Laboratory Manual: 1 Lmbty360

LABORATORY MANUAL
BTY360
BIOPROCESS ENGINEERING LABORATORY
Name of the Student:………………………………………………..

Registration Number/Roll No……………………………………….
Section and Group……………………………………………………..
School of Biotechnology and Biosciences
Session-term:
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General guidelines for the students
1. Student is expected to maintain the decorum of the laboratory by maintaining proper

discipline and adhering to the University rules and policies.
2. Before coming to the lab class, students should read the principle, procedure and
other related aspects from the lab manual provided to them. Having background
knowledge of the experiment, students will be able to perform it efficiently and
effectively.
3. Students are required to write each observation in the worksheet only while
performing the experiment and get is signed from the teacher. All students should maintain
the lab manual and worksheet in a clean and decent form.
Dress code: No specific dress code is required. However, wear lab coat all the time while
working in the lab.
Compulsory things to be carried by students in lab: Lab Manual and Lab Coat (Apron)
Safety Guidelines:
1. Students should always wear CLOSED-TOE SHOES/SANDALS or other footwear.
2. Girl students should tie long hairs for their own safety as well as for avoiding
contamination in microbiological experiments.
3. Autoclave and hot air oven may cause SEVERE BURN INJURIES so observe
special precautions and ask lab technician if you are not sure about anything.
4. Students should have basic understanding of chemical, fire, radiation, carcinogen

and biological safety.
Do’s:
1. Always listen carefully to instructions given by class teacher and STRICTLY follow
all such guidelines.
2. Report any safety risks or incidents immediately to class teacher and/or lab
technician.
3. Always record your observations in worksheet provided in lab manual.

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4. Always keep your mobile phones in the bags in switched off mode.
5. Return all the apparatuses or glasswares to the concerned lab technician after
completion of the experiment.
6. Clean your bench top after the experiment.
7. Do not throw tissue papers or cotton in the sink. DISCARD THESE PROPERLY.
Don’ts:
1. Never talk loudly or unnecessarily in the lab.
2. Do not attend mobile phones while working in the lab.
3. Do not eat or drink in the microbiology lab.
4. Never take any culture or chemicals outside the lab. However, if there is a need to
do so, take prior permission from the class teacher.
5. Never discuss unnecessarily in the culture room.
6. NEVER PERFORM MOUTH PIPETTING OF MICROBIAL CULTURES.
7. Never leave a microbial culture unattended or in open condition.
Other instructions:
In a microbiology laboratory, there are cultures which can be potential health hazards.
TREAT ALL MICROBIAL CULTURES AS POTENTIAL PATHOGENS OR

HEALTH HAZARDS.
Students, therefore, make it a habit to wash their hands with soap before leaving the
laboratory so other persons don’t get accidently infected.
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TABLE OF CONTENTS
S. No. Title of the Experiment Page
1 5-10
pH probe standardization
2 Determination of growth curve of a supplied microorganism 11-16

.
3 . Determination of Thermal Death Point (TDP) and thermal death 17-20

time
(TDT) of microorganism
4 Study of rheology of viscous polymers and microbial cultures. 21-25
5 Use of alginate for cell immobilization. 26-29
6 30-33
Isolation of industrially important microorganisms for microbial
Processes
.
7 Production and estimation of alkaline protease 34-37
8 . Microbial production of cellulase by cellulolytic microorganism 38-41
Determination of volumetric mass-transfer coefficient (KLa ) in a

9 fermenter 42-45
Demonstration of working of bioreactor and determination of

10 mixing 45-49
time in bioreactors.
Reference Books:
1. Experimental Process Biotechnology Protocol by S.N.Mukhopadhyay, Viva Books Pvt.
Ltd, 1st Deition, (2007).
2. Bioprocess Engineering Principles by P M Doran, Elsevier publications, USA, 1st

Edition, (2001)
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EXPERIMENT NO. 1
1. Experiment: pH probe standardization
Equipment Required: pH meter, Dissolved oxygen probe
Material Required: Buffer capsules of pH 4.0 and 7.0, unknown solution
2. Learning Objectives: To learn the handling and working of pH meter and dissolved
oxygen probe.
3. Theory/Principle/Background of the topic: Brief only (100-250 word) :

A pH meter is essentially a voltmeter with a high input impedance which measures the
voltage of an electrode sensitive to the hydrogen ion concentration, relative to another
electrode which exhibits a constant voltage. The key feature of the pH-sensitive electrode
is a thin glass membrane whose outside surface contacts the solution to be tested. The
inside surface of the glass membrane is exposed to a constant concentration of hydrogen
ions (0.1 M HCl). Inside the glass electrode assembly, a silver wire, coated with silver
chloride and immersed in the HCl solution, is called an Ag/AgCl electrode. This electrode
carries current through the half-cell reaction. The potential between the electrode and the
solution depends on the chloride ion concentration, but, since this is constant (0.1 M), the
electrode potential is also constant. A reference electrode is needed to complete the
electrical circuit. A common choice is to use another Ag/AgCl electrode as the reference.
The Ag/AgCl electrode is immersed in an 0.1 M KCl solution which makes contact with
the test solution through a porous fiber which allows a small flow of ions back and forth to
conduct the current. The potential created at this junction between the KCl solution and
the test solution is nearly zero and nearly unaffected by anything in the solution, including
hydrogen ions.
.
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4. Outline of the Procedure:
Standardization of pH meter
i. The teacher will demonstrate the students the handling and working of pH meter.
ii. Prepare buffer of pH 4.0 and 7.0 by dissolving the buffer tablets in 100 ml distilled
water.
iii. Calibrate the pH meter at 4.0 and 7.0
iv. Take the reading of the unknown sample provided .
i. First Calibration Point Remove the probe from the water and place the tip of the
probe into the Sodium Sulfite Calibration Solution.
ii. When the displayed voltage reading stabilizes, enter 0 (the known dissolved oxygen
value in mg/L).
iii. Second Calibration Point Rinse the probe with distilled water and gently blot dry.
iv. Unscrew the lid of the calibration bottle provided with the probe. Slide the lid and the
grommet about ½ inch onto the probe body.
v. Add water to the bottle to a depth of about ¼ inch and screw the bottle into the cap
Important: Do not touch the membrane or get it wet during this step. Keep the probe in
this position for about a minute.
vi. When the displayed voltage reading stabilizes, enter the correct saturated dissolved
oxygen value (in mg/L) using the current barometric pressure and air temperature values.
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5. Results: Students will be able to correctly measure the pH of given sample.
6. Scope of Result: Students will understand the working and construction of pH meter
and dissolved oxygen probe.
7. Results Required: Hand on training on pH meter and dissolved oxygen probe.
8. Cautions:
i. Wash the pH probe carefully before and after taking readings.
ii. Dry the probe prior to put in the KCl solution.
9. Suggested Literature
i. http://www.explainthatstuff.com/how-ph-meters-work.html
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Worksheet of the student
Date of Performance Registration Number:
Aim:
Observation Table1: pH and dissolved oxygen readings
S.No pH S.D
Result and Discussion:
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Error analysis:
Learning Outcomes (what I have learnt): to be written by the students in 50-70 words.
To be filled by faculty:
Parameters
S. No Marks obtained Maximum
Understanding of the student about the

1 Procedure /apparatus. 20
Observations and analysis including

2 learning outcomes 20
Completion of experiment, Discipline

3 and Cleanliness 10
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Signature Total:
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EXPERIMENT NO. 2
1. Experiment: Determination of growth curve of a supplied microorganism.
Equipment Required: Laminar air flow hood, Autoclave, Micropipette, Weighing

balance, Incubator (shaker), centrifuge, refrigerator, Spectrophotometer.
Material Required: E. coli (Log phase), Nutrient broth, conical flask (250 ml), test
tube, eppendorf tubes.
2. Learning Objectives: To determine the growth pattern and kinetics of microbial cells.
3. Theory/Principle/Background of the topic: Brief only (100-250 word) :

Bacterial population growth studies require inoculation of viable cells into a sterile
broth medium and incubation of the culture under optimum temperature, pH, and
gaseous conditions. Under these conditions, the cells will reproduce rapidly and the
dynamics of the microbial growth can be charted by means of a population growth
curve, which is constructed by plotting the increase in cell numbers versus time of
incubation and can be used to delineate stages of the growth cycle. It also facilitates
measurement of cell numbers and the rate of growth of a particular organism under
standardized conditions as expressed by its generation time, the time required for a
microbial population to double.
i. Log phase culture of E. coli. was taken and inoculated in 100 ml of autoclaved
NB with 1% (V/V) concentration.
ii. Culture was incubated at 37 C for 24 hours with regular sampling (5 ml) every
30 min.
iii. Centrifugation of the sample was done at 800 RPM for 5 min and cellular
pellet was discarded. Collection of sample at initial and one blank (broth
without cells) was done initially.
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iv. Samples were refrigerated till further use.
v. Samples were read in spectrophotometer at 600 nm with pure NB as blank.
vi. Curve was plotted between OD and Time of growth.
5. General Calculations
6. Result: The generation of given microbial sample is ………

7. Scope of Result: This experiment is designed to include only the lag, log and possibly
stationary phases of population growth. Upon completion of this experiment, you will
plot the data collected during this experiment by using two values for the measurement
of growth.
8. Results Required: Exponential growth curve.
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9. Cautions:
i. Standard curve to be prepared carefully.
ii. Chemical stocks to be prepared carefully.
iii. Measurements to be done carefully.
iv. Analysis to be done carefully.
i. http://iitd.vlab.co.in/?sub=63&brch=177&sim=1418&cnt=1
:
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Date of Perfomance: Reginstration Number:

Aim:
Observation Tables:
S. No.
Time OD
Calculation:
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Error analysis:
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Parameter Maximum
Marks
S. No obtained marks
1. Understanding of the student about the 20

procedure/apparatus.
Observations and analysis including learning

2. Outcomes 20
Completion* of experiment, Discipline and

3. Cleanliness 10
Signature Total:
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EXPERIMENT NO. 3
1. Experiment: Determination of Thermal Death Point (TDP) and thermal death time
(TDT) of microorganism.
Equipment Required: Laminar air flow hood, Autoclave, Micropipette, Weighing

balance, Incubator (static), water bath.
Material Required: E. coli. (Log phase), Nutrient broth, Agar, conical flask (250 ml),
test
tube, Petri plate.
2. Learning Objectives: To know the relation of time of and temperature of sterilization

with death kinetics of microbes..
3. Theory/Principle/Background of the topic: Brief only (100-250 word)

Thermal Death time - is the time required to kill all bacteria in a sample at a specific
temperature. Need to standardize the population size of your sample before determining
the thermal death time.
Thermal death point is the lowest temperature at which all microbes in a liquid will be
killed in 10 minutes. Thermal death time is a concept used to determine how long it
takes to kill a specific bacterium at a specific temperature. It was originally developed
for food canning and has found applications in cosmetics, producing salmonella-free
feeds for animals (e.g. poultry) and pharmaceuticals.
i. Two sets of NB were prepared. Inoculation was done in each and divided into 5 separate
test tubes.
ii. One set was put in water bath at 60 C for 5 different time periods viz. 2 min, 5 min,
iii. 10 min, 15 min, 20 min.
iv. Another set was put in different water bath with temperatures 60 C, 70 C, 80 C for 10
min.
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v. After treatment, each of the sample was spread over NA plates prepared in advance and
incubated at 37C for minimum 18 hrs.
vi. Observation for growth was done.
5. General Calculations: Observation of growth
6. Results: TDT of given microbial culture was found to be ……………….

TDP of given microbial culture was found to be ………… …….
7. Scope of Result: TDT and TDP will help in the control of harmful microorganisms
8. Results Required: Plates with no growth will help in determination of TDP and TDT.
9. Cautions:
i. Temperature of bath shall be confirmed with thermometer.
ii. Contamination shall be avoided.
iii. Test tubes shall be put properly in water bath.
i. http://www.fbs.leeds.ac.uk/institutes/ilse/movies/microbiology/transcript/Thermal_dea
th_point.html
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Date of Performance: Registration Number:
Aim:
Observation Tables:
S.No Plate Specifications Observation about Growth
Calculation:
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Error analysis:
Parameter Maximum
Marks
S. No obtained marks
1 Understanding of the student about the 20


2 Outcomes 20
Completion of experiment, Discipline and

3 Cleanliness 10
Signature Total:
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EXPERIMENT NO. 4
1. Experiment: Study of Rheology of viscous polymers and microbial

cultures.
Equipment Required: Viscometer, stop clock, burette stand.
Material Required: Distilled water, Fermentation broth, 10 % glycerin.
2. Learning Objectives: To calculate viscosity of the fluids.
3. Theory/Principle/Background of the topic: Brief only (100-250 words)

Bioreactor performance influenced by broth rheology, which is
determined by:
a. biomass concentration
b. morphology
c. biomass growth rate
d. extracellular components
As these parameters typically very throughout the course of a fermentation, so too will
the
rheology.
i. correlations which describe viscosity as a function of biomass concentration
only are of limited value
ii. polymer solutions, paper pulp suspensions often used to simulate flow
behaviour of fermentation broths, but they do not include effects of active
biomass on system performance
i. Density of water, glycerin and fermentation broth was measured.

ii. Equal and appropriate mass of each fluid which can fill the viscometer
appropriately was measured.
iii. Fluid was aspirated up-to upper mark the allowed to fall in viscometer.
iv. Time taken for passing between two marks was noted with stop clock.
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v. Viscosity of glycerin and fermentation broth was calculated with following formula :
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µ (g) t (g) density (g)
µ (w ) t (w) density (w )
5. Result: The viscosity of given sample was found to be ………… ………….
6. Scope of result: The fermentation broth too act as the medium for various physical,
biochemical and physical reactions to take place. The fermentation broth will be
implicated in all the mass and heat transfers that occur within the fermenter, and it
will be the medium that holds the fermentation products formed.
The nature and composition of the fermentation broth temporally and spatially will
affect the efficiency of the fermentation process. The interaction between the
fermentation broth and the various components is complex and affect both
directions
7. Results Required: Time taken by given fluid can be used to calculate viscosity.
8. Cautions:
a. Meniscus to be set accurately
b. Temperature and aeration should be maintained properly.
i. http://link.springer.com/chapter/10.1007%2F3-540-08557-2_1#page-1
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Aim:
Observation Tables:
S. No. Material Density Time taken Viscosity
1.
2.
3.
Calculation:
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Error analysis:
Parameter
Marks Maximum
S. No obtained Marks


2 Outcomes 20

3 Cleanliness 10
Signature Total:
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EXPRIMENT NO. 5
1. Experiment: Use of alginate for cell immobilization.
Equipment Required: Magnetic stirrer, centrifuge, weighing balance.
Material Required: Beaker, Syringe, sodium alginate (3% to 12 % depending upon

purity), Calcium chloride (0.2 M), microbial cells.
2. Learning Objectives: To learn technique of immobilization of cells.

Methods of yeast immobilization evaluated included collagen casting, acetic acid-
lactic acid cellulose microencapsulation, chitosan/glutar-aldehyde molding,
carrageenan-entrapping method, and calcium alginate gel-entrapping. Of these, the
calcium alginate gel-entrapping method was preferred because of its high enzymatic
activity, simple manner of preparation, and stability.
Preparation of a uniform calcium alginate gel, necessitated maintaining the viscosity

of the mixture of calcium alginate and yeast cells between 1000 and 2000 cps. The
addition of a nonionic surfactant and an unsaturated fatty acid at the time of gelling
was also found to improve cell retention and enzyme activity.
i. Active microbial culture was centrifuged and wet mass of cells was determined.
ii. Cells were suspended in 500 µ l of distilled water and mixed with 50 ml of
sodium alginate solution.
iii. The mixture was filled in syringe and dropped in ice cold calcium chloride
continuously stirred over stirred.
iv. Beads thus produced were kept for some time more in solution for curing.
5. General Calculations: Sodium alginate and calcium chloride to be weight exactly for
preparing desired solution.
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6. Results: Formation of alginate trapped immobilized cells
7. Scope of the result

The immobilized whole cell system is an alternative to enzyme immobilization. Unlike
enzyme immobilization, where the enzyme is attached to a solid support (such as
calcium alginate), in immobilized whole cell systems, the target cell is immobilized.
Such methods may be implemented when the enzymes required are difficult or
expensive to extract, an example being intracellular enzymes. Also, if a series of
enzymes are required in the reaction; whole cell immobilization may be used for
convenience.
8. Required Results: Solid beads having cells inside it.
9. Cautions:
i. Check for viscosity of alginate solution.
ii. Solutions shall be prepared carefully.
10. Suggested Literature.
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Worksheet of the Student
Aim:
Observation Tables:
S.No Observation Inference
Calculation:
Result and Discussion
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Error analysis:
Parameter
Marks Maximum


2 Outcomes 20

3 Cleanliness 10
Signature Total:
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EXPERIMENT NO. 6
1. Experiment: Isolation of industrially important microorganisms for microbial processes.
Equipment Required: Laminar air flow hood, Autoclave, Micropipette, Vortex,

Weighing balance, centrifuge, Incubator (static).
Material Required: Petri plates, test tube, spreader, Skimmed milk agar (SMA), CMC
agar, Starch agar, Nutrient agar. Sample from dumping ground soil. Czapek mineral salt
agar medium, 0.5 M sodium chloride, iodine solution.
2. Learning Objectives: To learn screening, isolation and pure culture of desired

organism from raw sample.
Casease is an exoenzyme that is produced by some bacteria in order to degrade casein.

Casein is a large protein that is responsible for the white color of milk. This test is
conducted on milk agar which is a complex media containing casien, peptone and beef
extract. If an organism can produce casein, then there will be a zone of clearing around
the bacterial growth.
Starch agar is inoculated with the species in question. After incubation at an

appropriate temperature, iodine is added to the surface of the agar. Iodine turns blue-
black in the presence of starch. Absence of the blue-black color indicates that starch is
no longer present in the medium. Bacteria which show a clear zone around the growth
produce the exoenzyme amylase which cleaves the starch into di- and monosaccharides.
These simpler sugars can then be transported into the cell to be catabolized. Bacillus
species are known to produce the exoenzyme, amylase.
i. Sample was collected and brought to laboratory.
ii. 1 gm of sample was dissolved in 10 ml of distilled water with vortexing.

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iii. Serial dilutions were prepared in distilled water.
iv. SMA, CMC agar and Starch agar were prepared, autoclaved and solidified in sterilized
petri plates. Composition of Czapek mineral salt agar medium for 1 litre:- Sodium
nitrate - 2g, potassium phosphate-1g, magnesium sulphate-0.5g, potassium chloride-
0.5g, CMC-5g,
peptone-2g, agar-20g, distilled water-1000ml.
v. Composition of skimmed milk agar for 1 litre: skim milk powder-100g, peptone-5g,
agar-15g, pH 7.2
vi. For each of the three types of agar 5 plates were spread with dilutions of soil sample.
vii. Plates were incubated at 37 C for 18-24 hours.
viii. Observed for zone of hydrolysis for SMA. CMC agar plates were flooded with congo
red dye 0.1 %, then terminated with 0.5 M sodium chloride. Starch plates were
flooded with iodine. Development of halos in CMC and Starch plates were observed.
5. General calculation: Measurement of zones of clearance.
6. Result: The zone of clearance was found to be…..
7. Scope of result: Zones of clearance indicates the preliminary information about enzyme
production by microbes.
8. Results Required: Zone of hydrolysis and halos in petri plates.
9. Cautions: Contamination has to be avoided.

i. http://microbiology.ukzn.ac.za/Libraries/MICR304/INDUSTRIAL_MICROORGANISM
S.sflb.ashx
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Worksheet of Student
Date of Performance:
RegistrationNumber:
Aim:
Observation Tables:
S.No. Microorganism/soil sample Zone of hydrolysis
1.
2.
3.
Calculation:
Error analysis:
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Parameter
Marks Maximum


2 Outcomes 20

3 Cleanliness 10
Signature Total:
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EXPERIMENT NO. 7
1. Experiment: Production and estimation of alkaline protease.

Equipment Required: Shaker incubator, centrifuge.
Material Required: Protese producing microbe culture (B. subtilis) and E. coli,
Skimmed milk powder agar, Nutrient broth.
2. Learning Objectives: Production of protease.
A protease (also termed peptidase or proteinase) is any enzyme that performs
proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link
amino acids together in the polypeptide chain forming the protein. Proteases have evolved
multiple times, and different classes of protease can perform the same reaction by
completely different catalytic mechanisms. Proteases can be found in animals, plants,
bacteria, archea and viruses. Proteases are involved in digesting long protein chains into
shorter fragments by splitting the peptide bonds that link amino acid residues.

Composition of skimmed milk agar for 1 litre: skim milk powder-100g, peptone-5g,
agar-15g, pH-7.2
i. Pour the autoclaved cooled medium of skimmed milk agar into sterile petri plates
and allow them to solidify.
ii. Label the 3 skimmed milk agar plates with the name of bacterial organism to be
inoculated and fourth as control.
iii. Make a single line streak inoculation from each culture into its labelled petri plates
iv. Across the surface of medium.
v. Incubate the plates (inoculated and uninoculated) for 24-48 hours at 37 degree
Celsius in an inverted position.
5. General calculations: Zone of hydrolysis to be determined accurately.
6. Results: Diameter of the zone of hydrolysis of given microbe sample is …………
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7. Scope of the result: More protein degradation more zone of hydrolysis will be
observed.
8. Results Required: Observe all the inoculated plate for any clearing around the line of
growth.
9. Cautions:
i. Avoid contamination.
ii. Prepare the stocks properly.

i. http://sciencejournal.in/data/documents/TLS-Vol-2-2-9.pdf
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Aim:
Observation Tables:
S.No. Microorganism Zone of hydrolysis
1.
2.
3.
Calculation:
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Error analysis:
Maximum
Marks
S. No Parameter obtained Marks


2 outcomes 20

3 Cleanliness 10
Signature Total:
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EXPERIMENT NO. 8
1. Experiment: Microbial production of cellulase by cellulolytic

microorganism.
Equipment Required: Shaker incubator, centrifuge.
Material Required: Cellulase producing microbe culture (B. subtilis), Aspergillus niger,
CMC, modified Czapek mineral salt medium, 0.5M sodium chloride.
2. Learning Objectives: Production of cellulases.
3. Theory/Principle/Background of the topic: Brief only (100-250 words)

Microbial cellulases have shown their potential application in various industries
including pulp and paper, textile, laundry, biofuel production, food and feed industry,
brewing, and agriculture. Due to the complexity of enzyme system and immense
industrial potential, cellulases have been a potential candidate for research by both the
academic and industrial research groups. Cellulases are inducible enzymes synthesized
by a large diversity of microorganisms including both fungi and bacteria during their
growth on cellulosic materials. These microorganisms can be aerobic, anaerobic,
mesophilic or thermophilic. Among them, the genera of Clostridium, Cellulomonas,
Thermomonospora, Trichoderma, and Aspergillus are the most extensively studied
cellulose producer. The biological aspects of processing of cellulosic biomass become
the crux of future research involving cellulases and cellulolytic microorganisms.
Cellulases are being commercially produced by several industries globally and are
widely being used in food, animal feed, fermentation, agriculture, pulp and paper, and
textile applications.

Preparation of Czapek mineral salt agar medium for 1 litre:- sodium nitrate -
2g,potassium phosphate-1g,magnesium sulphate-0.5g,potassium chloride-0.5g,CMC-
5g,peptone-2g,agar-20g,distilled water-1000ml
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i. Pour the autoclave cooled medium into sterile petri plates and allow them to modify.
ii. Label the plates each with the organism to be inoculated.
iii. Inoculate the appropriately labeled plates with respective organism.
iv. Incubate inoculated plates at 35 degree Celsius in inverted position for 2-5days
v. Flood the plates with 0.1% Congo red and then terminate with 0.3 M Sodium
chloride to observe the result.
vi. The above procedure is again done for soil sample, where serial dilution of soil
sample is being done and then they are spreaded on Czapek mineral salt agar plate
and the above procedure is followed from point 4-5.
5. General calculations: Protein estimation and enzyme activity of crude enzyme extract.
6. Result : The zone of clearance around cellulolytic microbial colony was found to be…
7. Scope of result: Successful utilization of cellulosic materials as renewable carbon

sources is dependent on the development of economically feasible process technologies
for cellulase production, and for the enzymatic hydrolysis of cellulosic materials to low
molecular weight products such as hexoses and pentoses.
8. Required Results: Observe the plates for the formation of zone around the growth.
9. Cautions:
i. Avoid contamination.
ii. Prepare the stocks properly.
i. http://www.turkjbiochem.com/2012/287-293.pdf
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Aim:
Observation Tables:
S. No. Microorganism/soil sample Zone of hydrolysis
1.
2.
3.
Calculation:
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Error analysis:
Parameter
Marks Maximum


2 Outcomes 20

3 Cleanliness 10
Signature Total:
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EXPERIMENT NO. 9
1. Experiment: Determination of KLa (mass transfer coefficient) of oxygen in water.
Equipment Required: Fermenter

Material Required: 0.5M Na2SO3, 1% starch solution, 0.1M Na2S2O7, 0.003
CuSO4.5H2O, Standard Na2SO3 (0.1M to 0.5M), 0.1M Iodine solution (20 g/l KI + 12.7 g/l
I2)
2. Learning Objectives: To determine the kLa in a fermenter
Oxygen acts a limiting nutrient for an aerobically growing culture in the bioreactor. Even in
a reactor having non limiting availability of normal Carbon, Nitrogen and other sources, the
limiting or no availability to aerobically growing culture may lead to death of the microbial
population in no time. Therefore the air (oxygen) has to be adequately supplied either by
sparger or in head space to enhance the dissolved oxygen so that microorganisms are able to
breathe through the pores. It is important to note that the capacity of water to retain oxygen
is rather limited. Under the normal condition of 25º C and 1 Atmosphere pressure, the
dissolved oxygen will be 8 mg/L. Air has to be continuously purged and agitated so as to
enhance the depleting oxygen in the bioreactor. For adequately aerated cultivation, the
supply of oxygen has to be greater than the demand of the culture to maintain the healthy
growth & metabolite production by the microorganisms at any point of time. Assume that
the bioreactor is in well mixed condition and dissolved oxygen concentration is constant
through-out the reactor, the following equation will hold true.
Rate of dissolution of dissolved oxygen (dCL/dt) = {Supply of oxygen (KLa (C*– CL)}
– {Demand by microorganism (rX)}.
Where KLa is termed as volumetric mass transfer coefficient or aeration efficiency, C*

saturation concentration of dissolved oxygen, CL is the dissolved oxygen concentration in
the bulk of the fermentation broth, “r” is respiratory rate constant and “X” is available
biomass (g/L).
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i. Fermentor is filled with 0.5M Na2SO3 and 0.003 M CuSO4. 5H2O (2.5 litre).
ii. Agitation is given at 400 rpm.
iii. After the aeration begins collect samples every 10 mins.
iv. Estimate the concentration of Na2SO3 by titration. Burette solution: 0.1 M Na2S2O7.
Flask solution: 500μl of Na2SO3 solution (sample or standard) + 15 ml iodine solution +
50μl starch.
v. During titration colour changes to straw yellow. Add the starch solution at this point only
and not before.
vi. Estimate the concentration of Na2SO3 in sample from standard curve.
5. General Calculations: Calculation for reagents preparation.
6. Results: Estimate the value of kLa
7. Scope of the result: Estimation of volumetric mass transfer coefficient kLa
8. Results Required: Value of mass transfer coefficient
9. Cautions:
i. Attention should be given to the readings of dissolved oxygen concentration
ii. Prepare the solutions properly.
10. Suggested Literature:

i. http://dl.uctm.edu/journal/node/j2013-4/4-Chaushev%20-%20351-356.pdf
43 LMBTY360
Aim:
Observation Table 1: (Estimation of KLa)
S.No
Time (in mins) Vol of Na2S2O7 consumed (in ml)
Calculations
44 LMBTY360
Error analysis:
Parameter
Marks Maximum


2 Outcomes 20

3 Cleanliness 10
Signature Total:
45 LMBTY360
EXPERIMENT NO. 10
1. Experiment: Demonstration of working of bioreactor and determination of mixing

time in bioreactors.
Equipment Required: Bioreactor.
Material Required: Methylene blue, stop watch.
2. Learning Objectives: To learn working and determine mixing time of bioreactor.

A bioreactor may refer to any manufactured or engineered device or system that supports
a biologically active environment. In one case, a bioreactor is a vessel in which a
chemical process is carried out which involves organisms or biochemically active
substances derived from such organisms. This process can either be aerobic or anaerobic.
These bioreactors are commonly cylindrical, ranging in size from litres to cubic metres,
and are often made of stainless steel.
A bioreactor may also refer to a device or system meant to grow cells or tissues in the
context of cell culture. These devices are being developed for use in tissue engineering
or biochemical engineering.
i. Working of bioreactor is taught with live demonstration. Control of

functions and subparts were observed carefully.
ii. For determination of mixing time at a particular speed of agitator,
bioreactor was run at 50 RPM and methylene blue was added in the
reactor with water in it.
iii. Time for complete homogenization was noted with stop watch.
iv. Step 2 and 3 were repeated with 100 RPM, 150 RPM and 200 RPM.
5. General Calculations: Demonstration.

46 LMBTY360
6. Results: Demonstration.
7. Scope of the result: A bio-reactor can be used in wastewater treatment and tissue
engineering.
8. Results Required: Time for mixing was determined.
9. Cautions: Mixing to be observed very carefully.
10. Suggested Literature:

47 LMBTY360
Aim:
Observation Tables:
S. No. Time for mixing Speed of agitator
1.
2.
3.
Calculation:
48 LMBTY360
Error analysis:
Parameter
Marks Maximum


2 Outcomes 20

3 Cleanliness 10
Signature Total:
49 LMBTY360
50 LMBTY360

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LABORATORY MANUAL

Name of the Student:………………………………………………..

1. Student is expected to maintain the decorum of the laboratory by maintaining proper

1. Students should always wear CLOSED-TOE SHOES/SANDALS or other footwear.

4. Students should have basic understanding of chemical, fire, radiation, carcinogen

3. Always record your observations in worksheet provided in lab manual.

6. Clean your bench top after the experiment.

1. Never talk loudly or unnecessarily in the lab.

2. Do not attend mobile phones while working in the lab.

3. Do not eat or drink in the microbiology lab.

5. Never discuss unnecessarily in the culture room.

6. NEVER PERFORM MOUTH PIPETTING OF MICROBIAL CULTURES.

7. Never leave a microbial culture unattended or in open condition.

TREAT ALL MICROBIAL CULTURES AS POTENTIAL PATHOGENS OR

S. No. Title of the Experiment Page

2 Determination of growth curve of a supplied microorganism 11-16

3 . Determination of Thermal Death Point (TDP) and thermal death 17-20

5 Use of alginate for cell immobilization. 26-29

8 . Microbial production of cellulase by cellulolytic microorganism 38-41

Determination of volumetric mass-transfer coefficient (KLa ) in a

Demonstration of working of bioreactor and determination of

2. Bioprocess Engineering Principles by P M Doran, Elsevier publications, USA, 1st

1. Experiment: pH probe standardization

Equipment Required: pH meter, Dissolved oxygen probe

Material Required: Buffer capsules of pH 4.0 and 7.0, unknown solution

3. Theory/Principle/Background of the topic: Brief only (100-250 word) :

7. Results Required: Hand on training on pH meter and dissolved oxygen probe.

Date of Performance Registration Number:

Observation Table1: pH and dissolved oxygen readings

Result and Discussion:

Understanding of the student about the

Observations and analysis including

Completion of experiment, Discipline

1. Experiment: Determination of growth curve of a supplied microorganism.

Equipment Required: Laminar air flow hood, Autoclave, Micropipette, Weighing

3. Theory/Principle/Background of the topic: Brief only (100-250 word) :

4. Outline of the Procedure:

6. Result: The generation of given microbial sample is ………

10. Suggested Literature

Date of Perfomance: Reginstration Number:

1. Understanding of the student about the 20

Observations and analysis including learning

Completion* of experiment, Discipline and

Equipment Required: Laminar air flow hood, Autoclave, Micropipette, Weighing

2. Learning Objectives: To know the relation of time of and temperature of sterilization

3. Theory/Principle/Background of the topic: Brief only (100-250 word)

4. Outline of the Procedure:

5. General Calculations: Observation of growth

6. Results: TDT of given microbial culture was found to be ……………….

10. Suggested Literature

Date of Performance: Registration Number:

Result and Discussion:

1 Understanding of the student about the 20

Observations and analysis including learning

Completion of experiment, Discipline and

1. Experiment: Study of Rheology of viscous polymers and microbial

2. Learning Objectives: To calculate viscosity of the fluids.

3. Theory/Principle/Background of the topic: Brief only (100-250 words)

4. Outline of the Procedure: