Module 2: Primary

separation and
Recovery process

Module II - Primary
separation and Recover
•1) Cell disruption process
methods
•2) Filtration
Cell disruption methods,
•3) Centrifugation
Filtration, Centrifugation
Cell Disruption methods
High-pressure homogenization, freezing, sonication,
bead milling and enzymatic disruption of cells, are
commonly used cell disruption methods
All cells have a cytoplasmic membrane that separates
the intracellular organelles of the cell from the
environment it is growing in.

In addition to the cytoplasmic membrane, some cells

contain a cell wall.

When producing substances in living cells, many

products are not secreted through the cytoplasmic
membrane to the growth media, but they stay inside
the cell.

In cell disruption, the cytoplasmic membrane and the

cell wall are broken down so the intracellular products
Various Cell Disruption methods
Mechanical Methods

The most common different mechanical

methods are sonication, high pressure
homogenization (HPH), high-speed
homogenization (HSH), Microfluidization.

Mechanical methods are nonspecific, but

they usually have higher efficiency than
non-mechanical cell disruption methods.
They also have more applications than
non-mechanical methods, mostly based
on cost and effectiveness.
High-pressure homogenization
High-pressure homogenization (HPH) is one of the most widely
used mechanical cell disruption methods. High pressure forces the
cells through a narrow hole or a channel, which creates a shear
force to the cells. Upon discharge from the channel, the cells
expand instantly. This instant expansion after the shear force
disrupts the cells. French press

[Link]
v=ZNrtjeGUoZY&list=PLOZ_QIhovrNTUs_CqTJ2JBR1GtpzLBtSa&index
High-speed
homogenization is a
process used to mix
substances thoroughly by
breaking down particles
and creating uniform
mixtures, often resulting in
stable emulsions. The
process relies on high-
speed rotation of a rotor
against a stationary stator,
generating shear forces
that disrupt particles and
reduce their size.

It's commonly used in

various industries like
food, pharmaceuticals, and
cosmetics to improve
product texture and Lab High Speed Shear Dispersing
Homogenizer Emulsifier Mixer
appearance. Also Essential
with 200-11000r/min
Sonication

In sonication method, pressure waves are generated

by ultrasound. It is suitable especially for small, 1
mL to 2 L, reactors. The mechanism is based on
around 25 kHz sonic waves that generate stable and
transient cavitation within the reactor. Transient
cavitations will begin to oscillate, generating bigger
bubbles and collapse by implosions.

The implosions cause a localized shock wave and

temperature increase. The increased temperature
ionizes its surroundings and the wave causes
enough force shear and disrupt the micro-
organisms’ outer membranes. Stable cavitations are
continuous oscillates that cause micro-scale eddie
currents that can disrupt micro-organisms cell.
Sonochemical
Sonication doesn't lead to the protein denaturation because cell
disruption can be performed at relatively low temperatures.
Chemicals or beads, which have to be removed afterwards, are
not needed in the method.
Ultrasonicators can also be scaled-up. However, when
ultrasonication is used for a long time radicals may be produced,
which can affect quality of the product. Therefore sonication
conditions should be optimized.
Bead milling

Bead mill is composed of grinding chamber filled

with cell suspension.
Agitation shaft transfers kinetic energy to the
grinding elements, such zirconia beads. Shear
forces and collisions of the beads disrupt the cells.

Smaller beads are used to release enzymes in

cytoplasm whereas larger beads release enzymes
in cytoplasmic membrane. Increased bead loading
improves efficiency of cell disruption.

However, also heating and power consumption

are increased. Also impeller tip speed and milling
time have an effect on the level of cell disruption.
Higher disruption is achieved with longer milling
time.
Bead Mill
Microfluidization is a high-pressure homogenization technique in which the
sample is pressurized and modified as a result of high shear rate and impact
forces .

Microfluidization is a unique technique applying high-pressure homogenization to

product streams. In this method, the sample is pressurized and passed through
two geometrically identical micro channels and then two streams are collided
with each other at very high velocities resulting in deformation and breakage of
structures of samples.
Physical methods include Freezing, Osmotic shock,
Thermolysis/ Autoclaving, Microwaving.
Freezing
Freezing and thawing are linked mechanical disruption methods
in which the cell disruption is continuous. The formation and
melting of the ice crystals are the driving forces. If the freezing
is carried out slowly and gradually, the ice crystals become
larger and the cell damage is also higher and might cause
severe damage to the organism e.g. by rupturing the cell walls.
If the product is intracellular, grinding can also be
introduced in combination with freezing to further
improve cell breakage. Freezing method is not too largely
applied because of the low yields and high energy
requirements.
Osmotic shock is also greatly related to thawing,
because of the sudden change in solute
concentration within and around the cell. The
osmosis will also cause shear stress to disrupt the
cell walls.
Enzymatic disruption
In enzymatic cell disruption, enzymes are used
to break down the cell walls and membranes.

The enzymes used can be produced by the cell

itself (autolysis) or foreign (lytic).

Autolysis can be achieved with for example

antibiotics, but foreign lytic enzymes are more
extensively studied.

Enzymatic cell disruption is gentle, specific

and easy to scale up but often time-
consuming.
Schematic overview of the major industrial applications of CWDEs. Specific roles
of the various CWDEs in the different industrial sectors are shown.
The process conditions have to be suitable for the
enzymes, which means that for example temperature,
pH and pressure have to be adjusted carefully.

Specific activity is a major factor in choosing

enzymes for cell disruption. Specific enzyme activity (usually stated simply as 'specific activity') is
the number of enzyme units per ml divided by the concentration of protein in mg/ml.

The enzymes should target the cell wall and

membrane but they should not affect the product.
The enzyme of choice depends on the product and
microbes used in the production.
For example lysozyme enzymes are often used for
bacterial cell disruption. Lysozymes are one of the
most studied and used lytic enzymes, but they
usually can not affect Gram-negative bacteria,
because of the outer cell membrane of the
bacteria…then how??
Cell Wall Degrading Enzymes (CWDEs)
are a heterogeneous group of enzymes
including glycosyl-hydrolases,
oxidoreductases, lyases, and
esterases. Advances in genetic
engineering allowed the expression of
recombinant CWDEs also in lab-
domesticated organisms such as E. coli,
yeasts and plants, dramatically
increasing the available options. Fig:
Plant protoplast using Macerozyme
Enzymatic Cell Lysis

Enzymatic lysis is a biological cell lysis method in which enzymes such as

lysozyme, lysostaphin, zymolase, cellulose, protease or glycanase are used.
Most of these enzymes are available commercially and can be used for large
scale lysis.

One advantage of enzymatic lysis is its specificity. For example, lysozymes are
used for bacterial cell lysis whereas chitinase can be used for yeast cell lysis
and pectinases are used for plant cell lysis. Lysozyme reacts with
peptidoglycan layer and breaks the glycosidic bond. For that reason, gram-
positive bacteria can be directly exposed to lysozyme, however, outer
membrane of the gram-negative bacteria needs to be removed before exposing
the peptidoglycan layer to the enzyme. Lysozyme treatment is generally
conducted at pH 6–7 and at 35-37 °C .

For gram-negative bacteria, lysozyme is used in combination with detergents

to break the cell wall and membrane. Another example is proteinase K which
is used for isolating genomic DNA.
The cells can be pretreated with a detergent,
which dissolves the outer membrane, or the
lysozyme itself can also be modified to be
effective in lysing Gram-negative bacteria.

For example, labiase and achromopeptidase are

commercially available enzymes.

The yeast cell wall is composed mainly of

mannoproteins, glucans and chitin, so several
enzymes, including glucanases, mannanases
and chitinases, are needed.
Enzymes in industry: some examples

•In recent years, calf rennets are replaced by microbial rennets (e.g.
Mucor michei). They are acid aspartate proteases for coagulation of
milk to make cheese
•Invertase from yeast and lactase in food industry
•Cellulase and amylase to remove waxes, oils, and starch
coatings on fabrics and to improve the look of the final
product
•Amylase and protease for baking
•Lipases in fruit juices to break down cell membranes for
increased yield
•Proteases, lipases, amylases, oxidases, peroxidases, and cellulases
in detergents to help break down stains and chemical bonds
•Carbohydrase to convert starch into corn syrup
•Zymase to convert carbohydrates into ethanol in alcoholic
beverages
•Cellulases are used to convert cellulose into glucose to improve
biofuel
•Lipase and phospholipase are used in the production of
biodiesel by converting free fatty acids to fatty acid methyl
esters
•Dextranase, penicillin amidase, penicillin acylase, and subtilisin are
a handful of enzymes used for pharmaceutical products
Chemical cell disruption

In chemical cell disruption, chemical agents,

such as urea, butanol or isopentanol, are used
to disrupt membrane structures of the cells. The
chemicals interact with molecules in the cell wall or
cell membrane resulting in bond breakage in the
structures.

Chemical treatment is often followed by a

mechanical homogenization method and
separating the product with for example
chromatography or gel electrophoresis. These
necessary steps are time consuming and therefore
make the disruption method less efficient. There is
also the danger of toxic residues left in the product.
Here are some common chemical cell disruption agents:

[Link]: Detergents like Triton X-100, Tween 20, and SDS

(sodium dodecyl sulfate) are used to disrupt cell membranes by
disrupting lipid bilayers and promoting the solubilization of membrane
proteins. Detergents are effective for breaking down cell membranes
and releasing intracellular components.
[Link] and Bases: Strong acids (e.g., hydrochloric acid) and bases
(e.g., sodium hydroxide) can be used to disrupt cells by breaking
down their structural components. However, this method can be harsh
and may result in the denaturation of proteins and other
biomolecules.
[Link] Solvents: Organic solvents like ethanol, methanol, and
acetone can disrupt cell membranes and denature proteins. They are
often used in conjunction with mechanical methods to enhance
disruption efficiency.
[Link] Agents: Chelating agents like EDTA
(ethylenediaminetetraacetic acid) can be used to disrupt cells by
chelating metal ions that stabilize cell structures.
[Link] Shock Agents: Substances like sucrose or sorbitol can
create osmotic imbalances that cause cells to swell and burst. This
method is often used in combination with mechanical disruption
techniques.
[Link]: Surfactants, like octanoic acid or guanidine
Alkaline Lysis

In alkaline lysis, OH− ions are the main component used for lysing
cell membrane. The lysis buffer consists of sodium hydroxide and
sodium dodecyl sulphate (SDS).

The OH− ion reacts with the cell membrane and breaks the fatty
acid-glycerol ester bonds and subsequently makes the cell
membrane permeable and the SDS solubilizes the proteins in the
membrane. The pH range of 11.5–12.5 is preferable for cell lysis.
Although this method is suitable for all kinds of cells, this process
is very slow and takes about 6 to 12 h. This method is mostly used
for isolating plasmid DNA from bacteria.
Apart from ionic and non-ionic detergents, chaotropic agents can also be used
for cell lysis. These include urea, guanidine and Ethylenediaminetetraacetic
acid (EDTA) which can break the structure of water and make it less
hydrophilic and there by weakening the hydrophobic interactions. An
additional purification step has to be in cooperated into the cell lysis protocol
when using detergents
 Filteration
Typically in the field of bioprocess technology, the
highest equipment and operating cost exist in the
downstream processing of fermentation products.
Manufacturing costs, including capital and operational
expenses, of downstream processing part can be up to
50 % of the overall costs.
Often the situation is that operational costs exceed clearly all
other expenses and thus any improvements in the efficiency of
downstreamof
Principles processing can have significant effects in the
filtration
profitability of the process.
Filtration is one of the most common methods for the
downstream processing of fermentation products and it
is used at all production scales.
Basic principle in filtration is to separate the suspended
particles or larger molecular mass components from bulk
liquid with porous medium. Driving force can be pressure
difference, vacuum, concentration gradient, valence or
electrochemical affinity