Downstream Processing: Cell disruption method

Downstream processing: Cell
disruption methods
By: Khushboo Mishra
M.Sc. 4th
semester
Department of microbiology

INTRODUCTION:
 Downstream processing refers to the recovery and purification of biosynthetic
products, particularly pharmaceuticals from natural sources such as animal or
plant tissue or fermentation both including the recycling of salvageable
components and the proper treatment and the disposal of waste.
 Downstream processing is the recovery and purification of biochemical
products with proper treatment. It is a series of events which includes cell
separation, filtration, product recovery, extraction of product and purification
and then treatment of product by chemical, physical and biological means. This
is a necessary step in order to obtain a purified form of the product.
 Downstream processing is the process of isolation, purification, and separation
of products from recombinant DNA technology (RDT).
 This is an essential step in the manufacture of pharmaceuticals such as
antibiotics, hormones for example insulin and human growth hormone,
antibodies for example infliximab and abciximab, and vaccines; and antibodies
and enzymes are used in diagnostics; industrial enzymes and natural fragrance
and flavor compounds.

WHAT IS COMMON IN DOWNSTREAM TECHNOLOGIES?
• The product is in aqueous solution.
• Multiphase system: water, +solid, +oily, (+air bubbles)
• Complex system: many organic and inorganic substances, in solute, colloid
and dispersed form
WHAT IS DIFFERENT?
• Wide range in product concentration: 100 ppm → 10%-ig.
• Wide range in production scale: 100 g/year → 1.000.000 t/year.
• Many different operations (more than in chemical industry)

OPERATIONAL SEQUENCE
There are no fixed operational sequences but general guide-lines:
1. Separation of cells: If the presence of the biomass or cells causes trouble,
they have to be removed. The very first step in downstream processing is
solid-liquid separation, in which the cells (and other solids: medium pellets,
CaCO3, product crystals) are separated from the fermentation broth. This can
be done by
• filtration,
• centrifugation,
• sedimentation,
• flocculation or
• gravity settling.
Each of these processes has unique principle by which it works to separate the
solid content from liquid broth.
2. Disintegration of Cells:
• Disruption of microbial cells is usually difficult due to their small size, strong
cell wall and high osmotic pressure inside cells.
• Generally, cell disruption is achieved by mechanical means, lysis or drying.

• The method of cell disruption must not damage the product of interest; the
suitability of the methods is usually assessed in terms of recovery of a cellular
enzyme activity following cell disruption:
Mechanical Cell Disruption:
• This approach uses shear, e.g., grinding in a ball mill, colloid mill etc., pressure
and pressure release, e.g., homogenizer and ultrasound.
• A widely used method is as follows; the cell suspension is forced through a fine
nozzle; the cells disintegrate due to hydrodyanic shear and cavitation.
Drying:
• The cells may be dried, e.g., by adding the cells into a large excess of cold
acetone and subsequently extracted using buffer or salt solutions.
• Drying induces changes in cell wall structure which facilitates extraction. This
method is widely used.
Lysis:
• Microbial cells may be lysed by chemical means, e.g., salts or surfactants,
osmotic shock, freezing, or by lytic enzymes, e.g., lysozyme etc.
• In general, recovery of enzyme activity is the best following cell disruption
using enzymes or ultrasound, followed by thermal and osmotic methods, while
mechanical methods are the least desirable.
• However, ultrasound method is confined to laboratory mainly due to difficulties
in heat removal on a large scale.

3. Extraction:
• The process of recovering a compound or a group of compounds from a mixture
or from cells into a solvent phase is called extraction.
• Extraction usually achieves both separation as well as concentration of the
product. It is especially useful for the recovery of lipophilic substances, and in
antibiotic recovery; it is often an early step after cell separation.
Liquid-Liquid Extraction:
• It employs two immiscible liquids into which the product is differentially
soluble.
• Usually successively smaller volumes of the solvent are used for repeated
extraction of a given sample; back-extraction also tends to increase the
selectivity of extraction.
• The extraction may be performed in a single step, by multi-stage parallel-flow
extraction, or by counter-current extraction (most complex but most effective).
Whole Broth (Medium + Cells) Extraction:
• It should be used wherever possible since it reduces the number of steps as well
as product loss.
• The effectiveness of extraction may, however, be reduced due to the presence of
cells.

Aqueous Multiphase Extraction:
• It is used for separation of enzymes from cells/cell debris.
• The enzymes are extracted in an aqueous polyethylene glycol-dextran
mixture which form separate phases.
• Recovery of enzymes from these phases is rather easy and free from some of
the difficulties encountered in centrifugation.
4. Concentration:
Some concentration of the product may occur during the extraction step.
Further concentration may be achieved by:
(i) Evaporation,
(ii) Membrane filtration,
(iii) Ion exchange methods and
(iv) Adsorption methods.
Evaporation:
• It is generally used in cases of solvent extraction using various devices, e.g.,
continuous flow evaporators, falling film evaporators, thin film evaporators,
centrifugal thin film evaporators and spray- dryers.
• Efficient arrangements must be made for recovery of the evaporated solvent
to reduce costs. For low grade products, often evaporation of the whole broth
is undertaken using a spray-drier.

Membrane Filtration:
• It generally achieves both concentration and separation of the products usually
based on the size of molecules.
• The different processes of membrane filtration are: microfiltration,
ultrafiltration, reverse osmosis and electro-dialysis.
• Micro- and ultrafiltration work as sieves and separate molecules of different
sizes, but reverse osmosis can separate molecules of similar size. Microfiltration
can be used for cell separation as well.
Ion Exchange Resins:
• These are polymers having firmly attached ionizable groups (anions or cations)
which ionize under a suitable environment.
• These may be solid, e.g., dextran, cellulose, polyamine, acrylate etc., or liquid,
e.g., a solvent carrying a functional group like phosphoric acid mono- or diester
etc.
Solid ion exchangers may be used in two ways:
(i) They may be packed in columns or
(ii) They may be added to the extract and removed by decantation.

• Liquid ion exchangers dissolve only in non-aqueous solvent carrier and the
separation is similar to liquid-liquid extraction.
• Some antibiotics are recovered directly from the whole broth using ion
exchange resins. The product is recovered from the ion exchangers by ion
displacement; this also regenerates the ion exchanger.
Absorption Resins:
• These are porous polymers without ionization. Most compounds are adsorbed
to the resins in non-nonized state.
• The porosity of the resin determines the surface available for adsorption.
• These resins may be apolar (e.g., styrene-divinyl bcneze), polar (e.g., sulfoxide,
amide etc.), or semipolar (e.g., acrylic ester). The products are recovered from
such resins by solvent (organic) extraction, changed pH etc.
5. Purification:
• The final step in the recovery of a product is purification which aims at
obtaining the product in highly purified state.
• The earlier steps will have achieved variable degrees of purification which may
determine the degree of resolution necessary during the purification step.
• The degree of resolution will mainly depend on the similarities to the
metabolite of other molecules present in the concentrate, and the degree of
purity required in the final product.

Purification is achieved by:
(i) Crystallization and
(ii) Chromatographic procedures.
Crystallization:
• It is mainly used for purification of low molecular weight compounds like
antibiotics, e.g., penicillin G is usually extracted from fermentation broth in
butyl acetate and cystallized by the addition of potassium acetate in ethanolic
solution.
• Crystallization is the final stage in purification of products like citric- acid,
sodium glutamate etc.
Chromatographic Methods:
• These are used for purification of low molecular weight compounds from
mixtures of similar molecules, e.g., homologous antibiotics, and of
macromolecules, especially enzymes, which are similar in properties.
• The materials used for chromatography are generally coated on particulate
carriers which are packed in columns through which the liquid containing the
product is pumped either upward or downward.
• The separated product is recovered in some sort of fraction collector. On a
large, scale organic solvents are used for collection; therefore, the whole system
has to be installed in a flame-proof and explosion- proof room.

The different chromatographic procedures are:
(i) Adsorption,
(ii) Ion exchange,
(iii) Gel filtration,
(iv) Hydrophobic,
(v) Affinity,
(vi) Covalent and
(vii) Partition chromatography.
• Adsorption chromatography separates molecules due to their differential affinities
for the surface of a solid matrix, e.g., silica gel, alumina, hydroxyapatite (all
inorganic) or an organic polymer.
• In case of ion exchange chromatography, resins or polysaccharides, e.g., cellulose,
sepharose, having attached ionized functional groups are used for a high resolution
separation of macromolecules, e.g., proteins.
• Gel filtration uses molecular sieves, composed of neutral cross-linked carriers
(e.g., polymers like agarose, dextran’s), of different pore sizes. Molecules smaller
than the pore size enter the carrier and are retained; they are later eluted (in order
of molecule size) and collected.
• Gel filtration is used in aqueous systems. Hydrophobic carriers are used for
purification of hydrophobic molecules, e.g., many enzymes and other proteins.

6. Drying:
• Drying makes the products suitable for handling and storage. It should be accomplished
with a minimum rise in temperature due to heat sensitivity of most products.
• Addition of sugars or other stabilizers improves the heat tolerance of some
products like enzymes and pharmaceutical preparations.
The most common approaches to drying are as follows:
(i) Vacuum drying,
(ii) Spray drying and
(iii) Freeze drying.
 In spray drying, the solution or slurry to be dried is atomized by a nozzle or a
rotating disc. A current of hot (150-250°C) air is passed; the drying is so rapid
that the temperature of particles remains very low.
 Spray drying is used for enzymes, antibiotics, and food products. Vacuum
drying uses both heat and vacuum for drying; it can be applied both in batch
mode (e.g., chamber dryers) or in continuous mode (e.g., rotating drum vacuum
dryers).
 In freeze drying, the liquor to be dried is first frozen and the water is sublimed
from the frozen mass. A very low pressure (partial vacuum) is maintained to
promote sublimation of water. The energy needed for sublimation is provided
by heated plates and radiation on to the surface.

 The temperature of solid is regulated by regulating the pressure in the drying chamber.
This is the most gentle method of drying, and is used for many pharmaceutical products,
e.g., viruses, vaccines, plasma fractions, enzymes etc., and in food industries.

Cell Disruption Methods
 Cell disruption is the process of obtaining intracellular fluid via methods that
open the cell wall.
 The overall goal in cell disruption is to obtain the intracellular fluid without
disrupting any of its components.
 The method used may vary depending on the type of cell and its cell wall
composition.
 Irrespective of the method used, the main aim is that the disruption must be
effective and the method should not be too harsh so that the product
recovered remains in its active form.
 Cell disruption methods can be categorised into mechanical methods and
non-mechanical methods.
 Mechanical methods are divided into solid shear methods and liquid shear
methods.
 Non-mechanical methods can be divided into physical methods, chemical
methods and enzymatic methods.

Mechanical Methods of Cell Disruption
Mechanical methods are those methods that required some sort of force to separate
out intracellular protein without adding chemical or enzyme.
1. Mortar & Pestle
• It involves the grinding of the cells such that they are disrupted.
• This does not have to be in suspension and is often done with plant samples
frozen in liquid nitrogen.
• When the material has been disrupted, metabolites can be extracted by adding
solvents.
2. Blenders
• The use of blenders which employ high speed can be used to disrupt cell walls.
• It is the same process used by centrifugation, which separates or concentrates
materials suspended in a liquid medium.
3. Bead beating
• Glass or ceramic beads are used to crack open cells
• The kind of mechanical shear is gentle enough to keep organelles intact.
• It can be used with all kinds of cells, just add beads to an equal amount of cell
suspension and vortex.

Bead beating
4. Ultrasonication
• Ultrasonic homogenizers work by inducing vibration in a titanium probe that is
immersed in the cell solution.
• A process called cavitation occurs, in which tiny bubbles are formed and explode,
producing a local shockwave and disrupting cell walls by pressure change.
• This method is very popular for disruption of plant and fungal cells.

 High frequency vibration (- 20 kHz) at the tip of an ultrasonication probe leads to
cavitation, and shock waves thus produced cause cell disruption. The method can be
very effective on a small scale, but a number of serious drawbacks make it unsuitable
for large-scale operations.
 Power requirements are high, there is a large heating effect so cooling is needed, the
probes have a short working life and are only effective over a short range. Continuous
laboratory sonicators with hold-up volumes of around 10 cm3 have been shown to be
effective

5. Homogenization
• Liquid-based homogenization is the most widely used cell disruption technique for small
volumes and cultured cells.
• Cells are lysed by forcing the cell or tissue suspension through a narrow space
• Homogenizers use shearing forces on the cell similar to the bead method.
• Homogenization can be performed by squeezing cells through a tube that is slightly
smaller than beads beating.

Non-Mechanical Methods of Cell Disruption
Non mechanical methods are further divided into three classes which are following:
A. Physical methods:
1. Freeze-Thaw
• It is suitable when working with soft plant material and algae.
• Disruption is achieved via a series of freezing and thawing cycles.
• Freezing forms ice crystals, which expand upon thawing, and this ultimately
causes the cell wall to rupture.
2. Microwave/ Thermolysis
• Microwave (along with autoclave and other high temperature methods) are used
to disrupt the bonds within cell walls, and also to denature proteins.
• However, uncontrolled amount of heat can easily denature or damage target
proteins and subtances.

3. Osmotic Shock
• Through the process of osmosis, water can be moved into the cell causing its volume to
increase to the point that it bursts.
• The method however, can only work with animal cells and protozoa, since they do not
have cell walls.

4. Electric Discharges
• It is also possible to achieve cell disruption via electrical discharges in
mammalian and other cells that are bounded by plasma membranes only.
B. Chemical methods
 They are often used with plant cells (and sometimes in combination with
shearing).
 Organic solvents such as toluene, ether, benzene, methanol, surfactants, and
phenyl ethyl alcohol DMSO can be used to permeate cell walls.
 EDTA can be used specifically to disrupt the cell walls of gram negative bacteria,
whose cell walls contain lipopolysaccharides that are stabilized by cations like
Mg2+ and Ca2+.
 EDTA will chelate the cations leaving holes in the cell walls.
DETERGENTS
• A number of detergents will damage the lipoproteins of the microbial cell
membrane and lead to release of intracellular components.
• The compounds which can be used for this purpose include quaternary
ammonium compounds, sodium lauryl sulphate, sodium dodecyl sulphate (SDS)
and Triton X-100.

• Unfortunately, the detergents may cause some protein denaturation and may
need to be removed before further purification stages can be undertaken.
• Pullulanase is an enzyme which is bound to the outer membrane of Klebsiella
pneumoniae.
• The cells were suspended in pH 7.8 buffer and 1% sodium cholate was added.
The mixture was stirred for 1 hour to solubilize most of the enzyme.
• The use of Triton X-100 in combination with guanidine-HCl is widely and
effectively used for the release of cellular protein obtaining greater than 75%
protein release in less than one hour from Escherichia coli under fermentation
condition.

Enzymatic methods
• Another strategy to achieve cell lysis is to use digestive enzymes which will
decompose the microbial cell wall.
• Different cell types and strains have different kind of cell walls and membranes,
and thus the used enzyme depends on microbe. For example, lysozyme is
commonly used enzyme to digest cell wall of gram positive bacteria. Lysozyme
hydrolyzes β-1-4-glucosidic bonds in the peptidoglycan.
• The cell wall of yeast and fungi differs significantly from the cell wall bacteria.
One commonly used enzyme mixture for degradation of cell wall of yeast and
fungi is Zymolyase.
• It has for example β-1,3 glucanase and β-1,3-glucan laminaripentao-hydrolase
activities (Zymolyase , Yeast lytic enzyme).
• In addition, the enzymes that are commonly used for degradation of cell wall of
yeast and fungi include different cellulases, pectinases, xylanases and chitinases.
• Enzymes such as beta(1-6) and beta(1-3) glycanases, proteases and mannase can
also be used to disrupt the cell wall.

Downstream Processing: Cell disruption method

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