Ghasem D. Najafpour. Biochemical Engineering and Biotechnology

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184 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

7.9.2 Continuous Extraction Column Process,

Rotating Disk Contactors

Organic solvents are used to extract antibiotics. The characteristics of antibiotics and prod-

uct from extraction processes are summarized below:

• water immiscible

• multistage extraction

• purification, provides highly concentrated products

Stages of the extraction process using fermentation broth for product recovery are listed below:

• 20–35 g/l antibiotic

• filtration: to remove mycelia with addition of polymer to get clear filtrate

• neutralisation: penicillin, pH about 2–3

• solvent extraction: using suitable solvent such as amyl acetate, a salt solution of penicillin

is obtained

• precipitation of antibiotics is done with butyl acetate, a isobutyl ketone and mineral ions

such as Na



• crystallisation: solid product can easily be filtered

Erythromycin is extracted by an organic solvent such as pentyl acetate. Similarly, steroids,

vitamin B

, morphine and codeine are extracted with organic solvents.

Counter-current extraction columns are used. Figure 7.12 shows the counter-current

extraction column with a ternary diagram for material balance and equilibrium curve.

The mixture of feed and solvent has been identified as the crossing lines of material

balance and tie line connecting the desired product in organic and aqueous phases.

(7.9.2.1)

MSF M

 and 

Solvent

0 1.0

1.0

S′

FIG. 7.12. Countercurrent extraction column with material balance in the ternary diagram.

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Equilibrium data must be obtained for material balance showing raffinate and extracted

phases. A simple separation funnel for single-stage extraction using amyl acetate as organic

solvent is shown in Figure 7.13.

The equilibrium constant is defined by K, K  y/x, and the portion coefficient is also

defined by K as a ratio of C

which is constant. Based on the value of K, single or

multi-stage extractions are performed to do a perfect job in bioseparation.

Another parameter used to characterise two-phase partitioning is the purification factor,

defined as:

(7.9.2.2)

 C

, where product is shown in the lower phase and d

 C

, where product

partitions to the upper phase. When single-stage extraction does not give sufficient recovery,

repeated extraction can be carried out in a chain or cascade of contacting and separation units.

7.10 ADSORPTION

In general, adsorption is a surface phenomenon, where gas or liquid is concentrated on the

surface of solid particles or fluid interfaces. There are many adsorption systems.

7.10.1 Ion-Exchange Adsorption

Adsorption beds of activated carbon for the purification of citric acid, and adsorption of

organic chemicals by charcoal or porous polymers, are good examples of ion-exchange

adsorption systems. Synthetic resins such as styrene, divinylbenzene, acrylamide polymers

activated carbon are porous media with total surface area of 450–1800m

g

1

. There are a

few well-known adsorption systems such as isothermal adsorption systems. The best known

adsorption model is Langmuir isotherm adsorption.

Concentration of product in the preferred phase

Initial



pproduct concentration

Extract

Raffinate

Amyl acetate

FIG. 7.13. Separation of extract and raffinate layers.

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186 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

7.10.2 Langmuir Isotherm Adsorption

Adsorption, like extraction, depends on equilibrium relationships. Isothermal adsorption is

projected by Langmuir isotherms. The model is shown in Figure 7.14, which is based on

the linear model of the following equation:

(7.10.2.1)

where, C

is equilibrium concentration (kg solute/kg solid) and C

ASm

is the maximum

loading of adsobate.

7.10.3 Freundlich Isotherm Adsorption

Another useful model for isothermal adsorption is the Freundlich model, which is pre-

sented by the following equation.

(7.10.3.1)

where k

and n are constants based on the characteristics of the particular adsorption system.

If adsorption is favourable, the value of n is greater than 1. For n less than 1, the adsorption

process is not favourable.

Figure 7.15 shows the adsorption of ion exchangers, downflow pattern. The bed has an

adsorption zone with respect to time and is saturated with solute.

7.10.4 Fixed-Bed Adsorption

Fixed-bed adsorption may give a higher adsorption area per unit volume than any other type

of adsorber. The point of saturation of the bed is called the breakthrough point. By knowing

this point one can determine operation schedules. In designing fixed-bed adsorbers, the

CkC



CkC

ASm A A



1

ASm

Equilibrium concentration, C

Langmuir Isotherm

FIG. 7.14. Langmuir isotherm adsorption model.

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quantity of resin and the time required for adsorption of a given quantity of solute must be

estimated.

The overall rate of mass transfer from liquid to internal surface is given by

(7.10.4.1)

where C

is the concentration of ‘A’ at equilibrium, and the void function is defined as

a fractional volume, that is the ratio of the empty space volume in the bed and the total

volume (V

 V

)/V

. V

is the total volume and V

the volume of resin in the packed

bed.

7.11 CHROMATOGRAPHY

Chromatography is a separation procedure for resolving mixtures and isolating components.

The basis of chromatography is differential migration, and the selective retardation of solute

molecules during passage through the bed of resin particles. The fluid carrying the solutes

through the column used for elution is known as the mobile phase. The material that stays

inside the column and effects the separation is called the stationary phase. In gas chromatog-

raphy (GC), the mobile phase is a gas. GC is widely used as an analytical tool to separate rel-

atively volatile components. For instance, separation of three solutes from a mixture that is

injected into a column would lead to three separate peaks identified by the detectors on the

outlet analysis.









()

FIG. 7.15. Ion exchange adsorption bed.

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188 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

Chromatography is a high-resolution technique; therefore the method is suitable for recov-

ery of high-purity therapeutics and pharmaceuticals. Different chromatographic methods are

available for purification of proteins, amino acids, nucleic acids, alkaloids, vitamins, steroids

and many other biological materials. These methods are adsorption chromatography, partition

chromatography, ion-exchange chromatography, gel chromatography and affinity chromatog-

raphy. These methods differ in the principal mechanism by which the molecules are retarded

in the chromatography column. There are several distinct methods of chromatography:

• Adsorption chromatography

• Partition chromatography

• Ion-exchange chromatography such as carboxy methyl cellulose (CMC), agarose and/or

dextrins

• Gel chromatography or molecular sieve chromatography such as polyacrylamide gels

• Affinity chromatography, which is the binding of bio-molecules with the matrix bed, often

used for antibodies and antigens

In adsorption chromatography, the bed has special characteristics to adsorb solutes. The recom-

mended beds for adsorption chromatography are silica gel, alumina and charcoal.

In ion-exchange chromatography agarose, dextrose and carboxy methyl cellulose

(CMC) are used as the media beds for separation. In gel chromatography the bed is mainly

molecular sieves such as polyacrylamide gels. In affinity chromatography, the separating

bed is a binding biomolecular matrix which is able to attract antibodies or antigen, so there

is good affinity for separating the product. The capacity of the column is given by:

(7.11.1)

where k is known as the capacity of the column, and the solvent volume for eluting the bed

is defined with V

; also V

is the void volume, the free volume, outside of the bed particles;

is known as internal volume of liquid in the pores of the particles; and V

is the volume of

the gel itself. The relative retention, d, is the ratio of two capacities known as selectivity.

The total volume of the gel column is:

where V

is the volume of eluting solvent, V

is the void volume outside the particles, V

is the

internal volume of liquid in the pores of the particles, and V

is the volume of the gel itself.

The relative retention is d  k

; k

and k

are called selectivity, and k

is the gel partition

coefficient, k

 (V

 V

)/V

, where V

is the multiplication of mass of dry gel, ‘a’ with W

(7.11.2)

VaW







()

VVVV

VVkV

total o i s

eopi









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DOWNSTREAM PROCESSING 189

where W

is the water regain value that is the volume of water taken up per mass of dry gel,

is the density of wet gel, and r

is the density of water.

7.11.1 Principle of Chromatography

The basis of chromatography is in the differential migration of chemicals injected into a

column. The carrier fluid takes the solutes through the bed used for elution (mobile phase).

The bed is the stationary phase. Based on mobility, the retention-time detectors identify the

fast and slow-moving molecules. Based on internal or external standards with defined con-

centration, all unknown molecules are calculated in a developed method by software. GC

columns are installed in an oven which operates at a specified temperature. A diagram of

an oven with GC column is shown in Figure 7.16.

Example 1

A 30 ml sample of broth from penicillin fermentation is filtered in the laboratory on a 3cm

filter at a pressure drop of 5psi. The filtration time is 4.5 minutes. Previous studies have shown

that the filter cake of Penicillium chrysogenum is significantly compressible with S 0.5. If

500 litres of fermentation broth from a pilot plant have to be filtered in 1 hour, what size of

filter is required for a pressure drop of 10psi and 5 psi? Neglect the resistance of the filter

medium.

Solution

Since the filter resistance in the filter medium is neglected, the second part of equation

(7.3.1.5) is neglected. Let us define a new constant, C, equal to the mass of solid deposited

on the filter per volume of filtrate.

(E.1.1)





FIG. 7.16. GC column in an insulated box.

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190 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

Equation (7.3.1.5) was modified as the second part is neglected, so the filtration time per

unit volume is:

(E.1.2)

The obtained equation is written with respect to pressure drop:

(E.1.3)

The area for a laboratory-scale filter is calculated by:

The area for a pilot-scale filter is obtained by:

The value for area A

is:

For a pressure drop of 5psi the filter area is:

05 1

0210





.( ) (

500,000 cm )

120 min

37 m

(. )() ( )

0 20125 5

05 1 2



500,000 cm

20 min

37 m

cP V

05 1

0 20125 10









500,000 cm

120 m

()

(. )( ) ( )



iin

cm or 1.15 m

115 10

0.5 1 3

2 3 cm 4.5 min

5 psi 30 cm







()

()( )

()(



))

()

0.5

.20125

psi min

 0







ma

VPm



marv

v21

 ()-

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Having a differential pressure in the above filtration process, and reducing the pressure drop

from 10 to 5psi, increases the filter area by 19%. The main reasons why an increase in the pres-

sure drop results in less filter area are the compressed cake and the porosity of the filter cake.

Example 2

Filtration of 300 ml of fermentation broth was carried out in a laboratory-sized filter with

a pressure drop of 10 psi. The filtration took 20 min. Based on previous studies, the filter

cake obtained from Penicillium chrysogenum was compressible with the exponent ‘s’ in the

equation for calculation of filter area equal to 0.5.

(a) What size of filter is required to carry out filtration of 1 m

broth from a pilot plant in

20 hours and at a pressure drop of 20 psi?

(b) What is the percentage increase in filter area if the pressure drop is reduced to 10 psi?

Solution

(E.2.1)

An 18.9% increase in the area of filter was obtained.

025

1 189





cP V

V











()



05 2

0 140

5520

2260

10 1 14 10 1 1

642

()

(). .

.



 

cm cm 4 m

ma  



2 2 10 20

10 300

0 1405

05 1 2

()

()()

() ( )



aaP









ma ma



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192 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

(E.2.2)

The ratio of which is approximately a 19% in filter surface area.

Example 3

In a batch production of penicillin, 40 m

capacity of the plant, sterile air is required to be

supplied. The bioreactor requires 1vvm (volume of air/volume of broth. min). The incom-

ing air contains 3000 cells/m

of air, for 100 hours operation. Calculate the depth of filter,

if the penetration of bacteria is 1 in 1 million.

Solution

Assume D

filter

 60 cm, based on availability of filter

Air flow rate, F

air

 (40 m

)(1 vvm)(60 minh

1

)  2400 m

h

1

Microbial load  (3000 cells/m

)(2400)  7.2  10

cells

Cross sectional area of filter, A (/4)(0.6)

 0.028 m

Air velocity (2400)/(0.028)  8488 m/h

 N

kt

, (E.3.1)

ln(N

)  kL (E.3.2)

ln[(7.2  10

)/(10

6

)]  kL

 7.2  10

cells,

 1 cell in 10

cells  10

6

cells

L represents the length of filter, and k is the rate constant based on filter bed material 84

L  (1/k) ln 7.2  10

 (1/84) ln 7.2  10

 0.4 m.

Example 4

Microbial cells are separated from a culture broth at a flow rate of 3.35 10

3

/s.

Assume the cells are spherical with average diameter of 1 m. Select a centrifuge that can

perform this separation. Given data: r

cell

 1.1 r

water

, r

water

 997 kgm

1

at 25 C; r

broth



water

, and the viscosity of water is 0.9  10

3

Ns/m

Solution

(E.4.1)



1 356

114

1 189

05 05

220







(/)()

0 473

056

084

114

084





56 m

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DOWNSTREAM PROCESSING 193

(E.4.2)

(E.4.3)

(E.4.4)

Example 5

Microbial cell recovery is carried out in a continuous disc-stack centrifuge. The centrifuge

is operated at 5000 rpm for separation of baker’s yeast. At a feed rate of 60 l/min, 50%

of the cells are recovered. At constant speed, solid recovery is inversely proportional to

flow rate.

(a) What flow rate is required to recover 90% of cells if the centrifuge speed is fixed at

5000 rpm?

(b) What operating speed is required to recover 90% of cells if the flow rate in the centrifuge

is maintained at 60 l/min?

Solution

(E.5.1)

When we need to remove cells from fermentation broth, sedimentation is considered as a

downstream processing method. Alum, lime, polyelectrolyte are commonly used.

90 60

5000

()





33 l/min

33 33



08 rpm











p cell water

m /s)(18

()

335 10

  







09 10

110 1

()(

3 N s/m

kg m/s

m) (1.1 997 kg

//m ) 81 m/s

(. )

165 10

= 

S S

uDrZu





xutZu



S 83250 m

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