CULTURE MEDIA

UTILIZED IN THE

BIOTECHNOLOGY

INDUSTRY
On a large scale on must normally use sources of
cheap nutrients to create a medium which will
meet as many as possible the following criteria:

⚫ It will produce the maximum yield of product or biomass

per gram of substrate used.
⚫ It will produce the máximum concentration of product or
biomass.
⚫ It will permit the máximum rate of product formation.
⚫ There will be the mínimum yield of undesired products.
⚫ It will be cheap and of a consistent quality and be
readily available throughout the year.
⚫ It will cause minimal problems in other aspects of the
production process particularly aeration and agitation,
extraction, purification and waste treatment.
 Medium Formulation

Carbon & Energy Source Cell Biomass

+ +

Nitrogen Source Products

+ +

Other requirements CO2 + H2O + heat

 MAYOR NUTRIENTS
CARBON SOURCE NITROGEN SOURCE
Glucose Ammonium salts
Sucrose Nitrate salts
Lactose Meat extract
Mannitol Peptone
Glycerol Yeast Extract
Starch Soy meal
Molasses Corn steep liquor
etc. etc.
 What are yeast extracts?
▪ They are the result of the breakdown the yeast cell components
into smaller soluble molecules

▪ In order to breakdown the yeast cell, a process called autolysis is

used

▪ Autolysis is a process in which live active yeasts are degraded by

their own endogenous enzymes into much smaller molecules such
as peptides, amino acids and nucleic acids

▪ Important parameters for an optimal and standardized autolysis

process include pH and temperature control and the autolysis time
Production Flow Chart
Yeast extract and cell walls
OPTIMIZATION

OF CULTURE

MEDIA
RESPONSE SURFACES
RESPONSE SURFACES
RESPONSE SURFACES
 OPTIMIZATION STRATEGIES
⚫ Optimus: The best condition or quantity

⚫ Estrategies: Box-Wilson & RSM (Response

Surface Methodology)
⚫ Example: optimization of culture media by Box-Wilson
method
⚫ The critical factors of the process are known
⚫ Only a limited number of variables can be handled
(3-5) 2ⁿ
⚫ The factors should continuously vary during the
experimental tests
Fermentative production of streptomycin

Component Ingredient Concentration

(g/L)
A Yeast extract 17.0
B (NH4)2SO4 6.0
C K2HPO4 7.0
D NaCl 3.0
E MgSO4 1.0
F Glucose 60.0
G Ca2CO3 4.0
H Agar 2.0

It is known that the critical ingridients are: A, B, C y F

 ⚫ Theupper (+1) and lower (-1) levels are
established
⚫ Variation units: between 10 to 30% of the basal level

Component Basal (+1) (-1) Variation

Upper Lower units
A 17 20 14 3
B 6 8 4 2
C 7 9 5 2
F 60 70 50 10
 ⚫ The composition of the proposed media is:
Component Concentration (g/L)

A 20 14

B 4 8 4 8

C 5 9 5 9 5 9 5 9

F 50 70 50 70 50 70 50 70 50 70 50 70 50 70 50 70

⚫ It can be managed as a 2ⁿ factorial. In this case 2⁴

(four factors studied, each one at two levels)

⚫ Results of the proposed experiment (3 flasks

[replicates] for each treatment), randomly:
Treatment Component Yield (µg/ml)
(media) A B C F streptomycin

1 -1 -1 -1 -1 38
2 -1 -1 -1 +1 37
3 -1 -1 +1 -1 44
4 -1 -1 +1 +1 40
5 -1 +1 -1 -1 35
6 -1 +1 -1 +1 46
7 -1 +1 +1 -1 43
8 -1 +1 +1 +1 48
9 +1 -1 -1 -1 39
10 +1 -1 -1 +1 30
11 +1 -1 +1 -1 18
12 +1 -1 +1 +1 16
13 +1 +1 -1 -1 33
14 +1 +1 -1 +1 38 Media x= 36.4
15 +1 +1 +1 -1 32
16 +1 +1 +1 +1 45
⚫ Calculate regression coefficient for each component
A B C F
Up. Low Up. Low Up. Low Up. Low

39 38 35 38 44 38 37 38
30 37 46 37 40 37 40 44
18 44 43 44 43 35 46 35
16 40 48 40 48 46 48 43
33 35 33 39 18 39 30 39
38 46 38 30 16 30 16 18
32 43 32 18 32 33 38 33
45 48 45 16 45 38 45 32
250 331 320 262 286 296 300 282
250 − 331 − 81 320 − 262 58 286 − 296 − 10 300 − 282 18
= = = =
16 16 16 16 16 16 16 16
β= -5.06 β= 3.62 β= -0.62 β= 1.12
⚫ Higher values have a mayor influence in the
streptomycin yield
A B C F
Coefficient
of -5.06 3.62 -0.62 1.12
variation
3 2 2 10
units
-15.2 7.2 -1.2 11.2
⚫ Calculate modifications to the level of the components for a
2nd. experiment:
⚫ The component with the higher influence is taken as base and the
change of other components is determined with respect to it.

A -15.2/15.2 = -1
B 7.2/15.2 = 0.5
C -1.2/15.2 = -0.08
F 11.2/15.2 = 0.7
 A NEW EXPERIMENT IS DESIGNED

Concentration (g/L)

A B C F
Medio 17 6 7 60
base

Unid. De (-1) (+0.5) (-0.08) (+0.7)

var.

Medio 17 16 6.5 6.92 60.7

Medio 18 15 7.0 6.84 61.4
Medio 19 14 7.5 6.76 62.1
Medio 20 13 8.0 6.68 62.8
Comparison between original and optimized media

The streptomycin titre in the optimized medium was

compared experimentally with that in the original
medium. The increment in the optimized medium was
5 2%. In both cases, maximum production was reached
after 3 days (Fig. 7). Mycelial dry weight showed an increase of
approximately 10%, which gave an increase in specific production
of 32%. Specific growth rate calculated in the
exponential phase (μmax) was 0.065 h-1 in the original
medium and 0.096 h-1 in the optimized medium.
The pH profile was substantially modified due to the
increase in glucose concentration, and values were
consistently higher in the original than in the optimized
medium. This was due to the accumulation of lactate
and pyruvate in the optimized medium