The Recovery Process of

Products after Fermentation

Densel D.T Matambo
R212872H
Introduction
• After the production of the desired fermentation product, the next most important
step includes product recovery and purification.
• It is also known as the Downstream Processing.
• Downstream processing includes many technologies for laboratory and industrial-
scale separation of biological products.
• The Downstream Processing of fermentation products may be difficult and costly.
• The objective is to obtain a high quality product as fast as possible at an effective
recovery rate using least costs.
• The recovery costs of microbial products may vary from as low as 15% to as high
as 70% of the total manufacturing costs.
• The chosen process, and therefore its relative cost, will depend on the specific
product.
WHY RECOVERING PRODUCTS?
• At the time of harvesting, the product concentration may be low in an aqueous
solution, which may contain microorganisms, cell debris, soluble and insoluble
media components and other metabolic products.
• The product may also be intracellular, heat labile and easily damaged by
contaminating microorganisms which increase the difficulties of product recovery
SO,
• If product is heat labile, speed of operation may be the dominant factor in noble
product recovery.
• To ensure the proper processing of harvested broth within a reasonable time limit
processing equipment must be of the correct type and of correct size.
The choice of recovery process is based on the following criteria:
• The intracellular or extracellular location of the product.
• The concentration of the product in the fermentation broth.
• The physical and chemical properties of the desired product (as an aid to select
separation procedures).
• The intended use of the product.
• The minimal acceptable standard of purity.
• The magnitude of biohazard of the product or broth.
• The impurities in the fermenter broth.
• The market potential of the product.
It is an essential step in the manufacture of many products such as
• antibiotics
• hormones
• industrial enzymes
• natural fragrance
• flavor compounds
Possible problems in fermentation and product purification
• time of harvest
• pigment production
• ionic strength
• culture medium constituents.
Schematic process of recovering
fermentation products
Stages of downstream processing
Stage 1 -Removal of Insoluble
• The first stage for the recovery of an
extracellular product aims to remove large
solid particles and microbial cells.
• It is possible by various measures like by
centrifugation, filtration, sedimentation,
precipitation, flocculation, electro-
precipitation, and gravity settling.
Stage 2 -Product Isolation
• In this stage, the broth is extracted into major fractions using various techniques
like ultrafiltration, reverse osmosis, adsorption/ion-exchange/ gel filtration or
affinity chromatography, liquid-liquid extraction, two phase aqueous extraction,
precipitation etc.
• It allows the removal of unnecessary components whose properties differ
distinctly from that of the desired product.
Stage 3 -Product Purification
• Once, the product containing fraction is purified by fractional precipitation,
further more precise chromatographic techniques and crystallization is used to
obtain a highly concentrated product which is essentially free from impurities.
• Steps in this stage are expensive because it requires sensitive and sophisticated
equipment which contributes a major section of the entire downstream processing
expenditure.
Stage 4 Product Polishing
• It is the last processing steps which end with packaging of the product.
• It includes Crystallization, desiccation and spray drying.
• Sometime it also includes operations to sterilize the product by removing
contaminants which otherwise affect product safety.
Recovery of Penicillin G
The harvested broth from the fermenter is chilled
to 5-10 Degrees Celsius. The P.chrysogenum is
filtered off by the rotary vacuum filter. The
filtrate is acidified by sulphuric acid to pH 2.0-
2.5. Penicillin is extracted from the aqueous
filtrate into butyl acetate in a centrifugal counter-
current extractor where treatment and disposition
of the aqueous phase. Penicillin is extracted from
the butyl acetate into a aqueous buffer, in a
centrifugal counter-current extractor, butyl
acetate is recovered and recycled. The aqueous
fraction is acidified by sulphuric acid to pH 2.0-
2.5, penicillin is reextracted into butyl acetate.
Potassium acetate is added in the crystallization
tank along with the organic extract to crystallize
the penicillin as potassium salt. Crystals are
recovered in a filter centrifuge. Futhure
processing of the penicillin salt is carried out to
remain with penicillin.
Removal of microbial cells and other solid matter:
• Normally separated from the harvested broth by filtration or centrifugation
• It is common practice to use filter aids when filtering is slow.
• Charged properties of microbial cells to be exploited by electrophoresis and di-electrophoresis and
flocculation characteristics and magnetic separations to be improved by ultrasonic treatment .
Foam separation:
• It exploits differences in surface activity of materials.
• The material may be whole cells or molecular such as a protein or colloid
• The material is selectively adsorbed to the surface of gas bubbles rising through a liquid.
• Then it is separated and finally removed by skimming
• It may be possible to make some materials surface active by the application of surfactants such as
long-chain fatty acids, amines, and quaternary ammonium compounds.
• Materials made surface active and collected are termed colligends whereas the surfactants are
termed collectors
• When developing this method of separation, the important variables, which may need experimental
investigation are pH, air-flow rates, surfactants, and colligend-collector ratios.
• The recovery of surface active products is clearly an important potential application of this technique.
• Foam separation of E. coli using lauric acid, stearyl amine t-octyl amine as surfactants, it was shown
that up to 90% of the cells were removed in 1 minute and 99% in 10 minutes.
Precipitation:
• This is the chemical process in which solid gets formed in a solution or inside another solid.
• Precipitation may be conducted at various stages of the product recovery process.
• It is a particularly useful process as it allows enrichment and concentration in one step, thereby
reducing the volume of material for further processing.
• It is possible to obtain some products (or to remove certain impurities) directly from the broth by
precipitation, or to use the technique after a crude cell lysate has been obtained.
Typical agents used in precipitation render the compound of interest insoluble, and these include:
• Acids and bases to change the pH of a solution until the isoelectric point of the compound is reached
and pH equals pI, when there is then no overall charge on the molecule and its solubility is decreased.
• Salts such as ammonium and sodium sulphate are used for the recovery and fractionation of proteins.
The salt removes water from the surface of the protein revealing hydrophobic patches, which come
together causing the protein to precipitate. The most hydrophobic proteins will precipitate first, thus
allowing fractionation to take place. This technique is also termed “salting out.”
• Organic solvents: Dextrans can be precipitated out of a broth by the addition of methanol. Chilled
ethanol and acetone can be used in the precipitation of proteins mainly due to changes in the dielectric
properties of the solution.
• Nonionic polymers such as polyethylene glycol (PEG) can be used in the precipitation of proteins and
are similar in behavior to organic solvents.
• Polyelectrolytes can be used in the precipitation of a range of compounds, in addition to their use in
cell aggregation.
• Protein binding dyes (triazine dyes) bind to and precipitate certain classes of protein
• Heat treatment as a selective precipitation and purification step for various thermostable products and
in the deactivation of cell proteases
Filtration
• Filtration is one of the most common processes used at all scales of operation to separate suspended
particles from a liquid or gas, using a porous medium which retains the particles but allows the liquid
or gas to pass through
It is possible to carry out filtration under a variety of conditions, but a number of factors will obviously
influence the choice of the most suitable type of equipment to meet the specified requirements at
minimum overall cost, including:
• The properties of the filtrate, particularly its viscosity and density.
• The nature of the solid particles, particularly their size and shape, the size distribution and packing
characteristics.
• The solids to liquid ratio.
• The need for recovery of the solid or liquid fraction or both.
• The scale of operation.
• The need for batch or continuous operation.
• The need for aseptic conditions.
• The need for pressure or vacuum suction to ensure an adequate flow rate of the liquid.
• A simple filtration apparatus which consists of a
support covered with a porous filter cloth.
• A filter cake gradually builds up as filtrate passes
through the filter cloth.
• As the filter cake increases in thickness, the
resistance to flow will gradually increase.
• Thus, if the pressure applied to the surface of the
slurry is kept constant the rate of flow will
gradually diminish
• Alternatively, if the flow rate is to be kept
constant the pressure will gradually have to be
increased.
• The flow rate may also be reduced by blocking of
holes in the filter cloth and closure of voids
between particles, if the particles are soft and
compressible.
• When particles are compressible, it may not be
feasible to apply increased pressure.
Flow through a uniform and constant depth porous bed can be represented by the Darcy equation:

• µ=liquid viscosity
• L=depth of the filter bed
• ∆P=pressure differential across the filter bed
• A=area of the filter exposed to the liquid
• K=constant for the system. K itself is a term which depends on the specific surface area (s) (surface
area/unit volume) of the particles making up the filter bed and the voidage (S) when they are packed
together.
• The voidage is the amount of filter-bed area, which is free for the filtrate to pass through. It is
normally 0.3–0.6 of the cross-sectional area of the filter
K (Kozeny’s constant) can be expressed as:
• Unfortunately, s and S are not easily determined.
• In most practical cases L is not readily measured but can be defined in terms of:
• V = volume of filtrate passed in time t and
• ν = volume of cake deposited per unit volume of filtrate
Types of filtration
• Plate and frame filters
• Pressure leaf filters
• Continuous filters
• Cross filtration
Centrifugation
Microorganisms and other similar sized particles can be removed from a broth by using a centrifuge when
filtration is not a satisfactory separation method.
Although a centrifuge may be expensive when compared with a filter it may be essential when:
• Filtration is slow and difficult.
• The cells or other suspended matter must be obtained free of filter aids.
• Continuous separation to a high standard of hygiene is required.
Noncontinuous centrifuges are of extremely limited capacity and therefore not
suitable for large-scale separation.
• The centrifuges used in harvesting fermentation broths are all operated on a continuous or
semicontinuous basis.
• Some centrifuges can be used for separating two immiscible liquids yielding a heavy phase and light
phase liquid, as well as a solids fraction. They may also be used for the breaking of emulsions.
Cell Disruption
Disruption: the cell envelope is physically broken, releasing all intracellular components into the
surrounding medium
• It is a method for releasing intracellular molecules.
• To release the cellular contents from their extremely tough cell wall a number of methods have been
established. For example physico-mechanical methods such as liquid shear, solid shear, freeze thawing,
agitation with abrasives, and ultra-sonication.
• The chemical methods include osmotic shock, usage of detergents, alkali treatment, and enzyme
treatment.
Non Mechanical Cell Disruption
Chemicals:
• use chemicals to solubilise the components in the cell walls to release the product.
Chemical requirements:
• products are insensitive to the used chemicals.
• the chemicals must be easily separable.
Types of chemicals:
• Surfactants (solubilizing lipids): sodium sulfonate, sodium dodecyl sulfate.
• Alkali: sodium hydroxide, harsh- Organic solvents: penetrating the lipids and swelling the cells.
e.g. toluene.
• Bacteria were treated with acetone followed by sodium dodecyl sulfate extraction of cellular proteins. For
example: sodium sulfonate: NaHSO3, sodium dodecylsulfate: Sodium dodecyl sulfate (or sulphate) (SDS
or NaDS) (C12H25NaO4S)
• Enzymes: to lyse cell walls to release the product gentle, but high cost, i.e. lysozyme (carbohydrase) to
lyse the cell walls of
• Bacteria: Osmotic shock
• Osmosis is the transport of water molecules from high- to a low-concentration region when these two
phases are separated by a selective membrane.
• Water is easier to pass the membrane than other components.
• When cells are dumped into pure water, cells can swell and burst due to the osmotic flow of water into
the cells
Liquid-liquid extraction:
• It is a method to separate compounds on the basis of their relative solubilities in two different
immiscible liquids.
• So, an extraction of a substance from one to another liquid phase in which the desired product is
preferentially soluble is known as liquid-liquid extraction.
• It is also known as solvent extraction and partitioning.
• It is used separate inhibitory fermentation products such as ethanol and acetone-butanol from
fermentation broth.
• For example: antibiotics (i.e. solvent amyl-acetate)
Requirements of liquid extractants:
• nontoxic, selective, inexpensive, immiscible with fermentation broth and
• high distribution coefficient:
KD=YL/XH
• YL and XH are concentrations of the solute in light and heavy phases,
respectively
Chromatography
• To separate the solutes based on the different rate of movement of the solutes in the column with
adsorbent materials.
Principles:
• Chromatographic processes involve a stationary phase and a mobile phase.
• Stationary phase can be adsorbent, ion-exchange resin, porous solid, or gel usually packed in a
cylindrical column.
• Mobile phase is the solution containing solutes to be separated and the eluant that carriers the solution
through the stationary phase.
• Applicable for protein, organics separation.
Method:
• A solution containing several solutes is injected at one end of the column followed by the eluent
carrying the solution through the column.
• Each solutes in the original solution moves at a rate proportional to its relative affinity for the
stationary phase and comes out at the end of the column as a separated band.
• Liquid Chromatography
Different types of
• chromatography techniques are used like adsorption chromatography, ion exchange chromatography, gel
permeation chromatography, affinity chromatography, reverse-phase chromatography and high-
performance liquid chromatography.
Drying
• To allow minimum loss in viability, activity and nutritional value drying is necessary.
• It removes water from a heat-sensitive material assuring least damage.
Crystallization
• It is used for final purification of a diverse range of compounds including the recovery of organic acids
and amino acids.
It must be remembered that the upstream and downstream processing are integral parts of an overall
process.
• As they are interconnected, neither stage should be developed independently, as this might result in
problems and unnecessary expenditure.
SUMMARY
So, by taking care of some steps in the upstream process product recovery may be made easier. i.e.
• Selection of test strain: By selecting test microorganisms that do not produce any pigment and/or
undesirable metabolites.
• Environmental setup: By adjusting the production environments to allow least production of undesirable
secondary metabolites.
Besides there are some process parameters that should be checked and maintained like:
• Time of harvesting
• pH maintenance during fermentation and harvesting
• Temperature maintenance
• Use of suitable chemicals for flocculation and separation
• There are so many problems associated with the product recovery program.
• For example, the recovery of extra cellular enzymes might be difficult as it needs immediate processing.
• One another problem is pigment production by test organisms because sometimes the pigment binds to the
same resin as the enzyme.
• At the time of foam separation, selection of antifoam should be proper otherwise it affects ultrafiltration or
ion exchange resins used in recovery stages.
• In short it should always be remembered that good recovery starts in the fermentation by the selection of
proper upstream processing like the correct media and proper time of harvesting.
• The major problem currently faced in product recovery is transfer of small-scale preparative methods to
the production scale without disturbing yield of the process, quality of the product and purity level of the
product.
THANK YOU