Dr. Alka Gupta Lab Assistant and Dr. Vinitanjali Banerjee
(Researcher) Biochemistry Division, NSI, Kanpur, for their whole hearted help during the course
of investigation.
I also thanks to lab attendant Shri Rakesh Biochemistry Division for their attitude in carrying out
all my experiments in time with perfect result.
This keen interest through encouragement and helping attitude during the course of
investigation is highly appreciated.
I find no theoretical gems from the ocean of my heartfelt sense of gratitude to my parents Mr.
Mahim Kumar Shukla & Mrs. Anita Shukla for my education, spring of inspiration and
sacrifice since I saw light on this earth. Words would not be enough as to how to express
gratitude to beloved mother Mrs. Anita Shukla, father Mr. Mahim Kumar Shukla. My family
‗s moral support , encouragement , their love , affection and advise was a great help and
guidance for me during my dissertation work, which will ever remain in the depth of my heart
now, always and forever. My mother‘s blessing were the source of inspiration and power.
Place: Kanpur Vanistha Shukla
Date:
DECLARATION
I hereby declare that dissertation entitled ― comprises the work done by me submitted to SHRI
RAMSWAROOP MEMORIAL UNIVERSITY ,DEVA ROAD LUCKNOW, in partial
fulfillment of the requirement of the award of degree of Master of Science in
BIOTECHNOLOGY is my own, conducted under guidance and supervision of Dr.
SANTOSH KUMAR (Assistant Professor, Biochemistry) , National Sugar Institute , Kanpur
.
I further declare that to best of my knowledge that dissertation does not contain any part of work
, which has been submitted for the award of any degree or diploma either in this university or
elsewhere. The data mention in this report were obtained during genuine work and collected by
me and where other source of information is used, they have been duly acknowledged.
Place: Kanpur Vanistha Shukla
Date:
TABLE OF CONTENT
Chapter 1: Introduction……………………………………
Chapter 2: Review of Literature………………….................
Chapter 3: Materials and methods…………………….........
Chapter 4: Results and Discussions…………………...........
Chapter 5: Summary and conclusion………………………..
References……………………………………………............
LIST OF ABBREVATIONS
 %...........................................................Percentage
 ○C……………………………………..Degree Celsius
 g……………………………………….Grams
 L……………………………………….litre
 mg……………………………………..Milligram
 ml……………………………………...Milliliter
 DW…………………………………….Distilled Water
 h………………………………………..Hours
 FE………………………………………Fermentation Efficiency
 FS……………………………………….Fermentable Sugar
 RS……………………………………….Residual Sugar
 TRS……………………………………...Total Reducing Sugar
ABSTRACT
Roll No: 201610901010002
Name: Vanistha shukla
Semester & Year of admission: fourth semester & 2016-2018

Degree: M.Sc biotechnology
Thesis Title: “ALCOHOL FERMENTATION BY IMMOBILIZED

YEAST CELLS‖
The aim of this study is to synthesize alcohol from molasses by using immobilized yeasts cells
for optimized time period and compared the fermentation efficiency from the molasses.
Fermentation by saccharomyces cerevisiae immobilized by entrapment in alginate. The rate of

ethanol production by the entrapped cells was 20-25% higher than that of the free cells. The
different concentration of sodium alginate and yeast were used for the fermentation by different
types beads like , ordinary immobilized beads and immobilized glass beads and their alcohol
productin
the fermentation was done by immobilized ordinary beads and immobilized glass beads and the
dilution of molasses 3.5 times and after that separate the alcohol by the distillation technique.
Fermentation efficiency was also determined during fermentation process after asserting
parameters like TRS, RS and ethanol content
Various parameters showed encoring result with regard to fermentation efficiency and better
results shown the ordinary beads shown the 94% efficiency and the glass beads shown the 90%
efficiency ranges .
The maximum fermentation efficiency value is 94.74 by 6g alginate and 12g yeast ordinary bead
for 48 hours in 9 cycle and glass beads fermentation efficiency 90.44 by 10g alginate and 12 g in
48 hours. The better result given by ordinary immobilized beads rather than immobilized glass
beads, and the other concentration combination of yeast and alginate given less fermentation
efficiency compared to these combination.
CHAPTER 1 : INTRODUCTION
INDIA is the acknowledge original home of both sugarcane cultivation and sugar manufacture.
Indian mythology supports the above fact, as it contains some legends showing the origin of
sugarcane. The growth of sugar industry is full of tales of adventure and conquest. It received the
attention of builders of different Empires from time to time. ―Legend has it, that around 800 B.C.
sugarcane was taken to China where soil conditions were ideal for its development and that the
Greek invasion of the country under Alexander the Great marked the entry of sugarcane into
Europe. The actual cultivation of the crop in that continent began in 700 A.D.‖
Sugar manufacture was started in India between the fourth and sixth centuries and was initially
carried out through the simple process of crushing cut-pieces of cane by a heavy weight and
boiling the juice so obtained and stirring until solids formed. These solids being of uneven
shapes and sizes were called Sarkara, the Sanskrit term for gravel. The modern Word ―Sugar‖ is
derivative of the word Sarkara. The larger solids were called Khand from which the word candy
has descended. Knowledge of sugar-making spread to China and Persia.
Sugar industry is the second largest agro based industry in India. Its contribution to the Indian
economy is enormous. With a total turnover of around Rs. 20000 crore per-annum, the Indian
Sugar Industry is amongst the largest tax payers, contributing around Rs. 1800 crore per-annum
to the Central and State exchequers. Further, about 4.5 crore sugarcane farmers, their dependents
and a large number of agriculture labour and about 5 lakh skilled and semi-skilled workers
mostly from rural areas, earn their livelihood from the sugar industry. It is also employment
generated industry through its various ancillary activities, various agencies of distributive trade
and through subsidiary industries such as confectionery and alcohol. By way of sugarcane price
about Rs. 12000 crore are disbursed amongst cane farmers every year.
MANUFACTURE OF SUGAR-
The sugar commonly used is white crystalline in shape. The present system of sugar manufacture is
subdivided into following stages:
 Extraction of juice from sugarcane by milling
 Clarification of juice
 Concentration of juice by evaporation to syrup
 Crystallization of juice by vacuum pan boiling
 Centrifugal separation of sugar and molasses from the massecuites
 Drying and cooling of sugar
 Sugar grading and packing
The various by-products of sugar industry also contribute to the economic growth of the nation to
promoting the number of supplementary industries. Sugarcane has emerged as a multi-product crop used
as a basic raw material for the production of sugar, ethanol, paper, electricity via co-generation. The
feeding of cattle of sugarcane is important source of bio-energy, and more demand in rural area. Molasses
is an important nourishing stock for distilleries. In sugar industry, production of electricity using bagasse
was the typical option 3 and use of bagasse as an alternative raw material for wood pulp for economic and
environmental sustainability.
Sugar is a seasonal and agro-based industry. Sugar can be produced from sugarcane, sugar-beet
or any other crop having sugar content. But in India, sugarcane is the main source of sugar. ―The
crushing season of sugar industry ranges between 180 and 240 days in a year depending on the
location, climatic factors and cultivation patterns in area. In the manufacture of sugar, there are
two by-products- -bagasse and molasses.
BAGASSE-
Bagasse is the by-product or residue of milling or diffusing cane, the woody Fiber of the cane.
When sugarcane is crushed in the milling plants of the sugar. Factories for extracting its juice,
the fibrous residue left over after the extraction of juice is known as ―Bagasse‖. Bagasse contains
mainly fiber, pentosans, lignin, sugar and minerals. Bagasse Production generally amounts to one
third of the cane crushed. Bagasse comes from Saccharum officinarum after extraction of juice
from sugarcane which is perennial monocotyledon and belongs to the large grass family.
MOLASSES:
Molasses, also called treacle, syrup remaining after sugar is crystallized out of cane or beet
juice. Molasses syrup is separated from sugar crystals by means of centrifuging. Molasses is
separated from the sugar crystals repeatedly during the manufacturing process, resulting in
several different grades of molasses; that obtained from the first extraction contains more sugar,
tastes sweeter, and is lighter in color than molasses obtained at the second or third extractions.
The third and final extraction yields blackstrap molasses, a heavy, viscous, dark-colored product
that has had all the sugar removed from it that can be separated practically by ordinary
crystallization.(1)
Molasses can be used:
 In some cookies and pies

 In gingerbread (particularly in the Americas)
 In barbecue sauce
 In beer styles such as stouts and porters
 To stabilize emulsification of home-made vinaigrette (2)
 The principal ingredient in the distillation of rum
 An iron supplement
 A source for yeast production
 The main ingredient in the production of citric acid
 An additive in tobacco(3) smoked in a hookah (4)
Molasses is the mother liquor remaining after crystallization and removal of sucrose from the
cane juice. Molasses is a sweetener check that is formed as a byproduct of the sugar-making
process. First, sugar cane or sugar beets were crushed and the juice was extracted. The juice was
then boiled to form sugar crystals, which were removed from the liquid. Molasses is the thick,
brown syrup left after the sugar has been removed from the juice. This process is repeated
several times, and each time a different type of molasses is produced. Molasses defined as the
final mother liquor obtained in manufacture of sugar by repeated crystallization and
centrifugation. The TDS of the molasses have (Total Dissolved Solids) in the range of 95000 to
100,000 mg/l.70 to 80% of molasses is converted into alcohol in distilleries and the 6 to 8% of
molasses used in cattle feed and 3 to 6% production of many chemicals and organic acids
Sugarcane molasses is a viscous, dark and sugar-rich by-product of sugar extraction from the
sugarcane (Saccharum officinarum L.). It is a major feed ingredient, used as an energy source
and as a binder in compound feeds.
Cane molasses, major by-product of the cane sugar industry, has found use as cattle feed and also
as a raw material resource for the production of alcohol, yeast, and other fermentation products.
However, for the development of new or improved use of molasses, more complete knowledge
of the chemical composition of cane molasses is desirable.
Cane final molasses had been used as raw material for the production of alcohol, and
biochemical, for example, monosodium glutamate by fermentation, for the production of yeast,
and in livestock and poultry feeds. However, for the development of new or improved use of
molasses more complete knowledge is desirable. These, however, had been mainly concerned
with carbohydrates, vitamins, non-nitrogenous acids, pigments, waxes, sterols, lipids, a few
amino acids, and inorganic compounds.
COMPOSITION OF CANE MOLASSES-
AVERAGE COMPOSITION OF CANE MOLASSES-
 Composition of cane molasses depends on several factors – locality, cane variety, soil,
climate and cane processing
 Cane molasses pH-5.5 to 6.5 contains 30 to 32% sucrose and 15 to 20% reducing sugar
 Sugar content of molasses is 62%
Sucrose 32.0%
Glucose 14.0%
Fructose 16.0%
GRADES OF MOLASSES
There are three grades of molasses based on the TRS( Total Reducing Sugar )content
Grade A
Grade A molasses has a good flavor and good color. The Grade A is practically free from
defects, which means that any extraneous material does not affect the appearance or edibility.
Grade A must have a minimum of 88 to 90 percent Brix solids and 50% or more total reducing
sugar(TRS), a maximum of 12-13% ash .
Grade B
Grade B must have a minimum of 85 to 88 % Brix solids and44% to less 49.9% total reducing
sugar(TRS), a maximum of14-15% ash .
Grade C
Grade C is fairly free from defects, which means that any extraneous material does not seriously
affect the appearance or edibility. Grade C must have a minimum of 83-85% Brix solids and 40-
43.9% total reducing sugar(TRS), a maximum of 17-18 % ash.
Grades Brix TRS% Ash %
Grade A 88 to 90 50% more 12-13%
Grade B 85 to 88 44% to less 49.9% 14-15%
Grade C 83 to 85 40 to 43.9% 17 to 18%

Below grade Below 85 Less than 40% More than 18%
FERMENTATION-
The word fermentation comes from the Latin ―fermentare‖ means to boil. Fermentation stood for
the decomposition of foodstuff, generally accompanied by evolution of gas e.g. fermentation of
sugars to alcohols and carbon dioxide by yeast. Fermentation of fruits to alcohol and making of
beverage out of fruits and grain have been practiced for centuries. Now man is directing the life
process of microorganisms, yeasts, bacterial and moulds to the production of large number of
chemicals, like alcohol, acetone, acetic acid, lactic acid citric acid and many antibiotics which
are of great synthetic as well as industrial important.
Fermentation process by Molasses

MOLASSES FERMENTATION-
Ethanol being a renewable resource of energy is probably a cleaner alternative to fossil fuels. Demand
for Ethanol is increasing day by day due to its versatile application and utility. To meet the acing
demands, production of Ethyl Alcohol or Ethanol through fermentation is gaining momentum and
acclamation globally.
At industrial level, ethanol is prepared by molasses fermentation. A residue begotten from sugar
cane processing, molasses is the mother liquor left after crystallization of sugarcane juice. It is a
dark colored viscous liquid that contains approximately 40 to 50% fermentable sugar. Being one
of the earliest biotechnologies used by humans for the production of Ethanol, molasses
Fermentation is also the most cost-effective way.
Other than the formation of ethyl alcohol or ethanol, molasses is a microbiological energy source
that helps in the process of growing yeast, bacteria and moulds. Not only is it the most cost-
effective amongst the available energy sources for such industries but is also the easiest to
incorporate in fermentation processes. A sound source of B Vitamins and biotin in molasses
hastens fermentation processes that help in faster ethanol production.
Molasses Fermentation is a biological process in which sucrose from molasses is converted into
cellular energy that eventually produces ethanol and carbon dioxide. Molasses from sugarcane or
sugar imparts an appropriate substrate for ethanol production. They are the basic sources of
Sucrose (C12H22O11) which is present in them in concentrated sugar form. Only 50% of molasses
contains eminent reducing sugars that prove useful in the fermentation process. Molasses
constitutes many fermentable and un-fermentable sugars with traces of Nitrogen, Phosphorus,
Solid Sludge and Ash. Fermentable sugars are directly used during Molasses fermentation, while
the un-fermentable sugars like Starch, Cellulose or Pectins can be used only after being
hydrolyzed. With the help of apposite enzymes and reactions, Ethanol is formed. The Ethanol
begotten from the whole process is used in the form of absolute or rectified spirit.
TYPES OF FERMENTATION-
BATCH FERMENTATION-
Batch fermentations the population of microorganism goes through several distinct growth
phases: Lag, acceleration, exponential growth, deceleration, stationary and death in the lag phase
virtually no growth occurs and the microbial population remains relatively constant.
Nevertheless, it is a period of intense metabolic activity as the microbial inoculums adapts to the
new environment. When cells are inoculated into fresh medium they may be deficient in essential
enzymes, vitamins or cofactors, etc. that must be synthesized in order to utilize available
nutrients, prior to cell division taking place. The chemical composition of the fermentation media
influences the length of the lag phase. It is usually longer if the inoculum was grown up using a
carbon source different from that in the fresh medium, because the cells must synthesize
enzymes required to catabolize the new substrate. Physiological stress may also have an effect,
especially as cells are often transferred from an inoculums medium of low osmotic pressure (low
solute concentration) to fresh medium of higher osmotic pressure (high solute concentration).
Other factors influencing the length of the lag phase are the age, concentration, viability and
morphology of the inoculums. Generally, inocula prepared from cells harvested in the
exponential growth phase (period of most rapid growth) exhibit shorter lag phases than those
harvested from subsequent stages.
CONTINUOUS CULTURE FERMENTATION-

Continuous culture fermentations have many applications for both laboratory research and
industrial-scale processes. Studies can be performed on all aspects of cell growth ,physiology
and biochemistry. They are useful for ecological studies and as a genetics tool for the
examination of mutation rates, mutagenic effects, etc. Application in industrial fermentations
overcomes many limitations of batch processes. Initially, continuous fermentations start as batch
cultures ,but exponential growth can then be extended indefinitely, in theory, through the
continuous addition of fresh fermentation medium. The reactor is continuously stirred and a
constant volume is maintained by incorporating an overflow weir or other leveling device.
Fresh medium is continuously added and displaces an equal volume of spent fermentation broth
and cells at the same rate as fresh medium is introduced. Steady state conditions prevail, where
the rate of microbial cell growth equals the rate at which the cells are displaced
From the vessel. As with batch fermentations, the specific rate at which the microorganism
grows in continuous culture is controlled by the availability of the rate-limiting nutrient.
Therefore, the rate of addition of fresh medium controls the rate at which the microorganisms
grow .However; the actual rate of growth depends not only on the volumetric flow rate of the
medium into the reactor, but also on the dilution rate (D). This equals the number of reactor
volumes passing through the reactor per unittime and is expressed in units of reciprocal time, per
hour.
DISTILLATION –
Distillation is method of separation of components from a liquid mixture which depends on the
differences in boiling points of the individual components and the distributions of the
components between a liquid and gas phase in the mixture. The liquid mixture may have
different boiling point characteristics depending on the concentrations of the components present
in it. Therefore, distillation processes depends on the vapor pressure characteristics of liquid
mixtures. The vapor pressure is created by supplying heat as separating agent. In the distillation,
the new phases differ from the original by their heat content. During most of the century,
distillation was by far the most widely used method for separating liquid mixtures of chemical
Distillation unit
Components (Seader and Henley, 1998). This is a very energy intensive technique, especially
when the relative volatility of the components is low. It is mostly carried out in multi tray
columns. Packed column with efficient structured packing has also led to increased use in
distillation.
Distillation is a widely used method for separating the component substances from a liquid
mixture by selective evaporation and condensation. To separate a mixture of liquids, the liquid
can be heated to force components, which have different boiling points, into the gaseous state.
In industrial chemistry, distillation is a unit operation of practically universal importance, but it is
a physical separation process and not a chemical reaction. Distillation is used for many
commercial processes, such as the production of gasoline, distilled water, xylene, alcohol,
paraffin, kerosene, and many other liquids.
ALCOHOL AND ITS PRODUCTION-
Ethanol is a volatile, flammable, clear, colorless liquid. Ethanol is a good solvent. It is also used
as a germicide, beverage, and antifreeze, fuel, depressant and chemical intermediate. It can be
made by the fermentation process of material that contains sugar or from the compound which
can be converted to sugar. Yeast enzyme readily ferment sucrose to ethanol.
CHEMICAL REACTIONS:
The main reactions during fermentation of molasses by yeast are as follows:
In the first step, when the yeast is added to a solution of molasses, which contains sugar as well
as invert sugar (glucose), the entire sugar (disaccharide) is converted into invert sugar
(Monosaccharide) ,according to the following equation .This is by the action of enzyme
(invertase) content in the yeast.
INVERTASE:
C12H22O11 + H2O 2C6H12O6
(Sugar) (Invert Sugar)
It may be seen from the above reaction that according to the molecular weight of sugar and
invert sugar, 100 parts of sugar yields 105.26 parts of invert sugar, when sugar is inverted.
In the next reaction, the enzymes ‖Zymase‖ content in the yeast converts the sugar into Ethyl
Alcohol and Carbon dioxide according to the following equation:
C6H12O6 2C2H5OH + 2CO2
Ethanol, also called alcohol, ethyl alcohol, grain alcohol, and drinking alcohol, is a chemical
compound, a simple alcohol with the chemical formula C2H5OH. Its formula can be also written
as CH3−CH2−OH or C2H5−OH (an ethyl group linked to a hydroxyl group). Ethanol is
a volatile, flammable, colorless liquid with a slight characteristic odor. It is a psychoactive
substance and is the principal type of alcohol found in alcoholic drinks.
Ethanol is naturally produced by the fermentation of sugars by yeasts or

via petrochemical processes, and is most commonly consumed as a popular recreational drug . It
also has medical applications as an antiseptic and disinfectant. The compound is widely used as
a chemical solvent, either for scientific chemical testing or in synthesis of other organic
compounds, and is a vital substance utilized across many different kinds of manufacturing
industries. Ethanol is also used as a clean-burning fuel source.
CHEMICAL STRUCTURES OF ETHANOL
Ethanol is a 2-carbon alcohol. Its molecular formula is CH3CH2OH. An alternative notation is

CH3−CH2−OH, which indicates that the carbon of a methyl group (CH3−) 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. The properties of ethanol stem primarily
from the presence of its hydroxyl group and the shortness of its carbon chain. Ethanol‘s hydroxyl
group is able to participate in hydrogen bonding, rendering it more viscous and less volatile than
less polar organic compound of similar molecular weight. Ethanol, like most short- chain
alcohols, is flammable, colorless, has a strong odour and is volatile.
PHYSICAL PROPERTIES-
Ethanol is a volatile, colorless liquid that has a slight odor. It burns with a smokeless blue flame
that is not always visible in normal light. The physical properties of ethanol stem primarily from
the presence of its hydroxyl group and the shortness of its carbon chain. Ethanol's hydroxyl
group is able to participate in hydrogen bonding, rendering it more viscous and less volatile than
less polar organic compounds of similar molecular weight, such as propane.(5)
SOLVENT PROPERTIES-
Ethanol is a versatile solvent, miscible with water and with many organic solvents,
including acetic acid, acetone, benzene, carbon tetrachloride, chloroform, diethyl ether, ethylene
glycol, glycerol, nitromethane, pyridine, and toluene.(6) It is also miscible with light aliphatic
hydrocarbons, such as pentane and hexane, and with aliphatic chlorides such
as trichloroethane and tetrachloroethylene.
PROPERTIES OF ALCOHOL-
Molecular formula C2H5OH
Molecular weight 46.07
Density 0.791 at 20°C
Boiling Point 78.3°C

USES OF ALCOHOL / ETHANOL:
FEED STOCKS:
Ethanol derivatives have wide spread and important industrial applications besides as a base
chemical for other organic compounds. These include ethyl halides, ethyl esters , diethyl ether ,
acetic acid ,butadiene and ethyl amines.
ANTISEPTIC USE:
Ethanol is used in medical wipes and in most common antibacterial hand sanitizer gels at a
concentration about 62% (percentage by weight, not volume) as an antiseptic. Ethanol kills
organisms by
denaturing their proteins and dissolving their liquids and is effective against most bacteria and
fungi, and many viruses, but is ineffective against bacterial spores.
OTHER USES:
 Ethanol is easily miscible in water and is good solvent. Ethanol is less polar than water
and is used in perfumes, paints and tinctures.
 Ethanol is also used in design and sketch art markers , such as copic.
YEASTS AND ITS CLASSIFICATION AND USES-
Yeasts are unicellular eukaryotic fungi with completely different properties from those of
bacteria, which are Prokaryotic microorganisms. Yeast contains almost the same organelles of a
mature eukaryotic cell. Nucleus, Golgi apparatus, mitochondria, endoplasmic reticulum, vacuole,
and cytoskeleton are the most important one. Yeast cell particle size is typically of 5×10μm. The
primary method of reproduction is by budding, and occasionally by fission. Yeast can be
identified and characterized based on cell morphology, physiology, immunology, and using
molecular biology techniques. The natural habitat of yeast may be soil, water, plants, animals,
and insects with special habitat of plant tissues. Many commercial products contain a mixture of
varying proportions of live and dead S. cerevisiae cells are available for using as feed additives
in animals nutrition(7)
INTRODUCTION-
Saccharomyces cerevisiae yeast is unicellular fungi that divide asexually by budding or fission
and whose individual cell size with a large diameter of 5– 10μm and a small diameter of 1–7μm.
The cells of S. cerevisiae are pigmented, where cream color may be visualized in surface-grown
colonies (Walker and White, 2011). Yeast cell is completely deferred than bacterial cell in both
structure and function. Yeast Saccharomyces S. cerevisiae has an extensive history of uses in the
area of food processing. It is commonly known as baker‘s yeast or brewer‘s yeast. S.
cerevisiaehas been used for centuries as leavening for bread and as a fermenter of alcoholic
beverages and wine production. Yeast also has a new function as natural feed additives in
ruminant and non-ruminant animals for manipulating the gastrointestinal tract and the rumen
environment.
DESCRIPTION AND SIGNIFICANCE-
Yeasts are fungi, whose common characteristics are predominant or permanent unicellular state.
Yeasts are unicellular eukaryotic fungi with completely different properties than those of
bacteria, which are Prokaryotes. For example, yeasts have a resistant to antibiotics, sulfamides
and other anti-bacterial agents. This resistance is genetically and natural, and not liable to be
modified or transmitted to other microbes. Moreover, yeast particle size (5×10μm) is also
significantly higher than bacteria size (0.5×5μm).(8)
CLASSIFICATION OF YEAST-
Saccharomycetes belongs to the kingdom of Fungi and the division Ascomycota. It is the only
class in the subdivision Saccharomycotina, the budding yeasts. Saccharomycetes contains a
single order: Saccharomycetales(9)
Domain Eukarya
Kingdom Fungi
Phylum Ascomycota
Subphylum Saccharomycotina
Class Saccharornycetes
Order Saccharomycetales
Family Saccharomycetaceae
Genus Saccharomyces
Species S.cerevisiae
yeast are usually selected ,keeping in view the following characteristics-
 High multiplication rate and fermentation rate

 High yield of ethyl alcohol
 Tolerance to high glucose as well as ethyl alcohol concentration in the substrate
 Tolerance to high osmotic pressure in the substrate
 Tolerance to high pH and high temperature of the substrate
USES OF YEASTS-
The useful physiological properties of yeast have led to their use in the field
of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this
technology. Many types of yeasts are used for making many foods: baker's yeast in bread
production, brewer's yeast in beer fermentation, and yeast in wine fermentation and for xylitol
production.
So-called red rice yeast is actually a mold, Monascus purpureus. Yeasts include some of the
most widely used model organisms for genetics and cell biology..
IMMOBILIZATION-
Enzyme or cell immobilization is a technique specifically designed to restrict the freedom of

movement of an enzyme or cell . Immobilization of enzymes or cell is a common practice,
mainly in order to minimize enzyme or cell costs on the process economics by making it possible
to reuse the enzyme or cells many times and also minimize the operation cost as the
immobilization technique may be modify the enzyme or cell behavior, thus reducing the enzyme
,cell and product costs significantly. Many techniques have been used previously for enzyme
and cell immobilization, as entrapment, adsorption, covalent binding, encapsulation, and cross
linking. Immobilization is defined as the imprisonment of cell or enzyme in a distinct and
support or matrix on which the enzymes or cells are immobilized allows the exchange of
medium containing substrate. It‘s a method of air filtration and purification that uses whole cell
immobilization .It is a process whereby micro fine particulate matter is removed from the air by
attracting charged particulates in the air to a bio reactive mass, or bioreactor, which
enzymatically renders them inert(10).
WHOLE CELL IMMOBILIZATION –
The immobilized whole cell system is an alternative to enzyme immobilization. Unlike enzyme
immobilization, where the enzyme is attached to a solid support (such as calcium alginate or
activated PVA or activated PEI), in immobilized whole cell systems, the target cell is
immobilized. Such methods may be implemented when the enzymes required are difficult or
expensive to extract, an example being intracellular enzymes.(11,12)
Also, if a series of enzymes are required in the reaction; whole cell immobilization may be used
for convenience .This is only done on a commercial basis when the need for the product is more
justified.
Multiple enzymes may be introduced into the reaction, thus eliminating the need for
immobilization of multiple enzymes. Furthermore, intracellular enzymes need not be extracted
prior to the reaction; they may be used directly. However, some enzymes may be used for the
metabolic needs of the cell, leading to reduced yield of the cell.
The attachment of enzymes or whole cells within inert matrix or their entrapment in order to
confined or localize in a certain defined space, with retention of their catalytic activities ,which
can be used repeatedly and continuously, is known as their immobilization.
The advantages of immobilizing the whole cells as biological catalyst over that of a purified
enzymes preparation are numerous. The expanses of cell separation ,isolation and purification
of the enzymes are obviated. A wide scope of catalytic reaction is possible including multi step
reaction utilizing several enzymes, maintenance of the enzymes in their natural state enhances
their stability, and the presence of cofactors and continued protein biosynthesis within the cells
contributes to the longevity of enzymatic activity. The field of whole cells immobilization has
been revived extensively. There are, in principle four methods often used to immobilize a
biocatalyst
 Cross linking of the biocatalyst itself with try or multifunctional reagents.
 Covalent and coordinate bonding of the cell to an otherwise inert support.
 Adsorption or an inert support.
 Entrapment within practices, fibers or microcapsules in hollow fibers.
For ethanol production by immobilized living cells, carrier binding method was first used.
Afterwards, entrapment method has mainly been employed. Entrapment method offers a straight
forward mechanical procedure for the immobilization of whole cells. In this method, the size of
the immobilized cell species make it rather simple to prepare porus network which provides
complete cell retention with a high degree of porosity for subtract and product transport. It is
clear, however, that network has to be formed in a presence of cells to be entrapped and that the
network forming reactions should , therefore , be adapted to physiological requirements and
stability of the cells. No shedding of cells or progeny was reported for continuous operation of
system using entrapped whole cells. This is a problem often encountered in adsorption or
chemical immobilization methods.
Adsorption of cells on to a performed carrier is a classical method. A performed carrier of

variable structure is mixed with the cell suspension and the cells adhere to the surface in a more
or less complete way. (Navano and Durand) First immobilized yeast cells using porous glass
beads as the carrier and compared the metabolism of adsorbed cells with that of free cells .As a
result, it was found that adsorption of cells on the carrier led to acceleration of metabolism in
cells and conversion of glucose into ethanol yeast cells were also adsorbed onto the ceramic
rasching rings .which are coated with gelatin and sprayed with glutaralyhedye . Various ion
exchange resins have also been used for adsorption of yeast cells . recent studies on immobilized
yeast cells for ethanol fermentation confirm the finding that wood chips are the suitable material
which provide maximum adsorption surface .
In a overall evaluation of the method of cells adsorption, the advantage of simplicity and
negligible changes in cell physiology are overshadowed to some extent by the disadvantages of
limited cell loading the limited adhesion stability compared to cell entrapment. Thus application
in any reactor with higher shear fields caused by high fluid flow rates or intensive mixing is
prohibited due to cell washout possibility. On a cell number basis ,optimum value of 10 7 to 10
8
cells m1 -1 for yeast have been determined on porous glass carriers. This may be compared with
9
6*10 cells m1-1 for yeast cells entrapped in K-carragenan showing a much higher loading
capacity of the later method. There are various methods available to obtain particles containing
entrapped cells such as:
 Block polymerization with subsequent mechanical disintegration into particles .
This is a simple method but it results in particles of an irregular size distribution which
may show poor flow properties .Moldings of particles (beads) in a template.
This method results in a uniform preparation of immobilized cells but it is less suitable for the
preparation of large quantities.
CHAPTER 2: REVIEW OF LITERATURE
Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were
employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and
other sugar sources. Some improvements were made in order to avoid microbial contamination
and keep cell viability for stable long run operations.(14)
Yeast-related industries, continuous fermentation systems offer important economic advantages

in comparison with traditional systems. Fermentation rates are significantly improved,
especially when continuous fermentation is combined with cell immobilization techniques to
increase the yeast concentration in the fermentor. (15)
) fermentation and maturation processes use open fermentation and lager tanks. Although these
vessels had previously been considered indispensable, during the past decades they were in many
breweries replaced by large production units (cylindroconical tanks). These have proved to be
successful, both providing operating advantages and ensuring the quality of the final beer.
Another promising contemporary technology, namely, continuous beer fermentation using
immobilized brewing yeast, by contrast, has found only a limited number of industrial
applications. Continuous fermentation systems based on immobilized cell technology (16)
Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized

cell reactor (ICR) was successfully carried out to improve the performance of the fermentation
process. The fermentation set-up was comprised of a column packed with beads of immobilized
cells. The immobilization of S. cerevisiaewas simply performed by the enriched cells cultured
media harvested at exponential growth phase.(17)
Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the
continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to
selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast
beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as
drops to 0.05M CaCl2 solution.(18)
CO2 gas, evolved during alcohol fermentation using immobilized yeast, causes several
undesirable problems in a packed-bed bioreactor installed vertically as it increases the dead
space and causes hydrostatic pressure. The horizontal packed-bed bioreactor was 1.5 times
more productive than the vertical packed-bed bioreactor when operated continuously (19)
Continuous alcoholic fermentation of untreated crude sugar beet molasses has been studied. The
process was carried out in a vertical fluidized‐bed reactor with beads of calcium‐alginate
containing immobilized Saccharomyces cerevisiae. The influence of hydraulic residence time,
concentration of substrate and other variables have been studied.(20)
Sugar-cane stalks, 2.0 cm long, were used as a support for yeast immobilization envisaging
ethanol production. The assays were conducted in 38.5 L fermenters containing a bed of stalks
with 50% porosity. The operational stability of the immobilized yeast, the efficiency and stability
of the process, as well as the best dilution rate were evaluated. Molasses from demerara sugar
production was used in the medium formulation.(21)
Yeast is a unicellular eukarotic fungus, very common in the environment and is mostly
saprophytic. It has been classified as Ascomycetes or Basidomycetes underfungi taxonomy and
there are about 1500 species of yeast (22)
Because of yeast fermentative characteristic, there is always a need for yeast strains with better
features of fermentative especially high ethanol tolerance for production of ethanol as bio fuel on
commercial scale. (23)
CHAPTER 3:MATERIALS AND METHODS
GENERAL:
The immobilized yeast cells were later on utilized for their ability to ferment molasses for the
production of alcohol.
All the glasswares were cleaned with sulfuric and nitric acids , washed several times with tap
water and finally rinsed with distilled water. All chemicals used were of analytical or equivalent
grade and were obtained from Merck/ Loba/ BDH etc.
GLASSWARE AND HANDLING:

The glass wares were throughout experimental work was of Borosil, Schott and Dura make to
avoid chances of breakage during usage, sterilization and heating. Before use, the glass wares
were washed with chromic acid, sometimes with nitric acid and then by lab clean detergent
followed by distilled water.
Glasswares used in present study were Test-Tubes, Conical flasks , Beakers, measuring cylinder
,volumetric flask , burette ,round bottom flask ,distillation sets , glass beads and syringe without
needle
CHEMICALS AND GROWTH MEDIA:

All the chemicals used for preparation of reagents were of AR, EP grade of Sigma, Merck or
equivalent grade. The growth media used in microbiological work were readymade and supplied
by Titan Biotech and Thomas Baker. They were also of AR grade.
Instruments Used:
1. Micro and Other Balances: Almost all-weighing work was carried on analog and
microbalances (Sartorius and Mettler AC 200) having sensitivity 0.01mg and 0.1mg,
respectively.
2. Hot plate: hot plates are generally used to heat glassware or its contents. Some hot plates
also contain a magnetic stirrer, allowing the heated liquid to be stirred automatically.
In a student laboratory hot plates are used because baths can be hazards if they spill, overheat or
ignite, because they have a high thermal inertia (meaning they take a long time to cool down)
and mantles can be very expensive and are designed for specific flask volumes.
3. Alcohol meter: Alcohol Meter is a hydrometer which is used for determining the alcoholic
strength of liquids. It only measures the density of the fluid. Certain assumptions are made
to estimate the amount of alcohol present in the fluid. Alcohol meters have scales marked
with volume percents of "potential alcohol", based on a pre-calculated specific gravity.
4. Magnetic stirrer: magnetic stirrer or magnetic mixer is a laboratory device that employs a
rotating magnetic field to cause a stir bar immersed in a liquid to spin very quickly, thus
stirring it. The rotating field may be created either by a rotating magnet or a set of stationary
electromagnets, placed beneath the vessel with the liquid.
Prepration of substrate:
Molasses:
Molasses is the mother liquor left over after crystallization of sugar from concentrated cane
juice.it is not commercially possible to extract any more sugar from it and hence, used as raw
material in the distilleries for producing ethyl alcohol from the sugars and reducing sugars
contained in molasses.
Calcium alginate:
Calcium alginate is a water-insoluble, gelatinous, cream-coloured substance that can be

created through the addition of aqueous calcium chloride to aqueous sodium alginate. Calcium
alginate is also used for entrapment of enzymes and cells and forming artificial seeds in plant
tissue culture."Alginate" is usually the salts of alginic acid, but it can also refer to derivatives of
alginic acid and alginic acid itself; in some publications the term "algin" is used instead of
alginate. Alginate is present in the cell walls of brown algae, as the calcium, magnesium and
sodium salts of alginic acid.
Yeast( saccharomyces cerevisiae):
Yeasts are eukaryotic, single-celled microorganisms classified as members of

the fungus kingdom. The first yeast originated hundreds of millions of years ago, and
1,500 species are currently identified.(24,25,26) They are estimated to constitute 1% of all
described fungal species.[4]Yeasts are unicellular organisms which evolved from multicellular
ancestors,[5] with some species having the ability to develop multicellular characteristics by
forming strings of connected budding cells known as pseudohyphae or false hyphae.[6] Yeast
sizes vary greatly, depending on species and environment, typically measuring 3–
4 µm in diameter, although some yeasts can grow to 40 µm in size.[22] Most yeasts
reproduce asexually by mitosis, and many do so by the asymmetric division process known
as budding.
ANALYTICAL METHODS:
Fehling solution:
Fehling's solution is a chemical reagent used to differentiate between water-
soluble carbohydrate and ketone functional groups, and as a test for reducing sugars and non-
reducing sugars, supplementary to the Tollens' reagent test. The test was developed by German
chemist Hermann von Fehling in 1849.(27)
Fehling's solution is prepared by combining two separate solutions, known as Fehling's A and
Fehling's B. Fehling's A is aqueous solution of copper(II) sulfate, which is deep blue. Fehling's B
is a colorless solution of aqueous Potassium sodium tartrate (also known as Rochelle salt) made
in a strong alkali, commonly with sodium hydroxide. Typically, the L-tartrate salt is used. The
copper(II) complex in Fehling's solution is an oxidizing agent and the active reagent in the test.
The deep blue active ingredient in Fehling's solution is the bis(tartrate) complex of Cu2+. The
tartrate tetraanions serve as bidentate alkoxide ligands.(28)
Principle of Fehling’s test:
Fehling‘s test is one of the sensitive test for detection of reducing sugars. Fehling‘s reagents
comprises of two solution Fehling‘s solution A and solution B. Fehling‘s solution A is aqueous
copper sulphate and Fehling‘s solution B is alkaline sodium potassium tartarate ( Rochelle salt).
Rochelle salts (sodium potassium tartarate) present in the reagent acts as the chelating agent in
this reaction.
Reagents:
Fehling A:
34.64g CuSO4.5H2O + Distilled water make upto 500 ml
Fehling B:
173g sodium potassium tartarate + 300 ml Distilled water + 50g NaOH IN 100 ml 500ml
with Distilled water
Methylene blue indicator:
1g methylene blue 100 ml Distilled water
6N NaOH:
240g NaOH 1litre with Distilled water
1% glucose:
1g glucose 100 ml Distilled water
1:1 HCl:
100 ml conc HCl+100 ml Distilled water
method:
 10 ml fehling solution was taken in 250 ml conical flask (5 ml fehling A+ 5 ml

fehling B ) and 30 ml distilled water mixed into fehling solution(heat 80 degree
celcious, indicator methylene blue)
 Then the burette was filled 1%standerd glucose solution and titrated
 Brick red color precipitate was obtained (red precipitate cuprous oxide)
calculation :
Fehling factor = dilution factor * burette reading
Dilution factor = 1g glucose
100
=0.01
Total reducing sugar:
The sum total of sugar (C12H22O11) and reducing sugars like glucose (C6H12O 6) present in
molasses is known as total reducing sugars. The total reducing sugars in molasses is
obtained by titration with fehling solution after hydrolysis and expressed as total invert
sugar.
Principle:
Lane and Eynon method is based on the principle of reduction of Fehling's solution
by reducing sugars. ... Total sugars include reducing sugars and non-reducingdi - and
oligo- saccharides like sucrose, which on mild acid hydrolysis are converted into reducing
sugars.
method:
 12.5 molasses was taken in beaker and then volume make upto 100 ml distilled in
volumetric flask
 Then 10 ml diluted molasses was taken in test tube and then added 1:1 HCl in test
tube and mixed it properly
 Then the test tube was put in boiling water (inversion) for 15 minute and after the
inversion than cool it
 After the cooling the molasses 2 ml 6N NaOH was added into the test tube and then
make up the volume 100 ml
 Then the burette was filled with above make up volume into it
 250 ml conical flask was taken and 5 ml fehling A and 5 ml fehling B was added
with 30 ml water
 Then titrated it properly by adding 4-5 drop of methylene blue dye as an indicator
 As the red brick color was appeared and then reading of burette was note down.
Calculation:
Total reducing sugar= fehling factor *100*100*dilution factor
weight of sample *burette reading

Residual sugar:
Residual sugar (or RS) is the measure of the amount of sugar solids that remain unfermented in
finished wort.
Residual sugar= Fehling factor *100
Burette reading
Fermentation efficiency:
Fermentation efficiency is an expression of how much alcohol was usually produced in
beer relativce to the amount that could be theoretically produced
Method:
 100g molasses was taken in beaker and make up into 350 ml
 250 ml molasses was taken for fermentation and 0.5g urea and 0.5g phosphate was
added into substrate
 Active dry yeast was added into molasses.
 Than after conical flask was tightly pluged fermented at room temperature for 48
and 24 hours.
Calculated as the ration of practical yield to the theoretical multiplied by 100 and theoretical
yield was calculated as fermentable sugar percentage multiplied by 0.644
Fermentation efficiency= Actual ( Practical )% in wash
Theoretical Alcohol % in wash

Expressed as a percentage of theoretical yield that would be achieved if all present sugars
were absolutely transferred to ethanol and carbon dioxide.
Prepration of immobiliszed beads:

An immobilized is an enzyme and cell attached to an inert, insoluble material—such
as calcium alginate (produced by reacting a mixture of sodium alginate solution and
enzyme solution with calcium chloride). This can provide increased resistance to changes
in conditions such as pH or temperature. It also lets enzymes be held in place throughout
the reaction, following which they are easily separated from the products and may be used
again - a far more efficient process and so is widely used in industry for enzyme and cells
catalysed reactions. An alternative to enzyme immobilization is whole cell immobilization
(29,30)
 Sodium alginate was taken in beaker and dissolved in distilled water and Stirred
until all sodium alginate is completely dissolved and yeast also added into the
mixture
 And then Dripped the yeast-alginate mixture of cross linking solution. (The cross
linking solution is prepared by adding CaCl2 to the growth media. The calcium
cross linking solution is agitated on a magnetic stirrer
 The gel formation achieved at room temperature and the sodium alginate drops
come in direct contact with the calcium solution. A diameter of 0.5-2 mm can be
readily achieved with a syringe and a needle. The beads was fully harden in 1-2
hours.
 Then washed the beads with fresh calcium cross linking solution.
 .the immobilized yeast beads was formed and then beads was used for fermentation.
Immobilized beads
SOME TECHNICAL TERMS USED IN THE DISTILLERIES:
Molasses:
Molasses is the mother liquor left over after crystallization of sugar from concentrated cane
juice.it is not commercially possible to extract any more sugar from it and hence, used as
raw material in the distilleries for producing ethyl alcohol from the sugars and reducing
sugars contained in molasses.
Total reducing sugars:
The sum total of sugar (C12H22O11) and reducing sugars like glucose (C6H12O 6) present in
molasses is known as total reducing sugars. The total reducing sugars in molasses is
obtained by titration with Fehling solution after hydrolysis and expressed as total invert
sugar.
Fermentable sugars:
Fermentable sugars are those, which can be converted into ethyl alcohol by fermentation.
These can be arrived by deducting unfermentables sugars from total reducing sugars.
Brix :
In sugars and allied industries, the term brix is used to indicate the percentage of dissolved
solids. It is an indication of the density expressed on Brix Hydrometer Scale .Molasses has
a brix of 80 to 85 degree, which means the solid content of molasses is 80-85% and the
balance is 15-20 % of molasses in water.
Alcohol:
Alcohol usually means ethyl alcohol with the chemical formula of C2H5OH obtained by
fermentation of molasses with yeast or other methods .
Wort :
The term is used to denote a solution of molasses in water used either for yeast propagation
or fermentation with yeast into alcohol.
REAGENTS:
S.No. Name of chemical Name of company
1 Sodium alginate (C6H9NaO7) CDH
Calcium chloride (CaCl2 )

2 MERK
3 Copper sulphate(CuSO4 ) CDH
4 Sodium potassium CDH

tartrate(KNaC4H4.4H2O )
5 Sodium hydroxide (NaOH) MERK
 Sodium alginate:
Sodium alginate is a natural hydrophilic polysaccharide derived isolated from
marine brown algae. It has been widely investigated in the field of drug delivery
due to its biocompatible and biodegradable nature.
 Calcium chloride:
Calcium chloride is an inorganic compound, a salt with the chemical formula
CaCl2. It is a colorless crystalline solid at room temperature, highly soluble in
water. Calcium chloride is commonly encountered as a hydrated solid
 Copper sulphate:
Copper sulfate is a sulfate salt of copper. It is a potent emetic and is used as an
antidote for poisoning by phosphorus. It also can be used to prevent the growth of
algae. Cupric sulfate is a salt created by treating cupric oxide with sulfuric acid.
 Sodium potassium tartrate :

Sodium potassium tartrate tetrahydrate, also known as Rochelle salt, is a double
salt of tartaric acid first prepared (in about 1675) by an apothecary, Pierre
Seignette, of La Rochelle, France.
 Sodium hydroxide:
Sodium hydroxide, also known as lye and caustic soda, is an inorganic
compound with the formula NaOH.
CHAPTER 4: RESULTS AND DISCUSSIONS
Industrial fermentation is basically a process in which chemical changes are brought

about in organic compounds through the activities of enzymes recreated by
microorganisms. Gay lussac attributed this bubbling to the release of carbon dioxide and
fermentation of ethanol. Louis Pasteur described chemical changes in organic as life
without air. Fermentation is the process in which microbial biotransformation are
achieved and these can be achieved in the presence or absence of air and based on oxygen
requirement.
Fermentation can be aerobic or anaerobic. Alcoholic fermentation up to immobilized
yeast propagation stage is aerobic process while later on ethanol production is carried out
an aerobically.
Immobilized studies:
in a typical ionotropic gelation, a gel is formed by contacting a polyelectrolyte solution
with a low molecular weight multivalent counter ion. The solidification occurs by
polysalt formation such as alginate / Ca2+ . the major factor in selecting the most effective
system for cell immobilization are the pore size defined by molecular size and the
structure of the compound used as the carrier for the active biological material as well as
the molecular size of the substrate or product aspect to diffuse into or from the matrix . In
present study of sodium alginate / Ca2+ gel has been selected for whole cell
immobilization studies and the fermentation characteristics of cell immobilizates of
sacchromycess cerivasae prepared from two types of alginates by both the traditional
external gelation method as well as by internal gelation method. Effects of various factors
such as alginate concentration, calcium chloride concentration , molecular weight
(characterized by viscosity) of sodium alginate, chelating agents and sand on
fermentation have been studied.
Fermentation studies with using immobilzed yeast:

In present, the work which had been done, there are two types of immobilized beads had
been used for ethanol production as follows:
1. Immobilized Ordinary beads
2. Immobilized glass beads
Immobilized ordinary beads:
Immobilized ordinary beads are immobilized yeast beads which are prepared by as follows:
1. Sodium alginate and yeast dissolved in distilled water, clump formation should be
avoided. Stirred until all sodium alginate is completely dissolved.
2. Than Drip the yeast-alginate mixture of cross linking solution. (The cross linking solution
is prepared by adding CaCl2 to the growth media. The calcium cross linking solution is
agitated on a magnetic stirrer.
3. Gel formation could be achieved at room temperature as soon as the sodium alginate
drops come in direct contact with the calcium solution. A diameter of 0.5-2 mm can be
readily achieved with a syringe and a needle. The beads fully harden in 1-2 hours.
4. Washed the beads with fresh calcium cross linking solution.
5 The immobilized ordinary beads formed

Immobilized glass beads
1. In immobilized glass beads, hydrofluoric acid used to rough the surface of glass
beads because of so the mixture of alginate and yeast were attached.
2. After that, sodium alginate and yeast dissolved in distilled water, clump
formation should be avoided. Stirred until all sodium alginate is completely
dissolved.
3. Glass beads were added into the mixture and after that used forship to carried out
immobilized glass beads and put it into a cross linking solution.
4. Gel formation could be achieved at room temperature as soon as the sodium
alginate drops come in direct contact with the calcium solution. A diameter of
0.5-2 mm can be readily achieved with a syringe and a needle. The beads fully
harden in 1-2 hours..
5. Washed the beads with a fresh calcium cross linking solution
Performance of isolated immobilized yeast onto molasses:
for the purpose the molasses diluted to approximately three to four times and the pH was set at
4.5 using sulfuric acid, and then immobilized yeast were added into the different concentration of
alginate and yeast in same molasses media. Before added the beads the TRS content was checked
by lane & Eynon method. the sets were incubated at room temperature for regular interval of 24
and 48 hours and then after analyzed for residual sugar and alcohol percentage in wash alcohol
these the calculation of fermentation efficiency and ethanol yield was carried out. A control of
stanadard granular of sacchromyces cerevisiae was used. The results are obtained presented in
following tables.
Table 5: Recycling studies with immobilized yeast cells on ordinary

beads on molasses media for 24 hours
Cycle 1:
S.No. substrate TRS(%) RS FS(%) TH.Y (%) (PR.Y.) FE(%)

(%)
1. Control 14.8% 0.37 14.43 9.29 7.29 78.47%
2. 6/2 14.8% 0.37 14.43 9.29 5.33 57.37%
3. 6/6 14.8% 0.39 14.41 9.28 6.11 65.84%
4. 6/12 14.8% 0.38 14.42 9.28 6.08 65.51%
 6/2 - 6g alginate and 2g yeast

 6/12-6g alginate and 12g yeast
 TRS- Total Reducing Sugar(g/100)
 RS- residual sugar (g/100)
 FS- fermentable sugar
 TH.Y- alcohol percentage ( theoretical)
 PR.Y-alcohol percentage (actual)
 F.E- fermentation efficiency
Cycle 2:

(%)
1. 6/2 14.8% 0.34 14.46 9.31 5.36 57.57%
2. 6/6 14.8% 0.27 14.53 9.35 6.14 65.66%
3. 6/12 14.8% 0.25 14.55 9.37 6.20 66.16%
Cycle 3:

(%)
1 6/2 14.8% 0.29 14.51 9.34 5.32 56.95%
2 6/6 14.8% .031 14.49 9.33 6.10 65.38%
3 6/12 14.8% 0.27 14.53 9.35 6.25 66.84%
Cycle 4:

(%)
1 6/2 14.8% 0.39 14.41 9.28 5.38 57.97%
2 6/6 14.8% 0.28 14.52 9.35 6.13 65.56%
3 6/12 14.8% 0.26 14.54 9.36 6.26 66.88%
Cycle 5:

(%)
1 6/2 14.8% 0.33 14.47 9.31 5.45 58.53%
2 6/6 14.8% 0.31 14.49 9.33 6.34 67.95%
3 6/12 14.8% 0.35 14.45 9.30 6.45 69.35%
Cycle 6:

(%)
1 6/2 14.8% 0.30 14.5 9.34 5.49 58.77%
2 6/6 14.8% 0.35 14.45 9.30 6.45 69.35%
3 6/12 14.8% 0.28 14.52 9.35 6.68 71.44%
Cycle 7:

(%)
1 6/2 14.8% 0.29 14.51 9.34 5.42 58.02%
2 6/6 14.8% 0.31 14.49 9.33 6.58 70.52%
3 6/12 14.8% 0.32 14.48 9.32 6.79 72.85%
Cycle 8:

(%)
1 6/2 14.8% 0.37 14.43 9.29 5.81 62.54%
2 6/6 14.8% 0.32 14.48 9.32 6.45 69.20%
3 6/12 14.8% 0.28 14.52 9.35 6.90 73.79%
Cycle 9:

(%)
1 6/2 14.8% 0.26 14.54 9.36 6.01 64.20%
2 6/6 14.8% 0.28 14.52 9.35 7.04 75.29%
3 6/12 14.8% 0.24 14.56 9.37 7.10 75.77%
Cycle 10:

(%)
1 6/2 14.8% 0.26 14.54 9.36 6.23 66.55%
2 6/6 14.8% 0.32 14.48 9.32 6.98 74.89%
3 6/12 14.8% 0.31 14.49 9.33 7.30 78.24%
cycle 11:

(%)
1 6/2 14.8% 0.36 14.44 9.29 6.38 68.67%
2 6/6 14.8% 0.34 14.46 9.31 7.00 75.18%
3 6/12 14.8% 0.23 14.57 9.38 7.45 79.42%
Cycle 12:

(%)
1 6/2 14.8% 0.35 14.45 9.31 6.48 69.60%
2 6/6 14.8% 0.34 14.46 9.38 7.05 75.15%
3 6/12 14.8% 0.26 14.54 9.36 7.56 80.76%
Cycle 13:

(%)
1 6/2 14.8% 0.30 14.5 9.33 6.38 68.38%
2 6/6 14.8% 0.34 14.46 9.31 7.31 78.51%
3 6/12 14.8% 0.32 14.48 9.32 7.50 80.47%
Cycle 14:

(%)
1 6/2 14.8% 0.29 14.51 9.34 6.48 69.37%
2 6/6 14.8% 0.28 14.52 9.35 7.21 77.11%
3 6/12 14.8% 0.26 14.54 9.36 7.75 82.79%
Cycle 15

(%)
1 6/2 14.8% 0.33 14.47 9.31 7.02 75.40%
2 6/6 14.8% 0.32 14.48 9.53 7.26 76.18%
3 6/12 14.8% 0.27 14.53 9.35 8.01 85.66%

Cycle 1:
S.No. substrate TRS(% RS FS(%) TH.Y (PR.Y.) FE(%)

) (%) (%)
1. Control 14.8 0.39 14.41 9.28 6.38 68.75

2. 10/2 14.8 0.37 14.43 9.29 6.39 68.78
3. 10/6 14.8 0.38 14.42 9.28 6.18 66.59
4. 10/12 14.8 0.34 14.46 9.31 6.42 68.95

Cycle 2:

(%)
1. 10/2 14.8 0.35 14.45 9.30 6.36 68.38
2. 10/6 14.8 0.31 14.49 9.33 6.22 66.66
3. 10/12 14.8 0.26 14.54 9.36 6.52 69.65
Cycle 3:

(%)
1. 10/2 14.8 0.33 14.47 9.31 6.42 68.95
2. 10/6 14.8 0.26 14.54 9.36 6.38 68.16
3. 10/12 14.8 0.25 14.55 9.37 6.56 70.01
Cycle 4:

(%)
1. 10/2 14.8 0.31 14.49 9.33 6.50 69.66
2. 10/6 14.8 0.36 14.44 9.29 6.48 69.75
3. 10/12 14.8 0.22 14.58 9.38 6.68 71.21
Cycle 5:

(%)
1. 10/2 14.8 0.29 14.51 9.33 6.52 69.88
2. 10/6 14.8 0.32 14.48 9.32 6.53 70.06
3. 10/12 14.8 0.33 14.47 9.31 6.73 72.28
Cycle 6:

(%)
1. 10/2 14.8 0.32 14.48 9.32 6.58 70.60
2. 10/6 14.8 0.38 14.42 9.28 6.60 71.12
3. 10/12 14.8 0.36 14.44 9.29 6.84 73.62
Cycle 7:

(%)
1. 10/2 14.8 0.30 14.5 9.33 6.51 69.77
2. 10/6 14.8 0.32 14.48 9.32 6.62 71.03
3. 10/12 14.8 0.29 14.51 9.34 6.90 73.87
Cycle 8:

(%)
1. 10/2 14.8 0.28 14.52 9.35 6.54 69.94
2. 10/6 14.8 0.26 14.54 9.36 6.73 71.90
3. 10/12 14.8 0.24 14.56 9.37 6.93 73.95
Cycle 9:

(%)
1. 10/2 14.8 0.25 14.55 9.37 6.59 70.33
2. 10/6 14.8 0.28 14.52 9.35 6.78 74.65
3. 10/12 14.8 0.23 14.57 9.38 6.95 74.09
Cycle 10:

(%)
1. 10/2 14.8 0.29 14.51 9.34 6.70 71.73
2. 10/6 14.8 0.30 14.50 9.33 6.82 73.09
3. 10/12 14.8 0.30 14.50 9.33 7.03 75.34
cycle 11:

(%)
1. 10/2 14.8 0.31 14.49 9.33 6.84 73.31
2. 10/6 14.8 0.29 14.51 9.34 6.90 73.87
3. 10/12 14.8 0.28 14.52 9.35 7.01 74.97
Cycle 12:

(%)
1. 10/2 14.8 0.28 14.52 9.35 6.95 74.33
2. 10/6 14.8 0.26 14.54 9.36 6.92 73.93
3. 10/12 14.8 0.30 14.5 9.33 7.08 75.88
Cycle 13:

(%)
1. 10/2 14.8 0.35 14.45 9.30 6.98 75.05
2. 10/6 14.8 0.30 14.5 9.33 6.96 74.59
3. 10/12 14.8 0.26 14.54 9.36 7.16 76.49
Cycle 14:

(%)
1. 10/2 14.8 0.24 14.56 9.37 7.04 75.13
2. 10/6 14.8 0.23 14.57 9.38 7.19 76.65
3. 10/12 14.8 0.27 14.53 9.35 7.29 77.96
Cycle 15

(%)
1. 10/2 14.8 0.29 14.51 9.34 7.16 76.65
2. 10/6 14.8 0.24 14.56 9.37 7.28 77.69
3. 10/12 14.8 0.26 14.54 9.36 7.54 80.55

Cycle 1:

(%)
1. Control 14.8% 0.31 14.49 9.33 7.24 77.59
2. 20/2 14.8% 0.32 14.48 9.32 5.43 58.26
3. 20/6 14.8% 0.36 14.44 9.29 5.65 60.81
4. 20/12 14.8% 0.34 14.46 9.31 5.68 61.00
Cycle 2:

(%)
1. 20/2 14.8 0.30 14.5 9.34 5.44 58.24
2. 20/6 14.8 0.33 14.47 9.31 5.69 61.11
3. 20/12 14.8 0.31 14.49 9.33 5.73 61.41
Cycle 3:

(%)
1. 20/2 14.8 0.32 14.48 9.32 5.46 58.58
2. 20/6 14.8 0.34 14.46 9.31 5.78 62.08
3. 20/12 14.8 0.31 14.49 9.33 5.85 62.70
Cycle 4:

(%)
1. 20/2 14.8 0.36 14.44 9.29 5.58 60.06
2. 20/6 14.8 0.31 14.49 9.33 5.81 62.27
3. 20/12 14.8 0.34 14.46 9.31 5.92 63.58
Cycle 5:

(%)
1. 20/2 14.8 0.36 14.44 9.29 5.40 58.12
2. 20/6 14.8 0.33 14.47 9.31 5.90 63.37
3. 20/12 14.8 0.29 14.51 9.34 6.00 64.23
Cycle 6:

(%)
1. 20/2 14.8 0.31 14.49 9.33 5.60 60.02
2. 20/6 14.8 0.32 14.48 9.32 5.29 56.75
3. 20/12 14.8 0.28 14.52 9.35 6.04 64.59
Cycle 7:

(%)
1. 20/2 14.8 0.33 14.47 9.31 5.75 61.76
2. 20/6 14.8 0.34 14.46 9.31 5.30 56.92
3. 20/12 14.8 0.31 14.49 9.33 6.20 66.45
Cycle 8:

(%)
1. 20/2 14.8 0.36 14.44 9.29 5.50 59.20
2. 20/6 14.8 0.32 14.48 9.32 6.04 64.80
3. 20/12 14.8 0.34 14.46 9.31 6.25 67.13
Cycle 9:

(%)
1. 20/2 14.8 0.32 14.48 9.32 5.62 60.30
2. 20/6 14.8 0.29 14.51 9.34 6.12 65.52
3. 20/12 14.8 0.28 14.52 9.35 6.40 68.44
Cycle 10:

(%)
1. 20/2 14.8 0.30 14.5 9.33 5.72 61.30
2. 20/6 14.8 0.27 14.53 9.35 6.15 65.84
3. 20/12 14.8 0.29 14.51 9.34 6.46 69.16
Cycle 11:

(%)
1. 20/2 14.8 0.31 14.49 9.33 5.80 62.16
2. 20/6 14.8 0.26 14.54 9.36 6.22 69.87
3. 20/12 14.8 0.28 14.52 9.35 6.54 69.94
Cycle 12:

(%)
1. 20/2 14.8 0.26 14.54 9.36 6.01 64.20
2. 20/6 14.8 0.28 14.52 9.35 6.36 68.08
3. 20/12 14.8 0.29 14.51 9.34 6.60 70.66
Cycle 13:

(%)
1. 20/2 14.8 0.31 14.49 9.33 6.10 65.38
2. 20/6 14.8 0.32 14.48 9.32 6.48 69.52
3. 20/12 14.8 0.28 14.52 9.35 6.68 71.44
Cycle 14:

(%)
1. 20/2 14.8 0.34 14.46 9.31 6.20 66.59
2. 20/6 14.8 0.31 14.49 9.33 6.56 70.31
3. 20/12 14.8 0.28 14.52 9.35 6.79 72.62
Cycle 15

(%)
1. 20/2 14.8 0.32 14.48 9.32 6.40 68.66
2. 20/6 14.8 0.31 14.49 9.33 6.62 70.95
3. 20/12 14.8 0.26 14.54 9.36 6.95 74.25
Cycle 1:

(%)
1. Control 14.8 0.35 14.45 9.30 7.10 76.34
2. 6/2 14.8 0.30 14.5 9.33 5.20 72.66
3. 6/6 14.8 0.27 14.53 9.35 6.01 63.63
4. 6/12 14.8 0.25 14.55 9.37 7.01 74.81

Cycle 2:

(%)
1. 6/2 14.8 0.34 14.46 9.31 6.89 74.00
2. 6/6 14.8 0.26 14.24 9.17 6.95 75.79
3. 6/12 14.8 0.33 14.47 9.31 7.21 77.44
Cycle 3:

(%)
1. 6/2 14.8 0.34 14.46 9.31 6.92 74.32
2. 6/6 14.8 0.26 14.54 9.36 6.97 74.46
3. 6/12 14.8 0.25 14.55 9.37 7.26 77.48
Cycle 4:

(%)
1. 6/2 14.8 0.32 14.48 9.32 6.94 74.46
2. 6/6 14.8 0.23 14.57 9.38 6.91 73.66
3. 6/12 14.8 0.34 14.46 9.31 7.28 78.19
Cycle 5:

(%)
1. 6/2 14.8 0.21 14.59 9.39 6.98 74.33
2. 6/6 14.8 0.24 14.56 9.37 7.03 75.02
3. 6/12 14.8 0.26 14.54 9.36 7.32 78.20
Cycle 6:

(%)
1. 6/2 14.8 0.31 14.48 9.33 7.06 75.66
2. 6/6 14.8 0.27 14.53 9.35 7.15 76.47
3. 6/12 14.8 0.31 14.49 9.33 7.40 79.31
Cycle 7:

(%)
1. 6/2 14.8 0.27 14.53 9.35 7.18 76.79
2. 6/6 14.8 0.28 14.52 9.35 7.20 77.00
3. 6/12 14.8 0.26 14.54 9.36 7.52 80.34
Cycle 8:

(%)
1. 6/2 14.8 0.33 14.47 9.31 8.02 86.14
2. 6/6 14.8 0.31 14.49 9.33 7.48 80.17
3. 6/12 14.8 0.31 14.49 9.33 8.26 88.53
Cycle 9:

(%)
1. 6/2 14.8 0.21 14.59 9.39 8.36 89.31
2. 6/6 14.8 0.40 14.4 9.27 8.07 87.05
3. 6/12 14.8 0.30 14.5 9.33 8.84 94.74
Cycle 10:

(%)
1. 6/2 14.8 0.39 14.41 9.28 8.28 89.22
2. 6/6 14.8 0.34 14.46 9.31 8.01 86.03
3. 6/12 14.8 0.31 14.49 9.33 8.34 89.38
Cycle 11:

(%)
1. 6/2 14.8 0.38 14.42 9.28 8.30 89.43
2. 6/6 14.8 0.34 14.46 9.31 8.26 88.72
3. 6/12 14.8 0.31 14.49 9.33 8.45 90.56

Cycle 1:

(%)
1. Control 14.8 0.35 14.45 9.31 8.27 88.92
2. 10/2 14.8 0.35 14.45 9.30 6.65 71.50
3. 10/6 14.8 0.22 14.58 9.30 5.44 57.99
4. 10/12 14.8 0.34 14.46 9.38 7.58 80.81

Cycle 2:

(%)
1 10/2 14.8 0.30 14.5 9.33 6.70 71.81
2 10/6 14.8 0.26 14.54 9.36 6.66 71.15
3 10/12 14.8 0.34 14.46 9.31 7.60 81.63
Cycle 3:

(%)
1 10/2 14.8 0.33 14.47 9.31 6.74 72.39
2 10/6 14.8 0.28 14.52 9.35 6.75 72.19
3 10/12 14.8 0.32 14.48 9.32 7.63 81.86
Cycle 4:

(%)
1 10/2 14.8 0.28 14.52 9.35 6.84 73.15
2 10/6 14.8 0.29 14.51 9.34 6.88 73.66
3 10/12 14.8 0.26 14.54 9.36 7.66 81.83
Cycle 5:

(%)
1 10/2 14.8 0.23 14.57 9.38 6.87 73.24
2 10/6 14.8 0.29 14.51 9.34 6.97 74.62
3 10/12 14.8 0.36 14.44 9.29 7.68 82.66
Cycle 6:

(%)
1 10/2 14.8 0.28 14.52 9.35 6.99 74.75
2 10/6 14.8 0.34 14.46 9.31 7.14 76.69
3 10/12 14.8 0.36 14.44 9.29 7.72 83.10
Cycle 7:

(%)
1 10/2 14.8 0.27 14.53 9.35 7.09 75.82
2 10/6 14.8 0.31 14.49 9.33 7.32 78.45
3 10/12 14.8 0.32 14.48 9.32 7.80 83.69
Cycle 8:

(%)
1 10/2 14.8 0.28 14.52 9.35 7.20 77.00
2 10/6 14.8 0.31 14.48 9.33 7.40 79.31
3 10/12 14.8 0.46 14.34 9.23 7.85 85.04
Cycle 9:

(%)
1 10/2 14.8 0.41 14.39 9.26 8.16 88.12
2 10/6 14.8 0.42 14.38 9.26 8.20 88.55
3 10/12 14.8 0.39 14.41 9.28 7.84 84.48
Cycle 10:

(%)
1 10/2 14.8 0.36 14.44 9.29 8.26 88.91
2 10/6 14.8 0.42 14.38 9.26 8.18 88.33
3 10/12 14.8 0.35 14.45 9.30 7.90 84.94
Cycle 11:

(%)
1 10/2 14.8 0.40 14.4 9.27 8.24 88.88
2 10/6 14.8 0.35 14.45 9.30 8.11 87.20
3 10/12 14.8 0.38 14.42 9.28 8.15 87.82

Cycle 1:
S.No. substrate TRS(%) RS FS(%) TH.Y (PR.Y.) FE(%)

(%) (%)
1. Control 14.8 0.40 14.4 9.27 8.52 91.90

2. 20/2 14.8 0.27 14.53 9.35 5.65 60.42
3. 20/6 14.8 0.31 14.49 9.33 4.92 52.73
4. 20/12 14.8 0.30 14.5 9.33 5.32 57.02
Cycle 2:

(%)
1 20/2 14.8 0.31 14.49 9.33 5.68 60.87
2 20/6 14.8 0.31 14.49 9.33 5.12 54.87
3 20/12 14.8 0.29 14.51 9.34 5.34 57.17
Cycle 3:

(%)
1 20/2 14.8 0.36 14.44 9.29 5.72 61.57
2 20/6 14.8 0.28 14.52 9.35 5.25 56.14
3 20/12 14.8 0.24 14.56 9.37 5.45 58.16
Cycle 4:

(%)
1 20/2 14.8 0.27 14.53 9.35 5.85 62.56
2 20/6 14.8 0.36 14.44 9.29 5.45 58.66
3 20/12 14.8 0.30 14.5 9.33 5.56 59.59
Cycle 5:

(%)
1 20/2 14.8 0.34 14.46 9.31 6.18 66.38
2 20/6 14.8 0.34 14.46 9.31 5.65 60.58
3 20/12 14.8 0.29 14.51 9.34 5.64 60.38
Cycle 6:

(%)
1 20/2 14.8 0.22 14.58 9.38 6.62 70.57
2 20/6 14.8 0.25 14.47 9.31 5.75 61.76
3 20/12 14.8 0.30 14.5 9.33 5.80 62.16
Cycle 7:

(%)
1 20/2 14.8 0.32 14.48 9.32 7.09 76.07
2 20/6 14.8 0.33 14.47 9.31 6.00 64.44
3 20/12 14.8 0.36 14.44 9.29 6.95 74.81
Cycle 8:

(%)
1 20/2 14.8 0.25 14.55 9.37 7.10 75.77
2 20/6 14.8 0.28 14.52 9.35 7.05 75.40
3 20/12 14.8 0.29 14.51 9.34 7.00 74.94
Cycle 9:

(%)
1 20/2 14.8 0.31 14.49 9.33 7.86 84.24
2 20/6 14.8 0.28 14.52 9.35 7.25 77.54
3 20/12 14.8 0.29 14.51 9.34 7.08 75.80
Cycle 10:

(%)
1 20/2 14.8 0.34 14.46 9.31 7.95 85.39
2 20/6 14.8 0.29 14.51 9.34 7.40 79.22
3 20/12 14.8 0.27 14.53 9.53 7.20 75.55
Cycle 11:

(%)
1 20/2 14.8 0.31 14.49 9.33 8.01 85.85
2 20/6 14.8 0.40 14.4 9.27 7.84 84.57
3 20/12 14.8 0.36 14.44 9.29 7.52 80.94
Table 11: Recycling studies with immobilized yeast cells on glass
Cycle 1:

(%) (%)
1. Control 14.8 0.36 14.44 9.29 8.16 87.83

2. 6/2 14.8 0.31 14.49 9.33 4.13 44.26
3. 6/6 14.8 0.28 14.52 9.35 4.25 45.45
4. 6/12 14.8 0.32 14.48 9.32 5.01 53.75

Cycle 2:

(%)
1 6/2 14.8 0.34 14.46 9.31 4.16 44.68
2 6/6 14.8 0.29 14.51 9.34 4.28 45.82
3 6/12 14.8 0.27 14.53 9.53 5.08 53.30
Cycle 3:

(%)
1 6/2 14.8 0.24 14.56 9.37 4.23 45.14
2 6/6 14.8 0.26 14.54 9.36 4.39 46.90
3 6/12 14.8 0.28 14.52 9.35 5.47 58.50
Cycle 4:

(%)
1 6/2 14.8 0.34 14.46 9.31 4.65 49.94
2 6/6 14.8 0.26 14.54 9.36 4.65 49.67
3 6/12 14.8 0.25 14.55 9.37 5.52 58.91
Cycle 5:

(%)
1 6/2 14.8 0.33 14.47 9.31 4.85 52.09
2 6/6 14.8 0.31 14.49 9.33 4.95 53.05
3 6/12 14.8 0.35 14.45 9.30 5.62 60.43
Cycle 6:

(%)
1 6/2 14.8 0.39 14.41 9.28 5.02 54.09
2 6/6 14.8 0.41 14.39 9.26 5.00 53.99
3 6/12 14.8 0.38 14.42 9.28 5.24 56.46
Cycle 7:

(%)
1 6/2 14.8 0.41 14.39 9.26 5.12 55.29
2 6/6 14.8 0.28 14.52 9.35 5.14 54.97
3 6/12 14.8 0.33 14.47 9.31 5.34 57.35
Cycle 8:

(%)
1 6/2 14.8 0.29 14.51 9.34 5.22 55.88
2 6/6 14.8 0.31 14.49 9.33 5.44 58.30
3 6/12 14.8 0.33 14.47 9.31 5.48 58.86
Cycle 9:

(%)
1 6/2 14.8 0.25 14.55 9.37 5.36 57.20
2 6/6 14.8 0.26 14.54 9.36 5.48 58.54
3 6/12 14.8 0.32 14.48 9.32 5.66 60.72
Cycle 10:

(%)
1 6/2 14.8 0.41 14.39 9.26 5.45 58.85
2 6/6 14.8 0.28 14.52 9.35 5.75 61.49
3 6/12 14.8 0.33 14.47 9.31 5.85 62.83
Table 12: Recycling studies with immobilized yeast cells on glass
Cycle 1:

(%)
1. Control 14.8% 0.39 14.41 9.28 8.02 86.42
2. 10/2 14.8% 0.29 14.51 9.34 5.02 53.74
3. 10/6 14.8% 0.34 14.46 9.31 5.12 54.99
4. 10/12 14.8% 0.29 14.51 9.34 5.03 53.85

Cycle 2:

(%)
1. 10/2 14.8 0.32 14.48 9.32 5.10 54.72
2. 10/6 14.8 0.29 14.51 9.33 5.21 55.84
3. 10/12 14.8 0.30 14.5 9.28 5.75 61.96
Cycle 3:

(%)
1. 10/2 14.8 0.34 14.46 9.53 5.35 56.13
2. 10/6 14.8 0.30 14.5 9.33 5.49 58.84
3. 10/12 14.8 0.29 14.51 9.31 5.95 63.90
Cycle 4:

(%)
1. 10/2 14.8 0.29 14.51 9.31 5.66 60.79
2. 10/6 14.8 0.27 14.53 9.35 5.75 61.49
3. 10/12 14.8 0.27 14.53 9.35 6.16 65.88
Cycle 5:

(%)
1. 10/2 14.8 0.32 14.48 9.32 5.78 62.01
2. 10/6 14.8 0.29 14.51 9.33 5.79 62.05
3. 10/12 14.8 0.30 14.5 9.28 6.35 68.42
Cycle 6:

(%)
1. 10/2 14.8 0.28 14.52 9.35 5.85 62.56
2. 10/6 14.8 0.33 14.47 9.31 5.98 64.23
3. 10/12 14.8 0.35 14.45 9.30 6.74 72.47
Cycle 7:

(%)
1. 10/2 14.8 0.32 14.48 9.32 6.06 65.02
2. 10/6 14.8 0.29 14.51 9.33 6.21 66.55
3. 10/12 14.8 0.30 14.5 9.28 6.83 73.59
Cycle 8:

(%)
1. 10/2 14.8 0.31 14.49 9.33 6.12 65.59
2. 10/6 14.8 0.36 14.44 9.29 6.33 68.31
3. 10/12 14.8 0.22 14.58 9.38 6.92 73.77
Cycle 9:

(%)
1. 10/2 14.8 0.33 14.47 9.31 6.19 66.48
2. 10/6 14.8 0.26 14.54 9.36 6.46 69.01
3. 10/12 14.8 0.25 14.55 9.37 6.95 74.17
Cycle 10:

(%)
1. 10/2 14.8 0.35 14.45 9.30 6.25 67.20
2. 10/6 14.8 0.31 14.49 9.33 6.58 70.52
3. 10/12 14.8 0.26 14.54 9.36 6.99 74.67
Table13: Recycling studies with immobilized yeast cells on glass
Cycle 1:

(%)
1. Control 14.8% 0.36 14.44 9.29 8.20 88.26
2. 20/2 14.8% 0.32 14.48 9.32 5.24 56.22
3. 20/6 14.8% 0.34 14.46 9.31 5.22 56.06
4. 20/12 14.8% 0.31 14.49 9.33 5.30 56.80

Cycle 2:

(%)
1. 20/2 14.8 0.36 14.44 9.29 5.46 58.77
2. 20/6 14.8 0.31 14.49 9.33 5.72 61.30
3. 20/12 14.8 0.34 14.46 9.31 5.66 60.79
Cycle 3:

(%)
1. 20/2 14.8 0.36 14.44 9.29 5.66 60.92
2. 20/6 14.8 0.33 14.47 9.31 5.70 61.22
3. 20/12 14.8 0.29 14.51 9.34 5.75 61.56
Cycle 4:

(%)
1. 20/2 14.8 0.33 14.47 9.31 5.69 61.11
2. 20/6 14.8 0.34 14.46 9.31 5.72 61.43
3. 20/12 14.8 0.31 14.49 9.33 5.85 62.70
Cycle 5:

(%)
1. 20/2 14.8 0.36 14.44 9.29 5.70 61.35
2. 20/6 14.8 0.32 14.48 9.32 5.76 61.80
3. 20/12 14.8 0.34 14.46 9.31 5.95 63.90
Cycle 6:

(%)
1. 20/2 14.8 0.32 14.48 9.32 5.75 61.69
2. 20/6 14.8 0.29 14.51 9.34 5.85 62.63
3. 20/12 14.8 0.28 14.52 9.35 6.10 65.24
Cycle 7:

(%)
1. 20/2 14.8 0.21 14.49 9.33 5.80 62.16
2. 20/6 14.8 0.26 14.54 9.36 6.22 69.87
3. 20/12 14.8 0.28 14.52 9.35 6.54 69.94
Cycle 8:

(%)
1. 20/2 14.8 0.26 14.54 9.36 6.61 64.24
2. 20/6 14.8 0.28 14.52 9.35 6.36 68.02
3. 20/12 14.8 0.29 14.51 9.34 6.62 70.87
Cycle 9:

(%)
1. 20/2 14.8 0.31 14.49 9.33 6.56 70.31
2. 20/6 14.8 0.26 14.54 9.36 6.20 66.23
3. 20/12 14.8 0.28 14.52 9.35 6.79 72.62
Cycle 10:

(%)
1. 20/2 14.8 0.34 14.46 9.31 6.76 72.61
2. 20/6 14.8 0.31 14.49 9.33 6.56 70.31
3. 20/12 14.8 0.28 14.52 9.35 6.85 73.26
Cycle 1:

(%)
1. Control 14.8% 0.38 14.42 9.28 8.09 87.17
2. 6/2 14.8% 0.32 14.48 9.32 4.16 44.63
3. 6/6 14.8% 0.29 14.51 9.33 4.95 53.05
4. 6/12 14.8% 0.30 14.5 9.28 5.24 56.46

Cycle 2:

(%)
1. 6/2 14.8 0.24 14.56 9.37 4.76 50.80
2. 6/6 14.8 0.26 14.54 9.36 4.98 53.20
3. 6/12 14.8 0.28 14.52 9.35 5.30 56.68
Cycle 3:

(%)
1. 6/2 14.8 0.26 14.54 9.36 5.10 54.48
2. 6/6 14.8 0.25 14.55 9.37 5.21 55.60
3. 6/12 14.8 0.28 14.52 9.35 5.75 61.49
Cycle 4:

(%)
1. 6/2 14.8 0.34 14.46 9.31 5.38 57.78
2. 6/6 14.8 0.38 14.42 9.28 5.75 61.96
3. 6/12 14.8 0.31 14.49 9.33 5.95 63.77
Cycle 5:

(%)
1. 6/2 14.8 0.29 14.51 9.53 6.01 63.06
2. 6/6 14.8 0.30 14.5 9.33 6.32 67.73
3. 6/12 14.8 0.34 14.46 9.31 6.50 69.81
Cycle 6:

(%)
1. 6/2 14.8 0.28 14.52 9.35 6.34 67.80
2. 6/6 14.8 0.27 14.53 9.35 6.61 70.69
3. 6/12 14.8 0.27 14.53 9.35 6.94 74.22
Cycle 7:

(%)
1. 6/2 14.8 0.25 14.55 9.37 7.00 74.70
2. 6/6 14.8 0.26 14.54 9.36 6.81 72.75
3. 6/12 14.8 0.32 14.48 9.32 7.25 77.78
Cycle 8:

(%)
1. 6/2 14.8 0.41 14.39 9.26 7.61 82.18
2. 6/6 14.8 0.28 14.52 9.35 7.88 84.27
3. 6/12 14.8 0.33 14.47 9.31 7.66 82.27
Cycle 9:

(%)
1. 6/2 14.8 0.39 14.41 9.28 8.13 87.60
2. 6/6 14.8 0.41 14.39 9.26 8.20 88.55
3. 6/12 14.8 0.38 14.42 9.28 8.45 91.05

Cycle 1:
S.No. substrate TRS(%) RS FS(%) TH.Y (%) (PR.Y.)(%) FE(%)
1 Control 14.8 0.29 14.51 9.34 8.14 87.15

2 10/2 14.8 0.29 14.51 9.34 6.04 64.66
3 10/6 14.8 0.27 14.53 9.35 6.23 66.63
4 10/12 14.8 0.30 14.5 9.33 6.38 68.38

Cycle 2:

(%)
1 10/2 14.8 0.22 14.58 9.38 6.11 65.13
2 10/6 14.8 0.29 14.51 9.34 6.32 67.66
3 10/12 14.8 0.24 14.56 9.37 6.41 68.40
Cycle 3:

(%)
1 10/2 14.8 0.28 14.52 9.35 6.23 66.63
2 10/6 14.8 0.29 14.51 9.34 6.45 69.05
3 10/12 14.8 0.28 14.52 9.35 6.54 69.94
Cycle 4:

(%)
1 10/2 14.8 0.30 14.5 9.33 6.43 68.91
2 10/6 14.8 0.32 14.48 9.32 6.75 72.42
3 10/12 14.8 0.34 14.46 9.31 6.80 73.03
Cycle 5:

(%)
1 10/2 14.8 0.35 14.45 9.31 6.66 71.53
2 10/6 14.8 0.36 14.44 9.29 6.84 73.62
3 10/12 14.8 0.35 14.45 9.30 6.98 75.05
Cycle 6:

(%)
1 10/2 14.8 0.34 14.46 9.31 6.84 73.46
2 10/6 14.8 0.32 14.48 9.32 6.97 74.78
3 10/12 14.8 0.30 14.5 9.33 7.23 77.49
Cycle 7:

(%)
1 10/2 14.8 0.28 14.52 9.35 7.06 75.50
2 10/6 14.8 0.46 14.34 9.23 7.19 77.89
3 10/12 14.8 0.36 14.44 9.29 7.21 77.61
Cycle 8:

(%)
1 10/2 14.8 0.28 14.52 9.35 7.33 78.39
2 10/6 14.8 0.36 14.44 9.29 7.29 78.47
3 10/12 14.8 0.27 14.53 9.35 7.35 78.60
Cycle 9:

(%)
1 10/2 14.8 0.36 14.44 9.29 7.45 80.19
2 10/6 14.8 0.31 14.49 9.33 7.36 78.88
3 10/12 14.8 0.34 14.46 9.31 8.42 90.44
Cycle 1:

(%) (%)
1 control 14.8 0.29 14.51 9.34 8.18 87.58

2 20/2 14.8 0.27 14.53 9.35 5.65 60.42
3 20/6 14.8 0.31 14.49 9.33 5.38 57.66
4 20/12 14.8 0.34 14.46 9.31 5.60 60.15

Cycle 2:

(%) (%)
1 20/2 14.8 0.36 14.44 9.29 5.76 62.00

2 20/6 14.8 0.28 14.52 9.35 5.45 58.28
3 20/12 14.8 0.24 14.56 9.37 5.58 59.55
Cycle 3:

(%) (%)
1 20/2 14.8 0.32 14.48 9.32 5.78 62.07

2 20/6 14.8 0.34 14.46 9.31 5.56 59.72
3 20/12 14.8 0.30 14.5 9.33 5.65 60.55
Cycle 4:

(%) (%)
1 20/2 14.8 0.38 14.42 9.28 5.85 63.03

2 20/6 14.8 0.29 14.51 9.34 5.72 61.24
3 20/12 14.8 0.36 14.44 9.29 5.63 60.60
Cycle 5:

(%) (%)
1 20/2 14.8 0.22 14.58 9.38 6.03 64.28

2 20/6 14.8 0.25 14.47 9.31 5.86 62.94
3 20/12 14.8 0.30 14.5 9.33 5.84 62.59
Cycle 6:

(%) (%)
1 20/2 14.8 0.34 14.46 9.31 6.62 71.1

2 20/6 14.8 0.34 14.46 9.31 6.74 72.39
3 20/12 14.8 0.29 14.51 9.34 5.86 62.74
Cycle 7:

(%) (%)
1 20/2 14.8 0.25 14.55 9.37 6.75 72.03

2 20/6 14.8 0.28 14.52 9.35 6.45 68.98
3 20/12 14.8 0.29 14.51 9.34 5.99 64.13
Cycle 8:

(%) (%)
1 20/2 14.8 0.32 14.48 9.32 6.76 72.53

2 20/6 14.8 0.33 14.47 9.31 6.55 70.35
3 20/12 14.8 0.36 14.44 9.29 6.03 64.90
Cycle 9:

(%) (%)
1 20/2 14.8 0.34 14.46 9.31 6.86 73.68

2 20/6 14.8 0.32 14.48 9.32 6.74 72.31
3 20/12 14.8 0.28 14.52 9.32 6.15 65.98
Chapter 5: Summary & conclusion
In this study, different concentration of sodium alginate and yeast were taken for the production
of alcohol by immobilized yeast beads by different type‘s ordinary immobilized beads and
immobilized glass beads were taken for different time.
For the production of alcohol, the molasses were diluted in water and then transformed into the
liquid medium and left for 24 and 48 hrs. and production of ethanol was observed by the
distillation method. Once the production was observed, there were further transformed into fresh
liquid medium.
The process is repeated various times for alcohol production for different concentration of
various cycles. In ordinary immobilized beads for 24 hours, the beads used in 15 cycles and
given good production of alcohol in 6g alginate and 12g yeast combination had good
fermentation efficiency. in 48 hours the beads reuse till 11 cycles and they also given a good
fermentation efficiency.
The immobilized glass beads recycled 9 cycles for particular intervals and then the production of
alcohol and fermentation efficiency or reuse of immobilized beads were observed by distillation.
The molasses procured from the national sugar institute factory analyzed for TRS. The TRS
content of molasses was 52.4%. for the fermentation study , the molasses was diluted 3.5 times
in order to have initial TRS content of approximately 14.8%. the diluted molasses was fortified
with proper quantity of urea and phosphate and pH was adjusted to 4.5with sulfuric acid. It was
distributed into conical flask and then plugged the conical flask and incubated at room
temperature . The sample were drawn at regular time intervals and then analyzed for the residual
sugar , alcohol percent in wash. From these data fermentation efficiency were calculated.
It is concluded from the data that results with regards to fermentation efficiency but better results
were seen with immobilized yeast beads that beads reused various time and the reuse of the
beads the alcohol production is increased and the good efficiency were observed. The ordinary
beads were better than the glass beads because ordinary beads have no substrate to fermentation
and glass beads have substrate to stuck the mixer of alginate and yeast which gave less efficiency
of alcohol. The maximum efficiency value was 90-94% was found the ordinary beads and the
glass beads were did not give occurring results.
References
1. The Codex Alimentarius Commission. (2009; 2010). Codex Alimentarius – 212.1 Scope
and Description. Food and Agriculture Organization of the United Nations.
2. "Archived copy". Archived from the original on 2017-09-21. Retrieved 2017-09-20.
3. "The Hidden Chemicals in Hookah Tobacco Smoke". Hookah users inhale smoke, which
is generated by heating hookah tobacco that is fermented with molasses and fruits and
combined with burning charcoal.
4. "Hookah (Shisha, Narghile) Smoking and Environmental Tobacco Smoke (ETS). A
Critical Review of the Relevant Literature and the Public Health
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tobacco-molasses mixture is subjected: actually hundreds of degrees below that of
cigarettes. [...] Another team described the ―chillum‖ as being a pipe made of clay and in
which ―tobacco is burnt along with molasses and coal and smoked from the other end
either directly at the mouth or through a long pipe with the smoke passing through a
water container‖. [...] Mixing tobacco with molasses is a very ancient habit. A WHO
report dates back ―the addition of molasses to burley tobacco in the nineteenth century to
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Alcohol Fermenttion by Immobilised Yeast Cells

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Alcohol Fermenttion by Immobilised Yeast Cells

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“ALCOHOL FERMENTATION BY IMMOBILIZED YEAST CELLS”

Thesis submitted to the

Institute of Biosciences and Technology,

Shri Ramswaroop Memorial University,

Lucknow Deva Road, Uttar Pradesh

The partial fulfillment of the requirement for the degree of

Ms. VANISTHA SHUKLA

Under guidance and supervision of

Dr. SANTOSH KUMAR

NATIONAL SUGAR INSTITUTE

Ministry of Consumer Affairs, Food & Public Distribution,

Department of Food & Public Distribution ,Government of India, Kanpur-208017

This is to certify that the thesis entitled ―ALCOHOL FERMENTATION BY

Place: Barabanki Signature of Director

Place: Kanpur Vanistha Shukla

Place: Kanpur Vanistha Shukla

Chapter 2: Review of Literature………………….................

Chapter 3: Materials and methods…………………….........

Chapter 4: Results and Discussions…………………...........

Chapter 5: Summary and conclusion………………………..

Roll No: 201610901010002

Name: Vanistha shukla

Semester & Year of admission: fourth semester & 2016-2018

Thesis Title: “ALCOHOL FERMENTATION BY IMMOBILIZED

Fermentation by saccharomyces cerevisiae immobilized by entrapment in alginate. The rate of

 Extraction of juice from sugarcane by milling

 Crystallization of juice by vacuum pan boiling

 Centrifugal separation of sugar and molasses from the massecuites

 Drying and cooling of sugar

 Sugar grading and packing

Molasses can be used:

 In some cookies and pies

COMPOSITION OF CANE MOLASSES-

AVERAGE COMPOSITION OF CANE MOLASSES-

Grades Brix TRS% Ash %

Grade A 88 to 90 50% more 12-13%

Grade B 85 to 88 44% to less 49.9% 14-15%

Grade C 83 to 85 40 to 43.9% 17 to 18%

Fermentation process by Molasses

CONTINUOUS CULTURE FERMENTATION-

ALCOHOL AND ITS PRODUCTION-

The main reactions during fermentation of molasses by yeast are as follows:

C12H22O11 + H2O 2C6H12O6

(Sugar) (Invert Sugar)

C6H12O6 2C2H5OH + 2CO2

Ethanol is naturally produced by the fermentation of sugars by yeasts or

CHEMICAL STRUCTURES OF ETHANOL

Ethanol is a 2-carbon alcohol. Its molecular formula is CH3CH2OH. An alternative notation is

Molecular weight 46.07

Density 0.791 at 20°C

Boiling Point 78.3°C

YEASTS AND ITS CLASSIFICATION AND USES-

DESCRIPTION AND SIGNIFICANCE-

yeast are usually selected ,keeping in view the following characteristics-

 High multiplication rate and fermentation rate

Enzyme or cell immobilization is a technique specifically designed to restrict the freedom of

WHOLE CELL IMMOBILIZATION –

 Cross linking of the biocatalyst itself with try or multifunctional reagents.

 Covalent and coordinate bonding of the cell to an otherwise inert support.

 Adsorption or an inert support.

 Entrapment within practices, fibers or microcapsules in hollow fibers.

Adsorption of cells on to a performed carrier is a classical method. A performed carrier of

 Block polymerization with subsequent mechanical disintegration into particles .

and keep cell viability for stable long run operations.(14)

Total reducing sugar= fehling factor 100100*dilution factor