Ghasem D. Najafpour. Biochemical Engineering and Biotechnology

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174 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

This type of filter can be used for removal of Penicillium and Streptomyces mycelia in the pro-

duction of penicillin and streptomycin, respectively.

In these processes the rotary drum filter

is used with a pre-coated cloth filter with filter aid; the filter cake is removed by a knife blade

which scrapes the cake from the rotating drum.

7.3.1 Theory of Filtration

Assume laminar flow of filtrate of liquid through the filter cake. Rate of filtration is usually

measured as the rate at which liquid filtrate is collected. The filtration rate depends on the area

of the filter cloth, the viscosity of the liquid, the pressure drop across the filter and filter cake

resistance. At any instant during filtration, the rate of filtration is given by the equation:

(7.3.1.1)

where A is filter area, V

is volume of filtrate, t is filtration time, P is the pressure drop across

the filter, m

is the fluid viscosity, and W is the mass of solids in the filter cake. W is defined as:

(7.3.1.2)

where m is the ratio of mass of wet cake over mass of dry cake and  is the solid mass

fraction.

Also from (7.3.1.1), r

is the filter media resistance and a is the average specific cake resist-

ance. If the filter cake is incompressible, a is constant; for compressible cake a is defined as:

(7.3.1.3)

where S is cake compressibility, a is constant, depending on the size of particles in the cake.

For incompressible solids, S is about zero; for highly compressible solids, S is about 1. For

convenient integration, rewrite (7.3.1.1) in its reciprocal form:

(7.3.1.4)

The separation of variables is used with suitable integrations, which result in the following

equations:

(7.3.1.5)

mAP

Vddd

ÚÚÚ



marv



1 



aa()P

mass of solids in the cake

r



11-







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DOWNSTREAM PROCESSING 175

(7.3.1.6)

Using an initial condition for calculation for defining integration constant at t  0, V

 0,

gives the integration constant to be zero. By division of the above equation with V

we can

obtain a linear model, which we can plot and obtain the slope and intercepts. The modified

equation after division of (7.3.1.6) is:

(7.3.1.7)

Now the (7.3.1.7) is linear as the linearised model is (y  k

x  k

(7.3.1.8)

where, The slopes of the above lines depend on

physical properties of the fermentation broth such as viscosity, density, mass fraction and

pressure drop. The pH for the filtration of fermentation broth in the production of strepto-

mycin using Streptomyces griseus may show an additional effect; neutralising pH should

eliminate such an effect.

In general, fungal mycelia are filtered relatively easily, because mycelia filter cake has

sufficiently large porosity. Yeast and bacteria are much more difficult to handle because of

their small size. Alternative filtration methods, which eliminate the filter cake, are becoming

more acceptable for bacterial and yeast separation. Micro-filtration is achieved by develop-

ing large cross-flow fluid velocities across the filter surface while the velocity vector normal

to the surface is relatively small. Build up of filter cake and problems of high cake resistance

are therefore prevented. Micro-filtration is not discussed in this section.

7.4 CENTRIFUGATION

Centrifugation is used to separate materials of different densities when a force greater than

gravity has been implemented, such as centrifugal forces. Centrifugation may be used to

remove cells from fermentation broth; yeast, for example, is harvested in a centrifuge unit.

For dilute suspensions each cell may be treated as a single particle in an infinite fluid. In

the concentrated fluid with suspended solids, the particle’s motion is influenced by neigh-

bouring particles. A continuous process is commonly used in separation of solid parti-

cles from fermentation broth. The particle’s velocity correlates with the hindered settling

ccm

and





marv

()



versus

VA P m

fff

/()







marv

21





()()At

AP m

V







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176 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

particles (u

), the single particle’s velocity (u

) and the volume fraction of particles (e

). The

correlation is:

(7.4.1)

The empirical relations for b in the above equation derive for various ranges of 

are:

(7.4.2)

Another type of centrifuge is known as a scroll conveyer centrifuge. By comparing several

types of continuous centrifuges, the scroll type of centrifuge has an important feature of

the decanter, that is able to handle large solid particles without any clogging. The decanter

centrifuge is used for recovery of large mould pellets and the large throughput capacity of

the nozzle. The settled solid is easily removed with a scroll centrifuge.

7.4.1 Theory of Centrifugation

The particle’s velocity in a particular centrifuge is compared with the settling velocity that

occurs under the influence of gravity and the effectiveness of centrifugation. The terminal

velocity during gravity settling of a small particle in dilute suspension is given by Stoke’s law:

(7.4.1.1)

where u

is the sedimentation velocity under gravity, r

is the density of the particle, r

the density of fluid, m is the viscosity of the fluid, D

is the diameter of the solid particle,

and g is gravitational acceleration. In (7.4.1.1) the centrifugal force is implemented to

obtain terminal velocity in the centrifuge:

(7.4.1.2)

where u

is the particle velocity in the centrifuge, v is the angular velocity of the bowl in rad/s,

and r is the radius of the bowl or centrifuge drum. The ratio of velocity in the centrifuge to

velocity under gravity (u

) is called the centrifuge effect or g-number, and is denoted as Z;

therefore:

(7.4.1.3)



uDr



rr

uDg



rr

m18

b 

 



1 3 05 0 15 0 5

1 2 29 0

.. .,

2.84

3.43

irregular particles

.. . ,

205

12 015

 



spherical particles

dilute suspensionss





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DOWNSTREAM PROCESSING 177

The force developed in a centrifuge is Z times the force of gravity and is often expressed as

so many g-forces. Industrial centrifuges have Z factors from 300 to 16,000. In a small labo-

ratory centrifuge Z may be up to 500,000. The particle velocity in a given centrifuge can be

increased by increasing the centrifuge angular velocity (v); by increasing the particle diam-

eter (D

); by increasing the density differences between particle and liquid (r  r

 r

);

and decreasing suspension viscosity (m).

However, where the particles reach the walls of the bowl, the separation is also affected by

the time of exposure to the centrifugal forces. In continuous flow and devices such as the disc-

stack centrifuge, the residence time increases by decreasing the feed flow rate. Performance

of centrifuges of different size can be compared by using a parameter known as the sigma fac-

tor (S). For a continuous centrifuge, the sigma factor is related to the feed flow rate:

(7.4.1.4)

where Q is the volumetric flow rate and u

is the terminal velocity of the particles in a gravi-

tational field. The sigma factor physically represents the cross-sectional area of a gravity set-

tler with the same sedimentation characteristics as the centrifuge. For two centrifuges with

equal particle velocities, the performance of their effectiveness is related by their flow rate:

(7.4.1.5)

where, subscripts 1 and 2 denote the two centrifuges. Equation (7.4.1.5) can be used

to scale-up centrifuge equipment. The sigma factor depends on centrifuge design. Figure 7.4



S

Solid

Liquid

Feed

FIG. 7.4. Disc stack bowl centrifuge.

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178 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

shows a simplified stack bowl centrifuge. For a disc-stack bowl centrifuge, S is defi-

ned as:

(7.4.1.6)

where v is the angular velocity in rads/s, N is the number of discs in the stacks, r

is the outer

radius of the disc, r

is the inner radius of the disc, and u is half of the cone angle of the disc.

For a tubular bowl centrifuge the above equation has to be modified. Figure 7.4 shows the

disc stack bowl centrifuge.

7.5 SEDIMENTATION

When cells have a high tendency to aggregate closely (coagulate) or to form multicellular

flocs with the aid of polyvalent cations or extracellular polymers, the recovery of cell biomass

becomes simple and easily applicable by the sedimentation process. Such aggregation pro-

vides cell recycle streams in activate sludge wastewater treatment; and several highly floccu-

lent yeast strains are used in brewing beer and single-cell protein production. In fact, we need

to remove cells from fermentation broth, so sedimentation is considered as a downstream pro-

cessing method. Alum, lime and polyelectrolytes are commonly used to create macroflocs.

There are several natural and chemical coagulants used for aggregating suspended cells in

bioprocesses. Figure 7.5 shows that cells are removed from fermentation broth and the

sludge of coagulants with biomass settles as sludge. The clear solution is analysed for

enzyme activity and further process purification is needed for enzyme recovery.

Cell removal polyelectrolyte aggregated floc 

S



()

tan

()

Sludge

Broth

Clear

solution

FIG. 7.5. Sedimentation and settled sludge.

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DOWNSTREAM PROCESSING 179

The nature of chemical coagulants are such that the macrofloc may possess certain charges;

for example lime (CaO), alum (Al

) and flocculating polyvalent cations carry positive

charges, which interact with proteins. The interactions are simply illustrated in Figure 7.6.

It is necessary to define settleable solids and the settling velocity. Let us take barley and

use the polysaccharide content of it with a well-known yeast for brewing beer. It is ideal to

use a natural settling tank to have a clear solution; we can generate bioflocs that settle down

the biomass faster. Figure 7.7 shows the free-falling solid particles in a fluid. When cells

have a high tendency to aggregate, with the aid of polyvalent cations, cell biomass recov-

ery becomes possible.

In brewing beer and SCP production, several highly flocculent yeast strains are used. The

special yeast strains are easily separated without the use of any expensive separation

process.

The settling velocity is defined by:

(7.5.1)

ukC





Barley (starch) brewing be

Saccharomyces cerevisiae

æÆæææææææ eer)

. . . .

. ..

. .. ..

… . . . .. .

max

FIG. 7.7. Freely falling of solid particle.

- -

- - -

2−

−

R – CH – COOH

FIG. 7.6. Floc with positive or negative charges.

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180 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

where m is an empirical constant in the range 1.7–2.6. Once the cell concentration achieves

a large value, C

max

, further concentration occurs at a negligible rate as seen in an activated

sludge process. The relative abilities of sedimentation, centrifugation, filtration and drying

to achieve dewatering up to a desired level are important in determining which processes are

appropriate. For example, centrifugation at Z  3000 removes all the floc water, giving a pel-

let with 5% moisture; further dewatering may be required after cell lysis. Increased sedimen-

tation rates may be possible in inclined tubes or narrow channels, leading to evolution of a clear

fluid zone at the top of the channel or tube.

7.6 FLOTATION

Flotation is another method to remove solid (cells) from fermentation broth, using air bub-

bles to float protein. We may use different kinds of high shear-force devices to make homo-

geneous solutions for liberating intracellular enzymes. Figure 7.8 shows several types of

impeller for homogenisation.

Non-mechanical methods are also used to break down the cell wall and to release intra-

cellular enzymes or proteins. Listed below are several methods that fracture cell walls and

release cell content:

• Osmotic shock

• Freezing

• Solvent

• Detergents

• Based on shear forces: high pressure homogeniser

• Manton Gaulin homogeniser works at high pressure, about 550 atm

• Lysis of cells for intracellular products

• Cell wall lysed with organic solvent extraction

7.7 EMERGING TECHNOLOGY FOR CELL RECOVERY

Particulates can be removed from aqueous suspension by attachment to rising air bubbles.

This method is known as flotation, which is widely used for recovery of small particles

Ultrasound

FIG. 7.8. Various impeller shapes.

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DOWNSTREAM PROCESSING 181

from aqueous suspension minerals. This method is used in beer processing. A related tech-

nique, for flotation, uses air–water surface tension to strip out proteins from the broth solu-

tion and accumulate them in a high protein. English beer is prepared for cell separation by

air flotation. Flotation is applied to concentrate Acinetobacter cerificans for production of

SCP. Acinetobacter cerificans is a suitable microorganism used in the production of SCP.

The synthesised protein is concentrated by aeration using the flotation method.

7,8

Figure 7.9

shows an aeration vessel with agitation and mechanical foam breakers.

Application of charges and filtration may separate protein very efficiently. Electro-

kinetic deposition uses voltage gradients of 1050V/cm to produce solid biomass with

densities of up to 40% w/v.

7.8 CELL DISRUPTION

Downstream processing of fermentation broths usually begins with separation of cells by fil-

tration or centrifugation. In centrifugation, a vertical rotor, horizontal rotor or even disc types

are used. Sedimentation or coagulation are used in downstream processing. A combination of

bioprocesses is required for product recovery. Separation of products such as ethanol, citric

acid and antibiotics is extracellular but isolation of enzymes inside the cells requires cell rup-

ture. Cells are broken down by lysis of the cell wall. Cell disruption is used for downstream

product recovery. Removal of biomass from the extracted product is necessary. The choice

of process such as filtration, batch or continuous, vacuum filtration, cross flow, etc. is based

on the nature of the product. Biomass separated from the liquid is discarded or sold as a by-

product. For example the products of recombinant proteins, which remain inside the cells,

must be ruptured to release the intracellular products. Two categories of well-defined methods

for cell rupturing are: mechanical grinding with abrasives, high-speed agitation, high pres-

sure pumping and ultrasound; non-mechanical methods such as osmotic shock, freezing and

thawing, enzymatic digestion of cell walls and treatment with solvents and detergents.

A widely used technique for cell disruption is high-pressure homogenisation. Shear forces

generated in this treatment are sufficient to completely disrupt many types of cell. A common

air

SCP

FIG. 7.9. Aeration vessel with foam breaker for SCP production.

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type is the Manton–Gaulin homogeniser.

9,10

In this system, a high-pressure pump incorporates

an adjustable valve with a restricted orifice through which the cells are forced at a pressure of

up to 550 atmospheres. The homogeniser is of general applicability for cell disruption. The

homogenising valve can become blocked when used with highly filamentous organisms.

Stages involved for product recovery are:

• Lysis of cells for intracellular product recovery

• Extraction of lysed or ground cells

• Removal of unconverted soluble substrate

• Removal of biomass from extracted product

• The choice of filtration (batch, continuous vacuum, cross flow, etc.)

• Centrifuge (vertical rotor, horizontal rotor)

• Sedimentation (and/or coagulation)

The process depends on broth conditions (temperature, pH, ionic strength), medium com-

ponents and final state of the desired product.

7.9 SOLVENT EXTRACTION

Many antibiotics have excellent solubility in organic solvents and they are water immiscible.

A multistage extraction separates the aqueous phase from the organic phase. Extraction can

provide concentrated and purified products.

A typical penicillin broth contains 20–35 mg/l of antibiotic. Filtration is used to remove

mycelial biomass from fermentation broth. The filtration may be subjected to filter aided

polymers. Neutralisation of penicillin at pH 2–3 is required. Amyl acetate or butyl acetate is

used as an organic solvent to remove most of the product from the fermentation broth. Finally,

penicillin is removed as sodium penicillin and precipitated by a butanol–water mixture.

Extraction of penicillin from the fermentation broth is normally practised using organic

solvents such as butyl acetate, amyl acetate methyl isobutyl ketone and methyl ethyl ketone

(MEK). Isolation of erythromycin using pentyl or amyl acetate is another example of sol-

vent extraction. For recovery of steroids, solvent extraction is used. Purification of vitamim

and isolation of alkaloids such as morphine and codeine from raw plant materials are

used in solvent extraction methods. Two phases are formed: the separated organic and

aqueous phases. Vigorous mixing requires perfect contact of liquid phases and turbulences

to facilitate solute transfer from the aqueous phase to the organic phase. However, organic

solvents are undesirable for the isolation of proteins. Two phases are produced when a par-

ticular polymer plus salt are dissolved in water above certain concentrations. When bio-

molecules and cell fragments are distributed in the aqueous phase, one phase contains

protein and cell fragments are confined to other phase. The extracted phase goes to other

unit for precipitation or crystallisation. The partition coefficient (k C

) is constant,

where C

is the equilibrium concentration of component “A” in the upper phase and C

is the equilibrium concentration of “A” in the lower phase. If k 1, component “A” favours

the upper phase; if k  1, component “A” favours the lower phase. For effective separation

182 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

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DOWNSTREAM PROCESSING 183

in a single stage of extraction, k 3 is required, for low k, a large volume of solute is also

required. The yield is defined as the ratio solute extracted over the original amount of solute

in the feed:

(7.9.1)

where V

is the original volume, V

is the upper phase volume, and V

is the lower phase

volume.

7.9.1 Product Recovery by Liquid–Liquid Extraction

Solvent extraction is popular for recovery of fermentation products downstream. Antibiotics

are dissolved in an organic solvent, which may be precipitated by converting antibiotic to

salt form and separating it from the organic solvents. Simple alcohol (R-OH, CH

OH,

OH), propanol, butanol and ketones are used in pharmaceutical industries. A simple

separation funnel to show the two phases are clearly separated is shown in Figure 7.10.

Two sequential stages of extraction with fresh solvent are shown in Figure 7.11.

(7.9.1.1)

SFRE

VC VC

oAo

uAu

uAu lAl





Feed

Solvent

FIG. 7.11. Sequence of single stages of extraction with fresh solvent.

Organic

phase

Aqueous

phase

FIG. 7.10. Single-stage extraction in a separation funnel.

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