SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303 Experiment – 1 Aim: To prepare broth media for microbial growth. Theory: Bacteria in contrast to fungi are often cultured in a liquid broth (i.e. media lacking Agar). The most common constituents of basic media used in routine bacteriological laboratory are beef extract (a beef derivative which is a source of organic carbon, nitrogen, vitamins and inorganic salts) and peptone (a semi digested protein). These may be modified in a variety of ways by supplementing with some specific chemicals or materials to provide a medium suitable for the cultivation or demonstration of a reaction for specific types or groups of bacteria. Nutrient broth and glucose broth have been considered as basic liquid media for cultivation of bacteria. Materials Required: Constituents for Nutrient broth Peptone 5.0g Beef extract 3.0g Distilled water 1000.0ml Constituents for Glucose broth Peptone 10.0g Glucose 5.0g Sodium Chloride 5.0g Distilled water 1000.0ml
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SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Experiment – 1
Aim: To prepare broth media for microbial growth.
Theory:
Bacteria in contrast to fungi are often cultured in a liquid broth (i.e. media lacking Agar). The most common
constituents of basic media used in routine bacteriological laboratory are beef extract (a beef derivative which is
a source of organic carbon, nitrogen, vitamins and inorganic salts) and peptone (a semi digested protein).
These may be modified in a variety of ways by supplementing with some specific chemicals or materials to
provide a medium suitable for the cultivation or demonstration of a reaction for specific types or groups of
bacteria.
Nutrient broth and glucose broth have been considered as basic liquid media for cultivation of bacteria.
Materials Required:
Constituents for Nutrient broth
Peptone 5.0g
Beef extract 3.0g
Distilled water 1000.0ml
Constituents for Glucose broth
Peptone 10.0g
Glucose 5.0g
Sodium Chloride 5.0g
Distilled water 1000.0ml
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
1 N HCl
1N NaOH
pH meter
Distilled water
Hot plate or Heater
Autoclave
Beaker corning, 50ml, 250ml
Measuring cylinder
Cotton
Culture tubes
Glass rod
Weighing balance
Spatulas
Procedure:
Nutrient broth
1. Put the weighed amount of peptone (5g) and beef extract (3g) in 500ml of distilled water in a
beaker.
2. Heat with agitation to dissolve the constituents.
3. Add more distilled water to make the total volume as 1 liter.
4. Adjust pH of the medium to 7.0, using a pH meter, by adding either acid or alkali, as the case may
be.
5. Pour 10 ml of media in to 50ml corning flask.
6. Apply cotton plugs on the mouth of coning flasks
7. Cover the cotton plugs with aluminum foils tightly.
8. Autoclave at 121oC, 15 psig pressure for 20 minutes.
9. Put off the power supply for the autoclave and remove the plug from the socket.
10. Allow the autoclave to cool to room temperature (make sure that the pressure in side the
autoclave is shown zero).
11. Release the pressure valve completely and wait for a minute before opening the autoclave lid.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
12. Take out the nutrient broth corning flasks and label them using a marker pen and put your name
along with date and incubate at 37oC temperature in the incubator.
13. After 48 hrs check for any contamination or turbidity in the broth and if there is any turbidity noted
in the broth then repeat the above steps.
14. If no turbidity is observed then store it in the refrigerator for further use.
Glucose broth
Glucose broth can be prepared in the same way as nutrient broth.
Observations:
The media flasks are to be kept under observation / incubation for 48 hrs at room temperature or in an
incubator at 37oC and check weather there was any contamination in the broth media. If there is any
contamination then discard the media and repeat the experiment till you succeed to have a sterile broth.
Result: Briefly write about the conclusion and result of the experiment performed.
Note: Clean all your glassware and the work bench after finishing your experiment.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Experiment – 2
Aim: To culture the microbial organisms in a shake flask using orbital shaker incubator.
Theory:
Microbial culturing is performed by using broth as a liquid media in the laboratory on a small scale or in an
industrial fermenter on a large scale. The media termed broths, milks, or nutrient solutions are made by
dissolving various solutes in distilled water and later sterilizing (autoclaving) it. Appropriate physical and
chemical environments are required for growing microorganisms in a broth. Temperature is the most vital
requirement for growing cultures which is supplied by incubator in the laboratory. Many cultures also require
oxygen, a certain amount of which is absorbed in to the surface of broth in the flask placed in an undisturbed
rack on the incubator shelf; this condition is termed as static incubation. Now a shaker incubator having
shaking platform fitted with conical flask holders, where the culture flasks can be put up, are used to provide
oxygen to broth cultures and at the same time maintains the desired or set temperature. For large volumes of
broth, air is filter sterilized and pumped in to large broth filled vessels, fermentors.
Growth of microorganism occurs throughout the container and may present a dispersed cloudy or particulate
appearance. Some microorganisms uniformly increase the turbidity (milkiness or cloudiness) of broth, due to
increase in their numbers. Pellicle, a mat of cells, is formed by some microorganisms which grow only at the
surface of a static broth culture. Other microbial cultures may settle to the bottom of the flask/ tube to form a
sediment or button of cells that stick together. Filamentous fungi form intertwined mycelia (clumps) suspended
within a broth.
The prime use of broth cultures is to study the characteristics of microorganisms and for the production of
various industrial products by the microorganisms, e.g. production of antibiotics, mass production of bakers
yeast etc.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Materials Required:
Sterile broth media ( Nutrient broth / Glucose broth )
Transfer loop
Non adsorbent cotton
70% alcohol
Microbial culture ( Bacillus subtilis )
Spirit lamp
Match box
Laminar hood
Shaker incubator
Tissue paper
Marker
Procedure:
1. Take the transfer loop, non-adsorbent cotton, 70 % alcohol, Bunsen burner & tissue paper and
place them in the laminar air flow chamber.
2. Put on the UV light in the laminar hood and wait for 15 minutes. Make sure that the laminar hood
is closed properly before the UV light is on.
3. Put off the UV light after 15 minutes and put on the fluorescent (white) light and the laminar air
flow.
4. Open the lower lid (part) of the laminar hood and place the sterile media and the culture microbe
on the work bench of laminar hood.
5. Fold your both hand clothes beyond the elbows and wipe the palms and hands up to the elbows
with 70% alcohol.
6. Spray the 70% alcohol on the surface of work bench and further wipe the surface using non
adsorbent cotton or tissue paper and keep the cotton /tissue paper at a corner. If required take the
assistance of the instructor.
7. Light the spirit lamp / Bunsen burner and adjust the flame to an appropriate level.
8. Flame the transfer loop to red hot condition and then wait for a minute to cool the loop.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
9. Open the cotton plug of the microbial strain in the vicinity of flame and take a loopful of the culture
and close the culture tube with the same cotton plug. Caution, do not keep the loop on the work
bench and hold it in your hand for the next step. If required, take the help of the instructor.
10. Take the sterile media flask and open the cotton plug in the vicinity of flame and transfer the
loopful of microorganisms in to the media by dipping and shaking of the loop in the media. Then
immediately close the mouth of the media flask with the cotton plug in the vicinity of the flame
only.
11. Now flame the transfer loop to red hot and then keep it on the work bench.
12. Before closing, put off the burner flame, clean the work bench and wipe the spills using the tissue
paper and drop it in to the dust bin.
13. Take out the inoculated media and the culture strain from the laminar and put off the laminar air
flow and white light and then close the laminar lid.
14. Place the microbial strain in the refrigerator
15. Label the inoculated media flask with your name and put the date and time using marker pen and
place the flask on the plat form of shaker incubator.
16. Set the temperature at 30oC and shaker speed at 90 rpm. If required take the help of instructor.
17. Wait for 24 hours to observe the growth of microbial culture.
Observations:
Observe the turbidity of the broth as the indication of growth of microorganisms. Store it in the fridge
for future use.
Result: Briefly write about the conclusion and result of the experiment performed.
Note: Clean all your glassware and the work bench after finishing your experiment.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Experiment – 3
Aim: To estimate the Microbial biomass produced through shake flask culturing.
Theory: Estimation of bacterial growth requires inoculation of a sterile broth medium and incubation of the
culture under optimum temperature and gaseous conditions. The bacteria will reproduce rapidly under these
conditions and the microbial growth may be measured by variety of techniques like wet weight determination,
dry weight determination, determination of total nitrogen by Kjeldahl method, direct counting of cells under a
light microscope, or indirectly by viable count (plate count) which detects living organisms by their ability to
form colonies on agar surfaces, and turbidity measurements, which relate cell number to the turbidity
(cloudiness) of a broth culture.
Here we will estimate the biomass concentration by wet weight method and it can be extended to dry weight
as well. Initially it is better to start with the determination of wet weight of biomass using centrifuge to make a
pellet of the particle like bacterial cells suspended in the broth. To weight the biomass simple digital balances
can be used with higher sensitivity as the biomass collected would be of very small quantity of mg order.
Materials Required:
Microbial broth culture
Pipettes ( 5/10 ml )
Centrifuge tubes
Centrifuge
Weighing balance
Blotting paper
Tissue paper
Marker pen
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Procedure:
1. Take the shake flask containing the broth from the refrigerator and keep it in on the work bench to
bring it to normal room temperature.
2. Take the centrifuge tubes and label them as 1, 2, 3 & 4 on the tube as well as on the cap and then
weigh them using the digital balance. Note the readings.
3. Evenly distribute the broth from the shake flask using the pipette in to centrifuge tubes (10 ml in each
tube).
4. Put the centrifuge tubes in to the centrifuge rotor and make sure that the tubes are arranged in the
opposite holdings / holes.
5. Close the centrifuge lid and make sure that it is locked.
6. Put the power plug in the socket and switch it on.
7. Put on the mains switch of centrifuge. Here you can observe the indicator glow as an indication to
power supply to the machine and the time display will be blinking.
8. Switch on the rotor by rotate the speed controlling knob clockwise and observe the speed display for
the rotor speed and then finely keep rotating the knob till you get the speed of 5000 rpm.
9. Now set the time for the centrifugation period by pressing the time button step by step till you get 15
min display.
10. Now, wait till the centrifuge is stopped after 15 minutes and observe the displays of both time and
speed as zero as an indication for the completion of centrifugation process.
11. Put off the centrifuge mains and power supply, then open the centrifuge lid. If required take the help of
the instructor.
12. Take out the tubes and decant the supernatant (liquid portion) in to the sink and make sure that the
biomass pellet is retained in the tube undisturbed.
13. Use the blotting paper to remove the droplets from the tube with out touching the biomass pellet.
14. Close the centrifuge tubes with caps and weight them using the digital balance and note the
corresponding readings.
15. Clean the centrifuge tubes using the detergent water and put them in the hot air oven at 50oC for
drying. If required take the help of instructor.
16. Wash the pipette also using the detergent water and put up in the hot air oven.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Observations
S. No Sample Sample
Volume
(ml)
Initial
weight
(gm)
Final
weight
(gm)
Wet weight
of biomass
(gm)
Microbial
concentration
(mg/ml)
1 Microbial
broth culture
10
2
3
Initial weight: Weight of empty centrifuge tube with cap
Final weight: Weight of centrifuge tube with microbial biomass pellet after centrifugation
Wet weight of biomass = (Final weight) – (Initial weight)
Microbial concentration = (wet weight of biomass) / Sample volume
Result: Report the final result as the average concentration of the microbial culture in the shake flask for
24hrs culturing using the shaker incubator.
Note: Clean all your glassware and the work bench after finishing your experiment.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Experiment – 4
Aim: To plot Microbial growth curve for shake flask culturing using turbidity method.
Theory: Estimation of bacterial growth requires inoculation of a sterile broth medium and incubation of the
culture under optimum temperature and gaseous conditions. The bacteria will reproduce rapidly under these
conditions and the microbial growth may be measured by variety of techniques and one of the indirect
methods of measure is turbidity measurement, which relates cell number to the turbidity (cloudiness) of a
broth culture. Turbidity or optical density (OD), being an indirect measure of bacterial number, the increase in
turbidity indicates bacterial growth. OD is directly proportional to cell concentration. To measure the OD
spectrophotometer can be used. Bacterial growth curve plotting requires the samples of a 24hr shake flask
culture to be measured for population sizes at regular time intervals during the incubation period. The bacterial
growth curve shows four distinct phases of growth, lag phase, logarithmic phase, stationary phase and death
phase, the actual length of each phase varies with the organism and with environmental conditions.
Materials required:
Sterile Nutrient broth media (100ml)
Inoculum
Laminar hood
Spirit lamp
Match box
Micro Pipettes (1000μl)
Sterile micro tips (1000μl)
Centrifuge( SPINWIN )
Distilled water
Blotting paper
Tissue paper
Marker pen
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Procedure:
1. Prepare 100ml of nutrient broth in a 250ml of conical flask and sterilize it using autoclave.
2. Inoculate the sterile media with the given inoculum of 2ml aseptically in the laminar hood and shake
well for uniform distribution of inoculum.
3. Aseptically remove 1.5ml of culture broth from the shake flask using sterile micropipette and add it to a
centrifuge tube. (Make duplicate of samples.)
4. Incubate the conical flask in the shaker incubator at 30oC and 110 rpm. (Here initially note the time to
be zero.)
5. Centrifuge the broth solution at 10000 rpm to get the pellet of biomass for 10min.
6. Decant the supernatant and add 1.5 ml of distilled water to the pellet.
7. Place the centrifuge tubes in to the holes of vertex mixer platform and spin it vigorously for two
minutes to suspend the microbial pellet in the water.
8. Switch off the vertex mixer and take out the tubes and here you can observe the turbid solution of
cells.
9. Take the OD of this turbid solution at 600nm using spectrophotometer and note it in the record.
10. After one hour stop the shaker of the orbital shaker and take out the shake flask carefully and put it in
the laminar hood.
11. Repeat steps 3-10 till 12 hrs of incubation time.
12. Plot the growth curve with time on X-axis and OD on Y-axis.
Observations
S. No Time
(hr)
Sample
volume
Ce
ntr
ifu
gati
on
@
10
00
0
rpm
fo
r 1
0
min
Addition of distilled
water to pellet
OD @
620nm
Remarks
1 0 1.5ml 1.5 ml
2 0.5
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
3 1.0
1.5
10hr
Plot the graph between OD600 & Time (hr) and identify the lag phase, log phase etc.
Result: write about the growth curve and mention about lag period and log period etc. and also put your
remarks if any.
Note: Clean all your glassware and the work bench after finishing your experiment.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Experiment – 5
Aim: To Estimate the Monod Parameters for microbial growth kinetics
Theory:
Kinetic equations, which describe the growth of microorganisms on a particular substrate, are crucial in
understanding many phenomena in biotechnological processes. So, quantitative experimental data is used for
the design and optimization of biological transformation processes. A variety of mathematical models have
been proposed to describe the kinetics of substrate nutrients exposed to pure cultures of microorganisms or
microbial populations of natural environment. Among those models, Monod model has been widely used to
describe growth-linked substrate utilization as shown in the following equation:
max
s
s
K s
Where, μ = Specific growth rate of microbial cells
μmax = Maximum specific growth rate of microbial cells
Ks = Substrate constant
S = Concentration of substrate
Characterization of the microbe-substrate interactions involves estimation of several parameters in the kinetic
models from experimental data. In order to describe the true behavior of the system, it is important to obtain
accurate estimates of the kinetic parameters ( μmax , Ks ) in this model.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
16
Materials required:
Sterile Glucose broth media (100ml)
Inoculum ( E.coli )
Laminar hood
Spirit lamp
Match box
Micro Pipettes (1000μl)
Sterile micro tips (1000μl)
Centrifuge( SPINWIN )
Distilled water
Blotting paper
Tissue paper
Marker pen
Spectrophotometer
Procedure:
1. Prepare 100ml of glucose broth in a 250ml of conical flask and sterilize it using autoclave.
2. Inoculate the sterile media with the given inoculum of 2ml aseptically in the laminar hood and shake
well for uniform distribution of inoculum.
3. Aseptically remove 1.5ml of culture broth from the shake flask using sterile micropipette and add it to a
pre-weighed centrifuge tube. (Make duplicate of samples.)
4. Incubate the conical flask in the shaker incubator at 30oC and 100 rpm. (Here initially note the time to
be zero.)
5. Centrifuge the broth solution in the centrifuge tubes at 10000 rpm for 10min to get the pellet of
biomass.
6. Collect 1ml of supernatant in a vial and discard the remaining supernatant.
7. Weigh the pellet along with centrifuge tube using a balance. (note the reading for calculating the wet
weight)
8. Use the collected supernatant in the test tube as a sample to estimate the glucose concentration using
anthrone method.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
9. After one hour stop the shaker of the orbital shaker incubator and take out the shake flask carefully and
put it in the laminar hood.
10. Repeat steps 3-7 till 12 hrs of incubation time.
11. Plot the growth curve with substrate consumed (mg/ml) on X-axis and specific growth rate (min as well
as wet weight of Biomass on Y-axis.
Observations:
Make note of all your readings and the observations in the following two tables.
Table - 1
S. No Time Sample
volume
(ml)
Ce
ntr
ifu
gati
on
@ 1
000
0 r
pm
fo
r 1
0 m
in
Wet weight
of pellet
(mg)
Cell
concentration
^, X (mg/ml)
Cell growth
rate, (∆X/∆t)
Sp. Cell
growth rate
µ, (∆X/∆t)/X
1 1.5ml
2
3
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
^ Wet weight of cells per ml of sample volume
∆X = Xi – Xi-1 ; ∆t = ti - ti-1
Table -2
Sl.
No
Time
(hr)
Sample
volume
(ml)
Ce
ntr
ifu
gati
on
@ 1
000
0 r
pm
fo
r 1
0 m
in
Supernatant
volume (ml)
Follo
w t
he
anth
ron
e m
eth
od
to
det
erm
ine
the
glu
cose
co
nce
ntr
atio
n
OD620
Reading
Substrate
concentration
(mg/ml)*
1 1.5 1.0
2
3
* The values must be obtained from the standard plot of glucose concentration corresponding to
the obtained OD values.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Result: Write about the monad’s growth curve and mention the values of monad parameters i.e.
µmax and Ks from the graph.
Note: Clean all your glassware and the work bench after finishing your experiment.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Experiment - 6
Aim: To get familiarized with the lab scale fermenter (bench top fermenter)
Theory: Fermenters are the reactors modified and designed for the operation and maintenance of
fermentation process using microbial organisms. The vessel used for the fermentation process is generally of
cylindrical shape with a round bottom and a round top made up of stainless steel at pilot and industrial scale. In
case of laboratory bench top fermenters the body of the vessel is ideally made up of transparent glass of
appropriate thickness with a stainless steel top accommodating the provisions for the monitoring of process
parameters such as temperature, pressure, dissolved oxygen concentration, pH, antifoam agents etc. and
maintenance of sterility on the fermenter. The process parameters are usually monitored through probes
inserted from the top stain less steel plate (in case of bench top fermenter) and these probes are in turn
connected to an electronic control panel where the potentiometric signals of the probes converted in to digital
signals for the display during the process for real time monitoring. In addition, the fermenter is also connected
with an air sparger through which the sterile air is spurged in to the fermenter. This sterile air is generated using
a compressor which sucks the air from the atmosphere and pumps it through an air filter, and this sterile air
pipe line is connected to a flow regulation unit, where the air flow rate can be regulated through a knob fitted to
the rotameter. For the regulation of the temperature, a chilled water supply unit is connected to the fermenter
externally through a rectangular coil, and the control is provided by the flow control unit as it also connects the
chilled water supply unit and the fermenter. And these all parameters are controlled automatically trough the set
values of process parameters in the control panel. The bench top fermenter can be represented
diagrammatically as shown in the figure below.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Figure: Bench top fermenter
Figure: Control panel and stocking rack of Bench top fermenter
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Required Materials
Bentch top fermenter
o Cylindrical glass vessel
o Control panel
o Compressor
o Chilled water supply unit
o Flow regulatory unit
o Sensors
o Silicone rubber Piping
o Buffering bottles ( Acid /Base)
o Peristatic pumps
o Distilled water etc
Procedure:
1. Take a lab note book, pencil, eraser and a pen and go to the bench top fermenter.
2. With the help of the instructor identify the major parts of the fermenter and familiarize yourself.
3. Note all the probes present in the fermenter and identify the connections with the fermenter and control
panel
4. Draw and Label the fermenter unit along with the control panel, compressor, flow regulator and Chilled
water supply units. ( Ask the instructor for any clarification and suggestions)
5. Write briefly about the major parts of the fermenter in your observations.
SPSU UDAIPUR LAB MANUAL- BIOPROCESS ENGINEERING Department of Biotechnology BT -303
Observations:
Draw the neat and labeled diagram of the bench top ferment and give a brief description about the major parts
of the different units in the bench top fermenter.
Result: Briefly write about your familiarization with the bench top fermenter.