VITU N I V E R S I T Y

(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.MOHANASRINIVASANProf. CHITRA KALAICHELVAN

Mrs. M. THENMOZHI

School of Bio Sciences and Technology

(For Private circulation only)

VIT – A Place to learn; A chance to grow

VITU N I V E R S I T Y

(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

S. NO. DATE TITLE OF THE EXPERIMENTSPAGE

NO.REMARKS

INITIALS

1.

LABORATORY PRACTISES

2BIOREACTOR AND ITS STRUCTURE

3.ISOLATION AND CHARACTERIZATION OF

Lactobacillus FROM FERMENTED MILK

PRODUCTS

4.ISOLATION AND CHARACTERIZATION OF

Leuconostoc FROM RICE FLOUR

5.

ISOLATION AND CHARACTERIZATION OF

Saccharomyces cerevisiae FROM GRAPE

JUICE

6.PRODUCTION OF GRAPE WINE

7. ALCOHOL FERMENTATION BY YEAST

8.ISOLATION AND CHARACTERIZATION

OF BACTERIAL PIGMENTS

9.ISOLATION AND CHARACTERIZATION

OF Monascus FROM SOIL

10.PIGMENT EXTRACTION FROM

Monascus

11.BIOCATALYSIS OF PRECURSORS TO

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

reaction

/Shape

Catalase Oxidase Sugar fermentation/gas

production

Arginine

hydrolysis

Growth

at 15°C

and

45°C

Dextrose Sucrose Lactose

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

reaction/

Shape

Catalase VP

test

Citrate

utilization

Sugar fermentation

/gas production

Esculin

hydrolysis

Dextran

production

Dextrose Lactose sucrose

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

Staining

/Shape

Nitrate Urease Sugar fermentation/gas

production

Ascospore

Dextrose Sucrose Lactose

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.

2.

3.

4.

5.

6.

Colour

Aroma

pH

% Tartarate

% Acetate

% 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 BROTHYeast Extract – 0.5 gBeef extract – 1 gPeptone – 1 gGlucose – 2 g

Tween 20 – 0.5 gK2HPO4 – 0.2 gSodium acetate – 0.5 gDiammonium citrate – 0.2 gMgSO4 – 0.02gMnSo4 – 0.02gDistilled water – 100 mlpH – 6.2 - 6.5

LEUCONOSTOC AGARCaCO3 – 5 gMalt extract – 5 gNaCl – 0.25 gBeef extract – 0.1 gPeptone – 0.1 gAgar – 2 gDistilled water – 100 mlpH – 6.4

YEPD BROTH Yeast extract – 1 gPeptone – 2 gDextrose – 2 gPhenyl alanine – 0.5 gDistilled water – 1000 mlpH – 6.2 – 6.5

M9 MINIMAL MEDIUM

Preparation of M9 Salts aliquot 800ml H2O and add

i. 64g Na2HPO4-7H2Oii. 15g KH2PO4

iii. 2.5g NaCliv. 5.0g NH4Clv. Stir until dissolved

vi. Adjust to 1000ml with distilled H2Ovii. 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