Criteria for good medium

• It will produce the maximum yield of product or

biomass per gram of substrate used
• It will produce the maximum concentration of biomass
or product
• It will permit the maximum rate of product formation
• There will be minimum yield of undesired products
• It will be of consistent quality and available
throughout the year
• It will cause minimal problems during medium
sterilization
• Other aspects of production process such as aeration,
agitation, downstream processing, waste treatment
 Medium designed will affect the design of
fermenter ex oxidation of hydrocarbons highly
aerobic process –air lift reactor

 Problems will be encountered in scaling up.

Since large reactors will have low mass transfer
rate

 High viscous medium will consume more power.

 Besides growth and product formation medium

will influence the pH variation, foam formation,
morphological form of organism etc.,
 Use of complex nutrients will influence
downstream processing

 Variation in complex nutrients will result in

batch to batch variations.

 Medium cost has to be considered depending

on the product type. Eg. For single cell protein
production medium cost is more than 50 % of
production cost. In the case of pencillin it is
30% and in recombinant products it is less than
10 %.
 Medium formulation
Medium formulation is essential stage in
manufacturing process

Carbon & Nitrogen other

Energy + sources + O2 + nutrients
Sources

Biomass + products + CO2 +H2O +heat

 Elemental composition of microorganisms may

be taken as guide

 Design of medium will influence the oxygen

requirements
 Elemental composition
Element Bacteria Yeast Fungi

Carbon 50-53 45-50 40-63

Hydrogen 7 7 7
Nitrogen 12-15 7.5-11 7-10
Phosphorus 2-3 0.8-2.6 0.4-4.5
Sulphur 0.2-1.0 0.01-0.24 0.1-0.5
Potassium 1.0-4.5 1-4 0.2-2.5
Sodium 0.5-1.0 0.01-0.1 0.02-0.5

Calcium 0.01-1.1 0.1-0.3 0.1-1.4

Magnesium 0.1-0.5 0.1-0.5 0.1-0.5
Chloride 0.5 -- --
Iron 0.02-0.2 0.01-0.5 0.1-0.2
 WATER
Assessing suitability of water
- pH
- dissolved salts
- effluent contamination
In olden days mineral content is important
- High Ca for dark beers
- High carbonate for stouts
Nowadays
- Deionisation of water
Reuse of water is important
- It reduces water cost by 50%
- Effluent treatment cost by 10 fold
 Carbon sources
Factors influencing the carbon source
- Cost of the product
- rate at which it is metabolized
- geographical locations
- government regulations
- cellular yield coefficient
Methane - 0.62
Alkanes - 1.03
Glucose - 0.51
Acetate - 0.34
 Examples of carbon sources
Carbohydrates
Starch – max 2%
Molasses (Beet – sucrose 48.5% Raffinose
1.0% Invert sugar 1.0% same in cane
molasses 33.4%, 0%, 21.2%)
Sucrose
Glucose
Malt (Barley grains germinated and heat
treated)
Other materials of plant origin like soy bean
meal, pharmedia
Oils and fats
Oils are first used as antifoams and later
used as carbon sources (soya oil, olive oil,
maize oil, linseed oil etc.,)

Factors favouring oil

2.4 times energy than glucose
Hence volume advantage of 4 times.
some organisms can use only oils for
efficient production Eg. antibiotics (Methyl
oleate is used in cephalosporin)
Hydrocarbons and their derivatives

Now it is expensive
two times carbon and three times
energy than that of carbohydrates
 Nitrogen sources

Inorganic

Ammonia gas, ammonium chloride,

ammonium sulphate, ammonium nitrates,
sodium nitrates

Ammonia gas used for pH control

Ammonium salts produces acid conditions

when ammonia is utilised. pH drift

Sodium nitrate produces alkaline drift

Organic

Organic nitrogen may be supplied by

amino acids, protein, urea

Growth will be faster. These are commonly

added as complex nitrogen sources such
as soy bean meal, corn steep liquor etc.,
(During storage these sources are affected
by moisture, temperature and ageing)
Factors influencing choice of nitrogen
source

- Nitrate reductase enzyme is repressed by

ammonium ion. Hence ammonia or
ammonium salts are preferred

- Ammonium ions represses amino acid

uptake in fungal cultivations

- also ammonia regulates acid and alkaline

protease production

- antibiotic production by many fungi is

influenced by the nitrogen source.
- soy bean meal is preferred in polyene
antibiotics production due to slow hydrolysis
which prevents ammonia accumulation and
in turn aminoacid repression by it

- in gibberellin production, nitrogen source

influence production of gibberellins

- some complex nitrogen sources may not be

utilised by some microorganisms which may
cause problem in downstream processing
 Minerals
All microorganisms require minerals for
growth and product formation
Magnesium, phosphorus, potassium,
sulphur, calcium, chlorine are essential
components
Cobalt, copper, manganese, iron,
molybdenum, zinc are also essential but in
traces.
Also depending on product analysis apart
from biomass minerals will be decided. E.g
sulphur in pencillins, cephalosporins,
chlorine in chlortetracyclin etc.,
Concentration of phosphate in medium is normally
required in excess for buffering the medium.
Phosphate concentration in the medium are
critical in antibiotic production since some
enzymes of biosynthesis are influenced by
phosphate

Other metal ions influence the production of

secondary metabolites

The functions of each vary from serving in

coenzyme functions to catalyze many reactions,
vitamin synthesis, and cell wall transport.

Citric acid & Penicillin production – Fe, Zn, Cu

Protease production – Mn
 Chelators
Many media cannot be prepared without
precipitation during autoclaving. Hence some
chelating agents are added to form complexes
with metal ions which are gradually utilised by
microorganism

Examples of chelators: EDTA, citric acid,

polyphosphates etc.,

It is important to check the concentration of

chelators otherwise it may inhibit the growth.

In many media these are added separately after

autoclaving Or yeast extract, peptone complex
with these metal ions
Mandel and Weber, 1969 (g l-1)
Urea = 0.3 g
(NH4)2 SO4 = 1.4 g
K2HPO4 = 2g
MnSO4. 7H2O = 1.6 mg
CoCl2.6H2O = 2 mg
CaCl2. 2H2O = 0.4 g
Mg SO4.7H2O = 0.3 g
FeSO4. 7H2O = 5 mg
ZnSO4. 7H2O = 1.4 mg
Peptone = 1g
Yeast extract = 0.25 g
Maize / steep liquor= 10 g
 Growth Factors
• Some microorganisms cannot synthesize a
full complement of cell components and
therefore require preformed compounds
called growth factors
• Eg.: vitamins, aminoacids, fatty acids or
sterols
• Complex media sources contain most of these
compounds. Careful blending of these will
give the required growth factors.
• For vinegar production – Calcium
Pantothenate
• For Glutamic acid – Biotin
 Precursors

• Some chemicals when added to certain

fermentations are directly incorporated
into the desired product.

• Eg: Improving the yields of Pencillin

production
 Inhibitors
• When certain inhibitors are added to
fermentation more of a specific product
may be produced
• Eg : Glycerol fermentation
• Glycerol production depends on modifying
ethanol fermentation by removing
acetaldehyde
• Addition of sodium bisulphite forms
acetaldehyde bi sulphite. Acetaldehyde is
no longer available and dihydroxy acetone
is formed.
 Inducers
• Majority of the enzymes are inducible

• Substrates or substrates analogues are

used as inducers.

• Enzymes are produced in response to the

presence of these compounds in the
environment.

• Heterologous protein production in E.coli,

yeast etc.,
 Antifoams
• Most fermentations foaming is major
problem.
• It may be due to component in the
medium or some factor produced by
the microorganism.
• Foaming can be controlled by
• Modification of medium
• Mechanical foam breakers
• Chemical agents antifoams are added
Eg: Fatty acids, silicones, PPG 2000
• Antifoams are surface active agents
reducing the surface tension in the
foam and destabilising the protein
films
• An ideal antifoam should have the
following properties
• Disperse readily and have fast action
• Active at low concentrations
• Long acting in preventing new foam
• Should not be metabolized
• Should not be toxic to m.o, humans etc
• Cheap, should not cause problem in
fermentation
Medium Optimization
When considering the biomass growth
phase in isolation, it must be recognized
that efficiently grown biomass produced by
an ‘optimized’ high productivity growth
phase is not necessarily best suited for its
ultimate purpose, such as synthesizing the
desired product.
 Classical
design
Changing one variable at time
Total no of experiments will be xn
x – no of level
n - no of variables or factors
For ex 3 levels and 6 variables have to
be tested then the number of
experiments will be 36=729
 Statistical
optimization technique
Plackett Burman design
Response surface methodology

 Optimization through modelling

 Design of Experiments (DOE)
oHelp you improve your processes. You
can screen the factors to determine which
are important for explaining process
variation.

oAfter you screen the factors, Minitab /

Design expert software helps you
understand how those factors interact and
drive your process.
 Plackett Burman design
 More than five variables it is useful
 It will be useful in screening the
most important variable
 Here n no of experiments will be
conducted for n-1 variables
 Where n is the multiples of 4 like
8,12,16,20…100
 Authors give a series of
experimental design known as
balanced incomplete blocks
 Variables which is not having influence
in the process is designated as dummy
variables
 Dummy variables are required to
estimate the error in the
experimentation
 Minimum one or two dummy variables
should be included in the experimental
set
 More can be included if the real
variables are less
Row f1 f2 f3 f4 f5 f6 f7
r1 + + + - + - -
r2 - + + + - + -
r3 - - + + + - +
r4 + - - + + + -
r5 - + - - + + +
r6 + - + - - + +
r7 + + - + - - +
r8 - - - - - - -
Row f1 f2 f3 f4 f5 f6 f7
r1 + + + - + - -
r2 - + + + - + -
r3 - - + + + - +
r4 + - - + + + -
r5 - + - - + + +
r6 + - + - - + +
r7 + + - + - - +
r8 - - - - - - -
Row f1 f2 f3 f4 f5 f6 f7
r1 + + + - + - -
r2 - + + + - + -
r3 - - + + + - +
r4 + - - + + + -
r5 - + - - + + +
r6 + - + - - + +
r7 + + - + - - +
r8 - - - - - - -
Row f1 f2 f3 f4 f5 f6 f7 Y
r1 + + + - + - - 1.1
r2 - + + + - + - 6.3
r3 - - + + + - + 1.2
r4 + - - + + + - 0.8
r5 - + - - + + + 6.0
r6 + - + - - + + 0.9
r7 + + - + - - + 1.1
r8 - - - - - - - 1.4
ΣH 3.9 14.5 9.5 9.4 9.1 14.0 9.2

ΣL 14.9 4.3 9.3 9.4 9.7 4.8 9.6

ΣH-ΣL -11.0 10.2 0.2 0.0 -0.6 9.2 -0.4

Effect -2.75 2.55 0.05 0.00 -0.15 2.30 -0.10

Mean sq 15.12 13.01 0.005 0.000 0.045 10.58 0.020

Error mean sq 0.033 0.033 0.033 0.033 0.033 0.033 0.033

F test 465.4 400.2 3.255 0.000 - 325.6 -

Fungal system experimented for exopolysaccharide
production

Variable High Low

f1:Corn steep liquor 1% 0.5%

f2:Sucrose 3% 1.5%

f3:K2HPO4 0.2% 0.1%

f4:MgSO4.5H20 1.0% 0.5%

f5:FeSO4.7H20 0.01% 0%

f6:KNO3 0.2% 0.1%

f7:Dummy Variable NaCl 0.2% 0.1%

  f1 f2 f3 f4 f5 f6 f7 Biomass Polysac

1 + + + - + - - 17.15 2.290

2 - + + + - + - 15.34 1.968

3 - - + + + - + 14.89 1.004

4 + - - + + + - 15.02 1.557

5 - + - - + + + 15.32 1.765

6 + - + - - + + 14.35 1.872

7 + + - + - - + 17.70 2.563

8 - - - - - - - 12.82 0.556
  f1 f2 f3 f4 f5 f6 f7 Biomass
1 + + + - + - - 17.15
2 - + + + - + - 15.34
3 - - + + + - + 14.89
4 + - - + + + - 15.02
5 - + - - + + + 15.32
6 + - + - - + + 14.35
7 + + - + - - + 17.7
8 - - - - - - - 12.82
EH 64.22 65.51 61.73 62.95 62.38 60.03 62.26  
EL 58.37 57.08 60.86 59.64 60.21 62.56 60.33  
EH-EL 5.85 8.43 0.87 3.31 2.17 -2.53 1.93  
Effect 1.46 2.11 0.22 0.83 0.54 -0.63 0.48  
Mean square 4.28 8.88 0.09 1.37 0.59 0.80 0.47  
Ftest 9.18 19.06 0.20 2.94 1.26 1.72 -  
   f1 f2 f3 f4 f5 f6 f7 Polysac
1 + + + - + - - 2.290
2 - + + + - + - 1.948
3 - - + + + - + 1.004
4 + - - + + + - 1.557
5 - + - - + + + 1.765
6 + - + - - + + 1.872
7 + + - + - - + 2.563
8 - - - - - - - 0.556
EH 8.28 8.57 7.11 7.07 6.62 7.14 7.20  
EL 5.27 4.99 6.44 6.48 6.94 6.41 6.35  
EH-EL 3.01 3.58 0.67 0.59 -0.32 0.73 0.85  
Effect 0.75 0.89 0.17 0.15 -0.08 0.18 0.21  
Mean square 1.13 1.60 0.06 0.04 0.01 0.07 0.09  
Ftest 2.43 3.43 0.12 0.09 0.03 0.14 -  
The first row for Plackett-Burman designs.

n k String

11 12 + + - + + + - - - + -

15 16 + + + + - + - + + - - + - - -

19 20 + + - - + + + + - + - + - - - - + + -

23 24 + + + + + - + - + + - - + + - - + - + - - - -
 Plackett-Burman Design in 12 Runs for up to 11 Factors

Pattern X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11

1 +++++++++++ +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1
2 -+-+++---+- -1 +1 -1 +1 +1 +1 -1 -1 -1 +1 -1
3 --+-+++---+ -1 -1 +1 -1 +1 +1 +1 -1 -1 -1 +1
4 +--+-+++--- +1 -1 -1 +1 -1 +1 +1 +1 -1 -1 -1
5 -+--+-+++-- -1 +1 -1 -1 +1 -1 +1 +1 +1 -1 -1
6 --+--+-+++- -1 -1 +1 -1 -1 +1 -1 +1 +1 +1 -1
7 ---+--+-+++ -1 -1 -1 +1 -1 -1 +1 -1 +1 +1 +1
8 +---+--+-++ +1 -1 -1 -1 +1 -1 -1 +1 -1 +1 +1
9 ++---+--+-+ +1 +1 -1 -1 -1 +1 -1 -1 +1 -1 +1
10 +++---+--+- +1 +1 +1 -1 -1 -1 +1 -1 -1 +1 -1
11 -+++---+--+ -1 +1 +1 +1 -1 -1 -1 +1 -1 -1 +1
12 +-+++---+-- +1 -1 +1 +1 +1 -1 -1 -1 +1 -1 -1
 When to use PB
 Screening multi components at 2 levels
 It will give the range at which you have
to optimize the experiments further

Limitations:

 Itwill not give optimum concentration of

the variable
 Response Surface
Methodology
 Response surface methodology is a
method of optimization using statistical
techniques based upon the special
factorial design of Box and Behnken etc.,

 It is a scientific approach to determine the

optimum conditions which combines the
special experimental designs and Taylor
first order and second order equation
Sequential nature of RSM
 How to Proceed
 Select critical factors and regions to be tested
 Designthe experiment based on box behnken
or central composite design
 Do the experiment
 Fit the data to Taylor series, determine
coefficients to build model
 Validate model by selecting values in the
region tested
 Draw the contour plot and find optimum
concentration
Design of experiments

High
Variable 2

Low

Low Variable 1 High

 Coding the variables
Value of the variable - Middle point
Coding =
Difference/2

Glucose = 10 – 30 g/l
Coding 10 g/l glucose = [10-20]/(20/2) = -1
Coding 30 g/l = ?
Coding 20 g/l ??
 Taylor series
Yield Y = β0 + β1 X1 + β11 X12

Constant term + Linear term + Quadratic term

Y= β0 + β1 X1 + β2 X2 + β11 X12 + β22 X22 + β12 X1 X2

α = [2n]1/4
Design of experiments
[0,+1.414]
[-1,+1]
Variable 2 [+1,+1]

[-1.414,0] [0,0] [+1.414,0]

[-1,-1] [+1,_1]

[0,-1.414,0]

Variable 1
 Design the experiments for the
following variable concentrations

Corn steep Liquor = 0.5% to 1.5 %

Sucrose = 1.5% to 4.5 %

Write the coding equation for both Corn

Steep Liquor and Sucrose

For CSL = (Value-10)/5

For Sucrose = (Value -30)/15
 CSL Sucrose
Run
No Coded Uncoded Coded Uncoded
1 -1 5 -1 15
2 -1 5 +1 45
3 +1 15 -1 15
4 +1 15 +1 45
5 -1.414 2.93 0 30
6 +1.414 17.07 0 30
7 0 10 -1.414 8.79
8 0 10 +1.414 51.21
9 0 10 0 30
10 0 10 0 30
11 0 10 0 30
 run
order Csl (g/l) Sucrose (g/l) response
1 15 15 1.748
2 10 30 2.572
3 15 45 1.464
4 17.07 30 1.678
5 10 51.21 1.326
6 10 8.79 1.604
7 5 45 1.533
8 10 30 2.584
9 10 30 2.543
10 10 30 2.564
11 2.93 30 1.846
12 5 15 1.089
13 10 30 2.558
• 13 equations will be obtained from 13
experiments.
• Resulting equations will be solved by least
square method of matrix solving
• All the equations will be represented in the
form of
Y = βX
β = (X’X)-1 (X’Y)
  VARIABLE ESTIMATE ERROR
       
β0 Intercept 2.564219 0.070235
β1 X1 0.044063 0.05553
β2 X2 -0.029141 0.05553
β11 X1*X1 -0.43992 0.059558
β22 X2*X2 -0.588465 0.059558
β12 X1*X2 -0.182 0.078526

Standard Error of Mean = 0.043558

R-SQUARED 0.9529

ADJ R-SQUARED 0.9193

C.V. 8.13%

Y = β0+β1* X1+β2* X2+β11* X12+β22* X22+β12* X1*X2

Y = 2.564 + 0.044 X1 - 0.029 X2 - 0.44 X12 - 0.589 X22 - 0.182 X1 X2
http://www.itl.nist.gov/
div898/handbook/index.htm
• Using the actual values makes it
easy to calculate the response from
the coefficients since it is not
necessary to go through coding
process

• The reason for coding the variables

is to eliminate the effect that the
magnitude of the variable has on
the regression coefficient
• Prob>F is less than 0.05 indicated
significant model terms

• The standard error of estimate yields

information concerning the reliability of
the values predicted by the regression
equation. The greater the standard error
of estimate, the less reliable the
predicted value.

• Coefficient of variation less than 10 %

indicate high degree of precision and
reliability of experimental values
• The mathematical model is reliable with R2
value. Closer the value to 1 is the more
reliable the model.

• R2 value 0.9529 suggests that the model was

unable to explain 4.71% variations occurred

• R2 Value can be increased by including model

terms. Sometimes even higher value may
result in poor predictions.

• Adj R2 value will be verified. If this value

differs dramatically then insignificant model
terms have been included in the model
Ord VALUE VALUE RESIDUAL
Run ACTUAL PREDICTED  
1 1.748 1.791037 -0.043037
2 2.572 2.564219 0.007781
3 1.464 1.368755 0.095245
4 1.678 1.746948 -0.068948
5 1.326 1.346438 -0.020438
6 1.604 1.428849 0.175151
7 1.533 1.644629 -0.111629
8 2.584 2.564219 0.019781
9 2.543 2.564219 -0.021219
10 2.564 2.564219 -0.000219
11 1.846 1.622339 0.223661
12 1.089 1.338911 -0.249911
13 2.558 2.564219 -0.006219
 Residuals Vs Run order

0.3

0.2

0.1
residuals

-0.1

-0.2

-0.3
0 2 4 6 8 10 12 14
run order
 CSL Vs Residual

0.3

0.2

0.1
Residual

-0.1

-0.2

-0.3
0 5 10 15 20
CSL
 Sucrose Vs residuals

0.3

0.2

0.1
Residuals

-0.1

-0.2

-0.3
0 10 20 30 40 50 60
Sucrose
 Contour plot
• A contour plot is a graphical
technique for representing a 3-
dimensional surface by plotting
constant z slices, called contours,
on a 2-dimensional format.

• That is, given a value for z, lines

are drawn for connecting the (x,y)
coordinates where that z value
occurs.
Stationary ridge
RISING RIDGE
Y = β 0 +β 1 * X 1 +β 2 * X 2 +β 11 * X 12 +β 22 * X 22 +β 12 *
X 1 *X 2

Y = β 0 + X’ b + X’ B X

X= X1 b = β1 B = β 11 β 12 /2
X2 β2 β 12 /2 β 22

∂y/∂x =0

X s = -1/2 B -1 b
 Application of response surface
methodology to cell immobilization
for the production of palatinose
Design based on
Alpha factor = 1
• Optimum alginate concentration, cell
loading and bead diameter were 5%,
15 g /l and 2.25 mm, respectively.

• R2 value of 0.9259

• A very low value of coefficient of the

variation (C.V.) (4.46%)
 Residuals Vs run order

6
4
2

Residuals
0
-2
-4
-6
0 5 10 15 20
Run order