Ethyl Alcohol Production by Hydrolysis

www.final-yearproject.com | www.finalyearthesis.
com
ETHYL ALCOHOL PRODUCTION BY FERMENTATION

OF CANE SUGAR MOLASSES
PROJECT REPORT
SUBMITTED TO
SHRI GURU RAM RAI INSTITUTE OF TECHNOLOGY &
SCIENCE
(H.N.B. GARHWAL UNIVERSITY, SRINAGAR, GARHWAL)
SGRRITS
In Partial fulfillment of the Degree of
Master of Science in Biotechnology
By
SHRIYA TIWARI
INDUSTRIAL WORK CONDUCTED

AT
MOHIT PETRO CHEMICALS (P) LTD. BIJNOR
(U.P.)
www.final-yearproject.com | www.finalyearthesis.com
ACKNOWLEDGEMENT
With an overwhelming sense of legitimate pride and genuine obligation, I

seize this jubilant occasion to express my deep sense of gratitude and personal
regard to my supervisor Mr. Anil Lathian, Mohit Petro chemical P. Ltd, Bijnor,
U.P. valuable guidance, constant supervision tireless inspiration, and affectionate
attitude during the entire work from him I got help without even asking for it.
Again and over I salute the titanic heart.
I also express my earnest gratitude to Mr. Suresh Pal Panwar, General
Manager, Mohit Petro chemical P. Ltd, Bijnor, and U.P. for his regular
academic help and providing necessary facilities for carrying out the work.
I extend my grateful thanks to Mr. Kuldeep Jain (M.D) up this
assignment Mohit Petro chemical P. Ltd, Bijnor, and U.P.
At this junction I owe my deep regards to Mr. Anirudh Sharma & Mr.
Vipin Kumar for their constant encouragement.
My special thanks to Mr. Dilip Kumar, Mr. Awesh Tyagi, & Mr. Pankaj
Kumar.
I convey my sincere thanks to Mrs. Divya Juyal, Head of Department of
Biotechnology SHRI GURU RAM RAI INSTITUTE OF TECHNOLOGY &
SCIENCE for her valuable suggestion during my project work.
My heartful thanks to Dr. Kunal Kishor, Dr. Manisha, Dr. Keerti Katiyar
and Mrs. Shweta, for their cooperation during my project work.
I must express my sincere thanks to all other person who helped directly and
indirectly within lab giving their valuable advice and suggestion specially
Mr.bharat Singh, Mr. Lalit Rajput, Miss R. Rajput, Mr. sandeep Rajput and
Miss.Minakshi joshi.
I must mention that it would have been an uphill task for me accomplishes,
what little work I have done, if I had got the blessing as well as the mental,
financial support and endless love and care from my family members.
And ultimately I wish to thank almighty “GOD” the master of this universe.
Place: Bijnor Shriya Tiwari

CANDIDATE’S DECLARATION
I hereby declare that the project work entitled ETHYL ALCOHOL

PRODUCTION BY FERMENTATION OF CANE SUGAR MOLASSES is my
own work and that, to the best of my knowledge and belief, it contains no
material already submitted in any other University/Institute for the award of any
Degree/Course.
PLACE: DEHRADUN SHRIYA TIWARI

DATE:
CONTENTS
 INTRODUCTION TO DISTILLERY
 FERMENTATION
 MOLASSES
 DISTILLATION
 LABORATORY CONTROL
 PROBLEMS OF DISTILLERY
 SCALING PROBLEM
 HEAT ECONOMY
 RECOVERY OF FUSEL OIL
 SUGGESTION TO SISTILLERY
 BOILER & POWER PLANT
 REFERANCES
INTRODUCTION
The Mohit Petrochemicals (P) Limited (MPPL) is located in Bijnor; Uttar

Pradesh uses the molasses from sugar to produce alcohol and rectified spirit. The
molasses required for the process is sourced from sugar industries located in the
vicinity. The installed capacity of the unit is 40 kilo litre per day (KLPD) which
generates approximately 320 m3 per day of effluent called spent wash. The
MPPL has installed thermophilic biomethanation reactor based on anaerobic
methanation process for treatment of spent wash. The spent wash is subsequently
sent to the storage tank where the Biomethanation plant has been generating
biogas, which is being utilized for generating power through steam for meeting
captive consumption. By extracting and capturing methane enriched biogas in the
anaerobic digester the project reduces CH 4 emission that would have otherwise
emitted from the existing open lagoon system in the absence of the project
activity. Also the use of bio-gas for steam/power generation saves equivalent
amount of fossil fuel which would have been used in absence of project activity
for steam/power generation.
Purpose:
The purpose of project activity is extraction and capture of methane enriched bio-
gas generated from treatment of spent wash and to utilize it for generation of
steam/power required for the operating process.
The project activity is anaerobic treatment of distillery effluent (spent wash) and
collection of methane enriched biogas in a closed digester system. This reduces
methane emissions into the atmosphere that would have otherwise been emitted
from the existing open lagoons. And secondly, combustion of methane enriched
biogas captured and other biomass in a boiler for generation of power through
steam, thus converting its methane content into carbon dioxide thereby reducing
its greenhouse gas effect.
Distillery is the allied of the sugar industry, as the bye-product of sugar

factory (molasses) is the raw material for distillery. In view of the above,
distilleries are the sugar factories to facilitate easy transportation of molasses
which is an important factor in the cost of production.
Meerut-Muzaffarnagar region is quite rich in sugarcane. Nearly a dozen sugar

factories are functioning in the Districts of Meerut and Muzaffarnagar,
considering all the points for the Bijnor in the year 2003-04. The distillation plant
was established by ALFA-LAVAL Company.
The plant was into production in 2003, at a capacity of rectified spirit per day,
30,000 liters of Extra Neutral Alcohol and 20,000 liter of Absolute alcohol. Extra
Neutral plant was installed by KBK, Rectified spirit Plant and Spirit Plant and
Absolute alcohol was installed by ALFA-LAVAL 2003-04.
Name of Organization MOHIT PETRO CHEMICALS

(P) LTD. BIJNOR
Address 8km Nagina road Dist. - bijnor

state - Uttar Pradesh
Name of contact person Mr. Suresh Pal Panwar
Plant Supplied By ALFA-LAVAL\ KBK, Pune
Capacity 40,000 Rs. / 30,000 ENA
Year of establishment 2003-04
Raw Material Cane Molasses
Average F.E. 89-90%
Average D.E. 96-98%
PROCESS CHEMIST
Composite analysis of molasses sample (process) and wash analysis, spent

wash analysis, maintaining of P.D.-8, P.D.-9 Register, daily report, weekly
reports, monthly report etc, with the guidance of production Manager and Shift In
charge. He also maintains the hygienic condition of fermentation house to control
the pH of prefermentation, regular sterilization of wort line to control the specific
gravity desired and inoculation and transfer timing. To see proper temperature is
maintained in all distillation plant.
In this way each chemist is responsible for their work.

FERMENTATION
Fermentation is a bio-chemical reaction where the substrate is converted into

desired product with the help of micro-organism which may be yeast, Mould or
bacteria and the enzyme present in the Micro- organism actually acts in the
reaction.
Reaction: C6 H12 O6 2 C 2H5OH + 2 CO2
(sugar) (alcohol)
The yeast Saccharomyces cerevisiae that is used for the culture for Alcohol
fermentation is having two kinds of enzymes, one’s INVERTASE and other is
ZYMASE. Invertase converts sucrose to invert sugar and Zymase converts invert
sugar into Ethyl Alcohol.
Fermentation is a dynamic process involving a series of reactions.
These are:
(i) Enzymes added after cooling continuous to convert the gelatinized starch
to dextrin’s and fermentable sugars.
(ii) The yeast metabolizes sugar to acetaldehydes and then to ethanol. The
cycle is known as Glycolysis.
The lactic acid bacteria produce small amounts of a variety of products, some of
which are volatile and contribute to the congeners in the distillate. Molasses is
transported in the cells via specific carrier proteins called Permeases where it is
hydrolysed into 2 molecules of glucose. The Permeases are substrate and require
metabolic energy for operation.
C6H12O6 2 C2H5OH +2 CO2 + 2 ATP + heat
Two moles of ATP (Adenosine Triphosphate) are also produced which are
used to supply energy for cell maintenance and growth. Theoretically conversion
of 1gm of glucose via fermentation yields 0.511grms of ethanol. This theoretical
value is never obtained due to carbohydrate utilization for cell maintenance,

growth and formation of small amounts of glycerols and higher alcohols.
Fermentation efficiency also depends upon factors such as yeast strain and
environmental parameters. In practice efficiency is 60-80%.An increase in temp.
within a certain range increases activity. The condition in Fermentation vats are
such obtained so as to avoid bacterial action. During a normal Fermentation heat
is produced from active yeast growth and metabolism which causes a rise in
temp. Then this temp rise can drastically affect yeast metabolism and ethanol. An
average upper limit temp for growth is around 40 oC with an optimum temp of
about 30’C. Increased heat tolerances are obtained with media containing oleic
acid. The maximum concentration of ethanol that yeast can produce depends
upon the yeast strain used. In general, yeast cell growth is inhibited around 10 to
12% wt/vol. ethanol while 20% ethanol will terminate cellular metabolism.
Alcoholic fermentation of cane molasses is carried out by adopting anaerobic
fermentation process in India. Generally two types of fermentation performed in
distillery i.e. only process to obtain alcohol from strap molasses. Fermentation
are of following types.
1. Continuous Fermentation
2. Batch Fermentation
CONTINUOUS FERMENTATION
Continuous Fermentation Process is the process in which cells are maintained

in continuous culture in the exponential growth phase by the frequent addition of
fresh medium that is exactly balanced by the removal of cell suspension from the
bioreactor.
This process recently following in India, due to high technology it is not

working properly.
Distilleries not only want maximum recovery of alcohol from molasses,

But also want to economic on time space, labour & cost of production.
Whole period of cellular activity of yeast can be divided into three stages:
1. Multiplication
2. Production of alcohol
3. Other products
Better yield is obtained when fermentation is carried out at optimum activity of

all types. Number of cells is wash is kept constant.
If fermentation is carried out in such a manner that wort is fed at the same rate at
which the gravity is lost, values of three variable sugar, alcohol organism are
almost constant. Consequently the speeds of changes become nil.
1. Single stage
Single stage continuous fermentation is that in which the entire operation is

completed under the steady6 stage conditions in one vessel only where the
nutrients at the same continuous rate. This Process simple one and specially
adapted to the process involving only cell growth.
2. Double stage continuous fermentation
Here instead of carrying out the fermentation in single fermentation, it is taken

to another fermentation, which allows the secondary fermentation to complete.\
3. Multistage continuous fermentation
It employs a better way of vessels in series. Fresh medium being feed into first
the effluent from it passes to the second and so on through as many successively
increased resulting in their more effluent us in substrate and higher yield in
product. In this fermentation, the components phase column be handled
separately.
The first one day is for growth phase, second and success ending one may be for
production of product and so on.
4. Semi continuous fermentation
This fermentation process in the modification single stage continuous
fermentation and occasionally multistage operation, in which feeding & drawing
are accomplished intermediately rather than continuously.
Batch fermentation
Batch fermentation process refers to the process that starts with the
inoculation and end with the retrieval of the product happens inside a single
fermenter with no intermediate steps.
A tank of fermenter is filled with the prepared mash of raw materials to be

fermented. The temperature and pH for microbial fermentation is properly
adjusted, and occasionally nutritive supplements are added to the prepared mash.
The mash is steam sterilized in a pure culture process.
Growth of microorganisms during batch fermentation confirms to the

characteristic growth curve, with a lag phase followed by a logarithmic phase.
This, in turn, is terminated by progressive decrements I in the rate of growth until
the stationary phase is reached. This is because of limitation of one or more of
the essential nutrients.
In this process, number of vats was charged with fresh yeast in column from
prefermenters in the ratio 1:15. These fermenters are separately filled with diluted
molasses of desired sp. Gravity.
ADVANTAGES OF BATCH FERMENTATION:

 Contamination and Salts don’t recycle.
 Cleaning and Sterilization is regularly maintained.
 Any kind f mishandling leads to minimum losses.
DISADVANTAGES OF BATCH FERMENTATION:

 While transferring the material, there is some loss of material
 And thus efficiency reduces.
 As the losses increases efficiency decreases.
 Time taken is more during Batch Fermentation.
The batch process is completed in the following steps:
 Preparation of isolation pure culture of yeast and making the slant.

 Maintain the Lab-culture.
 First stage in the development of yeast in the yeast vessels.
 Final stage in the development of yeast into the substage (prefermentor).
 Conversion of the sugar in alcohol in the main fermentor.
MOLASSES
Molasses is a viscous by-product of the processing of sugar cane, grapes or

sugar beets into sugar. The word molasses comes from the Portuguese word
melaço, which ultimately comes from mel, the Latin word for "honey". Cane
molasses is an important by-product in the manufacture of cane sugar. Molasses
is a thick, viscous dark brown in colour and easily miscible with water. It is
obtained in process of manufacturing cane sugar, Molasses is used in
fermentation industries for the production of Ethyl alcohol, Yeast production,
acetone, Acetic Acid production and some other organic acid production, but the
largest utilization of molasses in India at present is in the Alcohol Industry.
COMPOSITION OF MOLASSESS
The average chemical composition of molasses is given below:
1. Brix
2. Sugar as
T.R.S %
 Sucrose%
 Glucose
 fructose
 other reducing substance
3. Moisture
4. Nitrogenous component
 Protein
 Amino acid
5. Non Nitrogenous organic acid
6. Wax
7. Ash
Molasses has a high pollution as it contains a large amount of total dissolved
solids, high sulphates with no dissolved oxygen and a high bio-chemical oxygen
demand (B.O.D.). Its pollution characteristics are given below:-
PARAMETER VALUE
1. pH (unit) 3.5-4.1
2. Colour dark brown
3. Total dissolved solids 200000-320000
4. B.O.D. (Mg/Ltr) 440000

5. C.O.D. (Mg/Ltr) 960000
6. Chloride (Mg/Ltr) 32000
7. Sulphates (Mg/Ltr) 15000
8. Dissolved Oxygen Nil
Quality of molasses
Quality of molasses is generally depends upon the brix, total reducing sugars
invert sugar percentage and ash percentage. Based on this parameter I.S.I. has
prescribed the following three grades of molasses.
Sl.no characteristics ISI requirement For cane sugar

Grade I Grade II Grade III
1 Density in Degree 85 80 80
2 Ash sulphated by 14% 17.5% 17.5%
wt.(calculated for 100
degree Bx Max)
3 Total reducing 50%& 44%& 40% to
matters (as invert
sugar by weight min.) Above above 43.9%
Molasses control in distillery

The molasses produced by sugar factory is dispatched to distillery as allotted
to the distillery from various sugar by the Excise commissioner/Free sale
purchased anywhere. Transportation of molasses is done under type control of

Excise Inspector posted at Sugar Factory and Distillery.
DECONTROL OF MOLASSES (Government Notification)

50% control & 50% free sale of total production of molasses of sugar factory.
The alcoholic based chemical industry lifting 65% control which distillery used
only control molasses in C.L.
STORAGE OF MOLASSES
The production of cane sugar in our country is seasonal; therefore, it is
necessary to store the molasses .The quantity of molasses for at least 3 month is
needed for storage according to the capacity of distillery and the quantity of
molasses. For a factory producing 1000 gallons of rectified spirit per day quantity
of 50 tones of molasses is needed per day. Sugar factory which is crushing 3000
tones cane per day can supply on 9-12% molasses for a distillery, so the storage
of such a large amount of molasses is necessary.
PRECAUTION IN STORAGE OF MOLASSES

1. The storage tank i.e. steel tank should be painted to stop the reaction (for
metal corrosion).The dia & height should be in 1:1 proportion.
Thickness of wall should be 8mm.
2. The steel or masonry tanks should be covered to prevent the atmospheric
moisture; it should have sufficient ventilation at the top of the tank.
3. The distance between the two tanks should be at least 20 feet.
4. The tank should be cleaned every year to prevent any contamination
5. There should be accurate weighing arrangement of molasses.
6. No. leakage seepage, over flow or any other accident likely to damage the
quality of molasses stored in the factory.
7. To have the proper arrangement to prevent the missing up to water with

molasses, or old deteriorated molasses with fresh molasses.
CONTROL MEASUREMENT OF STORED MOLASSES
1. DIAL THERMOMETER
Dial thermometer are placed in tank at different levels with the help of which
molasses can be stored without little defects. Normally 85 degree to 100 degree F
is the range at which storage of molasses becomes satisfactory. If temperature of
molasses rises at different heights, the temperature control is done by an air
compressor and perforated aeration is carried out. Bubbles present in molasses,
thus frothing is also automatically controlled.
2. COOLING COILS
Through the tanks cooling are passed through which cold water is circulated.
These coils bring down the heat description.
Yeast
A commonly employed micro-organism for carrying out the
fermentation is yeast strain “Saccharomyces- cerevisiae”. This is commonly
known as ‘Distiller’s yeast’.
The yeast belongs to the sub-division of the thallophytic designated as
the Bumyctes, of true fungi, since they posses no chlorophyll.
The individual yeast cells are generally spherical, oval or ellipsoid in
morphologically.
A healthy yeast cell may vary from a little over 1 to 9 or more microns in
which and from about 2 to 20 micron in length.
Yeast contains 68 to 83 moisture. The yeast cell is composed of nitrogen

containing compounds, carbohydrates, lipids, vitamins, minerals and other
substrate.
METHOD OF ISOLATING PURE CULTURE OF YEAST
In our lab we do single cell isolation according to the following sequence.
1. The yeast containing culture is transferred to the 10 Bx solution of molasses

containing nutrients at a pH of 4.8 incubate it for two days.
2. Now place it in fridge for settling was settling with 1% oxalic acid and 1%
sodium chloride solution several times, now inoculate the test tubes
containing nutrients with these settling.
3. Incubate for 24 hours at 25-30 degree C.
4. After this pour the content of test tube with 1000 times diluted on the
petriplates containing sterilized molasses, again incubates for 24 hours.
5. After this period examine the colonies under microscope.
6. The loop and transferred in a test tube containing liquid media over flame.
7. Incubate the tube for some day at 25-35 degree C. Now cool it for settling of
yeast.
8. Wash the settling with 1% sodium chloride in a tube containing 10 ml of
10% Ethyl Alcohol Centrifuge it for 10ml of 15% ethyl settling and again put
in a test tube containing 10 ml of 20% Ethyl Alcohol Centrifuge it for 10
minute and get setting.
The advantage of using alcohol is that alcohol will kill if
there is any contamination and in the same time the low alcohol tolerant yeast
will also not survive. Only the high alcohol tolerant pure yeast strain will be
exist.
MEDIA USE TO ISOLATE YEAST
Liquid Media
a. Malt Extract 0.3%

b. Yeast Extract 0.3%
c. Peptone 0.5%
d. Molasses (sterilized) 10 Degree Bx solution
e. Urea 400 Mg/Ltrs.
f. Sulphuric Acid pH-4.8
MAINTENANCE
Once we have isolate and purified the yeast it is necessary to preserve it
so as to maintain its quality. Generally we preserve these yeasts on slants for
couple of months. After 10-15 days if yeast is not in use we inoculate a separate
liquid media by this and again make the slant of it. By this, yeast does not lose its
qualities and activities. Sometimes we use liquid media for preservation kept in
refrigeration. But in this also it is necessary to transfer this yeast in second media
of same composition after and interval of 2 to 3 months to avoid inactivity.
SLANT TUBE PREPARATION

1. Mix and prepare the nutrient agar as in the preparation of Petri dishes.
2. Pour 5 ml of the nutrient into a test tube.
3. Plug the test tube with a cotton ball or a cap. Culture tubes with screw caps
may also be used.
4. After autoclaving the media for 20 minutes, the tubes are placed in a slanted
position to allow the agar to solidify.
5. After cooling, place the yeast colonies from liquid media with the help of
loop over flame.
6. Incubate at 30 hours. After this period the growing yeast colonies will be
prepared on slant. Now preserve it in fridge.
LAB CULTURE
Propagation of yeast is done in laboratory for fermentation. We propagate
yeast in 10 degree brix molasses solution with nutrients. (About 400mg/liter of
urea) and maintain the pH 4.8 by adding sulphuric acid. Now transfer this
molasses media in test tube, 250 ml conical flasks, 500 ml conical flask, 1 liter
volumetric flask, 3 liter volumetric flask, 5 liter these flasks contain in autoclave
for 15 minutes under a pressure of 15Lb/inch.
Now after cooling we inoculate the test tube front slant. Incubate it for 8
hours, after 8 hours 250 ml conical flask will be inoculate with the contents of
test tubes. Again incubate the flask for 8 hours at 30 degree C. This flask, which
in turn be used to inoculate 1 liter flask and 1 liter is for 3 liter & finally this 3
liter for 5 liter capacity flask. On this series start then it proceeds a head
continuously. We do first transfer in morning at about 8.00 A.M. and second
transfer in evening at about 6.00 P.M. The yeast culture is very active, viable,
budding with concentration of 107 to 10-10 cells/ml.
FERMENTATION HOUSE
Fermentation house is well equipped arrangements of steam coils, cooling
coils and air coils in yeast vessels and bobs (Pre-fermentor) we have following
equipments in fermentation house.
DILUTER
Having three connections, two are inlet for water and molasses and third one
is outlet for molasses of desired brix. It is drum shaped and situa6ted on ground
floor Diluted molasses comes in fermentation house through gravity.
STERILIZER
Sterelizer is made up of stainless steel having a capacity of 1800 liters,
inside the sterilizer are steam coils and water coils. It is used for culture media
sterilization.
PURE YEAST VESSELS

M/s Mohit Petro Chemicals have 4 number of stainless steel yeast vessels of
following capacity.
Yeast vessels No. 1 91 Liters

All the vessels are having stainless steel heating and cooling coils
concentrated in one zone only. All the vessels are provided with top flanges but
no light and sight glasses and wort inlet arrangement has been made .these
vessels are provided with air sparger pipe made of copper.
Yeast solution prepared in carboy is passed in yeast vessels. The capacity of
yeast vessels are so adjusted that the quantity of yeast is increased 4-5 times
greater than the previous one.
BUB TANKS /PREFERMENTERS

At present there are two bub tanks (prefermenters) are in order. The details of
bub tanks are given below:
Sl.no. Tanks No. Height Capacity

1 Bub Tank No. 1 7.40m 47183Lts
2 Bub Tank No.2 7.40m 47183Lts
Each bub tank is provided with air sparger pipe made of copper . The bub is
connected to yeast vessels through a 2’’ pipe line for transfer of yeast solution.
Before prefermenters stage yeast culture is developed in the covered vessels and
on the sterilized medium but at this stage the propagation is done in the open
vessels. With the unsterilized wort before taking transfer to the prefermenters
from the yeast vessels. It limed steam and finally washed with water. After this
yeast culture is taken into prefermentor and gradually filling with wort is started.
for each 1000 liters of wort 100gms urea and about 100 ml H2SO4 concentrated is
added to solution and sterilize air a palled through solution for proper yeast
propagation thus a bulk of yeast culture is obtained after 8-10 hours according the
requirement for fermentation house and sent to wash balk (fermenter)
WASH BALK OR FERMENTER
1. These are mild steel big capacity tank i.e. 250000lts to 3 lakh liters.
2. All the fermenters are having flat bottom.
3. There are four fermenters working in fermentation house.
The details of working fermenters are given below:
Sl.no. Tanks No. Height Capacity

1 W.B. No.1 886cm 303157Ltrs
2 W.B. No.2 886cm 303157Ltrs
3 W.B. No.3 900cm 249498 Ltrs
4 W.B. No.4 900cm 249498 Ltrs
4. Fermenters are equipped with cooling coils around it and arrangement for
filling, draining out sludge.
5. Actual fermentation tank placed in this tanks and dilute solution not 8-9.5 %
alcohol is produced here as a result of fermentation , A part of bub tanks solution
is tank in the wash back and dilute molasses solution of about 20-24 degree brix
(1.100-1.120 sp. gravity) having 13-16 % sugar is added gradually .
6. The wash back and one half of the sugar are fermented to alcohol within 8-10
hours.
7.After pitching whereas total fermentation takes place , about 32 hours to
complete ,the total time cycle require to ferment a wash back is given below :
Table
For efficient working and securing good result constant watch of the condition of
fermentation is necessary.
TEMPERATURE CONTROL DURING FERMENTATION PROCESS

Optimum temperature for alcohol fermentation is 32-35 degree C and heat
released during fermentation process. In summer seasons the atmospheric
temperature raises 38-40 degree C to take good result of fermentation temp,
control is necessary. It is done by spraying water through cooling coils outside
the vat. Recently submerged tube cooler are used, these coolers can be made S.
S-304 or mild steel and are being utilized to control the high temperature of
fermenter.
PH CONTROL
The optimum pH range for alcohol fermentation is 4.5-4.8 it is adjusted
adding the conc. Sulphuric acid in wort.
FOAMING CONTROL
During fermentation co2 evolved which came out from the fermentation tank
in form of forming resulting wastage of yeast and wash, so anti-foam is spread of
the wash back. Actually anti foam out down the surface tension of upper layer
and release of co2 gas . Anti foam is ulphonted castor oil. Generally it made from
sulphanation of ground out oil since it is cheaper.
Use of anti infectant

In Mohit petrochemical s Bijnor, we are using penicillin doses in propagation
stage of yeast vessels, and ammonium bifloride is added in small quantity (1 Kg
for 10,000 liters) n prefermenters. In fermenters we are using concentrated
sulphuric acid to maintain- the ph 4.5-4.8 accurately.
CONTAMINATION IN DISTILLERY
The contamination in distillery is mainly from bacteria , they may be due to
fungus contamination but they are rare .Spherical and rod shaped bacteria are
present in fermenter and prefermentor in the distillery , bacteria are widely
deteriorated in air, Bacteria are introduced into the air by various forces , the
principle source being from dust particle containing negative of contamination .
SOURCE OF CONTAMINATION
Contamination may got into the production suture from the stock culture of
the production microorganism from incorrect manipulation of the culture in
laboratory or in seed tanks from insufficiently sterilized fermentation medium
from air used for aeration from insufficient deforming during fermentation and
finely by commission of some of the basic rules pre venting outside
contamination are the important point.
The following are the important point to reduce the contamination:
1. There must be no direct connection between the sterile and non-sterile parts of
the Piping and armatures
2. Where connection and flanges are indispensable, this must be made of first
grade rubber without a textile layer
3. Welded construction should be used wherever possible.
4. The types of valves and armature must be so selected as to be easily
sterilizable and able to maintain sterility throughout the whole process.
5. After sterilization all parts of the equipment and piping junction which are to
remain sterile have to be maintained under constant over pressure of sterile air
from which bacteria do not get chances to develop.
6. Every part of the equipment must be capable of independent sterilization
which must not interfere with the function of the rest of the equipment. In the
production of ethanol, ammonium bifloride can be used as an antiseptic. The
application of penta chlorophenol in the production of ethanol was suggested.
AVOID CONTAMINATION
1. Maintain yeast culture on nutrient agar media at 0.5 degree C.

2. Isolate and screen single cell yeast culture by dilution plate technique.
3. Periodically is necessary. If contamination found and it is major risk for

draining the material from the vessel and follow all precautions.
4. Check air lines air filter s to avoid air born contamination
5. All the cutting lines. Wort lines should be thoroughly cleaned and
steamed.
6. Transfers from bub are done when brix falls 12 degree to 8 to 9 degree
Bx.
7. Maintain the pH of prefermenters and wash backs (fermenters) between
4.5 to 4.8 accurately.
ANNEXURE
1. ATTENUATION
The difference between the initial gravity & final gravity called attenuation.
46 degree attenuation is corresponds to produce 10 degree proof alcohol of
8.05 attenuation is corresponds to produce 1 % v/v alcohol.
2. OVER PROOF = (100+O.P.) Proof
3. UNDER PROOF= (100+O.P.)Proof
4.1 degree PROOF=4/7% v/v or 0.571 v/v
5.1 degree LONDON PROOF= 1 degree PROOF OR U.B. PROOF
6. TOTAL REDUCTION SUGAR:

Fermentable Sugar+ Residual Sugar
7. GAYLUSECC’S REACTION’
This reaction has relation between the alcohols produced with fermentable
sugar.
C6H12O6 degree 6=2C2H5OH + 2CO2
(180gms) (92gms) (88gms)
So 180 gms F.S. =92 gms Ethanol

100 gms will produce =51.1 gms of Ethanol
Specific gravity of 100% Ethanol =0.79
So 51.1/0.79 = 64.4ML/100 gms of F.S.
Fermentation Recovery= Total Fermented Wash X AL.%

In wash
(In A1.QTL or B.L. /QTL)
DISTILLATION HOUSE
M/s Mohit Petro Chemical Pvt.ltd have installed three plants namely R.S.
ENA and ABSOLUTE ALCOHOL plant was installed in the year 2003.
ANALYSER COLUMN WITH DEGASSIFYING COLUMN

One analyzer column was built in segments counting 20tunnel cup type plates.
5 tunnel cup type plates in the Degasifying column.
The working of degasifying column in separate to the Analyzer column. The
preheated wash from Beer-heater and then to heat Exchanger in fed to
degasifying column where wash is slightly heated i.e. up to 70 degree C. The
vapour is generated from degasifying column is passed to Aldehyde feed tank
than feed to Aldehyde column. Vapour which are generated by Aldehyde column
is passed to Aldehyde main and vent condenser from where condensate is taken
as a reflux to the column and from this product about 20% is stored separately as
on impure alcohol and the loose of the Aldehyde column is send to rectifier feed
tank than feed to rectifier column.
DISTILLATION
Distillation consists in separation more or less completely a volatile liquid
from less volatile substances with it is associated. The design of distillation
equipment is based upon the practical experience gained from the plant working
in the distillation plant.
THEORY OF DISTILLATION:
In the simplest mixture of two mutually soluble liquids, the volatility of each
is undisturbed by the presence of the other. In such a case, the boiling point of a
50-50 mixture, for example, would be halfway between the boiling points of the
pure substances, and the degree of separation produced by a single distillation
would depend only on the vapor pressure, or the volatility, of the separate
components at this temperature. This simple relationship is called as the Raoult's

Law. Raoult's law applies only to mixtures of liquids that are very similar in
chemical structure, such as benzene and toluene. In most cases wide deviations
occur from this law. Thus, one component is only slightly soluble in the other; its
volatility is abnormally increased. In the example above, the volatility of alcohol
in dilute aqueous solution is several times as great as predicted by Raoult's law.
In extremely concentrated alcohol solutions, the deviation is even more striking:
The distillation of 99 percent alcohol produces vapor that has less than 99 percent
alcohol. For this reason, alcohol cannot be concentrated by distillation beyond 97
percent, even by an infinite number of distillations
Distillation takes place in the following:-
ANALYSER
As the name is analyzer, this analyses the more volatile vapours from the
fermented wash leaving behind less volatile liquid at the bottom. The fermented
wash from the wash charger is pumped to passing though beer heated (pre heater)
and heat exchanger and after heated to an extent is fed to the feed plate in the top
of the analyzer column, the temperature in the bottom about 80-81 degree cen.
Gr. A t this temperature under satisfactory working condition, there is no loss of
alcohol within the spent wash there are 17 to 20 plates in the column, wash from
the feed plate, the strips to the next down plate though down pipe and spread over
the plate and similar to the rest plates of the column . vapour rich in alcohol a
send to the upper side of the column finally reached to the vapour to the vapour
line. The liquid going down from plates to plates ultimately nil in alcohol reached
a bottom of column and is send to the heat exchanger for heat recover. This
column is takes place in vaccum. Temperature is maintained by circulation the
bottom product through analyzer reboiler. In analyzer reboiler ethanol vapour
from rectifier column top comes maintain the temperature. The vapours
condensed by maintaining the bottom temperature is feed to efflux tank which is

feed to rectifier column.
RECTIFIER
Alcoholic vapours coming out from the analyzer column are some less richer
(about 34-40%) in alcohol these are passed through rectifier by rectifier feed tank
. higher boiling fraction leaves behind and vapours quite rich in alcohol (say 93-
94%) reached to the top of the rectifier, from here these are twisted to the
analyzer reboiler. A very important and valuable by product named as ‘fusel oil’
is collected from the lower portion of the rectifier , if it is not separated out then
it is a contamination for the final product as it is very pungent and bad smelling
substances it is a mixture of higher alcohol and its constituents are used as
solvent perfumes and sources of various chemicals the bottom temperature is
maintains 19-130 degree cent by applying steam through reboiler.
BEER-HEATER OR PRE HEATER

Beer heater is metal hollow cylindrical body fitter metal tubes wash from
charger though control value is passed through the tubes and alcohol vapour
coming out from the Analyzer column are passed through the shell side in
opposite direction the condensed liquid is collected in rectifier feed tank which is
feed to rectifier column. A direct latent heat transfer takes place with a partial
condensation of alcohol vapour and partial heating of wash. Condensed alcohol is
send to 20 plate of the Rectifier.
CONDENSER
Function of condenser is the same as that of Beer Heater with a different that
coolent used in water in place of wash is passed in the tubes. some distillers in
India use two condensers and one eer heater while others to beer heaters and two
condensers, one ordinary condenser=one total condenser having vent pipe. The
condensers, one ordinary condenser is sent to the top place of rectifier as in case
of beer heater(for making the same strength because the strength after condensing
that beer heater and Ist and IInd condenser are different) so to make the same
strength send in the rectifier top again and then send to cooler .
HEAT EXCHANGER
The heat exchanger plays an important role in the economy of the heat. The
wash preheated in the beer heater is passed through the beer heater on one side of
the plate and the spent was (68-70 Deg. Cen.) coming out from the Analyzer
column in passed through the other side thus heat transfer takes place and the
coming out wash is heated approximately to a temp. of 80-85 dec. cen. The spent
wash coming out to the cannel. Spent wash will be taken to Biogas plant.
E.N.A. PLANT
E.N.A. PLANT is supplied by M/s KBK pune . Its installed capacity is 30000
per Ltrs. Per day. In this plant we distilled further rectified spirit with a mixture
of water is ratio 1:2 or 1:3 Distillation in this plant taker place in the following
steps.
PURIFIER COLUMN
1. This column is made of copper & plays an important role to purify the quality
of spirit & water mixture is feed on plate.
2. This column consists of 10 segments of 60 plates with tunnel types cups.
3. This column is in vaccum. We collect some impure from the final condenser of
purity & reflux further feed to column then its lease back to the rectifier column.
4. The bottom temperature maintained 68-70 dec. cen. Bottom temperature is
maintained through reboiler by passing vapours from top of rectifier column.
RECTIFIER COLUMN
1. Alcoholic mixture coming from the bottom of purifier on 15th plate. Rectified
column consist of 67 plates.
2. The mixture feed to rectification column and higher boiling fraction leaves
behind and vapours quite rich of ethanol (95%) reached to the top of column
from where they are twisted to condenser.
3. A very important y-product named ‘fused oil’ is collected from the bottom and
it is essential to remove, due to presence of F.Oil the bad smelly final product
drawn from 60 to 33 Nos. of plates is obtained.
CONDENSERS
1. In ENA plant four condensers are present two on rectifier column and two are
a purifier column.
2. In shell side the vapors are present and in tubes water is passing.
3. The condensate of condenser’s is send to the top of rectifier column. From the
both column vent condenser’s some impure (about 10-20 %) spirit can be drawn
in the from of heads.
COOLERS
The taping drawn from rectifier and this is send to cooler s from cooling it is
necessary to cool the product before storage. It is done with the help of coolers.
LABORATORY CONTROL
METHODS & MATERIALS

All glass wares are cleaned before use with moderately hot chromic acid was
several times with rap water and finally rinsed with distilled water. All colour
tube and flak used are of Pyrex glass to avoid the chances of breakage during
sterilization. Sterilized non absorbent cotton is used for plugging the culture tube
& flasks.
Standard burette and pipette are used throughout the investigation.
STERELIZATION OF APPARATUS
All test tubes flask, bottlers and other glassware used in the cultivation of
cultures are dept over night at 15 deg. Cen. to 17 deg. Cen. for sterilization.
INCUBATION TEMPERATURE
All the test tubes, flasks and bottles used in the cultivation of culture are
incubating at 37 deg. Cen.
INSTRUMENTS EMPLOYED FOR ESTIMATION
A) MECHANICAL & ELECTRICAL INSTRUMENTS
i) pH meter
A pH meter is an electronic instrument used to measure the pH (acidity or

alkalinity) of a liquid (though special probes are sometimes used to measure the
pH of semi-solid substances). A typical pH meter consists of a special measuring
probe (a glass electrode) connected to an electronic meter that measures and
displays the pH reading.
All the pH measurement is carried out on an ELICIT –pH meter. The

instrument is standardized with standard buffers of pH and using glass electrodes.
ii) ANALYTICAL BALANCE
The basic tool in all quantitative analyses is the ANALYTICAL

BALANCE, used for the accurate weighing of samples and precipitates.
For usual analytical work the balance should be able to determine
difference in mass of 0.1milligram (about 0.000004 ounces). In
microanalyses the balance must be about 1,000 times more sensitive and
for special work, balances of even sensitivity have been citations.
iii) HOT AIR OVEN
All the glass ware’s sterilized by incubating them in over for 2 hours at 37 deg.
cen.
iv) AUTOCLAVE
An autoclave is a device to sterilize equipment and supplies by

subjecting them to high pressure steam at 121° C or more. It was
invented by Charles Chamberland in 1879, although a precursor known
as the steam digester was created by Denis Papin in 1679, it is used for
sterilization of liquid media and glass and apparatus at a pressure of 15
ph for one present.
v) LAMINAR AIR FLOW

A laminar flow cabinet or laminar flow closet or tissue culture

hood is a carefully enclosed bench designed to prevent contamination of
semiconductor wafers, biological samples, or any particle sensitive
device. Air is drawn through a HEPA filter and blown in a very smooth,
laminar flow towards the user. The cabinet is usually made of stainless
steel with no gaps or joints where spores might collect.
Such hoods exist in both horizontal and vertical configurations, and

there are many different types of cabinets with a variety of airflow
patterns and acceptable uses. NSF49 is the commonly accepted
regulatory standard for these cabinets.
Laminar flow cabinets may have a UV-C germicidal lamp to sterilize

the shell and contents when not in use. (It is important to switch this light
off during use, as it will quickly give any exposed skin sunburn and may
cause cataracts
vi) MICROSCOPE
Microscope is used study the morphology in yeast and to detect any

contamination if present.
vii) BOD INCUBATOR
The incubator is double walled chamber with glass wool/ PUF insulation
interior surface is made up of stainless steel and exterior surface is made up of
mild steel powder coded.
The incubator is equipped with Eurotherm temperature controller

which enables the easy temperature settings. With reference to safety the
equipment is installed with over shoot and under shoot temperature alarm.
The system is provided with the heating system with the help of SS heater in
order to achieve the required temperature
viii) REFRIGERATOR
A 165 liter refrigerator is used to preserved and store slants and liquid media.
ix) CHEMICAL BALANCE
Laboratory is equipped with two chemical balance used for accurate weighing
of chemicals.
x) HOT PLATES
Three hot plates are used for distillation and boiling of solution.
xi) GLASS WARES
Flasks (250ML 500ML),BEAKERS(100 ML),test tubes, measuring

cylinders(50ML,500ML & 1000 ML),Distillation assembly, separating funnel,
volumetric flasks (100ML,500ML & 1000ML), Burette & pipettes
(1ml,2ml,5ml,10ml,20ml& 50ml), and micropipettes(0,1ml,0.2ml & 0.5ml) etc.
EXPERIMENT
Determination of brix of molasses
APPARATUS
Brix spindle, measuring cylinder, thermometer etc.
PROCEDURE
Weight 50 gms of molasses and dissolve it in 450 distilled water and take the
solution in a cylinder for calculation of brix and float 0-10 Dec. Cen Brix spindle
in it. Take the temperature of the solution.
CALCULATION
Calculate the brix of molasses with following manner:
Hydrometer reading (A) = 8.4
Temperature of the solution (B) = 32Deg. Cen.
Temperature correcting =0.3384+0.33
Brix of molasses (A+C) X10 = 87.3 Bx
TOTAL REDUCING SUGAR

Now we see the % total reducing sugar in molasses. Take 10 ml of the above
solution in 10 ml measuring flask and add 5 ml concentrated HCL. For inversion,
add some water (app. 30 ml.). Heat this mixture on a water bath for 10 minutes at
70 deg. Cen. Cool it and neutralize with NaOH soln. (app. 6N strength using 1%
W/V phenolphthalein indicator and make up to the mark 100 ml and titrate again
5 ml Fehling solution A=% Fehling solution ‘B’ using 2% m/v) Methylene blue
as an indicator. Note the burette reading.
CALCULTATION
%total reducing sugar =100Xdilution factor %(T.R.S.)
Fehling Factor
Burette Reading
(Fehling factor is estimate and found 0.058)
=100X500/50~100/10X0.058 Burette Reading
Burette Reading =12.6
=100/10X0.058/12.6
T.R.S. =45.03%
DETERMINATION OF RESIDUAL SUGAR

Residual sugar is determined is wash after complete fermentation of wort, it is
determined as follows:
Take 5ml fehl’s A+5fehl’s soln. B and titrate against fermented was using
Methylene blue as an indicator.
% of Residual Sugar =100Xfehling factor
Burette reading
(Burette reading-5.0ml)=100X0.058
5
=1.16% in fermented wash
DETERMINATION OF % ALCOHOL IN FERMENTED WASH
Take 250 ml spent wash 27.5 Deb. Cen. Add 250 ml of water in a distillation
flask with using glass weeds. Now distill it and collect exactly 250 ml of distillate
and find out the alcohol percent in same manner.
Alcohol meter reading 98.6
Temperature 86F
Alcohol % in spent wash 0.1 %
ANALYSIS OF SPENT LEESE

About 250 ml spent lease collected from out let of exhaust column at a
temperature of 102 dec. cen. I8n the 500ml conical flask fitted with cotton plug.
Cool it to room temperature and pour in a cylinder. Now know the reading by
dipping the alcoholmeter and temperature see the alcohol % by alcoholmeter
table.
TO DETERMINATION OF FERMENTATTION EFFICIENCY OF

GIVEN STRAIN OF S.SEREVISIAE IN LABORATORY
Fermentation efficiency can be expressed as fermentation capacity of

particular strain of yeast related to production of alcohol. It can be determined by
noting down fermentable sugar percentage and alcohol percentage over
theoretical yield.
Prepare 600ml solution of molasses of about 20.0 Bx. Take a flat bottom flask
add urea’s in the ration of 200 mg/Ltr. Now adjust pH at 4.8 and is tribute as
follows.
1. 5 ml in test tube.
2. 15 ml in boiling tube
3. 150ml conical flask
4. 450ml in 1liter flat bottom flask
Now sterilize in autoclave at 15 PSI pressure for 30 minutes. Remaining

solution is used for determining TRS in molasses.
Inoculate the test tube yeast with the help of loop .keep it in incubator at 30 Dec.
Cen. For 24 hours. Then transfers the solution in 150 ml conical flask under
flame inoculates at 30 dec. cen. For 24 hours. Finally transfer the solution to 450
ml solution under flame this flask should be kept at least 15-20 hours at 30 min
the unfermentable sugar. For unfermentable sugar no need to invert in with HCL.
The difference between total sugar and unfermentable sugar will fermentable
sugar. Take 100ml of capacity, Make up to the volume of necessary shake it and
make homogeneous mixture. Find out the specific gravity of distillate. Determine
the alcohol percentage by using table. Compare it with that of theoretical
percentage using Gay-Lussac formula for alcohol (63 lit of alcohol per 100 kg of
fermentable sugars).
OBSERVATION
Given Fehling’s factor 0.058
1. Total reducing sugar of 20 Bx molasses media used for fermentation. 20 ml
sample is to be diluted to 250 ml inversion with HCL and neutralize with NaOH
and make up the volume 100 ml.
Titrate vale of diluted sample to neutralize 10 ml Fehling’s soln.

Total reducing sugars in molasses 250X058X100
Used for fermentation 20X5.1
=14.215 % (A)
Total reducing sugar in molasses when fermentation is over:
40 ML fermented wash is to take. Inverted with HCL Neutralize with NaOH and
volume make up to 100 ml.
Volume required for neutralization of 10ml of Fehling’s soln. 8.9 ml
Hence unfermented sugar’s =100X0.058X100
40X8.9
=14.215-1.629
=12.586%
 Estimation of alcohol
 Weight of pyknowmeter +alcohol=63/657 gms
 Weight of pyknowmeter +water =64.193 gms
 Weight of pyknowmeter only=10.078 gms
Temperature 15.5 deg. Cen.
CALCULATION
Weight of alcohol= 63.657-10.078

= 53.579gms
Weight of waters = 64.193-10.078
= 54.115gms
Density of alcohol= weight of alcohol
Weight of water
= 53.576
54.115
Percentage of alcohol = 0.990
At this density =7.17%v/v (from ADAC tables)
Theoretical yield of alcohol
100gms of fermented sugar
Yield alcohol =64.48%
Hence 12.586gms of fermented
Sugar yield alcohol =64.48/100 X12.586
=8.115%
Fermentation efficiency =7.17X100/8.115
=88.335%
Results
Fermentation efficiency of given stain is 88.335%

Total loss of fermentation efficiency -16 % so the fermentation efficiency 84 %
has been fixed based on above mentioned norms, a fermenter was set under
controlled and following results have been obtained.
1. Molasses taken in fermenter 200qtls

2. Total reducing sugar contents 47.0%
3. Total wort taken in fermenters 66.776ltr.
Including yeast form P.F.
4. Initial gravity 1.084
5. Temperature of setting 25 Dec.Cen.
6. Fermentation time 22Hrs
7. Raise in temp. observe 37 Dec.Cen.
8. Cooled with external water supply 33 Dec.Cen.
9. Final gravity collected at 25 Dec. 1.032
Cen.
10. Alcohol contents (by hydrometer) 7.0%
11. Final volume of wash in fermenter 66.309Ltr
12. Residual sugar content
13. (non fermentable sugar) 1.15 %
CALCULATION
1. Molasses taken 200qtls

2. Total reducing sugar 47%
3. Total reducing sugar in wt. 200X47X100/100
9400Kgs
4. Total unfermentable sugar 66309X1.15/100
=762.55Kgs
5. Fermentable sugar 9400-762.55
=8637.45Kgs
6. Alcohol to produced at 84 % 8637.45X84/100
fermentation efficiency 4781.49AL
7. %Fermentation efficiency 4641.6X84/4781.49
=83.29or83.3%
8.%loss of Fermentation efficiency 84.0-83.3
=0.7%
The norms for Fermentation efficiency has been fixed at 84%on the following
premisis
1. Loss of efficiency due to Pasteur’s 5%

coefficient
2. Loss of efficiency due to sugar 6%
consumption for yeast propagation
3. Loss of efficiency due to sludge 1.8-2%

formation
4. Loss of efficiency due to loss of 1.0-1.6%
dioxide depending on temperature of
fermenting wash
5. Loss of efficiency due to unknown
factor such as temperature , resulting in 0.50-1.4%
formation of side reaction

ANALYSIS OF RECTIFIED SPIRIT /LIQUOR

Weight of pyknowmeter in chemical balance .now fills this with alcohol and
again weight. Finally weigh with water
OBSERVATION
1. Weight of empty phyknowmeter = x gms

2. Weight of phyknowmeter + alcohol = y gms
3. Weight of phyknowmeter +water = z gms
CALCULATION
1. Weight of alcohol = x-y gms

2. Weight of water = x-y gms
Specific gravity of alcohol = weight of alcohol

Weight of water
= x-y
z-y
Now we can get the strength after seeing specific gravity against temperature
from ADAC table
But for routine laboratory work we adopt hydrometer method. For this method
pour the sample in the cylinder then dip the hydrometer to know the reading and
note the temperature. Now see the strength using table of strength of
alcoholment.
DETERMINATION OF TOTAL ACIDITY/REAGENTS
1. Standard sodium hydroxide solution 0.05N

2. Phenolphthalein indicator 1%v/v
Take 50ml sample in a conical flask 250 ml capacity add 50 ml neutralize

distill water using phenolphthalein indicator , note the titrate value.
Observation –titrate value =0.3ml
CALCULATION
Total acidity (in terms of tartaric acid) =
Where;
V=volume in ml of sodium hydroxide solution (0.05N STRENGTH)
V1=Alcohol percentage by volume
So,
V2=94v/v
V=0.3ml
Total acidity = 750XV/V1
=750X0.3/94.2
=2.388OR2.39gms/100ltr.Or absolute alcohol
DETERMINATION OF FIXED ACIDITY
Reagents
1. Standard Sodium hydroxide solu. 0.05N

2. Phenolphthalein Indicator 1%v/v
3. Neutralized distilled water
PROCEDURE
Take 50 ml of sample and evaporate to dryness. Dilute with

Neutralized distilled water to 100 ml and titra against standard sodium
hydroxide solution using Phenolphthalein Indicator.
Observation-titrate value = 0.1ml
CALCULATION
Fixed Acidity (in terms of Titaric Acid) = 0.00375X100X1000X2XV
Strength of alcohol
gms/100ltr A. Alcohol
V= Volume of Sodium hydroxide solu. Consumed
(1mg. of Sodium hydroxide solu. Is equivalent to 0.00375 gms
Of Tartaric acid)
= 0.00375X100X10000X20.1/94.2
=750X0.1/94.2
=0.796or0.8 gms/100ltr of Absolute Alcohol
DETERMINATION OF VALATILE ACIDITY

Reagents:
1. Standard Sodium hydroxide solu. 0.05N

2. Phenolphthalein Indicator 1%v/v
PROCEDURE
Take 50 ml of the distillate/alcohol sample. Obtain From the determination of
methyl alcohol add 50ml of neutralized distilled water; titrate against Standard
Sodium hydroxide sol. Using Phenolphthalein Indicator. (1 ml liter of Standard
Sodium hydroxide solu. Is equivalent to 0.003gms of acetic acid).
CALCULATION
Volatile acids express as= VX100X1000X0.003X2

Strength of alcohol in sample
V = Volume of St. Sodium Hydroxide Solution ml.
OBSERVATION
Titer Volume = 0.25ml

Volatile acidity= 0.25X100X1000X0.003X2/94.2
= 150/94.2= 1.59gms/100ltr. Of absolute alcohol
DETERMINATION OF ALDEHYDE
QUANTITATIVE TEST
Reagents:
1. Sodium Bi-sulphate Solution 0.05N

2. Standard Sodium Thiosulphate 0.05N
3. Standard Iodine Solution 0.05N
4. Starch freshly prepared
PROCEDURE
Take 50 ml of sample in 250 ml flask in a Iodine flask add 50 ml of distilled
water, keep it in a dark place for 30 minutes with occasional shaking. Add 10 ml
sodium Bisulphate solution. Run a blank sample having 100ml water + 100
sodium BI-SULPHATE solution. Kept it in a dark place with occasional shaking.
Add 25 ml Iodine soln. In both samples. Titrate excess iodine against standard
sodium thiosulphate solution using starch as a indicator.
The difference in titration value in blank and simple in militires of sodium
thiosulphate solution give the equivalent Aldehyde (one ml of standard sodium.
Thiosulphate soln. Equal to 0.001 gms of acetaldehyde).
OBSERVATION
Blank reading =24.5ml
Sample reading =24.4ml

Difference =24.5-24.4 =0.1ml
CALCULATION
Aldehyde in term 0.0011X2X100X1000X0.1
Of acetaldehyde strength of alcohol
= 220x0.1
94.2
0.23gms/100lit of absolute alcohol
QUANTITATIVE TEST FOR ALDEHYDE CONTENT
Reagents:
Sodium hydroxide soln. 20% W/v in distilled water
PROCEDURE
Mix 10 ml of material with 5ml of sodium hydroxide solution and set aside for 2
minutes.
OBSERVATION
The limit prescribed for Aldehyde content (0.006 gms per 100ml) shall be taken
as not having been exceed if no yellow color is produced in 5 minutes.
Note: Aldehyde content is rectified spirit not more than maximum 0.006 gms
/100ml first grade and 0. 01gms/100ml in II grade.
QUALITATIVE TEST FOR ALDEHYDE CONTENT
(Only for Rectified sprit E.N.A. only)

(Potassium Permanganate time Reaction)
TO determine Esters
Reagent:
1. Standard Sodium Hydroxide soln. 0.05N

2. Standard Sodium Acid 0.1 %
PROCEDURE
Take 50ml of sample and neutralize it with 0.05N hydroxide solution and now
add 10ml of 0.1N sodium hydroxide solution. Reflux it on steam water both for
an hour. Cool and back titrate the excess of alkali with standard sulphuric acid
0.1N simultaneously run a black taking 50ML of distilled in place of sample in
the same way. The difference in titration value in militires.
OBSERVATION
Blank reading = 8.8ml

Sample reading = 8.6ml
Difference = 0.2ml
CALCULATION
Ester expressed in terms of ethyl
Acetate in grams /1000 litres of absolute alcohol
= 0.0088x100x1000x2x0.2/strength of alcohol
=3.73 gms /100litres of absolute alcohol
DETERMINATION OF HIGHER ALCOHOL
1. Quantitative Method
2. Qualitative Method
Quantitative Method
REAGENTS
1. Oxidizing Mixture =100 gms. potassium Di-Chromate dissolve in

100ml concentrated sulphuric acid (A.R.grage)
2. Standard sodium hydroxide
3. Carbon tetrachloride(tode distilled before use)
4. Satured soln. of sodium chloride
5. Sodium sulphate
6. Phenolphthalein indicator 1 % w/v
PROCEDURE
In the solution after determinations of esters content add 50 ml saturated
chloride solution. Mix four times with carbon tetrachloride using 40 ml, 30ml,20
ml and 10 ml respectively with vigorously shaking about 30 minutes combine the
extract and wash three time with saturated sodium chloride solution and twice
with saturated sodium sulphate solution.
Filtered carbon tetrachloride and add 50 ml of oxidizing mixture refluxed for
2 hours with 50 ml of water and transfer it to the distillation flask using about 50
ml as left over in the flask.
Titrate the distillate against standard sodium hydroxide solution using
phenolphthalein as an indicator. Perform a blank in the same way 5 ml distilled
water in place of spirit.
CALCULATION
Higher alcohol as Amyl alcohol/100ltr of absolute alcohol
=0.08*100*1000*V*2/strength of alcohol in sample
Where;
V= difference in ml of standard sodium hydroxide used for blank and sample.
TEST FOR FUSEL OIL

25 ML spirit evaporate in porcelain disc when little portion of sample residue
in disc cool down the sample at room temperature. Add 1 ml conc. H2SO4-
presence of red of brown pot of presence of foreign order is shown the presence
of fusel oil.
QULITATIVE TEST FOR HIGHER ALCOHOLS REAGENTS:
1. Salisaldehyde 1 % solution.
2. H2SO4 conc. (A.R.Grade)
10 ml spirit sample = 1ML of 1 % Salisaldehyde solution add 20ml conc. H2SO4

allow to stand at room temperature for 24 hours. Note the colour.
COLOUR H.A.LIMITS
Light yellow traces
Yellow to brown about 0.01gms/100ML
Brown 0.02to 0.02gms/100ML
Red about
0.05gms/100ML0.1gms/100ML
Bark red to black above 0.1gms/100ML
All other analysis is performed as per I.s. 3752 regarding the liquor analysis etc.
All other analysis is performed as per I.s. per 323 regarding the Rectified Spirit
All other analysis is performed as per I.s. regarding the E.N.A.
Two type of brix hydrometers are being used which are calibrated of hydrometers
SCALING PROBLEM
Almost every distillery in India faces scaling problem. It has been experience
that scaling few plates below in the analyses column. It is much more prominent
on the feed plate and bottom of the plate. Scaling is of 3 types.
SOFT SCALE
This type of scale occurred due to settling of sludge suspended in the wash
which gets deposited in the beer heater, heat exchanger and analyzer column.
COPPER AERATED SCALE
This scale deposited in rectifying column.
HARD SCALING
This type of scaling is generally 1” to 1.5 “size and it is mainly due to the
deposition of calcium contents.
CAUSES OF HARD SCALING
Molasses is one the main single factor contributing towards the increased
intensity of scaling, formation of scale may be due to the following reasons.
1. Irregular working of plant.

2. Frequent fluctuation in the steam pressure feed to the column.
3. Hardwater aggravated scaling
4. Distillation of unfermented wash
5. Distillation of high gravity wash.
6. Distillation of low temperature of wash.
ANALYSIS
Less of inanition% 23.46
Silica as S102% 0.30
Calcium CaO % 31.29
Sulphate as SO2 % 44.70
Iron as Fe2O3 % 0.25
Alumina % NIL
Phosphate % Traces
Chemical analysis of molasses during a period of high and low scales :
Molasses sample during a period of High scale Low scale

PH at 30deg.cen. 5.4-5.7 5.2-5.4
T.R.S.%by weight 48.0-49.0 50.0-53.46
Unforgettable matter by wt.% 4.0-5.0 3.0-3.6
Acid insoluble ash % by wt. 9.5-11.6 10.06-10.07
Total ash % by wt. 0.06-0.085 0.04-0.05
Sulphate ash % by wt. 12.0-13.33 12.10-13.15
CaO % by wt. 1.20-1.26 1.05-1.20
CaSO4 % by wt. 2.90-3.06 2.50-2.60
Gummy matter % by wt. 0.55-3.70 3.40-5.26
Suspended impurity % by wt. 0.22-0.69 0.28-1.80
Aconitic acid by wt. 0.915-0.950 NIL
ANALYSIS OF WATER
Total alkalinity as CaCO3 362PPM

Carbonate alkalinity as CaCO3 12PPM
Bicarbonate as CaCO3 312PPM
Present hardness NIL
REMEDIAL MEASURES
1. The final gravity of wash should not be allowed to exceed 1.035.

2. Settling of molasses in the molasses storage tanks for a period of above
3 to 4 months.
3. Use of soft water in dilution of molasses.

4. By removing the cup on the plates above feed plate thus short
circulating the vapour.
According to Dr. Gunu Rao , the scale formation is due to :
1. Inadequate preheating of feed wash.

2. Under admission of alcohol into the top plate in the form of reflux of
vapour.
3. Calcium contents of molasses.
REMEDIAL PARAMETERS
1. In wash backs, fermented wash is allowed to settle for above 8 hours
so that sludge is settled down and supernatant wash is pumped to wash
charge.
2. A feet high pump line has been provided in the wash charger
connection the section of the pump so that any sludge etc., is retained in
was charger and is no pumped in column.
3. Maintenance of high feed armature, as possible before the wash to
analysis column.
4. Plant should be so designed to create a high vapour velocity.
REMOVING OF SCALE
MECHANICAL METHOD
Generally the scale is removed by hammering the cups and plates.
CHEMICAL METHOD
Recently a natural decaling agent that sod, salt of Ethylene daimio tetracetic
acid has been reported to be quite efficient in action on all sorts or scale.
Generally, the quantity of fusel oil produced is above 0.01 to 0.05 alcohols
and it depends upon the deficiency of rectifying column. Old molasses give good
yield of fusel oil. Since fusel oil is formed during fermentation, it remains in the
fermented liquor and is separated from alcohol by fractional distillation. The
rectifying column is provided with number if cocks at the bottom from alternate
plates from the bottom. The boiling point range of fusel oil remains in between
90 to 150 deg. En. The temperature in rectifying column remains high at bottom
and collection of fusel oil is dine at temperature 104 deg. Cen.- 106 deg. Cen. In
which concentration of fusel oil is high. Fusel oil is completely soluble in ethyl
alcohol named being less soluble in water (less volatile also). It forces down to
the rectifying column add comes down to bottom plates. It becomes insoluble in
high water concentration solution. At this state steams coming from analyzer and
exhaust column at higher temperature distillate the fusel oil and forces it up
words. These two opposite forces equilibrium missed at certain plates at which it
should be collected and passed towards the decanter. Due to the low density of
fuse oil it floats on the water.
COMPOSITION OF FUSEL OIL

The composition of fusel of the fusel oil and the amount of the different
content formed varies in different type of alcohol. Fermentation depending upon
certain factor like the raw material used of fusel oil formed during the molasses
fermentation and some other are as follows:
Isopropyl Alcohol 24.3

Isobutyl Alcohol 7.1
Isobutyl Alcohol 8.4
N.Amyl Alcohol 4.3
Iso Amyl Alcohol 46.3
Active Iso Amyl Alcohol 55.3
FUSEL OIL DECANTER AND SEPARATION

The construction is very simple. Firstly the fusel oil comes form the 30th to
50th plate the bottom of rectified column. There are 6-8 outlet valves for taking
fusel oil in different position and also takes in different temperature.
The temperature of these plates maintained about 104 deg. Cen. -106 deg. Cen.
Fusel oil comes from the rectified column. Firstly it is cooled by passing through
a coil which is immersed in cold water, cooled fusel oil is taken into a hollow
cylinder vessel in which water connection is given. There is a side outlet valves
for taking the fusel oil water is added slowly and layer rise till fusel oil come out
and filled in a pot for fuelling. This fusel oil is not purified because the
purification process is not taken place. But in my idea this is impure fusel oil.
There is a process of purification of fusel oil.
USES
1. It is used as liquor solvent.

2. It is used in the manufacture of varnishes & thinners
3. It is used in the perfumery (alcoholic perfumes).
4. Manufacture of explosive and artificial fruit essences.
5. Superior variety of fusel oil which contains 65 % butanone is used in
the manufacture of Rubber.
STEAM ECONOMY
Efficient use of steam is very much important from the point of view of
economy and conservation of thermal energy in the distilleries in India and this is
applicable to the saving of fuel resources. Steam is the carrier of best. So steam
economy means that economy energy cannot be overlooked, under any context.
In the cost structure of industrial alcohol about 20/- of the total cost is
contribution by fuel. This is could be obvious that any reduction in the

consumption of fuel will be directly rejected on the cost structure
SOME IMPORTANTS VIEWS

To produce on litre of alcohol steam consumption is 2.2-2.5Kg.
1Kg press mud produced .5-.8 Kg steam
1Kg coal produced 4Kg steam
1Kg husk produced 3Kg steam
ALCOHOL COST ANALYSIS

Steam poser 39%
Raw material 15%
Chemical 1%
Salaries & Wages 16%
Depreciation Insurance29%
The reduction in the cost of production of alcohol would put Industry on a

better footing for future expansion and modernization of plant. Efforts are being
made to minimize the steam consumption in plant and increase the fuel efficiency
of boiler section up to the maximum possible. At present steam consumption per
liter of sprit very present steam consumption per liter of spirit very between 2.4
kg/cm 2, 2.6 kg/c, 2. It was represented that was up to 3.kg/cm2. The main
sources of heat losses in a distillery are with spent wash going the out and heat is
loosed from alcohol vapour during its condensation. For this following
arrangement are to be made.
BEER HEATER
There is only one beer heater for each plant, which is situated at the top of the
still house which is very common device for steam economy in distilleries. it is
used to reheat the fermented wash the vapour coming from the top of rectifying
column. The vapour in a shell and the liquid is in the tube through counter
curredt. The fermented wash is heated up to 65-70 deg. Cen. The vapours of
alcohol are condensed which are sent to the rectifier column as reflux.
HEAT EXCHANGER
In this distillery one heat exchanger for each plant is situated at ground floor
of the still hours. It is a plate type heat exchanger, having a capacity of 2000
gallons per hour. It has 56 stainless steel plates fitted in two cylindrical rods. The
plates are rectangular with round corners bordered with special type.
From one side of the heat exchanger fermented wash is entered. The exchange of
heat exchanger between two liquids during the passage through plate’s taker
place. Here the wash at 50-55 deg. Cen. From beer heater enters and the spent
wash at 80 Dec. From analyzer column through direct connection from pipe is
fed. The heat exchanger is most important in all distilleries. The wash passing the
heat exchanger is about 68-70 deg. Cen., is send to analyzer top. Here one more
heat exchanger is situated near the Ist heat exchanger. The working of this heat
exchanger is to incrade the temperature of 28-30 deg. Cen. Is passed through one
side and spent wash , which is coming from its heat exchanger, is passed through
the opposite side, thus the heat transfer takes place and the water after passing the
heat exchanger is heated up to 60-62 dec. cen.
BY CONDENSERS
The vapours of ethanol which is condensed by other condenser through water,
this water has a heat about 40-42 dec. cen. And it again fed to the boiler. In this
way here 3rd type of heat economic for boiler.
OTHER MEASURES
LOCKING OF STEAM LINES
1. Steam pope lines should be properly locked. Unlocked steam line wastes
the steam.
2. Proper steam trap should be given in pipe to remove only condensate of
steam.
3. Leakage of steam should be avoided. Valves & flanges in steam pipe
should be made leak proof.
BY HEAT RECOVERY UNIT

Fuel gases are having high temperature before allowing going atmosphere. Thus
heat is recovered by putting H.R.U. fuel gasses heat the water and further
increases the Boiler efficiency.
SUGGESTIONS AND IMPROVEMENTS

I am glad to state that the factory is running well. The plant has unique
features that it has fuel new and modern equipments. Inspires of smoothness of
running of plant some drawbacks have also attracted me which are as follows:
1. There is not instrument is Fermentation House for maintaining pH sometimes

the acid added is more or less quantity. It is not better for good fermentation. I
suggest that to proper maintenance of pH by pH paper or pH meter , automatic
pH control should be important in fermentation house.
2. Fermentation efficiency will increase if the filling of fermenters is done
carefully. For this principle of continuous fermentation may be applied.
3. To improve the fermentation efficiency , following parameters should be
followed:
1. Cooling of fermenter should be properly maintained. it has been found that at
40 deg. Cen. The alcohol loss from fermenters is about 1.6 % with carbon
dioxide. If the temperature can be maintained at 32-33 Dec. cen., the loss
of alcohol with carbon dioxide will be around 0.5 %.
2. The cooling coils holes get blicks if the water is bad (contaminated) and also
formation takes place, which is entering into fermentation tanks
decomposes the sulphates and from sulphur dioxide. This sulphur dioxide
appears with alcohol on distillation. It should be cleaned properly to avoid
the problem.
3. Cooling coils on the back side of fermenter is approachable and remain in
operated. It should be working properly. For proper cooling of fermenters
plate coolers and wash circulation, pump, submerged type coolers can be
adopted.
4. The most important factor affecting the fermentation efficiency is the high
calcium % in molasses, so it is reduced to optimum value by treatment if
molasses or clarification of molasses by any of the method.
5. Complete separation of fusel oil is most important. The separation of fusel oil
depends on the design of the plates. Firstly and secondly on the
temperature maintained. It is essential to maintain the temperature about
104 deg. Cen. To 106 deg. Cen. At the bottom of the rectified column to
give undisturbed separation of fusel oils. Thirdly the separation of fusel oil
depends upon the plant operator.
6. Water tank should be cleaned in every 15 days. The possibility of infections
should be cleaned in every 15 days. The possibility of infections should be
totally removed.
7. The leakage in valves, pipe line joints favours the microbial infection. This
should be removed.
8. Steam pressure should be constant, should not be in flexibility nature.
BOILERS & POWER PLANT

There is one fluidized bed combustion thermal boiler.
The details of the boilers are given below:
FLUIDIZED BED COMBUSTION BOILER

It is multi thermal boiler
Make thermax pvt ltd. Pune
Capacity 8 Tones /Hours
No. UP/4083
Maximum pressure 32 kg/cm2
Water used Soft water
EQUIPMENTS IN BOILER
De aeriator
Economizer
Forced –draft fan
Induced- draft fan
Multi-Cyclone dust collector
MOUNTING:
Guage glass, pressure gauge, main stop valve, safety valve, air clock, blow
down cock, air vent, feed check value, mud hole door.
FITTINGS:
Anti priming pipe, internal feed pipe, low water high pressure safety valve,
fusable plug.
Water: ordinary / condensed water.
POWER GENERATING PLANT

There is a Bellis and Marco Poser Generation plant with an operating pressure
of 120-40 psig. The said engine is coupled with an alternator of having capacity
of 250 K.W. at 440 volts 50 cycles.
DIESEL ENGINE
Make kerloskar electronic company ltd.
Bangalore
R.P.M. 1500
Capacity 110 K.W.
A.C. Volts 420
C.P.S. 50
A.C. Amp 151
M.C. No. 71015-4
R.P.M. 1500
Capacity 880 KVS
A.C. Volts 415
C.P.S. 54
M.C. No. 25294835
REFERENCES
 Text Book of Alcohol Technology

 Text Book of Biotechnology- B.D.Singh
 Text Book of College Botany –Ganguly
 Text Book of Organic Chemistry –Behl & Behl
 Text book of Pharmaceutical Biotechnology-S.P.Vyas & V.K. Dixit
 www.google.com
 www.andrew.cmu.edu/user/jitkangl/fermentation
 rcrec-ona.ifas.ufl.edu/mol.pdf
 www.studentsguide.in/microbiology/industrial-microbiology/vinegar-
acetic-acid-fermentation.html
 www.microbiolotyprocedure.com/industrial-
microbiologyprocedure.com/industrial-microbiology/ethyl-alcohol-
production.html
 www.wikipedia.com
 www.final-yearproject.com
 www.finalyearthesis.com
fermentation
efficiency(%)
100
50 fermentation
efficiency(%)
0
24 h 36h 48h 60h 72h
Fermentation fermentation
period (h) efficiency(%)
24 h 49.7
36h 71.9
48h 83.3
60h 72.3
72h 66.2

Ethyl Alcohol Production by Hydrolysis

Uploaded by

Copyright:

Available Formats

You might also like

Ethyl Alcohol Production by Hydrolysis

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Ethyl Alcohol Production by Hydrolysis

Uploaded by

Copyright:

Available Formats

www.final-yearproject.com | www.finalyearthesis.

ETHYL ALCOHOL PRODUCTION BY FERMENTATION

INDUSTRIAL WORK CONDUCTED

With an overwhelming sense of legitimate pride and genuine obligation, I

Place: Bijnor Shriya Tiwari

I hereby declare that the project work entitled ETHYL ALCOHOL

PLACE: DEHRADUN SHRIYA TIWARI

The Mohit Petrochemicals (P) Limited (MPPL) is located in Bijnor; Uttar

Distillery is the allied of the sugar industry, as the bye-product of sugar

Meerut-Muzaffarnagar region is quite rich in sugarcane. Nearly a dozen sugar

Name of Organization MOHIT PETRO CHEMICALS

Address 8km Nagina road Dist. - bijnor

Composite analysis of molasses sample (process) and wash analysis, spent

In this way each chemist is responsible for their work.

Fermentation is a bio-chemical reaction where the substrate is converted into

value is never obtained due to carbohydrate utilization for cell maintenance,

Continuous Fermentation Process is the process in which cells are maintained

This process recently following in India, due to high technology it is not

Distilleries not only want maximum recovery of alcohol from molasses,

Better yield is obtained when fermentation is carried out at optimum activity of

Single stage continuous fermentation is that in which the entire operation is

2. Double stage continuous fermentation

Here instead of carrying out the fermentation in single fermentation, it is taken

3. Multistage continuous fermentation

A tank of fermenter is filled with the prepared mash of raw materials to be

Growth of microorganisms during batch fermentation confirms to the

ADVANTAGES OF BATCH FERMENTATION:

DISADVANTAGES OF BATCH FERMENTATION:

The batch process is completed in the following steps:

 Preparation of isolation pure culture of yeast and making the slant.

Molasses is a viscous by-product of the processing of sugar cane, grapes or

4. B.O.D. (Mg/Ltr) 440000

Sl.no characteristics ISI requirement For cane sugar

Molasses control in distillery

purchased anywhere. Transportation of molasses is done under type control of

DECONTROL OF MOLASSES (Government Notification)

PRECAUTION IN STORAGE OF MOLASSES

7. To have the proper arrangement to prevent the missing up to water with

CONTROL MEASUREMENT OF STORED MOLASSES

Yeast contains 68 to 83 moisture. The yeast cell is composed of nitrogen

METHOD OF ISOLATING PURE CULTURE OF YEAST

In our lab we do single cell isolation according to the following sequence.

1. The yeast containing culture is transferred to the 10 Bx solution of molasses

MEDIA USE TO ISOLATE YEAST

a. Malt Extract 0.3%

SLANT TUBE PREPARATION

PURE YEAST VESSELS

Yeast vessels No. 1 91 Liters

BUB TANKS /PREFERMENTERS

Sl.no. Tanks No. Height Capacity

WASH BALK OR FERMENTER

The details of working fermenters are given below:

Sl.no. Tanks No. Height Capacity

TEMPERATURE CONTROL DURING FERMENTATION PROCESS

Use of anti infectant

1. Maintain yeast culture on nutrient agar media at 0.5 degree C.

3. Periodically is necessary. If contamination found and it is major risk for

2. OVER PROOF = (100+O.P.) Proof