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BT 3120 - Bioreaction Engineering Laboratory

Lab Manual

INSTRUCTOR: Dr. Smita Srivastava

Department of Biotechnology Indian Institute of Technology Madras

Chennai 600 036, INDIA

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List of Experiments

Experiment TA Phone no and email id

Experiment 1 – Batch/ Fed batch reactor studies

Uma umakizhuveetil@gmail.co m 8754557477

Experiment 2 - KLa in airlift reactor & KLa in CSTR

Sneha snehasudhakara@gmail.co m 8754407513

Experiment 3– Residence Time Distribution (RTD) measurement

Harshal mysharshal@gmail.com 9846974961

Experiment 4 – Determination of Mixing time

Rajalakshmi raji.biome@gmail.com 9486192520

Experiment 5 – Immobilized Enzyme Kinetics-calculation of VM & KM

Sreeja s.sreejadoss@gmail.com 9884702108

Experiment 6 – Immobilized Enzyme Kinetics-effect of enzyme loading and flow rate

Mandeep mandeepkalsi5@gmail.com 9498098084

Experiment 7 – Free enzyme kinetics with and without inhibitor – calculation of VM and KM

Narayani narayani@ymail.com 8754439639

Experiment 8 – Free enzyme kinetics- effect of pH and temperature on enzyme activity

Reshma reshujv@gmail.com 9789007226

Experiment 9- Enzyme stability studies

Umamahes- hwari

uma1swarna@gmail.com 9444900513

Experiment 10: Sucrose hydrolysis –Kinetic study

Neha nehachetlangia@gmail.com 9025843103

Appendix and format for laboratory report

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• Total no. of students: 56

Laboratory Guidelines:

• The class is divided in to twenty groups (G1-G20) and each group contain

three students except two where two students are in the group • Each of the ten groups engaged in experiments will have a different

experiment on the same day. Thus, there will be 10 experiments running in parallel on the same day.

• A brief viva will be conducted every week about the experiment • Each group should maintain proper laboratory note book for recording

the data (no loose sheets please). After concluding the experiments, the lab records will be signed by the respective TA’s. A copy of the observation data in a sheet should be given to the TA for evaluation.

• All students must submit a report on the experiment performed on the previous week, after the experiment is concluded. The completed report should be turned in by 5.00 pm to respective TA’s in room BT 116 Biotech Dept. Late submissions will not be considered for evaluation.

Report should be submitted individually (hand written not computer generated).

• Follow the report format given below:

Title Page (with name) Objective Introduction Background Materials Experimental Procedure Results and discussion Conclusions and recommendation Bibliography Appendix (sample calculation)

• Only handwritten reports and plots on graph sheets in pencil will be accepted. •

There will be a final laboratory exam along with viva-voce where student the needs to do experiment alone, analyze the data and do the necessary calculations based on questions

• Wearing Laboratory coats and shoes is required for all laboratory classes, without which a student will not be allowed to do experiments

.

• Report immediately equipment problems or spillage to Teaching assistants (or) to the instructors.

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• After the experiment is over, clean the equipment/surrounding before handing it over to the respective TA.

• Report to the respective TA’s before you leave the lab.

• Marks distribution will be as follows:

Lab Reports - 30% Attendance/Conduct/Attentiveness - 10% Final exam (expt, viva) - 60%

• Eating, gum chewing, browsing net, chatting, unnecessary fiddling with

laboratory equipment, and any kind of frivolous activity is prohibited during the laboratory class.

• Academic dishonesty and falsifying data is unacceptable. Students turning in

laboratory records with plagiarized or fudged data will be awarded a ‘U’ grade in the course.

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Expt. No: 1.

Batch / Fed-Batch Reactor studies

Learning outcome: • The students will be able to operate a fermentor under fed-batch mode of operation • The students will be able to identify appropriate substrate feeding strategy for

maximum biomass production in microbial fermentation

Objective To study the effect of feeding strategies in a fed-batch bacterial bioreactor

Methods

1. Prepare the medium for the pre-culture and the fermentation with the following composition: Glucose 2 g/l , KH2PO4 3.8 g /l, K2HPO4 12.5 g /l, Yeast Extract 24 g/l and Tryptone 12g/l.

2. Autoclave the glucose (concentrated solution) separately and add it to the medium aseptically prior to the experiment..

3. Prepare inoculum in shaking flasks at a temperature of 37°C by adding 2 mL of the

main bacterial suspension (stored at -20°C) within 200 mL of culture medium with a glucose concentration of 5 g/l. Incubate overnight at 200 rpm in the shaker.

4. Autoclave separately the reactor tubing’s, Concentrated glucose solution (for fed batch) 100g/l, Antifoam 10% (w/v)PPG, in a bottle fitted with hypodermic needle and silicone tube by covering the needle ends with cotton and aluminum foil.

5. Add glucose to make the final concentration as given above. Calibrate Dissolved

oxygen probe to 100 % after agitating the medium with maximum agitation and 2VVM aeration and after getting a stable DO reading.

6. Add 200 ml of grown inoculum aseptically with a help of a Bunsen burner.

7. Take samples every 15minutes and analyze for biomass and glucose.

8. Feeding of glucose has to be started in the logarithmic phase of batch culture, when

desired concentration of glucose reached 0.05g/l.

9. Feeding strategies will be decided while during experiments(different feeding strategies for different batches)

10. calculate specific growth rate, Yx/s .

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Expt. No: 2.

Estimation of Kla by static gassing out method Learning outcome:

• The students will be able to compare the oxygen mass transfer characteristics (KLa) of a stirred tank reactor.

• The students will be able to determine the effect of reactor operating parameters agitation/aeration on KLa

Objective:

To estimate the Volumetric Oxygen Transfer Coefficient (KLa) in a fermentation process by static gassing out method in batch reactor.

Experimental Procedure

1. Fill 1-2 liter of water in the fermentor. 2. Calibrate the dissolved oxygen probe for 0% and 100% saturation. 3. Adjust the aeration and agitation to the desired value. 4. Strip off the oxygen from water by using nitrogen, from 100% saturation to 0%

saturation. 5. Follow the decrease in dissolved Oxygen value with time. 6. Repeat the experiment for two aeration/agitation rates

Observations

Time(sec) Dissolved Oxygen (%)

Result

Find the KLa value and discuss the effect of aeration and agitation. The same procedure is followed for various solutions provided.

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Expt. No: 3. Residence Time Distribution (RTD)

Learning outcome:

• The students will be able to generate a residence time distribution (E) curve for a flowing fluid in a CSTR

• The student will be able to determine the effect of agitation speed on mean residence time (tE) in a CSTR

Objective To calculate the residence time distribution (RTD) of the given CSTR by pulse input at different agitator speed and to study the effect of agitator speed on RTD of the CSTR. Materials and Equipments

1. Inlet and outlet peristaltic pumps, both are calibrated with same flow rates. 2. Tubing for connecting the inlet pump into the reactor and outlet pump out of

the reactor. 3. Potassium dichromate or Potassium permanganate .(anyone salt at a time in

different concentration) 4. Eppendroff to sample the outlet fluid. 5. Spectrophotometer.

Experimental Procedure

1. First calibrate the inlet and outlet peristaltic pumps at the same flow rates. This can be done by adjusting the rotor speed of the pumps.

2. Then set the agitator speed of the reactor at given rpm. 3. Pulse a 4 ml of a particular concentration of potassium permanganate or

potassium dichromate into the reactor and take samples at the exit, every 5 minutes for 2 h.

4. Analyze the concentration of potassium permanganate (or potassium dichromate) in the samples by measuring the OD at their respective λmax.

5. Now plot the concentration time profile. This will give the concentration – time curve.

6. Calculate the area under the concentration – time curve, An Numerically by using Simpson’s 1/3rd rule.

7. Next, divide each concentration by An , to get the various values of C ≡ E. i.e. C ≡ E = C/ An

8. Now, plot these values against time to get the E curve. 9. Now calculate E(t) = tE and plot E(t) =tE against time to get E(t) Vs t curve. 10. Calculate the area under the E(t) Vs t curve by Numerical method in order to

get mean residence distribution, tE of the reactor. 11. Repeat the same experiment after setting agitator speed at 500 and 1000 rpm

respectively.

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Results Calibration of standard potassium permanganate or potassium dichromate solutions with O.D: Concentration of standard potassium permanganate (or potassium dichromate) solution

O.D Mean O.D 1st observation 1st observation

Plot the calibration curve and fit it to a straight line c=m* (O.D)+k Agitator speed: 100 rpm Time, t (Unit: min)

Mean O.D

Tracer con c=m*(O.D) +k (Unit: g/l)

Area under c vs. t curve, An (Unit: g-min/l)

Normalized conc. ≡ E = C/ An (Unit: min-1)

Mean RTD, tE = Area under E(t) Vs t curve. (Unit: min)

Calculate space time, mean residence time and variance Remarks Comment on the effect of Agitator speed on Mean Residence Time distribution (Mean RTD) of the CSTR.

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Expt. No: 4.

Determination of Mixing Time Learning outcome:

• The student will be able to find the mixing time in a stirred tank reactor (STR) • The student will be able to determine the effect of agitation speed on mixing time • The student will be able to correlate the mixing characteristics (dimensionless mixing

factor) with the fluid flow characteristics (Reynolds number) in a STR Objective To measure mixing time in stirred vessel with both Newtonian and Non Newtonian fluid and relate it to Reynolds number Materials and equipment 1. One stirred vessel equipped with a variable speed stirrer. 2. Phenolphthalein as a tracer. 3. Distilled water. 4. Carboxy Methyl Cellulose (CMC) or sugar or starch or pectin of different viscosity . 5. Stopwatch. 6. 5N NaOH Procedure

1. Fill the vessel to 2/3 of the total capacity with the distilled water and start the stirrer. 2. Add 250 µL of 5N NaOH solution to make the solution alkaline (pH =9.2) 3. Set the speed of the stirrer to a known value. 4. Inject an appropriate amount of Phenolphthalein tracer into the vessel. 5. Start the stop watch as soon as the tracer is added and note the time when the solution

in the vessel seems homogenous .This way mixing time is obtained as function of stirrer speed.

6. Repeat step 1 to 5 for 5 different stirrer speeds. 7. Empty the vessel and fill it with equal amount of 0.05 %w/v high viscosity grade

CMC solution (Pseudoplastic fluid) and repeat the steps 1 to 6. 8. Repeat the experiment with 0.05 %w/v low viscosity grade CMC. 9. Obtain a correlation of mixing time, for the given stirred vessel by plotting

dimensionless mixing factor ft vs impeller Reynolds Number /ReN .

Results

For the given stirred vessel, Diameter of the impeller, Da = Vessel diameter, Dt = Height of liquid level in the vessel, H =

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Type of liquid used in the vessel

Density of the liquid, ρ (unit: kg/m3 )

Viscosity of Newtonian liquid, µ (unit: kg/m s)

Power Law constants for Pseudoplastic fluid

Impeller speed. (unit:s-

1)

Dimensionless mixing factor

( )2/1

3/22a

Tt DHND

tf =

Impeller Reynolds number

µρND

N a2

/Re =

or ( )

( ) KND

N n

na

1

22/Re 11 −

−

=ρ

K n

Newtonian Fluid (water)

----

------

High viscous CMC soln.

-------------

Low viscous CMC soln.

-----------

Plot dimensionless mixing factor ft Vs Impeller Reynolds number /

ReN from the experimentally obtained data. N.B. Values of the power law constants (both K and n) will be given to you. Reference: 1. Geankoplis, C. J. (1993), Transport Processes and Unit Operations, 3 rd ed.’ Prentice Hall.

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Expt. No: 5.

Immobilized enzyme kinetics-estimation of VM and KM

Learning outcome

At the end of this experiment, students will be able to

• determine the rate of an enzyme-substrate reaction • determine the effect of initial substrate concentration on rate of an enzymatic reaction • develop a mathematical model to describe a given enzyme substrate reaction kinetics

Objective

To study the immobilized enzyme kinetics using hydrolysis of sucrose catalysed by invertase enzyme immobilized using calcium alginate method. Materials Required

1. Glass column 2. Clamp stand 3. Rubber tubing 4. Water bath 5. Hot plate 6. Peristaltic pump 7. Beakers 8. Test tubes 9. Graduated cylinders 10. Spectrophotometer 11. Thermometer 12. DNS reagent 13. 40% Sodium potassium tartarate 14. 0.02M Sodium acetate buffer at pH 4.5

Materials to be prepared 1. Calcium alginate beads with immobilized invertase enzyme. 2. Various concentrations of sucrose solution in 0.02M sodium acetate buffer pH 4.5

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Procedure a) Setting up the immobilized enzyme reactor: 1. Plug the bottom end of the glass column with the Teflon sieve and fill the column (maintained at 55°c) with immobilized enzyme beads. (Fill the reactor with buffer and then add the beads for proper packing). 2. Connect rubber tubing from the inlet of the column to beaker containing buffer. 3. Set the outlet flowrate of the column to a known value. The connection between the buffer reservoir and the column should be air tight, to ensure flowrate in and out of the column to be equal. b) Carrying out immobilized enzyme – substrate reaction using different initial substrate concentration keeping enzyme loading and substrate flowrate constant: 4. Replace the buffer reservoir with beaker containing substrate solution. 5. Collect sample from the outlet of the column after steady state is reached. 6. Repeat the above steps for other substrate concentrations. 7. Perform DNS method and find rate of the reaction. c) Estimating VM and KM: 8. Plot the graph of initial susbtrate concentration vs. rate of reaction. 9. Estimate the kinetic parameters VM & KM. Result 1. Effect of substrate concentration S.No Substrate

concentration (g/l)

Absorbance (AU)

Invertsugar concentration (g/l)

Conversion (%)

Productivity (g/l-hr)

2. Estimation of VM and KM

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Expt. No: 6. Immobilized enzyme kinetics-effect of flow rate and enzyme loading

Learning outcome:

• The student will be able to determine the role of external mass transfer effects via change in flow rate (substrate loading) on the rate of enzymatic reaction in immobilized system of enzyme

• The student will be able to determine the effect of total enzyme loading on rate of enzymatic reaction in immobilized system of enzyme

Objective To study the immobilized enzyme kinetics using hydrolysis of sucrose catalysed by invertase enzyme immobilized by calcium alginate method Materials needed

1. Immobilized invertase enzyme in the calcium alginate beads 2. Gate clamps 3. sucrose solution of various concentrations in 0.02M sodium acetate buffer

at pH 4.5 4. test tubes 5. DNS reagent

Equipment Packed bed reactor made of glass tubing (25mm Internal Diameter) Graduated cylinder Temperature bath Thermometer Spectrophotometer Setting up the immobilized enzyme reactor First plug the bottom end of the glass tubing and fill the reactor with immobilized enzyme beads. (Before filling the beads, fill the reactor with buffer and then add the beads for proper packing). Keep topping up the reactor with sucrose solution using a variable speed pump, and allow the effluent to drip out of the reactor for about 5 min, analyze the reducing sugars formed using DNS reagent, Use a batch system for studying conversion with time. Allow different flow rate of reactant through the system to assess the external mass transfer effect. Experiments Effect of flow rate Vary the volumetric flow rate through the column to study the effect of flow rate on conversion. Tabulate the results.

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Enzyme loaded per particle Vary the enzyme concentration from 0.015 g to 0.03 grams of enzyme and simultaneously pack the beads in another reactor to study the effect of enzyme loading on conversion. Immobilization efficiency Check out the protein content in the effluent by estimating the protein concentration by reading the absorbance at 280 nm. Method of analysis Reducing sugars can be estimated by DNS method. Results Effect of flow rate S.No FlowRate

(ml/min) Absorbance (AU)

Invert sugar concentration (g/l)

X(%) rate (g/l-hr)

Effect of Enzyme concentration S.No Enzyme

loading (g/l)

Absorbance (AU)

Invert sugar concentration (g/l)

X(%) Rate (g/l-hr)

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Expt. No: 7. Free enzyme kinetics with and without inhibitor – calculation of VM and KM

Learning outcome:

• The students will be able to calculate the rate of enzymatic reaction • The students will be able to determine the effect of inhibitors on rate of

enzymatic reaction • The student will be able to identify the type of inhibitor

Objective To determine the Michaelis Menten kinetic parameters of invertase enzyme and to study the effect of inhibitors on the enzyme activity. Enzymatic Activity For the free enzyme, one unit of enzymatic activity (U) corresponds to the quantity of enzyme that produces one micromole of glucose and fructose in the hydrolysis of a 5% (w/v) sucrose solution, at 55ºC and pH 5.0

Materials and Equipments

1) Beakers 2) Graduated cylinder 3) Pipettes, 0.1 ml, 1 ml, 5 ml 4) Test tubes 5) Temperature bath 6) Thermometer 7) Balance 8) Spectrophotometer 9) Stop watch

Reagents required

1) Invertase stock solution 5g/liter 2) Sucrose solution 50g/liter 3) DNS reagent (1g DNS, 1 g NaOH in 100 ml distilled water. Add 0.05g of sodium sulphite at the time of use, prepare the reagent as much as you require for your expt.) 4) 40% sodium potassium tartrate solution

Procedure

I) Determination of Michaelis Menten kinetic parameters

1. Pipette out 0, 0.1,0.2,0.3,0.5,0.6,0.7,0.8, and 0.9 ml of sucrose solution in a series of test tubes and make up the volume to 0.95 ml with buffer (pH 5.0).

2. Add 50 µl invertase enzyme solution of particular concentration. Put Para film or aluminum foil over the test tube and immediately incubate the mixtures at 55ºC in a water bath for 10 minutes.

3. After the incubation add 1ml of DNS reagent. Heat the test tubes at 95ºC for 5 minutes.

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4. Add 0.3 ml of 40% sodium potassium tartrate solution and 0.7 ml distilled water to each of the test tube.

5. Cool the mixture and measure the optical density at 540 nm against the blank. The blank should not have sucrose solution.

6. Calculate the enzyme activity (µM/min) 7. Draw the double reciprocal plot of substrate concentration Vs enzyme activity

and calculate the kinetic parameters Vm and KM.

Substrate Conc (S)

(g/L)

1/S Optical density

Enzyme activity (v)

(µmole/min) 1/v

2. Enzyme inhibition studies

1. Take different volumes (say 10, 20, 50, 100, 150, 200, 250, 300, 400, 500 µl) of 25 mM CuSO4 or aniline solution in different test tubes. And make up to 500 µl with buffer (pH 5.0).

2. Add 500µl of 50 g/l sucrose solution to these tubes. 3. Add 50 µl of invertase enzyme solution. Immediately incubate the mixtures at

55ºC in a water bath for 10 minutes. 4. After the incubation, add 1ml of DNS reagent. Heat the test tubes at 95ºC for

5 minutes. 5. Add 0.3 ml of 40% sodium potassium tartrate solution and 0.7 ml dis.water to

each of the test tube. 6. Cool the mixture and measure the optical density at 540 nm against the blank.

The blank should not have sucrose solution. 7. Calculate the enzyme activity (µM/min)

Determine what type of inhibitor? Estimate the value of VM and KM using LWB plot, Eadie Hoffstee plot and Hanes plot. Determine the value of KI using secondary plot.

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Expt. No: 8. Free Enzyme Kinetics Studies-effect of pH and temperature on enzyme activity Learning outcome:

• The students will be able to determine the effect of pH on enzyme activity • The students will be able to determine the effect of temperature on enzyme

activity Objective To study how activity of invertase varies with respect to pH and temperature and determine the optimum pH and temperature for activity

Enzymatic Activity

For the free enzyme, one unity of enzymatic activity (U) corresponds to the quantity of enzyme that produces one micromole of glucose and fructose in the hydrolysis of a 5% (w/v) sucrose solution, at 55ºC and pH 5.0

Materials and Equipments

1) Beakers 2) Graduated cylinder 3) Pipettes, 0.1 ml, 1 ml, 5 ml 4) Test tubes 5) Temperature bath 6) Thermometer 7) Balance 8) Spectrophotometer 9) Stop watch

Reagents required

1) Invertase stock solution 5 g/l 2) Sucrose solution 50 g/l 3) DNS reagent (1g DNS, 1 g NaOH in 100 ml distilled water. Add 0.05 g of sodium sulphite at the time of use, prepare the reagent as much as you require for your experiment) 4) 40% sodium potassium tartrate solution 5) Buffer solution of various pHs (pH 2, 3…8)

I) Study of thermal stability of Invertase enzyme 1. Take 500μl sucrose solution in a test tube and add 450 μl of acetate buffer pH

5.0. 2. Incubate the test tubes for 5 min at different temperatures ranging from 30

deg to 90 deg C. 3. Add 50μl invertase solution. 4. Incubate for 10 min.

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5. After the incubation add 1ml of DNS reagent. Heat the test tubes at 95ºC for 5 minutes.

6. Add 0.3 ml of 40% sodium potassium tartrate solution and 0.7 ml water to each of the test tube.

7. Cool the mixture and measure the optical density at 540 nm against the blank. The blank should not have sucrose solution.

II) Effect of pH on Invertase activity

1. Take 500μl sucrose solution in a test tube and add 450 μl buffer of pH from 1 to 12.

2. Incubate the test tubes for 5 min at 55 deg C. 3. Add 50μl invertase solution 4. Incubate for 10 min. 5. After the incubation add 1ml of DNS reagent. Heat the test tubes at 95ºC for

5 minutes. 6. Add 0.3 ml of 40% sodium potassium tartrate solution and 0.7 ml water to

each of the test tube. 7. Cool the mixture and measure the optical density at 540 nm against the blank.

The blank should not have sucrose solution. i) Effect of temperature on invertase activity

Temperature (⁰C) Optical Density Sucrose conc. Activity (U)

ii) Effect of pH on invertase activity

pH Optical Density Sucrose conc. Activity (U)

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Expt. No: 9. Enzyme stability studies

Learning outcome The student will be able to determine the half-life of an enzyme at a given temperature or pH Objective To study the enzyme deactivation kinetics at different temperatures and different pH

Enzymatic Activity

For the free enzyme, one unity of enzymatic activity (U) corresponds to the quantity of enzyme that produces one micromole of glucose and fructose in the hydrolysis of a 5% (w/v) sucrose solution, at 55ºC and pH 5.0

Materials and Equipments

1) Beakers 2) Graduated cylinder 3) Pipettes, 0.1 ml, 1 ml, 5 ml 4) Test tubes 5) Temperature bath 6) Thermometer 7) Balance 8) Spectrophotometer 9) Stop watch

Reagents required

1) Invertase stock solution 5g/liter 2) Sucrose solution 50g/liter 3) DNS reagent(1g DNS, 1 g NaOH in 100 ml distilled water. Add 0.05g of sodium sulphite at the time of use, prepare the reagt as much as you require for your expt.) 4) 40% sodium potassium tartrate solution 5) Buffer solution of various pHs (pH 1, 2, 3…8)

Effect of heat treatment of stability of enzyme: 1. Make a invertase concenraion of 100 ug/ml in a said pH buffer. Take about 1

ml of enzyme solution into 2 eppendorfs. Incubate these at 50 and 70o C. 2. Take enzyme samples from these for every 10 mins time and carry on the

enzyme reaction as said earlier. Plot the residual activity vs. time. Plot of first order deactivation kinetics and determine thermal deactivation rate constant and half-life of enzyme. Results i) Effect of heat treatment on stability of enzyme

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Time (min) Activity (U) Relative activity 10 20 30 40 50 60 70 80 90

Effect of pH on the stability of enzyme: Procedure:

1. Make a invertase concenraion of 100 ug/ml in 2 different pH buffers say 5 and 7. Take about 1 ml of enzyme solution into 2 eppendorfs. Incubate these at 55o C for 90 min.

2. Take enzyme samples from these for every10mins time and carry on the enzyme reaction as said earlier.

Plot the curve of activity vs. pH Results i) Effect of pH on the stability of enzyme (treatment at different pH)

Time (min) Activity (U) Relative activity 0 10 20 30 40 50 60 70 80 90

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Expt. No: 10. Sucrose Hydrolysis by Hydrochloric acid: A kinetic study

Learning outcome The student will be able to obtain kinetic data and determine rate constant and order of reaction by various methods Objective To determine the rate constant and order of reaction for sucrose hydrolysis by dilute hydrochloric acid using differential, integral, polynomial method. Reagents:

1. Sucrose (1M or 0.9M, or 0.8M or 0.7M or 0.6M or 0.5M) 2. Hydrochloric acid (1.2 M or 1.1 M, 1 M or 0.9 M or 0.8 M) 3. GOD-POD reagent 4. Glucose for standard graph 5. 0.1 M phosphate buffer pH 7

Procedure:

1. Aliquot 2.ml phosphate buffer in a set of glass test tubes marked as 0,2,5,10,15,20,25,30,40,50,60,70,80,90,100,110,120 mints.

2. Add 200 ul NaOH to each tube.

3. Start the hydrolysis reaction by adding known concentration of hydrochloric

acid with a known concentration of sucrose. Collect initial time sample immediately.

4. Start collecting 100 ul sample for the said time and mix with the buffer and

NaOH solution already taken.

5. After collecting all the samples and just before starting the glucose assay by GOD-POD reagent add 200 ul hydrochloric acid of one fourth strength of acid taken in the initial reaction.

6. Aliquot one ml of GOD-POD reagent in a separate set of smaller glass test

tubes and to each these tube add 10 ul of the above mixture.

7. Incubate for 15 mints. at 37 deg.C water bath. Take readings at absorbance 540 nm taking a blank of 1ml GOD-POD reagent with 10 ul buffer.

8. Prepare a standard curve by running a GOD-POD assay with standard

glucose of concentration 0.005,0.01,.0.02,0.03,0.04,0.045 and 0.05 moles/Lit. Construct a standard graph. From this concentration of glucose produced in the reaction can be calculated.

9. Use differential and integral method and determine the rate constant and order of reaction

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Appendix

Invertase enzyme entrapment in calcium alginate gel

Equipment Beakers Graduated cylinder Balance Pipepetts Syringe

Reagents

Alginic acid, sodium salt Calcium chloride Enzyme

Preparation of calcium alginate beads Procedure

1. Dissolve 30 g of sodium alginate in 1 litre to make 3% solution 2. Mix approximately 0.015g of enzyme with 10 ml of 3% (wt) sodium alginiate

solution. (avoid clump formation) 3. Beads are formed by dripping the polymer solution from a height of

approximately 20cm in to an excess (100ml) of stirred 0.2M calcium chloride solution with a syringe and a needle at room temperature. Leave the beads in the calcium solution to cure for 0.5 -3 hours

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Discussion section should include explanations of what the results means to you. The results and observations reported should be discussed in reference to the theory given earlier in the report. The experimental results should be compared with published results (standard texts and handbooks), if possible. Any abnormal

Format for a Laboratory Report Reports are to be typed on A4 size paper and should contain the following sections: Title Objective Introduction Background Materials Experimental procedure Results and discussion Error analysis Conclusion Bibliography Notation Appendices It should generally be written in the past tense and with passive voice. Title Title should be brief and descriptive of the experiment under consideration. The title Page should also include your name, date of submission of report and other relevant details. Objective This section should briefly describe the objectives of the current investigation; it should be clearly stated. Introduction It should contain a short introductory remark concerning the experiment under study. Background The theory underlying the phenomena and the computation method should be briefly outlined. This section should only contain materials which are relevant to the experiments and which may be used for the discussion of the results obtained. Experimental Procedure This section should clearly state what you did. Diagram of the experimental set-up and a brief description of the apparatus used should be given. The operational procedure, preparation of standard graph etc, should be given in fair detail. The scope and design of the experiment should also be given. Results and discussion This is the most important section of the report. This section should summarize all the results obtained in the form tables, graphs etc. You should include a sample calculation of one experimental run. If possible an ‘error analysis’ should be carried out to determine the errors associated with each calculated result. Main results should be tabulated and all subsequent analysis of the main results should be clearly illustrated graphically or otherwise. All other data and calculations should be given in the appendices.

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behavior noted should be pointed out and explained suitably. For clarity of the report you may take one variable at a time to present the results and discuss it immediately. Error analysis Student need to analyze the error involved in measurement and calculate % error in the measured quantity Conclusion The conclusions that you can draw from the experimental results should be given here. This section must also clearly indicate how far the objective stated at the beginning has been achieved. Bibliography All references should be listed in formats as used in scientific journals and textbooks of science and technology. An example is shown blow. 1. Biochemical Engineering Fundamentals, J.E. Bailey and D. Ollis, Second Edition,

Mc-Graw Hill (1986). 2. Benedetti, L., Bertucco, A. and Pallado, P. 1997. Production of Micronic Particles

of Biocompatible Polymer Using Supercritical Carbon Dioxide, Biotechnology and Bioengineering, Vol. (53):232-237.

Notation All symbols used should be defined when they first appear in the report, and should be listed alphabetically in this section, together with their meaning. Follow SI units. Appendices Appendices should contain materials that are too bulky and detailed to include in the main body of the report as inclusion of which may prevent the smooth flow of thought and presentation of your results. These include unprocessed data, detailed calculations, intermediate results etc. All materials in the Appendices should of course be referred to in the main body of the report.

The Bradford Hill Questions Introduction - Why did you start? Methods - What did you do? Results - What did you find? Discussion - What do the results mean?

****************************************

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bt3120 - Bioreaction Engg. Lab for JAN-MAY 2013 Group Group Name Gender Semester

G1 G1 Monish R M 07

1 1 Pagidipally Vishal M 07

G2 G2 Akhil Sai Valluri M 05

2 2 Aman Kumar M 05

2 2 Amit Kumar M 05

G3 G3 Armaan Brar M 05

3 3 Ashok Regar M 05

3 3 L Bindu Bhargavi F 05

G4 G4 Deepak Jhajharia M 05

4 4 Eddy Hudson M 05

4 4 Gaurav Munshi M 05

G5 G5 Gopi Krishna R M 05

5 5 Gothi Suyog Dilip M 05

5 5 Haroon Abdul Hakkim H M 05

G6 G6 Jayaseelan V M 05

6 6 Kanika Verma F 05

G7 G7 Karunya Pentapalli F 05

7 7 Ketan Chandra M 05

7 7 Kuthati Puneeth Kumar M 05

G8 G8 M Manoj Kumar M 05

8 8 Mayank Nk Choudhary M 05

8 8 Mitan Sutradhar M 05

G9 G9 Mukul Jha M 05

9 9 Naik Manali Madhav F 05

9 9 Namit Sunil Holay M 05

G10 G10 D Nandita F 05

10 10 Pallavi Chakravorty F 05

10 10 Pentapaty Monica F 05

G11 G11 Pranoy U S M 05

11 11 Rohan Pradeep Bendre M 05

G12 G12 Sagina Venkatasureshbabu M 05

12 12 C Sandhya F 05

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Group Group Name Gender Semester 12 12 G V V Satya Sai M 05

G13 G13 Shikha Jain F 05

13 13 Shivam Parmar M 05

13 13 Shubhranshu Goel M 05

G14 G14 Suresh Anand V M 05

14 14 Tolkappiyan P M 05

14 14 Waghmare Kanishka Purushottam M 05

G15 G15 Abhishek B M 05

15 15 B Balaji Anand M 05

15 15 Darshan V M 05

G16 G16 Gurram Chaitanya Sai M 05

16 16 Joola Vasavi F 05

G17 G17 Karra Arun Kumar Reddy M 05

17 17 Karthik Dasari M 05

17 17 R Keerthan M 05

G18 G18 Marri Chettu Manoj M 05

18 18 P V Nishita Mohan F 05

18 18 Pallavi Singh F 05

G19 G19 Shaik Jainuddin M 05

19 19 Shenoy Chetan Dinesh M 05

19 19 Siddharth Dialani M 05

G20 G20 Somagutta Maheswar Reddy M 05

20 20 V Sowmya F 05

20 20 Vadlamuri Ranjeeth M 05

27