Downstream Processing

Question 1

What is downstream processing

Answer 1

The various stages of processing that occurs after the completion of the fermentation or bioconversion stage, including separation, purification and packaging of the product.

Question 2

4 stages in downstream processing

Answer 2

• Removal of Insolubles
• Product Isolation
• Product purification
• Product purification

Question 3

What and how the removal of insoluble achieved (5 ways)

Answer 3

It is the separation of cells, cell debris or other particulate matter.
1) Filtration
2) Centrifugation
3) Sedimentation
4) Flocculation
5) Gravity settling

Question 4

What is Flocculation

Answer 4

A process where a solute comes out of solution in the form of flakes.

Question 5

Describe the process filtration

Answer 5

A mechanical operation used for the separation of solids from fluids (liquids or gases) by interposing a medium to porous membrane through which the fluid can pass, but solids in the fluid are retained.

Question 6

What is a filter cake

Answer 6

The solid particles deposited on the filter to form a layer is known as a filter cake. All solid particles from the feed are stopped by the cake, and the cake grows at the rate at which particles are brought to its surface. All of the fluid goes through the cake and filter medium.

Question 7

One of the most commonly used type of filter in fermentation.

Answer 7

Continuous Rotary Vacuum filter

Question 8

How does a continuous rotary vacuum filter work?

Answer 8

1) The drum is pre-coated prior of filtration.
2) A small agent of coagulating is added to the broth before it is pumped into the filter.
3) The drum rotates under vacuum and a thin layer cells sticks to the drum.
4) The thickness of the layer increases in the section designed for forming the cake.

Question 9

Points to be considered while selecting the filter medium

Answer 9

• Ability to build the solid
• Minimum resistance to flow the filtrate
• Resistance to chemical attack
• Minimum cost

Question 10

What is centrifugation

Answer 10

Used to separate particles of 100 - 0.1 micrometer from liquid by gravitational forces.
Use of centrifugal force for the separation of mixtures.
More-dense components migrate away from the axis of the centrifuge.
Less-dense components migrate towards the axis.

Question 11

What parameters does centrifugation depend on?

Answer 11

• Particle size
• Density difference between cells and the both and broth viscosity.

Question 12

What are the types of centrifuges?

Answer 12

-Tubular bowl centrifuge
-Multichamber centrifuge
-Disc Bowel centrifuge

Question 13

What is sedimentation

Answer 13

It’s a free settling process depends only on gravity.
Particles settling is a high particle density suspension (hindered settling).
Applicable only for large particles greater than 100 micrometers flocs.
It is a slow process and takes ~ 3 hours.

Question 14

Uses of sedimentation

Answer 14

Process like activated sludge effluent treatment.

Question 15

What is Flocculation

Answer 15

Process where a solute comes out of solution in the form of flocs or flakes.

Question 16

How does Flocculation work

Answer 16

Particles finer than 0.1 um in water remain continuously in motion due to electrostatic charge which causes them to repel each other.
Once their electrostatic charge is neutralized (use of coagulant), the finer particles start to collide and combine together.
These larger and heavier particles are called flocs.

Question 17

What is accomplished by product isolation

Answer 17

• remove those components whose properties vary markedly from that of the desired product.
• isolation steps are designed to remove it (dialysis)
• reducing the volume
• concentrating the product
• liquid - liquid extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved.

Question 18

Methods for product isolation

Answer 18

) liquid - liquid extraction
) Adsorption
) Ultrafiltration
) Precipitation

Question 19

What is Liquid-Liquid extraction

Answer 19

It is separation process that takes the advantage of the relative solubilities of solute in immiscible solvents.

Question 20

How does liquid-liquid extraction work

Answer 20

Solute is dissolved more readily and becomes more concentrated in the solvent in which it has a higher solubility.
A partial separation occurs when a number of solutes have different relative solubilities in the two solvents used.
Solvent should be non toxic, selective, inexpensive and immiscible with broth and should have a high distribution coefficient for the product.

Question 21

What is Adsorption and how does it work?

Answer 21

It is a surface phenomenon. It is the binding of molecules to the surface and different from absorption.
The binding to the surface is weak and reversible.
Compounds containing chromogenic group are usually adsorbed on activated carbon.
Common adsorbent used are activated carbon, silica gel, alumina because they present enormous surface areas per unit weight.

Question 22

What are common adorbents used adsorption and why they are used?

Answer 22

• Carbon
• Silica gel
• Alumina
  They present enormous surface areas per unit weight.

Question 23

What is Ultrafiltration?

Answer 23

Ultrafiltration is basically a pressure driven separation process.
Governed by a screening principle and dependent on particle size.
The membranes have a pore size between 1 nm and 100 nm, allowing retention of compounds with a molecular weight of 300 to 500 000 Dalton.

Question 24

What is Ultrafiltration suitable for?

Answer 24

Is suitable for retaining biomolecules, bacteria, viruses, polymers, colloidal particles and sugar molecules.

Question 25

What is precipitation?

Answer 25

Formation of a solid in a solution during a chemical reaction. Solid formed is called the precipitate and the liquid remaining above the solid is called the supernate.

Question 26

How does precipitation work?

Answer 26

Salts such as ammonium & sodium sulphate are used for proteins to precipitate.
Organic solvents methanol used to precipitate dextrans.
Chilled ethanol and acetone used for protein precipitation.
Non ionic polymer such as polyethylene glycol used in precipitation.

Question 27

What is product purification done for?

Answer 27

Done to separate those contaminants that resemble the product very closely in physical and chemical properties.
Expensive to carry out
Require sensitive and sophisticated equipment.
Significant fraction of the entire downstream processing expenditure.

Question 28

Product purification examples

Answer 28

• Affinity, size exclusion, reversed phase chromatography.
• Crystallization
• Fractional precipitation

Question 29

Chromatography

Answer 29

• Separation of mixtures
• Passing a mixture dissolved in a “mobile phase” through a stationary phase, which separates the analyte to measured from other molecules

Question 30

Ion exchange chromatography

Answer 30

Used charged stationary phase to separate charged compounds. Resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained.

Question 31

Define Ion

Answer 31

Ion is an atom or molecule which has lost or gained one or more valence electrons, giving it a positive or negative electrical charge. Anions are negatively charged ions, formed when an atom gains electrons in a reaction. Cations are positively charged ions, formed when an atom loses electrons in a reaction, forming an electron hole.

Question 32

What is Affinity chromatography

Answer 32

Separates the protein of interest on the basis of reversible interaction between it and its antibody coupled to a chromatography bead. With high selectivity, high resolution and high capacity for the protein of interest, purification levels in the order of several thousand-sold are achievable. The concentrating effect enables large volumes to be processed. The protein of interest can be purified from high levels of contaminating substances. Making antibodies to the protein of interest is expensive, so affinity chromatography is least economical choice for production chromatography.

Question 33

How does affinity chromatography work?

Answer 33

Biological interactions between the antigen and the protein of interest can result from electrostatic interactions, van der Waals’ forces and/or hydrogen bonding. To elute the protein of interest from the affinity beads, the interaction can be reversed by changing the pH or ionic strength. The protein of interest is collected in a purified, concentrated form.

Question 34

What is size exclusion chromatography

Answer 34

Known as gel permeation/filtration chromatography (GPC).
Separates molecules according to their size.
Low resolution “polishing”.
Tertiary/ Quaternary structure (native).

Question 35

What is Revered phase chromatography

Answer 35

Is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.

Question 36

Define polarity

Answer 36

The dipole-dipole intermolecular forces
between the slightly positively-charged end of one molecule to the negative end of another or the same molecule.
Molecular polarity is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound’s structure.

Question 37

What is liquid chromatogaphy

Answer 37

separates molecules in a liquid mobile phase using a solid stationary phase.
Carried out either in column or a plane.
In column liquid chromatography, as the liquid mobile phase passes through the column, components in the mobile phase interact to varying degrees with the solid stationary phase, also known as the chromatography media or resin. Molecules of interest in the mobile phase are separated based on their differing physicochemical interactions with the stationary and mobile phases.

Question 38

Types of Liquid Chromatography

Answer 38

Reversed-Phase Chromatography
Normal Phase Chromatography
Ion Exchange Chromatography
Size Exclusion Chromatography

Question 39

What is HPLC

Answer 39

High-performance liquid chromatography (HPLC) is a technique in analytical chemistry used to separate the components in a mixture, and to identify and quantify each component. The sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure

Question 40

What is crystallization

Answer 40

Process of formation of solid crystals precipitating from a solution, melt or more rarely deposited directly from a gas.

Question 41

How is the process of crystallization used as a separation technique.

Answer 41

It is a chemical solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs.

Question 42

What is done during product polishing

Answer 42

) End with packaging of the product in a form that is stable, easily transportable and convenient.
) Crystallization
) Desiccation
) Lyophilization
) Spray drying

It also may include:
Sterilization of the product
Remove or deactivate trace contaminants which might compromise product safety viruses or depyrogenation

Question 43

What is lyophilization

Answer 43

Includes freezing the material and reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.