Chapter 5

Downstr eam Pr ocessing

Downstream processing, what and why

Downstream processing is any treatment of culture broth after fermentation to concentrate and purify products.

Separation Processes

The challenge
Low product concentrations Large number of impurities Thermolabile bioproducts

An ideal bioseparation process should combine high throughput with high selectivity, and should ensure stability of product.

Genetic modification

Microb es

Happy growing

Nutrie nts Oxyge Fermentat n ion Drug producing

Downstream Processing
Smash the microbes Remove cells/debris Kill the microbes

Concentrate and Purify the product

Formulate product

Market

Biological products
Product
Alcoholic beverages: Beer, wine, spirits Organic acids: Acetic acid, citric acid Vitamins: Vitamin C, vitamin B12 Amino acids: Lysine, glycine, Antibiotics: Penicillins

Nature of required

bioseparation

Clarification, distillation Precipitation, filtration, adsorption Precipitation, filtration, adsorption Precipitation, filtration, adsorption Precipitation, filtration, adsorption

Enzyme,Polysaccharides Filtration, precipitation, centrifugation, adsorption, chromatography

Distillation
Separating components with different volatilities. Such as organic solvents , volatile flavors and aromas.

Distillation

Stages in Downstream Processing

Removal of insoluble's Product Isolation Product Purification Product Polishing

1. Cell removal (filtration, centrifugation , coagulation and flocculation) 2. Primary isolation (adsorption, precipitation)
Removing components with properties significantly different from those of the products Large volume, relatively non selective

3. Purification (chromatography, ultrafiltration)

Removing components with properties similar to those of the products Highly selective

4. Product preparation (crystallization, drying)

Removal of insoluble's
Capture of the product as a solute in a particulate-free liquid Example Separation of cells, cell debris or other particulate matter from fermentation broth containing an antibiotic.

Typical Operations
Filtration
A mechanical operation used for the separation of solids from fluids.

Type of filtration unit

Plate and frame filter

16

Rotary-drum vacuum filter

Centrifugation
Use of the 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

Centrifuges
t
t

Decanter centrifuge

Coagulation and Flocculation

Coagulation: Forming semisolid lumps in a liquid. Flocculation: Forming woolly cloudlike aggregations.

Product Isolation
Reducing the volume of material to be handled and concentrating the product.

Precipitation
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 supernatant.

1. Isoelectric Precipitation
GA+ GA= pH >12 pI <2 3.22 7.0 GA GA

2. Organic solvent precipitation

By adding an organic solvent to an aqueous fermentation broth, the dielectric constant will decrease causing the solubility to decrease.

Example
Xanthan gum is recovered from an aqueous fermented broth containing the gum by adding to the broth organic solvent.

3. Insoluble salt precipitation

Usually used in extraction of amino acid and organic acid.

e.g.
Citric acid extraction and purification process Heating (destroy the microorganisms ) Neutralizition (forming calcium citrate ) Acidifying (liberate the citric acid from its salt )

Disadvantages
high chemical cost relatively complex technology

Ion Exchange Resins

Resins
Organic

or inorganic polymer used to exchange cations ( positive ions) or anions ( negative ions) from a solution phase

General Structure
Polymer

backbone not involved in bonding group for complexing anion or cation

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Functional

Organic Resin Groups

SO3 H

Linkage group

Cation exchange

CH2 Cl

CH 2 N(CH3 )3 Cl

Chloride

Anion exchange

e.g.
Purification of Lactic Acid from Fermentation Broths by Ion-Exchange Resins First, a strong cation resin was used to reduce the broth pH and remove the cations present in the solution; Then the acidified broth was purified by an anion exchanger.

Membrane Separation
Function : clarification and sterilization

Advantages
No phase change Energy efficient A higher quality product Environmentally friendly Easy to scale-up

Limitations
Long-term reliability not proven Excessive pretreatment Concentration polarization and membrane fouling Replacement of membranes

Membrane Fraction Technologies

W at er Sa lts La ct o Pr se ot ei M n + icr fa ob t es

Nanofiltration (200 - 700 MW) Reverse Osmosis (<200 MW)

Ultrafiltration (2000 - 50,000 MW)

Microfiltration (100,000 MW)

Semi-permeable Membrane

Wine Filtration Why?

Filtration Objectives/Purposes Include Remove insoluble contaminants Improve clarity, brightness Extend shelf life

Electrodialysis

Product Polishing
Final processing steps which end with

packaging of the product in a form that is stable, easily transportable and convenient
Crystallization, concentration and drying

are typical unit operations

Crystallization
Formation of a crystalline phase from a parent phase, e.g. solution One of the oldest and most important unit operations, e.g. extracting salt crystals from sea water

Definition of Crystal
A substance in which the constituent atoms, molecules, or ions are packed in a regularly ordered, repeating three-dimensional pattern. The distinction between a crystal and an amorphous solid is that between order and disorder over large distances

Crystallization Process
Liquid Mixture Generation of Supersaturation :Driving force Nucleation: Birth of Solid Phase

Final Product

Crystal Growth

Supersaturation refers to a state in which the liquid (solvent) contains more dissolved solids (solute) than can ordinarily be accomodated at that temperature.

Solubility & Supersolubility Diagram

Generation of supersaturation
Solvent evaporation Solution cooling Altering pH

Crystallization
- by evaporation of saturated solution

Crystallization
- by cooling saturated solution

Concentration
Filtration Centrifugation Reduced pressure concentration

Drying
Drying involves the transfer of heat to the wet material and removal of the moisture as water vapor. Usually, this must be performed in such a way as to retain the biological activity of the product.

Drying Methods
1 Atmospheric drying 2 Vacuum drying (Decompression ) 3 Freeze-drying (lyophilization) 4 Spray drying

lyophilization
Freezing the material 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.

Review Questions
1. Show the importance and main procedures of the downstream processing of fermentation. State the principles and the characteristics of every separation methods. 2. Tell the difference between freeze drying and vacuum drying.