VIT

UNIVERSITY
(Estd. u/s 3 of UGC Act 1956)

Vellore 632 014, Tamil Nadu, India

LABORATORY CUM PRACTICAL MANUAL

FERMENTATION TECHNOLOGY

Name :
Reg. No. :
Batch :
Semester : III
Course code : 09MSM513 L
Programme : M.Sc. Applied Microbiology

Prepared and compiled by

Prof. V.MOHANASRINIVASAN
Prof. CHITRA KALAICHELVAN
Mrs. M. THENMOZHI

School of Bio Sciences and Technology

(For Private circulation only)

VIT – A Place to learn; A chance to grow

 VIT
UNIVERSITY
(Estd. u/s 3 of UGC Act 1956)

School of Bio Sciences and Technology

FERMENTATION TECHNOLOGY

CERTIFICATE

This is to certify that this is a bonafide record of work done by

________________________________ (Reg. No.) ___________________, a student of

M.Sc. Applied Microbiology during the III Semester of the Year 2010 – 11 at VIT

University, Vellore – 632 014.

This record is submitted for the practical examination held on

__________________.

Date: Faculty-in-charge

Internal Examiner External Examiner

 LIST OF EXPERIMENTS

PAGE INITIALS
S. NO. DATE TITLE OF THE EXPERIMENTS REMARKS
NO.
LABORATORY PRACTISES
1.

BIOREACTOR AND ITS STRUCTURE

2

ISOLATION AND CHARACTERIZATION OF

3.
Lactobacillus FROM FERMENTED MILK
PRODUCTS

ISOLATION AND CHARACTERIZATION OF

4.
Leuconostoc FROM RICE FLOUR

ISOLATION AND CHARACTERIZATION OF

5. Saccharomyces cerevisiae FROM GRAPE
JUICE

PRODUCTION OF GRAPE WINE

6.

7. ALCOHOL FERMENTATION BY YEAST

ISOLATION AND CHARACTERIZATION

8.
OF BACTERIAL PIGMENTS
 ISOLATION AND CHARACTERIZATION
9.
OF Monascus FROM SOIL

PIGMENT EXTRACTION FROM

10.
Monascus

BIOCATALYSIS OF PRECURSORS TO
11.
AROMA AND FLAVOR COMPOUNDS USING

Saccharomyces cerevisiae

APPENDIX
 GENERAL INSTRUCTIONS FOR USING THE LABORATORY

Good laboratory practices :

1. Never mouth pipette any solution (acids, phenols, peroxides, organic solvents, culture
of organisms, etc)
2. Wear laboratory coat (use gloves when necessary).
3. Do not blow infectious materials out of pipettes.
4. Wrap a lyophilized culture vial with disinfectant wetted cotton before breaking.
5. Do not eat, drink or smoke inside the laboratory.
6. Aseptic techniques should be followed rigorously at all times.
7. All microbial cultures are handled and treated as potential biohazards and dispose them
of after autoclaving or treating with 10% formalin for 30 min.
8. No chatting, gossiping and loud discussions inside the laboratory (especially in front of
laminar air flow) as it affects yours and colleagues work.
9. Do not store food materials and drinks in the labotatory refrigerator.
Preparation of reagents
1. Store chemicals/ solvents at appropriate temperatures as mentioned on the label
2. The highest purity chemicals and double distilled water should be used for reagents and
solutions. (Weight of the substance, brand, batch No. of the substance, type of water
used, amount of alkali or acid used to dissolve should be recorded then and there).
3. Label all the reagents. The label should contain the name of the solution reagent,
concentration (% molar), date of preparation of the reagent and initials of the person
who prepared. Unlabelled solutions/reagents should be discarded.
4. Store the reagents in appropriate bottles and at appropriate temperatures. (Light
sensitive reagents and solutions should be stored in brown/ dark bottles).
5. Keep record of the reagent preparation and experimental details correctly. Store all the
data sheets supplied along with fine chemicals and enzymes. Note down the date of
expiry, batch No. and catalogue No. for labile substances.
Experiments and record keeping
1. Make sure the laboratory and laboratory benches are clean (all spillover of cultures,
corrosive solvents should be cleaned immediately and appropriately).
2. Make sure that the balance is cleaned after your weighing.
3. Make sure the glasswares, plasticwares, media, etc are ready for the experiment (if
needed sterilize them previous day itself).
4. Make sure that you have studied and understood the principle and methodology of each
experiment. Discuss the experiment with your Professor in advance. Keep appropriate
controls. Copy the protocols of the experiment in the record notebook. Attach print outs
of instruction sheets and supporting materials to record.
5. Label all the plates, tubes, cultures, etc., correctly before starting the experiment.
6. Make sure that the equipments are in working condition and available for the
experiment. If it requires prior permission and reservation, make in advance.
7. Make sure that you know the operations of the equipments correctly or get apt
assistance.
8. Microbial counts, standard graphs, enzyme assays should be done in triplicates. For
standard graphs, plot all the points and draw the line, which passes through maximum
number of points.
9. Use a record notebook of permanent binding. Number all the pages before using the
record notebook. Use permanent ink in writing the record. Don’t rely on memory.
Record all data directly into the lab note book. Avoid abbreviations, code names and
code numbers in recording the data. Start new experiment on a new page. Every
experiment should have date, title and objective(s). All calculations should be done step
by step and record neatly. Do not overwrite on mistakes. Photographs, printouts,
chromatograms, etc., should be labelled to indicate what they are supposed to represent
and then attach them directly on to the note book as soon as the experiment is over. Do
not record data on loose sheets. Do not take the record note book outside the laboratory.
(Relationship between two variables in experiments can be best represented using
graphs rather than in tables. All graphs should be titled, dated and fixed in the record
book on the same day.
 BIOREACTOR AND ITS STRUCTURE

Louis Pasteur’s work on fermentation of wine laid the foundation for bioreactors
as we know them today, because once the process is identified and understood, it could
be controlled. And it is the control of the process that concerns chemical engineers first
and foremost. The scope of bioengineering has grown from simple wine-bottle
microbiology to the industrialization of not only beer, wine, cheese and milk production,
but also the production of biotechnology’s newer products - antibiotics, enzymes,
steroidal hormones, vitamins, sugars and organic acids. This has been possible due to the
invention of bioreactors.

Bioreactor

A research team led by Chaim Weizmann in Great Britain during the First World
War developed a process for the production of acetone by a deep liquid fermentation
using Clostridium acetobutylicum. This lead to the eventual use of the first truly large
scale aseptic fermentation vessels (Hastings, 1978). These were used in central Europe in
the 1930’s for the production of compressed yeast. The bioreactors consisted of large
cylindrical tanks with air introduced at the base via networks of perforated pipes.

A bioreactor is a system in which a biological conversion is effected. The

bioreactors include mechanical vessels in which organisms are cultivated in a controlled
manner and materials are converted or transformed through specific reactions.
Bioreactors are specifically designed to influence metabolic pathways. The models and
designs that are used for bioreactions include continuous stirred-tank reactors, continuous
flow stirred-tank reactors, singularly or in series, ebullized bed reactors and fluidized bed
reactors (Kajiwara et al., 1997).

Bioreactors support and control biological entities and provide a higher degree of
control over process upsets and contaminations, since the organisms are more sensitive
and less stable than chemicals.
Bioreactor Configuration

In designing and constructing a bioreactor the points to be considered are:-

1. The vessel should be capable of being operated aseptically for a number of days.
2. Adequate aeration and agitation should be provided. However, the mixing should
not damage the cells.
3. Power consumption should be as low as possible.
4. A system of temperature and pH control should be provided.
5. Sampling facilities should be provided.
6. Evaporation losses should not be excessive.
7. Minimum requirement for labor in operation, harvesting, cleaning and
maintenance.
8. The cheapest material which enables satisfactory results to be achieved should be
used.
9. There should be adequate service provisions for individual plants.

Of these, maintenance of adequate aeration, agitation and aseptic conditions is the

most important requirement for any bioreaction. The design of a bioreactor involves the
parameters such as temperature, pH, substrate concentration, water, availability of salts,
vitamins, oxygen (for aerobic processes), gas evolution and product removal. The
bioreactor to be designed should consider to promote the optimal morphology of the
organism and eliminate or reduce contamination by unwanted organisms or mutation of
the organism (Viktor Nedovic and Ronnie Willard, 1996).

Bioreactor Design
A bioreactor is a vessel which provides a controlled environment for the growth
of microbial cells to obtain a desired product. A schematic representation of a simple
bioreactor is as shown in Figure 1.
Figure : A stirred tank bioreactor with baffles and an agitator

a) Body Construction: The material used for body construction should withstand
repeated sterilization cycles. On a bench scale, fine glass can be used. Most of the
large scale and pilot scale bioreactors are constructed from ISI grade 316 stainless
steel which has 18% chromium and 10% nickel. The thickness of the construction
material will increase with scale. An effective seal is made between the bioreactor
sides. The top and bottom of the seal can be made with a compressible gas fit or
lip seal or V-ring seal.

b) Aeration and Agitation Systems: The type of aeration and agitation systems
used for a particular bioreactor depends on characteristic fermentation conditions.
The structural components involved in this are:
 (i) Agitator or impeller
(ii) Stirrer glands and bearings
(iii) Baffles
(iv) Aeration system

Agitator or Impeller: Agitator helps in mixing objects either by any of the following
types:
a) Disc turbines b) Vaned discs c) Open turbines
d) Marine propellers

Good mixing and aeration in high viscosity broth may be achieved by a dual
impeller. The lower impeller in a bioreactor acts as the gas depressor and upper impeller
area primarily acts as the device for circulating the vessel contents.

Stirrer Glands and Bearings: There are a number of shafts and glands to obtain
satisfactory aseptic seal. The earlier seal being described by Rirch Johenson and Peterson
in 1950 as a porous bronze bearing of 13 mm shaft fitted in the centre of the bioreactor
top.

Baffles: A typical bioreactor has four baffles and they prevent vortexing and improve
its aeration efficiency. Baffles are actually metal strips, N 1/10 of vessel’s diameter and are
attached radially to the wall.

Aeration System: Aeration inside the bioreactor is provided with a sparger. A sparger is
defined as a device for introducing air or gas into the liquid phase of the bioreactor.
Spargers may be of porous type, each being used depending upon the type of bioreactor.

c) Temperature probes and control: Temperature control is one of the most

important parameter to be monitored and controlled in any type of bioreactor.
Heat will be generated in the bioreactor due to agitation. In a large scale
 bioreactor, heating coils or heating jackets are provided, through which
circulation of water takes place to control the temperature.

d) Sampling ports: To prevent contamination when operating a bioreactor

requiring GILSP, it is essential to maintain it at a positive pressure and the
sampling port is to be provided with steam supply.

e) Feed ports: Addition of nutrients, acid or alkali to a small scale bioreactor is

normally done in silicon tubes which are autoclaved separately and pumped by
peristaltic pump often under aseptic conditions.

f) Valves: Valves are attached to the bioreactor and are used for controlling the flow
of liquids and gases. There may be:
(i) Sample on/off valves which are either fully opened or fully closed.
(ii) Valves which provide coarse control of flow rates.
(iii) Valves which may be adjusted precisely so that flow rates are accurately
controlled.
(iv) Safety valves which are constructed in such a way that they allow the flow
of liquid or gas only in one direction.

g) Steam traps: In steam lines it is essential that steam condensate which

accumulates in the pipe be removed to ensure optimum porous conditions. This is
achieved by steam traps which collect and remove automatically any condensate
at appropriate points in steam line. A typical steam trap has 2 elements, one being
the valve and steam assembly which provides an opening and the second valve for
measuring some parameter of the condensate.
Fermentation process and stages

Figure: A generalized, schematic representation of a fermentation process

(Reproduced with permission from P. F. Stanbury, A. Whitaker and S. J. Hall,
‘Principles of Fermentation Technology’, Pergamon Press, Oxford, 1995)
 ISOLATION AND CHARACTERIZATION OF Lactobacillus FROM
FERMENTED MILK PRODUCTS

AIM:
To isolate and characterize Lactobacillus species from curd.

BACKGROUND:
The genus Lactobacillus consists of both hetero fermentative and homo
fermentative lactic acid bacteria. The homofermentative lacticacid bacteria ferment
sugars chiefly to lactic acid with small amount of acetic acid, carbon dioxide and some
trace products. If they are hetero fermentative they produce appreciable amounts of
volatile products including alcohol in addition to lactic acid. Lactobacilli are commonly
used in fermented diary products and vegetables. They are also used in the manufactures
of industrial lactic acid.
Lactobacilli are gram positive bacilli occurring in singles, pairs or in short chains.
They are non acid-fast, non spore forming; microaerophilic and their nutritional
requirements are complex as optimal growth is obtained only in media containing
fermentable sugars and adequate growth factors.
REQUIREMENTS:
Curd sample
MRS agar
MRS broth
Sugars - glucose, lactose and sucrose
Arginine
PROCEDURE:
i. Isolation of Lactobacillus
a. The curd sample was serially diluted in (10-4) to (10-6)
b. Samples from last 3 dilutions were taken and pour plated.
c. All the plates were incubated at room temperature
d. Examined for large cream colored slightly mucoid colonies.
 ii. Characterization
Isolated colonies were characterized by
a. Gram staining
b. Catalase test
c. Oxidase test
d. Sugar fermentation test
e. Growth at 15˚Cand 45oC
f. Arginine hydrolysis

Sugar fermentation test

1. MRS broth without glucose and beef extract was prepared.
2. Sugars were added at the concentration of 1% and phenol red at the
concentration of 0.004%. Durham tubes were placed in inverted position to trap
the gas production.
3. After sterilization the tubes were inoculated with isolated culture and incubated
for 2 days at room temperature and observed for acid and gas production.

Growth at 15oC and 45oC:

1. MRS broth was prepared, sterilized and inoculated with the culture and
incubated at 15˚C and 45oC separately for 3 – 4 days upto 1 week.
2. After incubation the tubes were examined for turbidity.

Arginine Hydrolysis test:

1. MRS broth without Ammonium citrate was prepared. Arginine was added at the
concentration of 0.5% after sterilization the tube was inoculated with the culture and
incubated at room temperature for 1 – 2 days.
2. After incubation few drops of Nesseler’s reagent was added to test presence of
Ammonia (which will give brown colour with Nesseler’s reagent).
OBSERVATION:

Media Grams Catalase Oxidase Sugar fermentation/gas Arginine Growth

reaction production hydrolysis at 15°C
Dextrose Sucrose Lactose
/Shape and
45°C

MRS
media

RESULT:
ISOLATION AND CHARACTERIZATION OF Leuconostoc FROM RICE FLOUR

AIM:
To isolate and characterize Leuconostoc from fermented rice flour.

BACKGROUND:
The genus Leuconostoc contain hetero fermentative lactic Streptococci which
ferments sugar lactose to Lactic acid and considerable amount of acetic acid, ethyl
alcohol, carbon dioxide and other acids.
Leuconostoc sp. has the ability to initiate lactic acid fermentation in food stuffs more
rapidly than other lactic acid bacteria and enough acids to inhibit the growth of non lactic
acid bacteria. They produce an important flavor compound called diacetyl, therefore,
Leuconostoc sp. plays major role in most of the food fermentation. The most common
species are L.messenteroids and L.cremoris. Leuconostoc sp are mostly used in
production of fermented vegetables and some dairy products. They require nutrient rich
medium. It is a gram positive cocci occurring in pairs (or) short chains. They are non
spore forming, non motile and dextran producing organisms.
MATERIALS REQUIRED:
Fermented rice flour (Sample), Leuconostoc agar, Esculin hydrolysis agar
MR-VP broth, Simmon citrate agar, Sugar fermentation broth, H2O2
Sucrose agar.
PROCEDURE:
ISOLATION:
i. The sample (fermented rice flour) was serially diluted in (10-4) to (10-6)
ii. Samples from last 3 dilutions were taken and spread plated on leuconostoc agar in
duplicates.
iii. All the plates were incubated at room temperature
iv. After incubation the plates were examined for pale white slimy colonies.

CHARACTERIZATION:
i.The isolated culture on Leuconostoc agar are characterized by
a. Gram staining
b. Catalase test
c. VP test
d. Citrate utilization test
e. Sugar fermentation test
f. Esculin hydrolysis test
g. Dextran production

SUGAR FERMENTATION TEST

1. Leuconostoc broth without beef extract was prepared.
2. Sugars were added at the concentration of 1% and phenol red at the
concentration of 0.004%. Durham tubes were placed in inverted position to trap
the gas production.
3. After sterilization the tubes were inoculated with isolated culture and incubated
for 2 days at room temperature and observed for acid and gas production.

ESCULIN HYDROLYSIS:
The isolated culture are inoculated on esculin hydrolysis agar slant and incubated
at 37oC for 24-48 hrs. After incubation the slant was observed for colour change to dark
brown to black.
DEXTRAN PRODUCTION:
 The isolated culture was inoculated on Leuconostoc sucrose agar and incubated at
37oC for 1-2 days. After incubation, the plates were observed for gummy mucous whole
irregular colonies.

OBSERVATION

Media Grams Catalase VP Citrate Sugar fermentation Esculin Dextran

reaction/ test utilization /gas production hydrolysis producti
Dextrose Lactose sucrose
Shape

LA

LA- Leuconostoc agar

RESULT:
ISOLATION AND CHARACTERIZATION OF Saccharomyces cerevisiae
FROM GRAPE JUICE

AIM:
To isolate and characterize Saccharomyces cerevisiae from grape juice.
BACKGROUND:
Saccharomyces cerevisiae is a type of yeast, with many forms(round, oval (or)
elongated) pseudomycelium. They reproduce by budding (or) ascospore formation.
Ascospores are formed either by conjugation of two cells (or) from diploid cells. Four
ascospores are seen in one ascus by process of meiosis.
Saccharomyces cerevisiae is widely employed in many industries for leavening of
bread in bakeries, production of wine in breweries, production of distilled liquor in
distilleries, production of invertase used in confectionaries etc. It is also used to produce
industrial alcohol and recombinant proteins. It is also used to produce home made
fermented foods like bread, wine, cake and curd etc.
MATERIALS REQUIRED:
i. Grape juice
 ii. Potato dextrose agar (PDA)
iii. Ascospore agar
iv. Nitrate broth
v. Christinson ‘s urea agar
vi. α – Napthyl amine
vii. Sulphanilic acid
viii. Lactophenol cotton blue
PROCEDURE:
ISOLATION:
i. The sample (grape juice) was serially diluted (10 fold dilution) upto 10-4
dilution.
ii. Plates were prepared from all the dilutions using PDA agar by pour plate
method.
iii. Incubated at room temperature for 72 h.
iv. After incubation the plates were examined for medium sized creamy colonies
with yeasty odor.
CHARACTERIZATION:
LACTOPHENOL COTTON BLUE STAINING:
A loopful of isolated culture was emulsified in a drop of LPCB stain on a glass
slide. A coverslip was placed over the emulsion and the prepared wet mount was
observed, under low power objective for round, oval budding yeast cells.
NITRATE REDUCTION TEST:
Nitrate broth was prepared, inoculated with isolated culture and incubated at room
temperature for 1-2 days. After incubation few drops of α – Napthyl amine reagent and
sulphanilic acid reagent was added. The color change was observed (pink).
SUGAR FERMENTATION TEST:
Sugar fermentation broth with 1% sugar was prepared, inoculated with isolated
culture and incubated at room temperature for 24 hrs. Next day, the tubes were observed
for acid and gas production.
ASCOSPORE FERMENTATION:
 Ascospore agar plate was prepared and inoculated with isolated culture and
incubated at room temperature for 3-4 days. After incubation a wet mount with LPCB
was prepared and observed under low power objective for ascospores.
UREASE TEST:
Christinson urea agar slant was prepared and inoculated with the isolated culture
and incubated at room temperature. The color change in the slant was observed(pink).

OBSERVATION:

Media LPCB Nitrate Urease Sugar fermentation/gas Ascospore

Staining production
Dextrose Sucrose Lactose
/Shape
PDA

RESULT:

PRODUCTION OF GRAPE WINE

PRINCIPLE
Fresh grape juice (must) contain up to 30% sugar (Fructose + Glucose). Yeast
ferment the sugars to acetaldehyde and then to alcohol. Hygienically prepared grape juice
(must) is fermented to alcoholic wine by Saccharomyces at 22oC by the end of 21 days.
The resultant wine can contain alcohol up to 15%. The percent acidity is determined by
titration against NaOH with phenolphthalein indicator. Other parameters studied at
regular intervals of the fermentation include aroma, clarity and alcohol content.
PROCEDURE
A. PREPARATION OF YEAST INOCULUM
1. Select black grapes for red wine and white grapes (green) for white wine. (Peeled
black grapes may also be used for white wine).
2. Pluck and remove the stalks of 100g of fresh grapes. Wash with clean drinking
water and crush in blender.
3. Strain the juice with the help of a clean strainer and collect the juice in a sterile
250ml flask.
4. Add 5g glucose to the flask and dissolve. [some labs prefer pasteurization of this
juice at 60oC for 30 min. This is however optional].
5. Inoculate the must so prepared, with 3ml of fresh, washed cells of Saccharomyces
cerevisiae. Incubate the flask in shaking conditions, in a constant temp shaker
incubator at 25oC for 24 h or until a heavy inoculum is built.
B. PREPARATION OF WINE
1. Follow steps 1 to 4 above (procedure A) using 750 g grapes in order to obtain
approx 500ml of must in a 100ml flask. Amount of sucrose should be 50g. [As
mentioned earlier, the must may or may not be pasteurized].
2. Pour the entire contents of the inoculum prepared in ‘A’ above, into the freshly
prepared must.
3. Incubate the flask for 21 days at room temp without shaking. However, the
contents may be gently mixed daily.
4. At intervals of 3, 7, 12, 14, and 21 days, check the fermenting beverage for
various parameters such as color, odour, clarity, % tatarate (total acidity), %
acetic acid (volatile acidity), and % alcohol.
5. The prepared wine should be subjected to clarification on the 22 nd day by
centrifugation followed by filteration.
6. Age the clarified wine in a refrigerator at 4 – 8o C for a month or two.

C. Determination of total acidity (expressed as % tartarate) and volatile acidity

(expressed as % acetic acid).
1. 10 ml fermenting wine sample + 10 ml d / w + 5 drops of 1% phenolphthalein
solution. Mix and titrate to the first persistent pink colour with 0.1 N NaOH.
Calculate the total acidity and volatile acidity.

ml of alkali x Normality of alkali x 7.5*

 % Tartarate =
Wt of sample in grams (1 ml = 1 mg)

ml of alkali x 0.1 x 7.5

% Tartarate =
10

2. Volatile acidity:

ml of alkali x Normality of alkali x 6.0*

% Acetate =
Wt of sample in grams (1 ml = 1 g)

ml of alkali x 0.1 x 6.0

% Tartarate =
10
(*7.5 & 6.0 are standardized calculation factors)
OBSERVATION AND RESULTS

S. No Parameter Raw must 7 days 14 days 21 days

1. Colour
2. Aroma
3. pH
4. % Tartarate
5. % Acetate
6. % Alcohol
Conclude as: Red / White wine was prepared by fermenting grape juice with
yeast inoculum and physical and chemical characteristics were studied. The
fermented wine was clarified and stored on a refrigerator for further maturation
and aging.

Trouble shoot
 Appearance of white cottony mycelium on the surface during fermentation
indicates spoilage due to fungal contamination.
 Sour odor or flavor is indicative of bacterial contamination.
  The finished product is expected to have % tartarate & acetate around 0.7
& 0.6 respectively. % alcohol depends on various factors, particularly the
initial total sugar content.

ALCOHOL FERMENTATION BY YEAST

OBJECTIVE

To demonstrate the production of ethanol by yeast and quantification of ethanol

produced in yeast fermentation.

BACKGROUND

The yeast Saccharomyces cerevisiae converts the fermentable sugars (glucose,

fructose and sucrose) into ethanol and carbon dioxide. For example

Yeast
Sucrose ethanol + CO2.

In the large scale production of alcohol molasses is used as the substrate for yeast
fermentation molasses, by product in the sugar refinery industries, contains about 45 – 55
% wt / v of fermentable sugar as sucrose. Sucrose is metabolized by yeast through Emden
Meyerhoff Pathway (EMP) and alcohol and CO2 are the end products of fermentation.
MATERIALS REQUIRED
Test tubes, conical flask, fermentation jar, micro distillation unit, standard
volumetric flask, molasses, urea, DNS reagent, Dichromate reagent, YPD medium, slant
culture of yeast Saccharomyces cerevisiae.
PROCEDURE
1. Inoculate the slant culture of yeast S. cerevisiae into a 50ml of YPD medium.
2. Incubate at 30oC in a shaker.
 3. Prepare the inoculum for fermentation as follows:
a. Dilute molasses to 12 brix (1.1 kg of molasses in 8 lit of water).
b. Adjust the pH to 5.5 with 10 N H2SO4.
c. Sterilize at 10 pounds pressure for 30 minutes and then cool.
d. Inoculate this 250 ml molasses medium (2 flasks) with 10% of the yeast
culture grown in YPD.
e. Incubate the culture in a shaker for 12 hrs.
4. Prepare the fermentation medium as follows:
a. Dilute molasses to 22 brix (2.1 kg. of molasses in 8 lit of water).
b. Adjust the pH to 5.5 with 10 N H2SO4.
c. Add urea to the final concentration of 100 ppm.
d. Pasteurize this medium in 2 fermentation jars (10 lit capacity) and cool.
5. Fermentation conditions
a. Inoculate with 10% v/v inoculum. (add 400ml to inoculum 4 lit of the
fermentation medium).
b. Incubate at 30oC for 48 hrs to 72 hrs.
6. Withdraw 10 ml sample at every 12 hrs intervals and estimate alcohol and sugar
concentration.
7. Plot a graph with time on axis and alcohol and sugar concentration on Y axis.

ESTIMATION OF ETHANOL
Reagent: Potassium dichromate
Dissolve 34 gm of K2Cr2O7 in 500ml of distilled water in 1 litre flask. Add 325 ml
of concentrate H2SO4 slowly keeping the flask in an ice bucket.

PREPARATION OF STANDARD CURVE FOR ALCOHOL CONCENTRATIONS

1. Prepare 1 to 10% v/v Alcohol in various test tubes.
2. Take 1 ml of the various concentration of alcohol into 25ml of water in 100ml
distillation flasks fitted with liebig condenser.
3. Distill and collect 15 ml of the distillate in a 50 ml volumetric flask containing
25ml of K2Cr2O7 solutions. Make up the volume to 50ml.
4. Keep the alcohol – potassium dichromate complex at 60oC for 30 min.
5. Measure O.D. at 600nm.
6. Plot a standard curve with concentration of alcohol on X – axis and O.D. and 600
nm. On Y – axis.

DETERMINATION OF CONCENTRATION OF ALCOHOL FROM THE

FERMENTATION MEDIUM
 Take 1 ml of sample, mix with 25ml of water and distill as above and measure
O.D. Find the alcohol concentration from the standard graph.

PREPARATION OF STANDARD CURVE FOR REDUCING SUGAR

a. Prepare the glucose solution in varying concentrations (0.1 mg to 1 mg/ml)
b. To one ml of sample add 3ml of DNSA reagent.
c. Boil in a water bath for 20 min.
d. Dilute the sample to 25 ml.
e. Measure O.D. at 550 nm.
f. Plot the standard curve with concentration of glucose on X – axis and OD at 550
nm on Y axis.

ESTIMATION OF REDUCING SUGAR

a. Take 5 ml of the sample from the fermented mash centrifuge at 5000 rpm to
remove cells. To the clear supernatant add 5 ml of water and 1ml of 10 N HCl.
b. Boil it for 15 min. and neutralize with Na2CO3.
c. Dilute this sample suitably and estimate the sugar as above.
d. Find the concentration of sucrose interms of glucose equivalent from the standard
graph.
Note: If molasses could not be obtained for laboratory exercise, use YPD medium
with 50 g/l sucrose for inoculum development and with 150 g/l Sucrose for
preparation of fermentation broth.

Observation

Result

ISOLATION AND CHARACTERIZATION OF BACTERIAL PIGMENTS

OBJECTIVE
To demonstrate the production of different kinds of pigments by bacteria and to
isolate and characterize the pigments.
BACKGROUND
Many soil bacteria are chromogenic and their pigmentation has been used as one
of the key characters of identification. Methods for isolating and characterizing
carotenoids pigments of gram positive and negative cells are known.

MATERIALS REQUIRED
Erlenmayer flask with minimal medium, organic reagents; 6% KOH in methanol
ether, petroleum ether, benzene, sodium sulfate, spectrophotometer, water bath;
cultures (Pseudomonas aureginosa, xanthomonas compestris; Serratia marcesens;
Corynebacterium poinsettiae).

PROCEDURE
1. Inoculate 100ml of minimal medium with 1 ml of log phase culture of bacteria
known to produce pigments.
2. Incubate for 48 hrs at 30oC in a shaker.
3. Harvest the cells by centrifugation at 10,000 rpm for 15 min.
4. Resuspend the cells in 100ml of distilled water and recentrifuge.
5. Resuspend the cells in 50 ml of methanol and transfer the suspension an
Erlenmeyer flask.
6. Keep the flask containing the bacterial suspension in a boiling water for 5 -10
min.
7. Intermittently swirl the flask to facilitate the release of bacterial pigment.
8. Cool the suspension, centrifuge to remove the cells.
9. Transfer the supernatant to a flask and add equal volume of 6% KOH in methanol.
10. Warm the content at 40oC for 10 min. swirl the content intermittently.
11. Transfer the content to a separating flask and add two volumes of diethyl ether
and enough water to separate the layer.
12. Gently shake the flask. The bacterial pigment gets into the ether phases.
13. Allow to stand for few minutes for separation of two phases.
14. Remove the ether phase. If you see the water, then dehydrate by adding anhydrous
solid sodium sulfate.
15. Wash the ether phase gain with water then dehydrate by adding anhydrous solid
sodium sulfate.
16. Transfer the ether to flask evaporator and evaporate to dryness at 30oC.
17. Redissolve the material in 10ml of petroleum ether and filter through Wattmann
No.1 filter paper.
 18. Read the absorbance of the filtrate between 350 nm to 540 nm using a
spectrophotometer.
19. Record the peaks of maximum absorbance.

OBSERVATION

RESULT:

ISOLATION AND CHARACTERIZATION OF MONASCUS SP FROM SOIL

AIM
To isolate and characterize Monascus sp from soil

BACKGROUND
Monascus sp is a pigment producing fungi present in soil. They are filamentous fungi
reproduce asexually by forming conidia, sexually by ascospore formation.The common
species of Monascus are Monascus purpureus, Monascus pilosus and Monascus
rubber.The pigments produced by various Monascus sp are monascin, monascorubin and
monascotin. These pigments are used as coloring agent in poultry, fish, meat and other
food products.Also used in color wine, pickle, ice cream, chocolate etc. It is also used in
cosmetics and textile industries as natural coloring agents. These pigments possess
antimicrobial and immunosuppressive activity.
MATERIALS REQUIRED
Soil sample, SDA plates, Standard glassware’s, LPCB stain.
PROCEDURE

1. Soil sample was serially diluted (10 fold dilution) upto 10-4 dilution.
2. Plates were prepared from all the dilutions using SDA agar by spread plate
method.
3. Incubated at room temperature for 7 days.
4. During incubation the plates were examined for white color mycelium initially
which becomes orange with age. The pigment can be seen on the reverse side
of the plate.

CHARECTERIZATION
LACTOPHENOL COTTON BLUE STAINING:
A loopful of isolated culture was emulsified in a drop of LPCB stain on a glass
slide. A coverslip was placed over the emulsion and the prepared wet mount was
observed, under low power objective.

OBSERVATION

RESULT
MASS MULTIPLICATIONOF MONASCUS SP AND PIGMENT EXTRACTION

AIM
To mass multiply Monascus sp and to perform solid state fermentation for pigment
extraction.
BACKGROUND
Red pigments produced by Monascus sp are used as coloring agent in food industries,
textiles and cosmetics. Production of pigment on industrial scale requires low cost
procedure. Being a soil fungus Monascus sp can grow on readily available agro waste
material. Therefore agro industrial waste material can be used as substrate for solid state
fermentation using Monascus sp to produce pigments. The solid substrate provides
nutrients as well as anchorage to the organism.

MATERIALS REQUIRED
Raw rice, ethanol, standard glasswares, SDB, Standard glasswares.
PROCEDURE

INOCULUM PREPARATION
To fully sporulated agar slope culture of Monascus sp 5.ml of distilled water added
aseptically and the spores were scraped using inoculation needle. The obtained spore
suspension used as inoculum for fermentation.

SOLID STATE FERMENTATION

1. 40 g of substrate (raw rice) was taken in a 250 ml conical flask and mixed with
approximately 15 ml of SDB.
2. pH was adjusted to 4.5 – 6 (preferably acid pH)
3. The moisten substrate was steamed for 10 – 20 min and cooled at room
temperature.
4. Moisture content was adjusted to 60% using sterile water
5. 2 ml of spore suspension was transferred to the flask aseptically, mixed well and
incubated at room temperature for 7- 10 days with daily examination and mixing.
PIGMENT EXTRACTION AND MEASUREMENT
1. After incubation the pigmented substrate was steamed for 20 min to kill the fungi
and dried completely at 45°C.
2. The dried substrate was taken, powdered and the pigment was extracted using
90% ethanol. (5 ml of solvent for each gram of powdered substrate)
3. The mixture was kept in rotary mixture for 1h and allowed to stand for 15 min.
4. The extract was filtered through whatman filter paper No. 1 and O.D of the filtrate
was measured at 510 nm against solvent blank.
5. The solvent was completely evaporated and the obtained pigment was emulsified
in suitable emulsifier.

OBSERVATION:
 RESULT:

BIOCATALYSIS OF PRECURSORS TO AROMA AND FLAVOR COMPOUNDS

USING Saccharomyces cerevisiae

AIM
To produce diphenyl ethanol using Saccharomyces cerevisiae

BACKGROUND
Diphenyl ethanol is an aromatic alcohol with rosy odor. This compound was produced by
many bacteria and yeast in food fermentation. Saccharomyces cerivisiae is the common
producers of this aromatic alcohol. Therefore used as biocatalyst for the production of
diphenyl ethanol.

BIOSYNTHETIC PATHWAY
 PHENYLALANINE

ά keto glutarate transamination

ά keto glutamate

PHENYL PYRUVATE

Décarboxylation

PHENYL ACETALDEHYDE

Dehydrogenase NADH+ + H+
NAD

DIPHENYL ETHANOL

MATERIALS REQUIRED
Saccharomyces cerivisiae culture
YEPD broth
Phenyl alanine
Ferric ammonium nitrate
Standard glass wares

PROCEDURE
1. YEPD medium with phenyl alanine is prepared and sterilized
2. Saccharomyces cerevisiae culture (0.1 ml) was inoculated in 10 ml of YEPD
medium
3. The inoculated broth was incubated at room temperature for 24 to 48 h
4. After incubation the broth was centrifuged at 5000rpm for 15 min.
5. Supernatant was taken and tested for presence of diphenyl ethanol by mixing 1 ml
of ferric ammonium nitrate solution for observing red colorization within 2 min.
OBSERVATION

RESULT

APPENDIX

MRS BROTH
Yeast Extract – 0.5 g
Beef extract – 1g
Peptone – 1g
Glucose – 2g
Tween 20 – 0.5 g
K2HPO4 – 0.2 g
Sodium acetate – 0.5 g
Diammonium citrate – 0.2 g
MgSO4 – 0.02g
MnSo4 – 0.02g
Distilled water – 100 ml
pH – 6.2 - 6.5

LEUCONOSTOC AGAR
CaCO3 – 5g
Malt extract – 5g
NaCl – 0.25 g
Beef extract – 0.1 g
Peptone – 0.1 g
Agar – 2g
Distilled water – 100 ml
pH – 6.4

YEPD BROTH
Yeast extract – 1g
Peptone – 2g
Dextrose – 2g
Phenyl alanine – 0.5 g
Distilled water – 1000 ml
pH – 6.2 – 6.5

M9 MINIMAL MEDIUM

Preparation of M9 Salts aliquot 800ml H2O and add

i. 64g Na2HPO4-7H2O
ii. 15g KH2PO4
iii. 2.5g NaCl
iv. 5.0g NH4Cl
v. Stir until dissolved
vi. Adjust to 1000ml with distilled H2O
vii. Sterilize by autoclaving

Measure ~700ml of distilled H2O (sterile)

Add 200ml of M9 salts

Add 2ml of 1M MgSO4 (sterile)

Add 20 ml of 20% glucose (or other carbon source)

Add 100ul of 1M CaCl2 (sterile)

Adjust to 1000ml with distilled H2O

NITRATE BROTH

Beef extract – 0.3g

Peptone - 0.5g

Potassium nitrate – 0.1g

Distilled water – 100 ml

Dissolve the ingredients, distribute in 5 ml quantities in 15 X 125 mm tubes and

autoclave at 121ºC for 15 min.

Reagent A

Alpha napthalamine – 0.5 g

Acetic acid (5N) 30% - 100 ml

Reagent B

Sulfanilic acid – 0.8g

Acetic acid (5N) 30% - 100 ml

Store the reagents in brown bottles.

Carbohydrate fermentation

A. Base

Peptone – 1.0g

Beef extract - 1.0g

NaCl – 0.5g

D.Water – 100 ml

B.Carbohydrate solution

Carbohydrate – 10.0g
 D.Water – 100 ml

C.Indicator

Phenol red – 1.6g

Ethanol – 100 ml

A = 900 ml

B = 100 ml

C = 1 ml

Mix dispenses in 1 ml amounts in 12 X 100 mm test tubes and autoclave for

10 min. at 121ºC.

REFERENCES

Hasting, J. J. H. (1971). Development of the fermentation industries in Great

Britain. Adv. Appl. Microbiol. 16 (2), 1-45.

Kajiwara, S., Yamada, H., Ohkani, N. and Ohtaguchi, K. (1997). Energy

Conversion and Management. Elsevier, 38 (2), 529-532.

Viktor, N. and Ronie, W. (2002). Applications of cell immobilization technology.

Springer, 44 (6), 212-216.

Stanbury P. F, A. Whitaker and S. J. (1995). Hall,‘Principles of Fermentation

Technology’, Pergamon Press, Oxford, 9 – 10

Jayababu mudili .(2009). Introdutory practical microbiology, Narosa publishers, 9.2 – 9.3