VIVEKANANDHA

COLLEGE OF ENGINEERING FOR WOMEN

[AUTONOMOUS INSTITUTION AFFILIATED TO ANNA UNIVERSITY, CHENNAI]

DEPARTMENT OF BIOTECHNOLOGY

LAB MANUAL

U15BT410 – BIOPROCESS ENGINEERING LABORATORY

Prepared By
Ms. R. Arthe
Assistant Professor
Department of Biotechnology

Approved By
Dr. S. Karunakaran
Head, Department of Biotechnology
 VIVEKANANDHA COLLEGE OF ENGINEERING FOR WOMEN
(Autonomous Institution Affiliated to Anna University Chennai)
Elayampalayam, Tiruchengode – 637 205

Programme B. Tech Programme code 105 Regulation 2015

Semester
Department BIOTECHNOLOGY IV
Periods per week Credit Maximum Marks
Course code Course name
L T P C CA ESE Total
BIOPROCESS ENGINEERING
U15BT410 LABORATORY 0 0 4 2 50 50 100

The main objective of this course is to

 Develop practical skills in enzyme isolation.
Objective  Evaluate enzyme kinetics.
 Analyze BOD
 Optimize growth medium and parameters influencing it.
LIST OF EXPERIMENTS

1.Growth of Bacteria - estimation of biomass, calculation of specific growth rate, yield Medium optimization coefficient

2. Growth of Yeast - estimation of biomass, calculation of specific growth rate, yield coefficient

3. Plackett Burman design

4. Medium optimization - response surface methodology

5. Enzyme kinetics - Michelis Menton parameters

6. Enzyme activity - effect of Temperature and pH

7. Enzyme inhibition kinetics

8. Enzyme immobilization - gel entrapment

9.Preparation of bioreactor, utilities for bioreactor operation

10. Biological Oxygen Demand

Total Periods: 60

Outcomes
Students who complete this course successfully are expected to
 Solve complex bioprocess engineering problems
 Applying skills of reactors in chemical and bioprocess industries
 Develop bio separation techniques
 Design reactors for plant and animal cell culture
LABORATORY SAFETY - GENERAL RULES AND REGULATIONS
A rewarding laboratory experience demands strict adherence to prescribed rules for personal
and environmental safety. The former reflects concern for your personal safety in terms of
avoiding laboratory setting to prevent contamination of experimental procedures by
microorganisms from exogenous sources. Because most microbiological laboratory procedures
require the use of living organisms, an integral part of all laboratory session is the use of
aseptic techniques. Although the virulence of microorganisms used in the academic laboratory
environment has been greatly diminished because of their long-term maintenance on artificial
media, all microorganisms should be treated as potential pathogens (organisms capable of
producing disease). Thus, microbiology students must develop aseptic techniques (free of
pathogenic organisms) in preparation for industrial and clinical marketplaces where
manipulation of infectious organisms may be the norm rather than the exception.

The following basic steps should be observed at all times to reduce the ever-present microbial
flora of the laboratory environment.

1. Upon entering the laboratory, place coast, books, and other paraphernalia in specified
locations-never on bench tops.

2. Keep doors and windows closed during the laboratory session to prevent contamination from
air currents.

3. At the beginning and termination of each laboratory session, wipe bench tops with a
disinfectant solution provided by the instructor.

4. Do not place contaminated instruments, such as inoculating loops, needles, and pipettes, on
bench tops. Loops and needles should be sterilized by incineration, and pipettes should be
disposed of in designated receptacles.

5. On completion of the laboratory session, place all cultures and materials in the disposal area
as designated by the instructor.
6. Rapid and efficient manipulation of fungal cultures and materials in the disposal area as
designated by the instructor.

7. Rapid and efficient manipulation of fungal cultures is required to prevent the dissemination
of their reproductive spores in the laboratory environment.

To prevent accidental injury and infection of yourself and others, observe the following
regulations at all times:

1. Wash your hands with liquid detergent and dry them with paper towels upon entering
and prior to leaving the laboratory.
2. Wear a paper cap or tie back long hair to minimize its exposure to open flames
3. Wear a lab coat or apron while working in the laboratory to protect clothing from
contamination or accidental discoloration by staining solutions.
4. Closed shoes should be worn at all times in the laboratory setting.
5. Never apply cosmetics or insert contact lenses in the laboratory.
6. Do not smoke, eat, or drink in the laboratory. These activities are absolutely prohibited.
7. Carry cultures in a test - tube rack when moving around the laboratory. Likewise, keep
cultures in a test-tube rack on the bench tops when not in use. This serves a dual purpose
to prevent accidents and to avoid contamination of yourself and the environment.
8. Never remove media, equipment, or especially, bacterial cultures from the laboratory.
Doing so is absolutely prohibited.
9. Immediately cover spilled cultures or broken cultures tubes with paper towels and then
saturate them with disinfectant solution. After 15 minutes of reaction time, remove the
towels and dispose of them in a manner indicated by the instructor.
10. Report accidental cuts or burns to the instructor immediately.
11. Never pipette by mouth any broth cultures or chemical reagents. Doing so is strictly
prohibited. Pipetting is to be carried out with the aid of a mechanical pipetting device.
12. Do not lick labels. Use only self-stick labels for the identification of experimental
cultures.
 13. Speak quietly and avoid unnecessary movement around the laboratory to prevent
distractions that may cause accidents.

The specific precautions outlined below must be observed when handling body fluids of
unknown origin due to the possible imminent transmission of the HIV and hepatitis B viruses
in these test specimens.
1. Disposal gloves must be worn during the manipulation of these test materials.
2. Immediate hand washing is required if contact with any of these fluids occurs and also
upon removal of the gloves.
3. Masks, safety goggles, and laboratory coast should be worn if an aerosol might be
formed or splattering of these fluids is likely to occur.
4. Spilled body fluids should be decontaminated with a 1:10 dilution of household bleach,
covered with paper toweling, and allowed to react for 10 minutes before removal.
5. Test specimens and supplies in contact with these fluids must be placed into a container
of disinfectant prior to autoclaving.

I have read the above laboratory safety rules and regulations and agree to abide by them.
Ex. No : 01 GROWTH OF BACTERIA - ESTIMATION OF BIOMASS, CALCULATION
Date : OF SPECIFIC GROWTH RATE, YIELD MEDIUM OPTIMIZATION
COEFFICIENT

AIM

To Study the growth of Ecoli strain(bacteria) in batch culture and to estimate the
biomass , specific growth rate and yield medium optimization coefficient.

PRINCIPLE

The growth rate typically changes on a hyperbolic fashion, if the concentration of

essential medium component is varied while the concentration of other medium component are
kept constant and it follows the Monod growth Kinetics,

μ = μmax S / (Ks + S)

Where,
S - concentration of the essential medium components.
μ - specific growth rate
μmax - maximum specific growth rate achievable.
S >> Ks , Ks -monod constant and is equal to concentration of the essential medium
component at which the specific growth rate is half of its maximum value. Specific growth rate
is linearly dependent on the concentration of essential medium component at lower
concentration and it is independent at higher the concentration of the essential medium
component.
Batch culture is closed culture system,which contains an initial,limited amount of
nutrition.The inoculated culture will pass through a number of phases
a) After inoculation the time upto which no growth takes place is referrred as lag phase and
may be considered as the time of adaption.
b) Following lag phase there is a period during which the growth rate of cells gradually
increases, the cell growth at a constant maximum rate and the period is known as log or
exponential phase
At this phase,
dx/dt = μx -------------------- (1)
x - Biomass concentration
t - Time in hour
On Integrating (1),
xt = x0.eμt -------------------- (2)

This is the equation for microbial growth in the exponential phase.

x0 – initial biomass concentration
 xt – biomass concentration after time t
t – time

On taking „ln‟,
ln xt = lnx0 + μt ---------------------- (3)

A Plot of ln (optical density) versus time „t‟ gives straight line with slope μ , (cell mass is
proportional to optical density).

Specific growth rate during the exponential can also be calculated by using the formula.
μ = ln OD2 – ln OD1 ------------------------ (4)
t2 – t1

Where,
OD1 – The optical density at time t1
OD2 – The optical density at time t2

Doubling time of the strain,

td = ln (2) /μ --------------------------- (5)

Following log phase, the deceleration phase and stationary phase, where the growth is almost
constant with respect to time. Depletion of nutrients leads to declining growth phase where
growth occurs but the death rate is greater.
Yield co-efficient,
Yx/s = x/s

Yx/s = xt – x0 / s0 - st ----------------------------- (6)

Where,
x0 – Initial concentration of biomass
xt – Concentration of biomass at time „t‟
s0 – Initial substrate concentration
st – Residual substrate concentration at time „t‟

MATERIALS REQUIRED:

 Shaker flasks
 Shaker
 Spectrophotometer
 E.coli strain
 LB broth
 DNSA reagent
  1M NaOH

PROCEDURE:
 Add 5% of the inoculum grown overnight in 200ml of LB medium.
 Incubate the culture at 37C (optimum growth temperature of Ecoli) in a
shaker at 150 rpm.
 Take 1ml of sample from the culture for every 20 minutes (Starting from t=
0), monitor the growth by measuring its optical density (OD) at 600nm, use
media before inoculation as blank. Dilute the fermentation broth (media)
accordingly to ensure that OD does not exceed 1.0.
 Transfer 1ml sample to effendorf tube, centrifuge at 400rpm for 10 min
remove the supernatant and dry the cell mass pellet in oven at 105C for
overnight and note the dry cell weight.
 Estimate the residual glucose concentration in the above supernatant using
glucose estimation by DNSA method.
 Repeat the steps for different samples for different time intervals.
 Plot a graph between ln(OD) versus time and find the slope, which is equal
to the specific growth rate μ (from graph)
 Calculate the specific growth rate μ (from graph) using equation (4) (from
calculation) .
 Calculate the doubling time and yield coefficient using equation (5) and
(6).

PROCEDURE FOR GLUCOSE ESTIMATION:

 Six test tube is taken and all labelled as 1,2,3,4,5and 6 respectively.

 First test tube was taken as control
 In test tube add 0.2, 0.4, 0.6, 0.8 and 1 ml of glucose in tube 2,3,4,5 and 6
respectively.
 Add distilled water to each test tube to make the volume upto 1 ml.
 Add 1 ml of DNSA to all the test tube.
 Place all the test tube in boiling water bath at 90C for 1 minute.
CALCULATION

Time OD for ln (OD) Specific Dry cell OD for Residual Yield

(min) cell growth weight residual glucose coefficient
mass at rate (μ) (x) glucose (S) (gm) Yx/s
600nm min mg/ml at 540 (mg
nm cell/mg
sub)
0
20
40
60
Average specific growth rate(μ) = Average yield coefficient Yx/s =

Concentration Distilled water DNSA reagent Absorbance at

of glucose 540 nm
(mg/ml) Boiled for
90C in water
bath for a min
and observe
colour change

Model Calculation

Specific growth rate, μ = ln OD2-ln OD1/t2-t1

Doubling time, td = ln(2)/μ

Yield Coefficient, Yx/s = xt-xo/so-st

Model graph
OD of glucose (540nm)
ln OD (600 nm)

Time (min) Conc. of glucose (mg/ml)

RESULT
The batch growth characteristics of the given bacterial strain was studied and the
following parameters were found.
Specific growth rate (from calculation) = min -1
Specific growth rate (from graph) = min -1
Doubling time (from calculation) = min
Doubling time (from graph) = min
Yield coefficient Yx/x = mg cell / mg sub
Ex. No : 02 GROWTH OF YEAST - ESTIMATION OF BIOMASS, CALCULATION OF
Date : SPECIFIC GROWTH RATE, YIELD COEFFICIENT

AIM
To study the growth of Saccharomyces cerevisiae strain (yeast) in batch culture using
YPD medium and to estimate the biomass, specific growth rate, yield coeffiecient

PRINCIPLE
The growth rate typically changes on a hyperbolic fashion, if the concentration of essential
medium component is varied while the concentration of other medium component are kept
constant and it follows the Monod growth Kinetics,

μ = μmax S / (Ks + S)

Where,
S - concentration of the essential medium components.
μ - specific growth rate
μmax - maximum specific growth rate achievable.
S >> Ks , Ks -monod constant and is equal to concentration of the essential medium
component at which the specific growth rate is half of its maximum value. Specific growth rate
is linearly dependent on the concentration of essential medium component at lower
concentration and it is independent at higher the concentration of the essential medium
component.
Batch culture is closed culture system,which contains an initial,limited amount of
nutrition.The inoculated culture will pass through a number of phases
a) After inoculation the time upto which no growth takes place is referrred as lag phase and
may be considered as the time of adaption.
b) Following lag phase there is a period during which the growth rate of cells gradually
increases, the cell growth at a constant maximum rate and the period is known as log or
exponential phase
At this phase,
dx/dt = μx -------------------- (1)
x - Biomass concentration
t - Time in hour
On Integrating (1),
xt = x0.eμt -------------------- (2)

This is the equation for microbial growth in the exponential phase.

x0 – initial biomass concentration
xt – biomass concentration after time t
t – time

On taking „ln‟,
 ln xt = lnx0 + μt ---------------------- (3)

A Plot of ln (optical density) versus time „t‟ gives straight line with slope μ , (cell mass is
proportional to optical density).

Specific growth rate during the exponential can also be calculated by using the formula.
μ = ln OD2 – ln OD1 ------------------------ (4)
t2 – t1

Where,
OD1 – The optical density at time t1
OD2 – The optical density at time t2

Doubling time of the strain,

td = ln (2) /μ --------------------------- (5)

Following log phase, the deceleration phase and stationary phase, where the growth is almost
constant with respect to time. Depletion of nutrients leads to declining growth phase where
growth occurs but the death rate is greater.
Yield co-efficient,
Yx/s = x/s

Yx/s = xt – x0 / s0 - st ----------------------------- (6)

Where,
x0 – Initial concentration of biomass
xt – Concentration of biomass at time „t‟
s0 – Initial substrate concentration
st – Residual substrate concentration at time „t‟

MATERIAL REQUIRED
Shake flask, Shaker, Pipette, Spectrophotometer, saccharomyces cerevisiae , YPD,
DNSA reagent, 2M of NaOH
YEAST EXTRACT PEPTONE DEXTROSE (YPD)
1% yeast extract, 2% peptone, 2% glucose/dextrose

PROCEDURE:
 Add 5% of the inoculum grown overnight in a 150ml of YPD
 Incubate the culture at 30C (optimum growth temperature of S.cereviseae)
in a shaker at 150 rpm.
  Take 1ml of sample from the culture for every 20-30 minutes (Starting
from t= 0), monitor the growth by measuring its optical density (OD) at
600nm, use media before inoculation as blank. Dilute the fermentation
broth (media) accordingly to ensure that OD does not exceed 1.0.
 Transfer 1ml sample to effendorf tube, centrifuge at 400rpm for 10 min
remove the supernatant and dry the cell mass pellet in oven at 150C for
overnight and note the dry cell weight.
 Estimate the residual glucose concentration in the above supernatant using
glucose estimation by DNSA method.
 Repeat the steps for different samples for different time intervals.
 Plot a graph between ln(OD) versus time and find the slope, which is equal
to the specific growth rate μ (from graph)
 Calculate the specific growth rate μ (from graph) using equation (4) (from
calculation) .
 Calculate the doubling time and yield coefficient using equation (5) and
(6).

PROCEDURE FOR GLUCOSE ESTIMATION:

 Six test tube is taken and all labelled as 1,2,3,4,5 and 6 respectively.
 First test tube was taken as control
 In test tube add 0.2, 0.4, 0.6, 0.8 and 1mg of glucose in tube 2,3,4,5 and 6
respectively.
 Add distilled water to each test tube to make the volume upto 1 ml.
 Add 1 ml of DNSA to all the test tube.
 Place all the test tube in boiling water bath at 90C for 5-10 minutes.
 CALCULATION

Time OD for ln (OD) Specific Dry cell OD for Residual Yield

(min) cell growth weight residual glucose coefficient
mass at rate (μ) (x) glucose (S) (gm) Yx/s
600nm min mg/ml at 540 (mg
nm cell/mg
sub)
0
20
40
60
Average specific growth rate(μ) = Average yield coefficient Yx/s =


Concentration Distilled water DNSA reagent Absorbance at

of glucose 540 nm
(mg/ml) Boiled for
90C in water
bath for a min
and observe
colour change

Model Calculation

Specific growth rate, μ = ln OD2-ln OD1/t2-t1

Doubling time, td = ln(2)/μ

Yield Coefficient, Yx/s = xt-xo/so-st

Model graph
OD of glucose (540nm)
ln OD (600 nm)

Time (min) Conc. of glucose (mg/ml)

RESULT
The batch growth characteristics of the given bacterial strain was studied and the following
parameters were found.
 Specific growth rate (from calculation) = min -1
 Specific growth rate (from graph) = min -1
 Doubling time (from calculation) = min
 Doubling time (from graph) = min
 Yield coefficient Yx/x = mg cell / mg sub
 

Ex. No : 03 MEDIUM OPTIMIZATION –

Date : PLACKETT BURMAN DESIGN

AIM
To optimize the concentration and composition of various nutrients in the media using
Plackett Burman method.

PRINCIPLE
The statistical design of experiment is an efficient procedure for planning experiments
so that the data obtained can be analysed to yield valid and objective , conclusion, screening
and evaluation of nutritional requirements and operating conditions is an important steps for
bioprocess development . Plackett burman design (PB) is very economical, statistical methods
with the run numbers a multiple of four. The PB medium comprises one type of two levels. –
1(L) for low level t2(H) for high level . This design is practical when the investigator is faced
with the large number of factors and is ensured which settings are likely to products optimal
and near optimal response.
This techniques allow for the evaluation of (N – 1) variables by N – experiments . N
must be multiples of 4 two of three variables are designated a dummy variable. The
incorporation of dummy variable into a experiment make it possible to estimate the variance of
an effects (i.e) experimental error. A table is usually drawn with column for trial number the
various and dummy variables each trial has equal numer of high(H) low(L) as seen similarly
under each variable column. The trial are carried out in a randomized sequence plackettt
burman design experimental design is based on the first order model.

Y = β0 + Σβixi

Where Y is responsible (biomass/metabolic/enzyme produced ) β0 is the model intercepts, βi is

the variable estimate and xi are independent variables. These variables whose confidence
levels were higher than 90% were considered that significantly influence the biomass/
Metabolic concentrations.

MATERIALS RECQUIRED :
 Ecoli
 Yeast extract
 Peptone media component
 Phosphate buffer
 Shaker flask
 Shaker
 Pipette
 Spectrophotometer
PROCEDURE
50ml of the media was prepared and sterilized on PB experimental design for each trail
of appropriate high and low concentrations
Medium Variables low (L) high (H)
g/l g/l
A Dextrose 3.0 6.0
B Sucrose 5.0 10.0
C Yeast Extract 1.5 3.0
D Malt Extract 1.0 2.0
E Nacl 2.0 4.0
F H2PO4 0.75 1.75
G MgSO4 0.5 1.0

 5ml of the inoculums was added to all media in previous step.

 The initial OD was taken for all media.
 The culture was incubated at 37C (optimum temperature of Ecoli ) in a shaker at
150 rpm for 24 hours.
 The biomass concentration was measured at the end of 2 hours by withdrawing a
1 ml of sample from the culture and optical density was measured at 600nm with
a spectrophotometer, the medium before inoculation was used as blank. Ensure
the OD of the medium should not exceed 1.0 by diluting the broth accordingly.
 The OD of experimental ruins was analysed and the main effect of each variable
on biomass formation was found.
 Plot a graph between main effects versus variables.
 The significant variables was found whose confidence levels were higher than
90%.
TABLE
PLACKET BURMAN EXPERIMENTAL DESIGN FOR 8 RUNS WITH 7 VARIABLES

Trial Variables Response

A B C D E F G (OD)
1 H H H L H L L
2 L H H H L H L
3 L L H H H L H
4 H L L H H H L
5 L H L L H H H
6 H L H L L H H
7 H H L H L L H
8 L L L L L L L
H denotes a high level value
L denotes a low level value
 A B C D E F G
Σ(H)
Σ(L)
Difference
effects
Mean
Square
Mean
Square for
error
F-test

CALCULATION
Difference effects = 2(ΣH – ΣL) / 8
Mean Square = (ΣH – ΣL)2 / 8
Mean square for error = E [(ΣH – ΣL)2 / 8] – G [(ΣH – ΣL)2 / 8]
F-Test = Mean Square / Mean Square Error
RESULT

The effect of various factor on a bioprocess was studies and following were found to be
significant at the given level
A = , B= , C= , D=, E=, F=, G=.
Ex. No : 04a MEDIUM OPTIMIZATION –
Date : USING RESPONSE SURFACE METHODOLOGY BOX BEHNKEN
DESIGN

AIM
To find the optimum values of significant medium components operating conditions of a
bioprocess using three level box and Behnken (BB) Design and five level central composite
(CC) DESIGN.

PRINCIPLE
Response surface methodology is a collection of mathematical & statistical techniques
that are useful for modelling and analysing problems where the response of interest is
influenced by several variables and the objective is to optimise this response as many
variables could potentially affect the efficiency of the pre-treatment process
Using response surface methodology (RSM) , the experiments are designed to allow us
to estimate interaction and even quadratic effects and therefore gives us an idea of the (local)
shape of the response surface we are investigating, RSM design are used to find improved or
optimal process setting troubleshoot process problem and weak points make a product or
process more robust against external and non – controllable influences. central composite
design (CCD) design and BB are two types of RSM
BOX AND BEHNKEN DESIGN :-
After determining the preliminary range of the production variables through one factor
at a time method and PBD a box and behnken experiment design, with three variables was used
to study the response pattern and to determine the optimum combination of variables the effect
of the variables X1 (glucose) , X2 (malt extract ) , X3 ( ammonium chloride) and X4 (sodium
chloride) at your variations levels in the extraction process is shown in table. four test variables
well coded according to the equation
Xi = xi − xo /∆x : i= 1,2,3,4
Where ,
Xi – the coded value of an independent variable
xi − the actual value of an independent variable
xo − the actual value of an independent variable at centre point
x- the step change value of an independent variable
For predicting the optimal point, a second order polynomial model is fitted to co relate
relationship between independent variable and response (biomass yield). For the three factor
the equation is,
Ybiomass = β0 +Σj=1k βjxj +Σj=1k βijxj2+Σj=1k βijxixj
Tables of variables used in RSM
Variables Code Levels (g/l)
-1 0 1
Glucose X1 3 5 7
Malt Extract X2 1 2 3
Ammonium Chloride X3 1 2 3
Sodium Chloride X4 1 2 3

Box Behnken design of factors in coded level with biomass as response

Run X1 X2 X3 X4
1 1 -1 0 0
2 0 0 1 1
3 1 1 0 0
4 -1 -1 0 0
5 0 0 0 0
6 0 0 0 0
7 0 0 0 0
8 0 0 0 0
9 0 0 0 0
10 0 0 0 0
11 -1 1 0 0
12 0 0 -1 -1
13 0 1 1 0
14 -1 0 0 -1
15 -1 0 0 1
16 0 1 -1 0
17 0 -1 -1 0
18 0 -1 1 0
19 -1 0 1 0
20 -1 0 -1 0
21 0 0 -1 0
22 0 0 1 1
23 1 0 0 1
24 1 0 0 -1
25 0 -1 0 1
26 0 1 0 1
27 1 0 -1 0
28 0 1 0 -1
29 1 0 1 0

MATERIALS REQUIRED:
 Bacterial/fungal culture
 Medium components
  Phosphate buffer
 Shaker flask
 Shaker
 Pipette
 Spectrophotometer
PROCEDURE
 Select four significant media components operating conditions (variables) and their
higher (+1) and middle (0) lower (-1) levels, which influences the growth of the given
strain
 Develop a Box and behnken experiment design for four variables (n) with 29 number of
experimental run (note: box and behnken experimental design for 29 runs with four
variables are given
 Prepare and sterilise 50 ml of the media based on box and behnken experimental design
and keeping other components (factors at constant)
 Add 5 ml of the inoculum to the cell media prepare in the previous step
 Incubate the culture at 30C (optimum growth temperature of the given strain) in a
shaker at 150 rpm
 Measure biomass concentration at the end of 24 hours (culture time) by cleaving with 1
ml of sample from the culture and measuring its optimal density at 600nm with
spectrophotometer use medium before inoculation as blank
 Ensure the OD if the medium should not exceed 1.0 by diluting the broth accordingly (
use appropriate assay for metabolites)
 Analyse the cell mass concentration (OD) using to find the co efficient of the model
By using the statistical tool, the optimized nutrient medium of the three factor is
calculated based on the ANOVA table
X1 X2 ΣY X1 2 X2 2 (ΣY)2
1
2

29
435

Source of Sum of Degree of Mean Variance Total

variation Square freedom Square Volume at
5% level
Between SSC (C-1) MSC = FC = FC (1,56)
group SSC/C-1 MSC/MSE
(column
treatment)
Error SSE (N-1)(C-1) MSE = - -
(residue SSE/56
within
group)

CALCULATION

N= 29x2 = 58
Total = Σx = 435
T2/N = 3262.5
TSS = ΣX12 + ΣX22 – (T2/N)
SSC = (ΣX12)/N1 + (ΣX22) /N1 – (T2/N)
SSE = TSS-SSC

RESULT

The effect of various factor on bioprocessing was studied & the following results are analyzed
by ANOVA table and the significant level was ___________________
Ex. No : 04b MEDIUM OPTIMIZATION –
Date : USING RESPONSE SURFACE METHODOLOGY
CENTRAL COMPOSITE DESIGN

AIM
To find the optimum values of significant medium components / operations of a bio
process using three level box and behnken (BB) design and five level central composite (CC)
design

PRINCIPLE
Response surface methodology is a collection of mathematical and statistical
techniques that are useful for modelling and analysing problems where response of the interest
is influenced by several variables and the objectives its optimised this response many variables
could potentially response, many variables could potentially effect the efficiency of the pre-
treatment process
Using response surface methodology experiments are designed to allow us to estimate
interaction and given quadratic effects and therefore gives us an idea of the (local) shape of the
response surface. We are investigating response surface design method designs are used to find
improved or optimal process setting troubleshoot process moves rebust against external and
non-controllable, influences, central composite design (CCD) and BB design are types of
response surface methodology

CENTRAL COMPOSITE ROTABLE DESIGN (CCRD)

The central composite rotable design(CCRD) was employed to determine the
effect of independent variables on the response and factor interaction with different
combination of variables hour independent with different combination of glucose (X1), mall
extract (X2), ammonium chloride (X3) sodium chloride (X4) were studied at five levels with
repetition at the central point and 2 replicates at the acid and fractional points for each of the
five variables studied high coded (+1,+2) medium coded (0) and lower coded (-2,-1) set points
(CCRD) were analysed by using the expert at software version 6 .both the lines and quadratic
effects of the five variables interactions with the release d mass of glucose from the dry
biomass the significant of these variables are evaluated by variance analysis (ANOVA) .these 3
dimensional surface plot were drawn to illustrate the effect of the dependant variables
described the quadratic polynomial equal that was fitted to the experimental data . the fit
models was evaluated by determined the R-Squared co-efficient for the validation of the
models optimum values for the selected variable were obtained by solving the regression
equation using design expert software version
Using the design the significant factors are prescribed into 5 levels loaded -2,-
1,0,1,2 for low, middle and high values for predicting function was filled to relationship
between factor (variables) and biomass metabolic concentration (response)

y= b0+b1-x1+b1-x12+b2x2+b22-x22+b3 x3+ b33x32+ b4x4+ b44x42+ b12x1x2+ b13x1x3+ b34x3x4+

b23x2x3+ b24x2x4+ b34x3x4
Where,
y is the response(biomass or efficiency) , x are the coded independent variables
(Table) and b are the co efficient. After the validation by analysis of variance (ANOVA) the
model was used as process estimation.
In this work the optimum values for the independent variables in a way that is obtained
high amount TRS and maximum efficiency of hydrolysis simultaneously were determined by
using the response desirable profiling function of statistical 7.0. the durability function allow
inspecting the response surface produced by fitting by the observed references using the above
mentioned equation based on levels of independent variables
These equations were used to predict values for biomass response at different
combinations of levels at the independent variables, specify desirability function for the
independent variable that produces the most desirable response on the dependence variables.

Table of variables used in RSM

Variables Code Levels (g/l)
-2 -1 0 1 2
Glucose X1 1 3 5 7 9
Malt Extract X2 0.5 1 1.5 2 2.5
Ammonium Chloride X3 0.5 1 1.5 2 2.5
Sodium Chloride X4 0.5 1 1.5 2 2.5

Central Composite design of factors in coded level with biomass as response

Run X1 X2 X3 X4
1 -1 1 1 1
2 -1 -1 1 -1
3 1 -1 1 -1
4 -2 0 0 0
5 0 0 0 0
6 0 0 0 0
7 -1 -1 -1 1
8 1 1 -1 -1
9 0 0 0 0
10 1 -1 -1 1
11 0 0 0 0
12 -1 -1 1 1
13 -1 1 -1 1
14 0 0 2 0
15 -1 -1 -1 -1
16 -1 1 -1 1
17 0 0 0 2
18 1 -1 1 1
19 0 0 0 0
20 1 1 1 -1
 21 -1 1 1 -1
22 -1 1 -1 -1
23 0 0 -2 0
24 0 0 0 2
25 0 -2 0 0
26 1 -1 -1 -1
27 1 1 1 1
28 0 2 0 0
29 2 0 0 0
30 0 0 0 0

MATERIALS REQUIRED
 Bacterial/fungus culture
 Medium components
 Phosphate buffer
 Shaker flask
 Pipette
 Spectrophotometer

PROCEDURE FOR CENTRAL COMPOSITE DESIGN

 Select four significant media components operating conditions (variables) and their
higher (+2,+1) middle(0) and lower (-1,-2) level, which influences the growth of the
given strain
 Develop a CC experiment design for four variables (n) with 30 number of experimental
runs
 Prepare and sterilize 50 ml of the media based on CC experimental design and keeping
other components (Variables) at constant
 Add 5 ml of inoculum to all media prepared in the previous step
 Incubate the culture at 30C (optimum growth temperature of the given strain) a shaker
at 150 rpm
 Measure biomass concentration at the end of 24 hrs (culture time) by withdrawing 1 ml
of sample from the culture and measuring its optical density at 600nm with a
spectrophotometer used medium before inoculation as a blank ensure the OD of the
medium should not exceed 1.0 by diluting the broth (use appropriate assay for
metabolitics)
 Analyse the cell mass concentration COD using the co-efficient of the model
By using the statistical tool the optimized nutrient medium of three factors calculated
based on the ANOVA table
 X1 X2 ΣY X1 2 X2 2 (ΣY)2
1
2

29
30
465

Source of Sum of Degree of Mean Variance Total

variation Square freedom Square Volume at
5% level
Between SSC (C-1) MSC = FC = FC (1,58)
group SSC/C-1 MSC/MSE
(column
treatment)
Error SSE (N-1)(C-1) MSE = - -
(residue SSE/60
within
group)

CALCULATION

N= 30x2 = 60
Total = Σx = 465
T2/N = 3603.75
TSS = ΣX12 + ΣX22 – (T2/N)
SSC = (ΣX12)/N1 + (ΣX22) /N1 – (T2/N)
SSE = TSS-SSC

RESULT
The effects of various factor on bioprocess was studied and following results are
studied and analysed by ANIVA table and significance level was ____________
Ex. No : 05a EFFECT OF SUBSTRATE CONCENTRATION ON ENZYME ACTIVITY
Date :

AIM
To analyze the effect of substrate concentration on the activity of enzyme.

PRINCIPLE
The enzyme and amylase can catalyse the hydrolysis of internal α-1,4, glycosidic bond
present in starch with the production of reducing sugars in the study of substrate concentration
of enzyme kinetics, the enzyme is kept constant whereas the concentration of starch is taken in
increasing order. As the substrate concentration increases the amount of products produced in
every successive tube also increases. This was explained by Michaelis as an enzyme catalyzed
reaction at varying substrate concentration is diphase ie., at low substrate concentration. The
active sites on molecules (enzyme) are not occupied by substrate and the enzyme rate varies
with substrate molecules concentration (Phase I). As the number of substrate molecules
increases, the enzyme attains the saturation level, since there is no more active sites remaining
for binding. So the enzyme can work with full capacity and its reaction rate is independent of
substrate concentration (Phase II).
This enzyme substrate ran can be determined by measuring the increase in reducing
sugars using the 3,5, dinitrosalicylic acid reagent.
In an alkaline condition, the pale yellow colour of DNSA undergo reduction to yield orange
coloured 3-aminoNSA. The absorbance of the resultant solution is read at 540nm. The intensity
of the colour depends on the concentration of reducing sugar, reduced α-amylase.

Starch  Maltose + Glucose

MATERIALS REQUIRED
 10 ml of 0.5%,1%,2%,3%,4%,5% soluble starch solution.
 10ml of α-amylase solution.
 10ml of 2N sodium hydroxide
 15ml of dinitrosalicyclic acid
 Distilled water – 150ml
PROCEDURE
 Make different concentration of starch soluble (.5%,1%,2%,3%,4%,5% soluble starch)
solution
 Take 12 clean and dry boiling tubes, label tubes control “C” and test “T” for each
concentration add 0.5ml of starch solution to all the tubes.
 Preincubate the starch solution of all concentration and α-amylase solution for 10 min at
37C
 Add 0.5ml of α-amylase enzyme to the tube labelled T for respective concentration.
 After incubation immediately add 2N NaOH to test.
 Pipette out 0.5ml of the α-amylase to the test tubes containing control solution mix the
solution well.
 Add 1ml of DNS reagent to all the tubes mix the solution in the test tube well.
  Keep it in boiling waterbath for 15 mins at 100C and cool it, dilute all the tubes by
adding 9.5ml of distilled water.
 Mix in solution in each tubes by using vertex mixture.
 The absorbance of test solution was read at 540nm against the control.

S.No Test Starch Volume Volume Volume Volume Volume Vol. OD at

Tube Conc. of of of NaOH of of DNS of 540nm

Incubate at 37C for 10

Substrate enzyme Enzyme in ml dis.

Boiling waterbath at
in ml in ml in ml water

100C for 5 mins

1 Cont 0.5 0.5 - 0.5 0.5 1 9.5
rol
Test 0.5 0.5 0.5 0.5 - 1 9.5

mins
S0 S dS (S0-S) V(dS/dt) 1/S0 1/V
dt=5
1
2
3
4
5

MODEL CALCULATION
Enzyme Velocity = Microgram of the starch X total dilution factor/
(Molecular weight of the starch X incubation time X volume of enzyme)
Dilution Factor = Final volume / initial volume
Final volume = aliquot solution + make up solution
Molecular weight of the starch = 342

RESULT
The maximum effect of substrate concentration and enzyme activity was found to be
________.
The value of Vmax and Km from,
Michaelis Mentons kinetics, Vmax =______________
Km = _____________
Lineweaver Plot , Vmax =_____________
Km = ______________
Ex. No : 05b
ENZYME KINETICS – MICHELIS-MENTON PARAMETERS
Date :

AIM
To study the Michelis-Menton kinetics of α-amylase enzyme and hence to determine
Vmax and Km.

PRINCIPLE

Kinetics of simple enzyme catalyzed reactions is referred to as Michelis Menton kinetics or

saturation kinetics. These models are based on data from batch reaction weight constant
volume in which the initial substrate (So) and enzyme (Eo) concentration are known.
Saturation kinetics can be obtained from a simple reaction scheme that involves a reversible
step for enzyme – substrate complex formation and a dissociation step of the ES complex.

Michelis Menton equation for steady state kinetics approximation is

Where Vmax – velocity of enzyme reaction of saturating substrate concentration, S -

Substrate concentration, Km - Michelis Menton constant, measure of affinity of enzyme for
substrate, Km = [S] at V = Vmax /2 from the graph V versus [S].

LINEWEAVER – BURK PLOT

If is the reciprocal of Michelis Menton approximation, A plot of 1/V versus 1/[S] gives
slope of Km/Vmax; and Y-intercept of 1/Vmax and X intercept of -1/Km.

EDDIE – HOFSFEE PLOT

A plot of V versus s V/[S] results in a line of slope –Km and y-intercept of Vmax and X
intercept of Vmax /Km.

Hans – Woolf plot:

A plot of [S]/V versus [S] results in a line of slope 1/ V max ; Y intercept of Km/ Vmax ; X
intercept of –Km.

MATERIALS REQUIRED

1) α-amylase (250µg/ml)

2) standard starch solution (2mg/ml)

3) substrate starch solution(10mg/ml)

4) KI iodine solution (5mMiodine in 30gm KI/L)

5) Phosphate buffer (pH 5.5)

PROCEDURE

1. Stir 2g of starch in beakers with 5ml phosphate buffer and add 75ml of phosphate buffer
and boil the suspension for 2 min.

2. Cool and add sufficient phosphate buffer to produce 100ml.

3. Dilute 10ml of this solution to 100 ml with phosphate buffer. Each ml of this starch
solution contains 2.0mg of soluble starch.

4. Weigh accurate quantity of α-amylase 25mg (250µg/ml) and add sufficient amount of
phosphate buffer to make up the volume to 100ml.

5. Label the test tubes as B,S1,S2,S3,S4&S5 and add standard starch solution 0, 0.2, 0.4,0.6,
0.8& 1.0 ml.

6. Take 4ml of sample at different concentrations (1,2,4,6,8 g/l) respectively and add 2ml
of α-amylase . After 3 minutes , to 1 ml of this solution add 1ml of distilled water and
keep the test tubes immediately in boiling water bath for 5 minutes and cool it.

7. Add 1ml of iodine in potassium iodide and incubate for 15 minutes.

 8. Make up the volume of test tubes to 10ml with distilled water and mix it well.Then read
OD at 640 nm.

TABULATION

S.N Test Reaction Conc. Vol. of Incubate Vol. of Vol. of Incubate OD at

o Tub volume of amylas for 5 distille Iodine for 5 540n
e of starch Starch e mins & d solutio mins & m
(ml) Soluti Keep in water n Keep in
on waterbat (ml) waterbat
(ml) h for 10 h for 10
mins mins

STANDARD GRAPH

REAGENTS B S1 S2 S3 S4 S5

Standard stock
solution(ml)

Concentration of starch
in solution(mg)

Distilled water (ml)

Iodine(ml)
Incubate at room temperature for 15 minutes

Distilled water (ml)

OD at 640 nm

S0 S 1/S0 ds(S0-S) V(ds/t) 1/V

where t = 5

CALCULATION

Enzyme activity,V= concentration of the reducing sugar ( µmol)

180×3 ( ml.min)

RESULT

The value of Km and Vmax are,

1. From Michaelis menton plot-

2. From lineweaver burk plot-

Ex. No : 06a
EFFECTS OF pH ON ENZYME ACTIVITY
Date :

AIM
To determine the effect of pH on the activity of enzyme amylase.

PRINCIPLE

Amylase is a hydrolytic enzyme which breaks down many polysaccharides like

starch which is a polymer of glucose linked by α-1, 4 glycosidic bond to yield a disaccharide
maltose as an end product. α-amylase is also called as α-1,4 gluconohydroxylase.

Amylase

(C6H12O6 )n + nH2O n(C12H22O11)

starch maltose

Saliva is a good source of amylase. The activity of enzyme depends on the pH at which
reaction is carried out. The optimal pH required for the activity of amylase can be calculated by
this experiment.

The rate of reaction is monitored by measuring the amount of substrate consumed or by

measuring the product formed. Here the substrate is starch and the product is maltose. Since
both are colorless DNS is used.DNS when added with maltose and heated it reduces to 3-
amino, 5 nitrosalicylic acid is determined by the color change in this solution. The conversion
of DNS to 3-amino,5 nitrosalicylic acid varies based on maltose concentration. It gives pure
orange red for high concentration of maltose and shows very less color change by low
concentration. The color change is noted spectrophotmetrically at 520nm. The concentration of
maltose formed can be calculated from the absorbance.

REAGENTS REQUIRED:

1. Phosphate buffer at varying pH range from 4.5 to 8.0

2. Substrate 1% starch solution

 3. Working standard glucose(2mg/ml)

4. 1M NaoH

5. Dinitro salicylic acid (DNSA) reagent

Soln A: Dissolve 30g of sodium potassium tartarate in 500ml of distilled water

Soln B: Dissolve250mg of 3,4 dinitrosalicilyic acid in 500ml of distilled water

Mix 50 ml of solution A and 20ml of solution B and make up to volume to 100ml with distilled
water.

6. Enyzme α-amylase solution (250µg/ml)

PROCEDURE

1. Label the test tubes as B,(S1-S5) and add working glucose solution 0.5,1,1.5,2and 2.5 and
make up the volume to 4ml with distilled water.

2. Add 1 ml of 1M NaoH and 1ml of DNSA to the mixture and keep at boiling water bath
for 10 min and read OD at 540 nm to plot standard graph.

3. Add 1ml of substrate starch solution to all test tubes. Pipette out 2ml of buffer of
different range into the test tubes respectively. Add 0.5 ml of diluted enzyme to test tubes
and incubate at 37oC for 5 minutes.

4. Stop the reaction by keeping the test tubes in boiling water bath for 5 minutes and cool to
room temperature.

5. Add 1ml of 1M NaoH and 1 ml of DNSA reagent and incubated in boiling water bath for
10 minutes and make up the volume to 10ml by adding water.

6. Then measure O.D at 540nm

TABULATION

particulars T1 T2 T3 T4 T5 T6 T7

pH 4.5 5 6 6.5 7 8 9

Substrate 1 1 1 1 1 1 1
starch(ml)

Buffer (ml) 2 2 2 2 2 2 2

Enzyme(ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Incubate at room temperature and keep it in boiling water bath for 10 minutes

1M 1 1 1 1 1 1 1
NaoH(ml)

DNSA 1 1 1 1 1 1 1
reagent(ml)

Keep in boiling water bath for 10 minutes and make up the volume to 10ml water

OD at
540nm

RESULT

The amylase activity at different pH was determined. Maximum amylase activity was observed
at pH _______.
Ex. No : 06b
EFFECT OF TEMPERATURE ON ENZYME ACTIVITY
Date :

AIM

To determine the effect of temperature on the activity of enzyme amylase.

PRINCIPLE

Each enzyme is most active at a specific temperature called it as optimum

temperature. A increase in the temperature increases the rate of reaction. Since the atoms in the
enzyme molecular have greater energies and tendency to move. Reaction velocity almost
doubles with 10oc rise in many enzymes. As the temperature rises, denaturation processes
progressively destroy the activity of the enzyme molecules due to the unfolding of the protein
chain after breakage of weak bonds leads to overall reaction velocity drops,which is explained
by Arrhenius equation.

A=Aoe_E/RT

For many proteins, denaturation begins to occur at 45oC to 50oC.some enzymes are yery
resistant to denaturation at high temperature, especially the enzyme isolated from thermophilic
organisms found incertain hot environment.

REAGENTS REQUIRED:

1. Phosphate buffer (0.1M)

2. Substrate 1% starch solution

3. 1M NaoH

4. Dinitro salicylic acid (DNSA) reagent

5. Enyzme α-amylase solution (250µg/ml)

PROCEDURE:
 1. Add 2.5ml of buffer in test and control tubes.

2. Add 1ml of substrate starch solution to both test and control tubes.

3. Then add 0.5ml of distilled water only to the control test tubes.

4. Then add 0.5ml of enzyme only for test tubes and incubate it at respective temperature
(30, 37, 45, 60,80oC) for 15 minutes. Then add 1ml of NaoH to both test tubes and
control tubes.

5. Then add 1ml of DNSA to test and control tubes and heat in boiling water bath for 10
minutes. Measure OD at 540nm.

TABULATION

Mix well and incubate at respective for 15 min and keep boiling water
Tem Buffe 1%Starc Distille Amylase NaO DNS OD at
pºC r hsolution d water H A 540n
m
(ml) (ml) (ml)
(ml)
ml ml

C 2.5 1 0.5 - 1 1

30 T 2.5 1 - 0.5 1 1
Keep in boiling water bath for 10 min
C 2.5 1 0.5 - 1 1

37 T 2.5 1 - 0.5 1 1

C 2.5 1 0.5 - 1 1

45 T 2.5 1 - 0.5 1 1

C 2.5 1 0.5 - 1 1
for 5 min

Mix well

50 T 2.5 1 - 0.5 1 1
 C 2.5 1 0.5 - 1 1

60 T 2.5 1 - 0.5 1 1

OBSERVATION

The graph plotted between temperature and enzyme activity decreases as the temperature
increases.

Enzymes are proteinaceous substance. Denaturation begins to occur at 40-50oC. one physical
mechanism for the phenomenon is obvious as the temperature increases, the atom in the
enzyme molecule have greater tendency to move. Eventually the aquire sufficient energy to
overcome the weak interaction holding the globular structure together and deactivation follows.
This makes the enzyme to lose its activity at higher temperature.

RESULT

The effect of enzyme activity at different temperature was determined. Maximum enzyme
activity was observed at ________.
Ex. No : 07
ENZYME INHIBITION KINETICS
Date :

AIM

To study the effect of copper sulphate and determine the type of inhibition by copper
sulphate on activity of α- amylase enzyme.

PRINCIPLE

Certain compounds may bind to enzyme and reduce their activity . The compounds are
called enzyme inhibitors. They may be reversible or irreversible. Reversible inhibitors may
dissociate more easily from the enzyme after binding the S major class of reversible inhibitors
are competitive , non competitive and uncompetitive.

i ) COMPETITIVE INHIBITION:

Competitive inhibitors are molecular that binds to the same site as the substrate
preventing the substrate from binding as they doso.

But are not changed by enzyme . They are usually substrate analysis and complete with
substrate for active site of the enzyme in the presence of a competitive inhibitor it takes as
higher substrate concentration to achieve the same velocities that were reached in its absence .
So which Vmax can still be reached if sufficient substrate is a variable , one – half Vmax
requires a higher (S) than before and thus Km is larger.

E+S ES E+P

Kis

EI
V = Vmax / Km (1+I/Kis)+S ---------------------------------------------- (1)

Rate of Enzymatic conversion is given equation where, Vmax – velocity of enzyme reaction of
saturating substrate concentration S – Substrate concentration , Km – Michaelis menton
constant, Measure of affinity of enzyme of substrate.

Km = (S) at V=Vmax/2 from graph Vs Km

ii) NON COMPETITIVE INHIBITORS

They are not substrate analogs. Non-competitive inhibitor are molecules that bind to
some other site on the enzyme reducing its catalytic power. Physically usually found in
allosteric system, not so common except in text books. Usually find mined corresponds to
inhibitor binding to E & ES with identical constant with non-competitive inhibition enzyme.
Molecules that have been taken out of the game so enzyme rate (velocity) is reduced for all
values (S) including the Vmax & one half Vmax but Km remain unchanged because the active
site of those enzyme molecules that have not been inhibited is unchanged. Effect is composite
of competitive is Km unchanged but Vmax decreased by 1+x/Ki

E+S  ES  E+P

+ +

I I

EI+S  ESI E+P

V=Vmax / Km (1+R/Kis) + S(1+ R/Kis)

V= (V/(1+R/Kis))S/Km+S if Kii = Kis --------------------------------------------(2)

The rate of reaction for such a type of inhibition is given in the above calculation.

iii) UNCOMPETITIVE
 This type of inhibition does bind at the active site, but only after the S has bound to the
active site so it does not compete with the S. Thus even the S is saturating and all E is in Ks,
the inhibitor can still binding to the active site and produce an inactive ESR complex. The
inhibitor only bind to ES so inhibitor stimulates formulation of ES and increases binding of
substrate to enzyme, So Km is reduced . However the ESR complex is non-productive. SO
Vmax lowered the double reciprocal plot is series of parallel lines indicating lower Km and
decreasing Vmax and is characteristic of uncompetitive inhibition. The rate of such a reaction
is given by equation

E+S  ES  E+P

Kii

ESI E+P

V/Km = unchanged, ie both Vmax & Km changed to some extent.

REAGENTS REQUIRED:

1. 0.5% working enzyme (invertase) solution was prepared in 0.05M PBS at pH-7

2. Sucrose -20g/l

3. 0.1M CuSO4

4. 1M NaOH

5. DNSA reagent

6. Potassium Phosphate buffer-pH 7

PROCEDURE:

STANDARD PLOT:
 1. Pipette out 0.5-2.5 ml of standard glucose into different test tubes.

2. Make up to 4ml with distilled water.

3. 1ml of DNSA and 1ml of NaOH is add to all the test tubes.

4. Keep it in boiling water bath for 10 minutes and then cool it.

5. Then measure the OD at 540nm.

INHIBITION KINETICS:

1. Take 1, 2, 3, 4, 5, and 6ml of sucrose solution into six different test tubes and label it as
T1-T6.

2. Add distilled water to make up the volume to 7ml.

3. Add 0.5ml of CuSO4, 2ml of buffer and 2 ml of enzyme to each of these test tubes.

4. Allow the reaction mixture to react for 5 minutes.

5. Take 1ml of reaction mixture from the test tubes and add 3ml of distilled water.

6. Add 1ml of NaOH and 1ml of DNSA to each of these test tubes and Keep it in boiling
water bath for 10 minutes and cool the test tubes.

7. Then measure OD at 540nm.

8. Repeat the same procedure without adding copper sulphate as inhibitor and buffer
solution.

TABULATION

GLUCOSE STANDARD

PARTICULARS T1 T2 T3 T4 T5 T6

Glucose (ml) 0 0.5 1 1.5 2 2.5

Distilled water (ml) 4 3.5 3 2.5 2 1.5

1M NaOH 1 1 1 1 1 1

DNSA reagent 1 1 1 1 1 1

Keep in boiling waterbath for 10 mins

OD at 540nm

ESTMATION OF STARCH

PARTICULARS T1 T2 T3 T4 T5

Conc. of starch (ml) 1 2 4 6 8

Reaction volume of starch(ml) 4 4 4 4 4

Volume of α-amylase 2 2 2 2 2

Keep in room temperature for 2 mins & waterbath for 5 mins

Volume of distilled water 1 1 1 1 1

Volume of iodine solution 1 1 1 1 1

Incubate at room temp for 15 mins

OD at 610 nm

ENZYME KINETICS WITH INHIBITOR

Particulars T1 T2 T3 T4 T5 T6
Sucrose solution(ml)

Distilled water (ml)

CuSO4 (ml)

Buffer solution (ml)

Enzyme solution(ml)

volume of rxn mixture

Distilled water (ml)

NaOH (ml)

DNSA (ml)

Keep it in boiling water bath for 10 minutes

OD at 540nm

ENZYME KINETICS WITHOUT INHIBITOR

Particulars T1 T2 T3 T4 T5 T6

Sucrose solution(ml)

Distilled water (ml)

Buffer solution (ml)

Enzyme solution(ml)

volume of reaction mixture

Distilled water (ml)

NaOH (ml)
DNSA (ml)

Keep it in boiling water bath for 10 minutes

OD at 540nm

RESULT:

The effect of copper sulphate was studied and the kinetic parameters were determined

The type of inhibition by copper sulphate was identified as ……………………..

Ex. No : 08
ENZYME IMMOBILIZATION KINETICS GEL ENTRAPMENT
Date :

AIM

To study the effect of immobilization of α-amylase enzyme using calcium alginate. To find
maximum reaction rate for free and immobilized enzyme and the effectiveness facter (η)

PRINCIPLE

Entrapment is the physical enclosure of enzyme in a small space. Matrix entrapment and
membrane entrapment, imcluding micro encapsulation are the two major methods of
entrapment. The matrix used for enzyme entrapment are usually collagen. When immobilized
in a polymer matrix, enzyme solution is mixed with the polymer solution before polymerization
takes place. Calcium alginate is just a widely used polyacrylamide, which is most commonly
used in entrapment to form calcium alginate beads . This method does not alter the chemical
nature of enzyme.

Sodium alginate +enzyme+CaCl2Calcium alginate beads entrapped with enzyme

Unlike polyacrylamide gels, gelatine of calcium alginate does not depend on the
formation of more permanent covalent bond between polymer chains. Rather polymer molecule
are cross linked by calcium ions. Because of this calcium alginate beads can be formed in
extremely mild conditions which ensure that enzyme activity yield of over 80.1 can be
routinely achieved . However just as easily as calcium ions can be exchanged for Na ions. They
can also be displaced by other ions. This property can both be advantageous and
disadvantageous. If needed enzymes or microbial cells can be easily recovered by dissolving
the gel in a sodium solution. On the other hand proper caution must be taken to ensure that the
substrate solution does not contain high concentration of those ions that can disintegrate the
gel.

MATERIALS REQUIRED

 Conical flask
  Beaker

 Pipette

 Test tube

 Temperature bath

 Spectrophotometer

 Enzyme α-amylase

 0.2M Calcium Chloride

 3% Sodium alginate

 1% starch solution

 1M NaOH

 DNSA

PROCEDURE

 Dissolve 0.3g of sodium alginate in 10ml of distilled water to make 3% Sodium alginate
solution.

 After Sodium alginate is completely dissolved by gentle warming, leave the solution
undisturbed for 10mins to eliminate the air bubbles that can later be entrapped and cause
the bead to float.

 Prepare 50 ml of 0.2M Calcium Chloride solution in a beaker

 Mix 0.15g of α-amylase with 10 ml of 3% sodium alginate solution.

 Add sodium alginate and α-amylase mixture drop wise to CaCl2 solution in beaker
using 1ml micropipette or syringe at room temperature.

 Allow the beads in CaCl2 solution for 20 mins to deplete the excess calcium.
  Remove excess each on the surface of the beads by washing with distilled water.

 Transfer the beads to 50ml of 1% starch solution in a beaker. A equal quantity of α-

amylase to 50 ml of 1% starch solution in another beaker.

 Withdraw 1ml of sample at 3 mins interval from conical flask with immobilized and free
enzyme.

 Add 1 ml of sample to the test tube containing 1M NaOH, 1ml DNSA and add 1.5ml of
distilled water.

 Keep the test tube immediately in boiling waterbath for 10mins and cool it.

 Then measure OD at 540nm

 Find Vmax

 Find the effectiveness factor, which is the ratio of maximum reaction rate of
immobilized enzyme to the free enzyme.

TABULATION

FOR IMMOBILIZED ENZYME

PARTICULARS T1 T2 T3 T4 T5 T6

1M NaOH (ml) 1 1 1 1 1 1

DNSA (ml) 1 1 1 1 1 1

Distilled water (ml) 1 1 1 1 1 1

Immobilized enzyme 1 1 1 1 1 1

Keep in boiling waterbath for 10 mins & cool it.

OD at 540nm
 Amount of Product (mg)

Reaction rate

FOR FREE ENZYME

PARTICULARS T1 T2 T3 T4 T5 T6

1M NaOH (ml) 1 1 1 1 1 1

DNSA (ml) 1 1 1 1 1 1

Distilled water (ml) 1 1 1 1 1 1

free enzyme 1 1 1 1 1 1

Keep in boiling waterbath for 10 mins & cool it.

OD at 540nm

Amount of Product (mg)

Reaction rate

GLUCOSE STANDARD

PARTICULARS T1 T2 T3 T4 T5 T6

Glucose (ml) 0 0.5 1 1.5 2 2.5

Distilled water (ml) 4 3.5 3 2.5 2 1.5

1M NaOH 1 1 1 1 1 1

DNSA reagent 1 1 1 1 1 1
 Keep in boiling waterbath for 10 mins

OD at 540nm

CALCULATION

Effectiveness = Activity of Imm. enzyme / Activity of free enzyme x 100

RESULT

Effect of immobilization of α-amylase using sodium alginate was studied and the maximum
reaction rate for free enzyme = ____________μmol/ml.sec

Maximum reaction rate for immobilized enzyme = ____________μmol/ml.sec

Effectiveness factor = ____________

Ex. No : 09 PREPERATION OF BIOREACTOR UTILITIES
Date : FOR BIOREACTOR OPERATION

AIM

To study the preparation of bioreactor and utilities for bioreactor operation.

THEORY

 A cylindrical vessel either stirred or unstirred aerated or non aerated is called

bioreactor.It is used to cary out biological reactions which involves 3 phases
(gas,liquid,solid).These phase reactors are difficult to design because mass
transfer of components among these phases must be controlled.Non bioreactors
are constantly being developed for special applications.

 Provision of adequate mixing and aeration for the long proportion of the function
all the challenging factors is the design of bioreactor based n the application .

 Bioreactors of two types,

1. Aerobic Reactors

2. Anaerobic Reactors

 Anaerobic bioreactors are usually simple in construction without sponger and

agitator.Aerobic bioreactor are of these types

1. Stirred tank bioreactor(STR)

2. Bubble column bioreactor(BCB)

3. Air lift bioreactor

 The main advantages of STR are they all highly flexible and can provide
high RLA values for gas transfer stirred reactors of 400m all used for
antibiotic production with stirred power of 5k.
  Stirred tank reactor can make used commercial upto viscosity of about
1000cp .STF are also used for immobilized all culture in STF because high
speed impulses may damage or destroy the cells high levels of shear can also
change damage sensitive cells particularly in plant of animal cell culture.The
major components of fermentation are agitator, sparger, probes, controller
and jacket.

AGITATOR

Gas dispersion is mainly the function of impeller .The impeller must provide sufficient rapid
agitation to dispose bubbles throughout the tank to increase their residence time with in the
liquid and to shear large bubbles into small bubbles,crush ton impeller is disc. With 6-8 blades
designed to pump fluid in the radical direction. This is commonly found in industry and
laboratory fomenters. The axial flow hydro foil impeller can pump the liquid either down or up
they showed reduced maximum shear stress making them usable with sensitive culture such as
plant and animal all culture.They all capable of excellent performance with v is low my axial
sometimes controller of axial flow and surgon impeller are used a typical arrangement includes
four bottles ,cost shear damage with animal all culture in such cases axial flow impeller slightly
differ from enter all used to prevent sortex fermentation and switching in tel fumites
installation of multi impeller into mixing.

SPARGER

Sparger is usually either a ring with holes or a table with critics gas under pressure is applied to
the sparger.The size of the gas bubbles and their dispersion throughout the tank all the critical
paramites of the reaction performance although the smaller bubbles and better gas distribution
sparging tubes are after filled media high level suspended solids.

PROBES

The main parameter to the controlled in ferment all ph,temperature dissolved or stirred speed
and both level,ph probes is connected to the standard ph electrode which measure the ph inside
the fermentor. Thermistor are used as temperature probe for measuring the temperature in
bioreactors. the main compliment of no measuring device is colour electrode filled with
saturated kcl solution which senses the value of the no inside the bioreactor stirrer speed can
control by using variable speed motors typically only 70 – 80% of the reactor height is filled
with processed liquid this level can be controlled by level controller.

CONTROLLER

In foaming is the problem a supplementary impeller called a foam breaker may be installed
alternatively antifoaming agents are added to the both but they reduced the O2 transfer rate
mechanical foam disperse is generally preferred in laboratory fermentors. The antifoaming
agents are transferred by a peristaltic pump. If the PH go high then the flow of phase is
controlled by this peristaltic pump. If the PH is high then the flow of phase is controlled by
third peristaltic pump.

JACKET

Temperature control and heat transfer in stirred vessels can be accomplised by using jacket
vessels jacket consists of extended coil, internal helical coil, internal baffle type coil or
external heat exchanger. External jacket provide heat transfer area for laboratory and other
small scale fermentors internal jackets are preferred in industrial fermentors because they can
be operated with high liquid velocity and few disadvantages with internal jackets. They
interface with mixing the vessel and cleaning of reactor is difficult.

A material of construction ( MOC ) of bioreactors is usually a stainless steel ( SS ), SS316 is

used on a welded parts, SS304 are covers and jackets and d SS3161 for plant and animal cell
culture many laboratory scale fermentors are glass vessels are SS cover plates glass fermentor
are rarely used at the 50 litter scale and limit the maximum rate size about 500 litters. the use of
SS and Glass is called for by the need to sterilize the reactors.

Most fermentors are filled with height to diameter ratio ( nld ) of 2:3

Although bioreactors for animal cell culture tend to the closer to 1. Aspect ratio of about 1 has
the smallest surface are and requires the last material to constant for a given volume.
Typically only 70 – 80 % of volume of reactors to filled with processed liquids. Their allows
adequate head space for disengagement to droplets from the exhaust gas and to accommodate
the foms.

In large reactors to main limitation on size are ability of the design to provide an adequate
supply of O2 to remove metabolic heat efficient. Although copper coil have been better
thermal characteristics but SS coils are almost used however this choice depends on the nature
of medium and culture sterilise are prime designed consideration for fermentation hardware
steam under pressure is used for inside sterilization of reactors probes, values seats the number
of opening of the fermentors should be limited for most product of commercial interest
absolute sterility is required and contamination may cause the loss of product time and money.

Leakages should be avoided information small openings are made to leak with o-rings while
large openings with flat qlaskilo, although small fermentor prevention of contamination due to
the entry of foreign organism through a such a shaff is major challenge in the mechanical
design of fermentor.

PREPARATION OF BIO REACTION

1. Vessal preparation perior to autoclaving.

If is advisable to rinse the previously cleaned vessel prior to use. Remember that all
clamps must be open the value for the open position. If the glass wool is going to be replaced
for the run, then removal is and the rubber bulb of the sampler perior to rinshing. The
protective cap for the motor device adopter must also be in place, It will be necessary to hold
the protective cap for the agitation shaft in place if you plan to invest the head plate while
rinsing it . In this case hold the head plate on to the rest of the vessel, as failure to do so will
result in the rest of the vessel, as failure to do so will result in the falling out during inversion
the PH and dissolved O2 probes should not be in the head plate when it is rinsed all gas filters
must be removed prior to rinshing the sparger must in particular, be checked to ensure that it
cont clogged. The head plats must be oriented in combinati8on with the vessel and the internal
baffle so as to allow for the exhaust condenser rinses and the cooling jacket water lines to be
connected , also the baffle must be positioned so that it doesn‟t interface with the insertion of
the PH and dissolved O2 probes into their plates the sampling part must be positioned so that
sample room is available to take a sample. It is advantages to have the addition parts for acid,
base, etc, on the same side as the pumps. The old grease on the top of the glass cylinder should
be wiped on. The grease is applied by smearing a very thin layer around the top of the cylinder
usually with a finger tip.

All tubing connected to the head plate should be secured at the hand head plate
connection point as well as to any addition bottles or connections away from the head plate tie-
gun is useful for this purpose. The value for the samples must also be in the closed position.
Other those such as one attached to base on addition ports should be clamped to faciliate sterile
cook up. use easily removable metal clamps to actually close the line during autoclaving these
may be removed after the vessel has been autoclaved the open end of the tubing should be
covered with cotton which is then covered with aluminium foil. the clamp on tubing should go
below this sparger filter should also be covered , but not quite as tightly. The exhausted filter is
not usually covered. All tubing should be integrity.

The PH probe must be inspected prior to insertion to enough electrolytes are pleasant
and it good condition. The rubber stoppers must be checked for ensure that they are secure. The
PH probe must be properly calibrated prior to insertion in the head plate. Never insert the PH
probe until the head plate is properly secured.

The dissolved O2 probe must be checked to ensure the required amount of electrolyte
is present prior to inserting and we usually exchange electrolyte for each new run. The
dissolved O2 probes membrane must so be inspected prior to use. It is absolutely critical that
both the PH and dissolved oxygen, probes have their protein caps on prior to autoclaving and
these should infact be on except when the probe is being hooked up to the unit.

2. Vessel Sterilization :

When autoclaving the unit exhaust through the exhaust filters. So it is essential that
the lines be prevented from crimping and that the filter is good (unplugged). To insure that
crimping does not occur use a short piece of fairly rigid tubing. If the rigid tubing is not
available use a small splint to support the tubing the vessel is normally sterilized for 45
minutes. Note that certain media formulation cannot be sterilized for this length of time, as
degradation will occur. The process must never be autoclaved dry. If it becomes necessary to
sterilize the vessel without media, use a balanced salt (phosphate buffered saline) solution to
cover the ends of the probes. Aseptically remove the PBS prior to filling the vessel with the
desired media. Never place probes in distilled or de ionized water. This will cause your probs
to lose electrolyte. The maximum fill is 70% of the vessel‟s maximum volume. Autoclaving
should be done in a unit with a liquid on a slow exhaust setting ( see autoclave manufacturer‟s
instructions for autoclave liquids ). sterilize at 121‟c when sterilization is complete it is
important to check the exhaust line to ensure that it didn‟t crimp and the vessel‟s integrating
must also be as curtained.

3. POST STERLIZATION VESSEL SETUP:

The vessel must be gently handled when removed from the autoclave to present the
media from boiling up confirm that any unprotected vented lines are clamped upon removing
the vessel from the autoclave. The vessel‟s is then integrity must be again as curtained the
vessel is placed on the unit using the “guide posts” on the console base, the orientation must
allow for proper look up to the cooling jacket waterlines and the exhaust gas condensation line.
Note that this should be checked prior to autoclaving as indicated above connect to be stored in
a clean area in such a way as to protect the sensitive tip. Removing the PH probe is usually not
as difficult a process as inserting it because the shaft will be wet and thus should be relatively
easy to remove. However the damage of probe breakage is still very real and extreme care must
be taken when removing it. It is still necessary to use two hands with one hand at the top of the
part acting as a guide to ensure proper removal. A gentle place for this is required. If at any
point in this process the probe jams, it is essential to avoid trying to force it. It may be
necessary to use two hands with one hand at the top of the part acting at the3 top of the part
acting as a guide to ensure proper removal. A gentle place for this is required. If at any point in
this process the probe jams, it is essential to avoid trying to force it. It may be necessary to
reinsert it port in order to effect the probe‟s removal. In extreme cases it may be necessary to
remove the head plate with the probe still inside so that you can approach the problem from
both ends. In this case, it is critical to remove the head plate very carefully.
 Once the PH probe has been removed it should be immediately washed off with warm
water. If biomaterial has accumulated on the probe, using a sponge (as an equivalent that will
not scratch glass) and gentle pressure to clean the surface. The probe should be stored with the
tip immersed in either electrolyte on PH buffers. This electrolyte buffer can be reused, but it
should always be inspected prior to each use for precipitation or contamination. Now that the
vessel is detached from the unit, it remove any remaining cotton and foil covering the ports.
The rubber bulb on the sampler should be removed and rinsed separately the glass wool can be
removed at this point as well. The tube is detached and bevelled tips of interior portion of the
base type addition parts will often require special attention. All tubes and shafts must be
cleaned. Note that these may be same residual base or acid fest in those anes , so extreme
caution and the use of chemically resistant gloves is recommended water lines ensuring that the
water delivery and return lines are not inverted. Additionally it is necessary to connect the
outgoing lines prior to connecting the corresponding incoming lines. Insert the temperature
probe into the thermo cell. Check the water lines to the unit are open set the control to “ prime”.
If using an external recirculation chiller it must be tuned on at this time. The water level in the
chiller should be checked prior to use. After 2.5 mins the unit can be switched from “ prime” to
the desired PID temperature setting. This can be checked by making sure that water is truely
leaving the unit by observing the water drained through the “Drain” or “water Port” out.
Remove the protective caps from the PH and D.O probs and connect them to the unit. Be
careful with the PH probe to avoid the temperature to twist the probe into its connection to the
unit as this can comprise sterilizing. The connection must be screwed onto the probe. The PH
probe should also be checked to ensure that the secured also be rubber stopper in it water not
displace. Note the time that the D.O probe is connected, since the probe requires a minimum of
6 hours for polarization.

The protective tap on the agitation shaft should now be removed and the motor attached.
change the control panel on the unit to the gasses screen and set the air from “off” to “manual”
return to the master screen and make sure that “air flow” is one of the four parameters
displayed, use the knob on the sideof the machine to manually adjust the airflow to the desired
settings connect the airline from the unit to the terminal filter of the sponge a manner as
possible. Note that the filter will prevent external contamination but good techniques never
hurts. The clamp on the sponge line is then opened and the vessels can be visually abserved to
ensure that the air is flowing properly. Then set the agiation to the minimum deserved value.
After setup the unit should be carefully abserved in to ensure that there air no problems,
particularly as regarding water line leaks.

4. VESSEL OPERATION :

The vessel must be have any and all necessary addition bottles connected prior to use. If
a bottle such as the glucose feed is not initially executed. It can be hooked up later. The PH will
probably need to be adjusted setting the PH control to “PID” does their note that due to a
tendency for the unit to over heat the target PH during this initial adjustment it is desirable to
set the initial PH PID a little conservatively. Note that the PH reading must be taken from a
vessel that has already cooled down. Additional media components that are not autoclaved can
be added once the vessel has cooled sufficiently. The protocol for this is the same as for
inoculation as described below. Inoculation can be performed by aseptically pouring liquids
into the vessel through the inoculation part. However we normally use the harvest port to
inoculate. A peristaltic pump or gravity is used to introduce the inoculums. The shake flask is
disconnected. aAt this point, the harvest port terminus must be covered up against with sterile
cotton and foil. This must be performed in an aseptic manner. To harvest form the vessel, a line
can be attached to the harvest port and a peristaltic pump can be used to pump the culture broth.

5.VESSEL SHUT DOWN AND CLEANING :

When the fermentation run is complete, it is necessary to carefully shut the process down.
First all operating parameters ( agitation, temperature, D.O. level, PH and gas feed ) must be set
from their current control modes to the “off” mode. Note that the manner air value on the side
of the unit should also be closed as well additionally if supplemental oxygen feed was used, it
will be necessary to close the gas tank value and the lines leading from it to the unit. If a re
circulating chiller is in use, it should be shut off when the temperature control is shut off. Feed
lines from any addition bottles, which have been used, should be then removed. The next step it
to disconnect the vessel from the unit.
 The temperature probe needs to be removed from the thermo well. Remove the motor and
place the protective cap over the agitation shaft. Always disconnect the water lines by
disconnecting the incoming lines prior to the outgoing lines. The airlines prior to b e
disconnected from the sparger. Disconnect the PH and D.O probes from the unit and replace
their protective caps. The D.O probe presents an easy removed as you simply unscrew the
thread out and gently pull it out. It should be immediately rinsed off and then wiped dry gently
always remembering to avoid touching the membrane at tip. Some returns will result in an
accumulation of biomaterial on the probe and it may be necessary to wipe the probe down more
vigoursly but in no case should be the tip be touched. After cleaning the D.O. probe the tip can
be visually inspected for damage the probe for this procedure. It is often necessary to hand
wipe surface with a paper towel is order to fully remove residual traces of small particulate
debris.

The washing of the bottom portion of the vessel requires the same procedure as the head
plate. Note that the sides of the vessel particularly where the bottle was adjacent to it and side
ports ( plugged or unplugged ) may require special attention. The vessel can now be cleaned by
washing with detergent, or by using a cleaning solution. If the vessel is to be sterilized, or by
using a cleaning solution. if the vessel is to b e sterilized all the standard precautions must be
taken. Note that for this purpose the vessel dose not need to be seated except for those
previously cited values and tubing which run under the liquid level. It will be necessary under
the liquid level in the vessel. We recommend the use of DI water, and the fill should be at least
as high as your standard level for a run unless you have already specifically wiped the residual
grease off the top of the glass cylinder there should be enough so that the head plate can be
clamped to the lower position of the vessels. Instead the lightest possible pressure should be
used. The advantage to sterilization is that not only are residual viable organisms filled but
residual debits will loosen and become removable by washing after the vessel has cooled. If a
cleaning solution is required we recommend on 10% deduction of micro cleaning solution.
Alternatively if you are using the vessel for consecutive runs with the same media a ranching of
it with warm tap water and the DI water may suffice. Note that if water will run over a vessel
must be decontaminated prior to cleaning add water so that the liquid level reaches the
maximum working volume of the vessel. This will help prevent biological material from
adhering.

In large reactors to main limitation on size are ability of the design to provide an
adequate supply of O2 to remove metabolic heat efficient. Although coppercoil have better
thermal characteristics but SS coils are almost used. However this choice depends on the nature
of medium and culture sterilities are prime designed consideration for fermentation hardware
steam under pressure is used for inside the sterilization of reactors probes values seals the
number of opening of the fomenters should be limited for most product of commercial interest
absolute sterility is required and conformation may cause the loss of product, time and money.

Leakages should be avoided informant‟s small openings are made to leak with O-rings
while large openings with flat gaskets although small fermento prevention of contamination
due to the entry of foreign organism through a such a shaft is major challengein the mechanical
design of ferment.
Ex. No : 10
BIOLOGICAL OXYGEN DEMAND
Date :

AIM

To study the principle and procedure for determination of BoD in the testwater sample.

PRINCIPLE

BoD is the amount of oxygen utilized by Microorganism is a biological process that

breakdown organic matter into water. It is a measure of organic pollutant load. Creates the
oxidisible organic matter in water the greater will the BoD. Thus the strength of waste water
expressed in terms of BoD. BoD is important to know that the amount of organic matter present
in the waste and quality of oxygen required for its stabilization. The BoD values are thus very
useful in process designed to measure the treatment plant efficiency and operation.

The origin of organic compound are human criteria vegetable or organic compound.
Animal introduced into water body do not get the diluted sufficiently. Microorganism use up all
the O2 in water to oxidize organic matter drinking and for other.

The BoD value is measured by measuring the difference in dissolved oxygen of the
same water sample in 5 days. Dissolved oxygen present in water is calculated by using the
formula,

DO (mg/ml) = A.N.S/S.C X1000

where, A – ml of 0.025N thiosulphate solution consumed

N – Normality of standard thiosulphate solution

C – total volume of KMS alkali iodide oxide, potassium fluoride

S- volume of water sample

MATERIALS REQUIRED
  BoD incubator, Burette, Pipette, Conical flask, BoD bottles.
 Mn(II)S, Sodium azide, KI, NaOH, Starch, KF, Sodium thiosulfate.

PROCEDURE

 Collect the water sample in 2 different BoD bottles

 Incubate one BoD bottle in BoD incubator for 5 days at 20C, while determine DO of
another BoD on the 1st day
 After 5 days remove another BoD bottle from the incubator and determine the DO.
 Calculate the difference in DO, which measure of BoD.

RESULT

The principle and procedure for determination of biological oxygen demand in test
water sample was studied.