Downstream Processing & Product Recovery

Downstream processing
As the fermentation is complete necessary to recover the desired end product End products include
      Antibiotics Amino acids Vitamins Organic acids Industrial enzymes Vaccines

The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing (DSP) or product recovery.
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Downstream processing
The methodology adopted for DSP depends on Nature of end product Location of end product Concentration of end product Stability of end product Degree of purification required Presence of other products Product recovery yield expected to be higher if the number of steps in DSP lower
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Desired products for isolation by DSP are most frequently metabolites may be present as follows: 1. Intracellular metabolites: - products located within the cells - e.g. vitamins, enzymes. 2. Extracellular metabolites: - present outside the cells (culture fluids) - e.g. most antibiotics (penicillin, streptomycin), amino acids, alcohol, citric acid, some enzymes (amylases, proteases) 3. Both intracellular & extracellular: - e.g. vitamin B12, flavomycin
Sometimes the microorganism may itself be the desired end product for isolation, e.g. single-cell protein.
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Stages in downstream processing

Downstream processing of metabolites - a multistage operation - may be broadly divided into : 1. 2. 3. 4. 5. Solid-liquid separation Release of intracellular products Concentration Purification Formulation
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DESIRED PRODUCT IN CULTURE BROTH

INTRACELLULAR PRODUCT CELL DISRUPTION (physical, chemical enzymatic methods)

EXTRACELLULAR PRODUCT

BROTH WITH SOLIDS AND LIQUID SOLID-LIQUID SEPARATION (flotation, flocculation, filtration, centrifugation) CONCENTRATION (evaporation, liquid-liquid extraction, membrane filtration, precipitation, adsorption) PURIFICATION BY CHROMATOGRAPHY (gel-filtration, ion-exchange, affinity, hydrophobic interaction) FORMULATION (drying, freeze-drying, crystallization) FINAL PRODUCT
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Solid-liquid separation
First step in product recovery Separation of whole cells (biomass) and other insoluble ingredients from the culture broth Also k/a harvesting of microbial cells If desired product is an intracellular metabolite, it must be released from the cells before subjecting to solid-liquid separation by cell disruption Methods include flotation (foam separation), flocculation (precipitation), filtration and centrifugation

Flotation / foam separation

When a gas is introduced into the liquid broth, it forms bubbles Cells & other solid particles get adsorbed on gas bubbles These bubbles rise to the foam layer which can be collected (skimmed) and removed Presence of certain surfactant substances (k/a collector substances) facilitates stable foam formation e.g. long chain fatty acids, amines, quaternary ammonium compounds Materials made surface active and collected are termed colligends
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Flocculation / precipitation/ sedimentation

The cells (or cell debris) form large aggregates to settle down for easy removal The process depends on the nature of cells & the ionic constituents of the medium Addition of flocculating agents (inorganic salt, organic polyelectrolyte, mineral hydrocolloid) is necessary to achieve appropriate flocculation Allows enrichment & concentration in one step, thereby reducing the volume of the material for further processing Sedimentation achieved naturally with selected strains of brewing yeasts if chilled at the end of fermentation

Filtration
Most commonly used technique for separating the biomass and culture filtrate Efficiency of filtration depends on - Size & shape of the organism - Presence of other organisms - Viscosity & density of the medium - Temperature - Solid : liquid ratio - Scale of operation - Need for aseptic conditions - Need for batch/ continuous operation
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 Filtration is defined as the separation of solid in a slurry consisting of the solid and fluid by passing the slurry through a septum called filter medium. For filtration in some cases filter aids (diatomaceous earth) are used to improve porosity and faster flow rate.

Slurry Filter cake Filter cloth Support to filter cloth Filtrate

Diagram of a simple filtration apparatus

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Flow of slurry through a uniform and constant depth porous bed is ruled by Darcy equation, Rate of flow = dV/dt = KAP/ L
where, - liquid viscosity L depth of filter bed P pressure differential across the filter bed A area of filter exposed to the liquid K constant for the system (depends on surface area of filter bed, s and voidage volume when packed together) L, the depth of filter bed can be defined as volume of filtrate passed in time t and varies with volume of cake deposited per unit volume of filtrate. By carrying out small scale filtration trials, it is possible to determine K, and then the solved equation may be applied for large scale filtration calculation.
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Types of filters
Several filters such as depth filters, absolute filters, rotary drum vacuum filters, membrane filters are used Depth filters : - composed of a filamentous matrix such as glass wool, asbestos or filter paper - Particles are trapped within the matrix & the fluid passes out - Filamentous fungi can be removed using depth filters Absolute filters: - filters with specific pore sizes smaller than the particles to be removed - Bacteria from culture medium can be removed by absolute filters
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Types of filters
Rotary drum vacuum filters: - frequently used for separation of broth containing 10-40% solids (by volume) & particles in the size of 0.5-10 m - Successfully used for filtration of yeast cells & filamentous fungi Membrane filters: - membranes with specific pore sizes used - Clogging of filters major limitation - Two types of membrane filtrations- static filtration & cross-flow filtration

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Batch filters
1. Plate and frame filters: Plates and frames are arranged alternately assembled on a horizontal framework Plates covered with filter clothes & held together by hand screw to prevent leakage between frames Slurry is fed through the continuous channel by holes in the corners of the plates and frames Filtrate passes through the filter cloth or pad, runs down grooves in the filter plates and is then discharged through outlet taps to a channel. Most suitable for fermentation broths with a low solids content & low resistance to filtration

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Batch filters
2. Pressure leaf filters: - incorporates a number of leaves, each consisting of a metal framework of grooved plates which is covered with a fine wire mesh, or occasionally a filter cloth and often precoated with a layer of cellulose fibers - Slurry is fed into the filter which operated under pressure or by suction with a vacuum pump - Suitable for polishing large volumes of liquids with low solids content

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Three types based on the arrangement of filters. (a) Vertical metal-leaf filter consists of number of vertical porous metal leaves mounted on a hollow shaft in a cylindrical pressure level - solids from the slurry gradually build up on the surfaces of the leaves & the filtrate is removed from the plates via the horizontal hollow shaft (b) Horizontal metal-leaf filter consists of metal leaves mounted on a vertical hollow shaft within a pressure vessel - Filtration is continued until the cake fills the space between the disc shaped leaves or when the operational pressure has become excessive (c) Stacked disc filter this metal filter consists of a number of precision made rings which are stacked on a fluted rod - Filtrate passes between the discs & is removed through the grooves of the fluted rods, while solids are deposited on the filter coating.
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Continuous filters
1. Rotary vacuum filtration : Drum covered with diatomaceous earth matter, allowed to rotate under vacuum with half immersed in the slurry tank Small amount of coagulation agent added to broth & pumped into the slurry tank As drum rotates in the slurry tank under vacuum, thin layer of coagulated particles adhere to drum The layer thickens to from cake As the cake portion in the drum comes to the upper region which is not immersed in the liquid it is washed with water and dewatered immediately by blowing air over it Then before the dried portion is again immersed into the liquid it is cut off from drum by knife

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The mechanism of cake discharge is achieved by three ways: (a) String discharge Long lengths of string 1.5cm apart are threaded over the drum and round two rollers - The cake is lifted free from the upper part of the drum when the vacuum pressure is released and carried to the small rollers where it falls free (b) Scraper discharge by using a knife or scraper positioned accurately to slice off the cake (c) Scraper discharge with precoating to avoid blockage of filter cloth in the drum by cells a scraper which is coated with a layer of filter-aid 2 to 10 cm thick

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Continuous filters
2. Micro or Ultra Filtration: Filtration of suspended particles can be achieved by either dead end filtration or cross flow filtration. (a) Dead end filtration Solution poured over the membrane and the filtrate is collected at the bottom. On prolonged filtration pores become blocked which reduce the filtering capacity.

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(b) Cross flow filtration To prevent the blockage the solution is passed over the membrane - Cell suspension enters laterally and flows over the membrane - Filtrate gets collected at the bottom whereas the cells are pushed to the opposite end by the continuous flow of suspension which is sent out via an outlet at the opposite end - Liquid is again passed through a tube which recycles back to the flow - Since the cells do not block the pores the filtration process can be performed continuously

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Cross flow filtration

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Adsorption on filter aids: Filter aids - inert incompressible discrete particles of high permeability Solids such as wood pulp, starch powder, cellulose, inactive carbon, when added as filter aid enhances their filterability Filter aids absorb small particles, which otherwise clog the filter pores Filter aids also reduce the compressibility of the accumulated biomass by adsorbing the colloidal particles
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Centrifugation
Based on the principle of density differences between the particles to be separated and the medium. Mostly used for separating solid particles from liquid phase May be essential when
- Filtration is slow and difficult - Cells or other suspended matter must be obtained free of filter aids - Continuous separation to a high standard of hygiene is required

Non-continuous centrifuges are of extremely limited capacity and hence not suitable for largescale separation
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Centrifugation
o Employs centrifugal force to promote accelerated settling of particles in a solid-liquid mixture o Separation is achieved by means of accelerated gravitational force by rapid rotation o Microorganisms and other cells from the fermented slurry can be removed by using centrifuge when filtration is not a satisfactory separation method o Particle size that can be separated range from 0.1 m to 100 m

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Separation is based on Stokes law, which states that the rate of Newtonian viscosity characteristics is proportional to the square of the diameter of the particles which is expressed as, Vg = d2g (
P

L)

/ 18

where, Vg rate of sedimentation, d particle diameter, g gravitational force, P particle density, L liquid density, - viscosity
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Types of centrifuges
Tubular bowl centrifuge:
A simple and small centrifuge used in pilot plants Can be operated at a high centrifugal speed Can run in both batch or continuous mode The solids are removed manually Used to separate particle size of 0.1 - 200 m Simple machine made of a tube rotating between bearings at each end - Suspension enters at the bottom of centrifuge and high centrifugal forces act to separate the solids and liquids - The bulk of solids will adhere to walls of the bowl, while the liquids exit at the top of the centrifuge 27

Tubular-bowl centrifuge

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Disc centrifuge:
- Consists of several discs that separate the bowl into settling zones - The feed/slurry is fed through a central tube - The clarified fluid moves upwards while the solids settle at the lower surface
Disc-bowl centrifuge

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Multichamber centrifuge:
- Modification of tubular-bowl centrifuge - Has a number of tubular bowls arranged coaxially - The main bowl contains cylindrical inserts that divide the volume of the bowl into a series of annular chambers, which operate in sequence Fluid in - The feed enters the centre of Clarified fluid out the bowl and passes through each chamber - The solids settle on the outer walls of each chamber and the clarified liquid overflows from the largest diameter chamber

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Decanter centrifuge:
- Also k/a solid-bowl scroll centrifuge - Used for continuous handling of fermentation broths, cell lysates & coarse materials like sewage sludge - Consists of a horizontal cylindrical bowl tapered at one end, rotating at a high speed, with a helical extraction screw placed co-axially - The screw perfectly fits the internal contour of the bowl, only allowing clearance between bowl and scroll - The differential speed between the screw and scroll provides the conveying motion to collect and remove solids that accumulate at the bowl wall
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Solid-bowl scroll centrifuge

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The centrifugation theory

The terminal velocity during gravity settling of a small spherical particle in dilute suspension is given by Stokes law:

ug !

Vp  V f 18Q

2 Dp g

Where ug is sedimentation velocity under gravity, p is particle density, f is liquid density, is liquid viscosity, Dp is diameter of the particle, and g is gravitational acceleration. In the centrifuge:
uc ! Vp  Vf 18Q
2 D p[ 2 r

uc is particle velocity in the centrifuge, is angular velocity in rad/s, and r is radius of the centrifuge drum.
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The centrifugation theory

The ratio of velocity in the centrifuge to velocity under gravity is called the centrifuge effect or G-number.

[ 2r Z! g
Industrial Z factors: 300-16 000, small laboratory centrifuge may up to 500 000. The parameter for centrifuge performance is called Sigma factor

Q 7! 2u g

Q is volumetric feed rate. The Sigma factor explain cross sectional area of a gravity settler with the same sedimentation characteristics as the centrifuge. If two centrifuge perform with equal effectiveness

Q1 Q2 ! 71 7 2
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The centrifugation theory

Disc-stack bowl centrifuge

2T[ 2 N  1 3 3 r2  r1 7! 3 g tan U
N is number of disc, is half-cone angle of the disc. The r1 and r2 are inner and outer radius of the disc, respectively. Tubular-bowl centrifuge

T[ 2b 2 2 7! 3r2  r1 2g
b is length of the bowl, r1 and r2 are inner and outer radius of the wall of the bowl.
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Recovery of intracellular products

Several biotechnological products (vitamins, enzymes) located within the cells Need to be first released (maximally and in active form) for further processing and final isolation Microorganisms or other cells can be disintegrated or disrupted by physical, chemical or enzymatic methods Selection of a particular method depends on nature of cells (as there is a wide variation in the property of cell disruption or breakage)
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Major methods for cell disruption to release intracellular products

CELL DISRUPTION

Physical methods Ultrasonication * Heat shock (thermolysis) High-pressure * homogenisation Impingement *

Chemical methods Osmotic shock Alkalies Organic solvents Detergents

Enzymatic methods Lysozyme Glucanase, Mannanase, and protease

Grinding with glass beads *

( * indicate mechanical methods)

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Cell disruption
Physical-mechanical methods
Ultrasonication: - ultrasound waves of frequencies greater than 20 kHz ruptures the cell wall by a phenomenon known as cavitation - passage of ultrasound waves creates alternating areas of compression and rarefaction - cavities formed in the areas of rarefaction rapidly collapses as the area changes to one of compression - bubbles produced in the cavities collapse creating shock waves which disrupt cell walls
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Ultrasonic Homogenizer (Sonicator)

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Cell disruption
Physical-mechanical methods
High pressure homogenization: - Involves forcing of cell suspension at high pressure through a very narrow orifice to come out to atmospheric pressure - Sudden release of pressure creates liquid shear that can break the cells - cell suspension is pumped through the homogenizing valve at 200 1000 atmospheric pressure depending on microbes and cell concentration
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High Pressure Homogenizer - French Press

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Cell disruption
Physical-mechanical methods
Impingement: - Impinge means to strike or hit - A stream of suspended cells at high velocity and pressure are forced to hit either a stationary surface or a second stream of suspended cells - Cells are disrupted by the forces at the point of contact - Microfluidizer a device developed based on the principle of impingement - Successfully used for breaking E. coli cells - Advantage: - can be effectively used for disrupting cells even at a low concentration
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Microfluidizer

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Cell disruption
Physical-mechanical methods
Grinding with glass beads: - Cells mixed with glass beads subjected to a very high speed in a reaction vessel - Cells break as they are forced against the wall of the vessel by the beads - Factors influencing cell breakage
Size & quantity of glass beads Concentration & age of cells Temperature Agitator speed

- Under optimal conditions, a maximal breakage of about 80% 0f the cells

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Cell disruption
Physical-non-mechanical methods
Heat shock: - Breakage of cells by subjecting them to heat - Relatively easy and cheap - Used only for a few heat-stable intracellular products Freeze-thawing: - Freezing and thawing of microbial cell paste - Causes the ice crystals to form - Their expansion followed by thawing lead to disruption of cells
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Cell disruption
Chemical methods
Osmotic shock: - Osmotic shock caused by a sudden change in solute concentration will cause disruption of a no. of cell types - Involves suspension of cells (free from growth medium) in 20% buffered sucrose - Cells are then transferred to water at about 4C - Used for release of hydrolytic enzymes & binding proteins from Gram-negative bacteria

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Cell disruption
Chemical methods
Alkali treatment: - Used for hydrolysis of microbial cell wall - Alkali stability of desired product very crucial - i.e. to tolerate a pH of about 11.5 to 12.5 for 20-30 min - Used for extraction of some bacterial proteins - e.g recombinant growth hormone efficiently released from E. coli by treatment with sodium hydroxide at pH 11

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Cell disruption
Chemical methods
Organic solvents: - Water-miscible organic solvents- used to disrupt cells e.g. methanol, ethanol, isopropanol, butanol - These compounds are flammable, hence require specialized equipment for fire safety - Frequently used toluene - Dissolves membrane phospholipids - Creates membrane pores for release of intracellular contents

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Cell disruption
Chemical methods
Detergents: - Detergents that are ionic in nature can denature membrane proteins and lyse the cells - Cationic: cetyl trimethyl ammonium bromide - Anionic : sodium lauryl sulfate - Non-ionic detergents also used e.g. Triton-X-100 or Tween (less reactive than ionic) - Problem with detergents- affect purification steps, esply. salt precipitation - Overcome by using ultrafiltration or ion-exchange chromatography for purification
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Cell disruption
Enzymatic methods
Enzymatic digestion is involved in two stages (i) cell wall disruption resulting in the release of cell wall proteins leaving the protoplast intact and (ii) digestion of organelle membrane to release the organelle proteins Advantages: Lysis of cells occurs under mild conditions Thus, advantageous for product recovery

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Cell disruption
Enzymatic methods
Lysozyme most frequently used enzyme - Hydrolyses -1,4- glycosidic bonds of the mucopeptide in bacterial cell walls - Gram-negative bacteria (high content of cell wal mucopeptides) - more susceptible for lysozyme action - Other enzymes also used - For lysis of yeast cell walls, glucanase & mannanase in combination with proteases used

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Concentration of biological products

The filtrate that is free from suspended particles contains 80-98% water Desired product very minor constituent Water to be removed to achieve product concentration Commonly used techniques: - evaporation
liquid-liquid extraction membrane filtration precipitation adsorption

Procedure adopted depends on the nature of desired product and the cost factor
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Evaporation
Water in broth filtrate can be removed by simple evaporation process Evaporators have
a heating device for supply of steam, unit for separation of concentrated product and vapour a condenser for condensing vapour accessories and control equipment

Capacity of equipment variable ranging from small lab scale to industrial scale

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Evaporation
Types of evaporators: Plate evaporators:
- Liquid to be concentrated flows over plates - As steam is supplied, liquid gets concentrated and becomes viscous

Falling film evaporators:

- Liquid flows down long tubes, which gets distributed as a thin film over the heating surface - Suitable for removing water from viscous products of fermentation

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Evaporation
Types of evaporators: Forced film evaporators:
- Liquid films are mechanically driven - Suitable for producing dry product concentrates

Centrifugal forced film evaporators:

- Evaporate the liquid very quickly (in seconds) - Suitable for concentrating even heat-labile substances - A centrifugal force is used to pass on the liquid over heated plates or conical surfaces for instantaneous evaporation

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Liquid-liquid extraction
a classical method for recovery as well as concentration of various products Solvent extraction involves extraction of compound in a liquid phase to another liquid
Separation of a component from a liquid mixture by treatment with a solvent in which the desired product is preferentially soluble is k/a liquid-liquid extraction

The solute originally present in aqueous phase gets partitioned in both the phases distribution between the two immiscible liquids and solubility in two liquids decide the efficacy of extraction
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Batch extraction
Single stage batch extraction - the aqueous feed is mixed with the organic solvent - after equilibration, the extract phase containing the desired solute is separated out for further processing - In some cases, a single stage extraction may not be enough and multi stage process is required wherein fresh volume of solvent is contacted with the raffinate.
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Double-stage extraction process

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Continuous extraction
Co-current extraction - there are n mixer vessels in line - the raffinate goes from vessel 1 to vessel n - Fresh solvent is added to each stage - the extracting solvent pass through the cascade in the same direction - At every stage the extract is recovered
Feed Solvent Mixer Separator 1 Raffinate Mixer Separator 2 Raffinate Mixer Separator n Raffinate

Solvent Extract Extract

Solvent Extract

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Continuous extraction
Counter-current extraction - the extracted raffinate passes from vessel 1 to vessel n - while the product-enriched solvent is flowing from vessel n to vessel 1 - the most efficient method of extraction.
Feed Raffinate Mixer Separator 1 Mixer Separator 2 Raffinate Mixer Separator n Depleted raffinate

Solvent

Solvent enriched with product

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Extraction is achieved by three mechanisms viz., physical extraction, dissociative extraction and selective extraction Physical extraction - involves preferential dissolution of
the desired solute in a chosen organic solvent

Dissociative extraction - involves the modification of

the physical property of the solute to increase the solubility in organic phase

Selective extraction - involves modifying the solute

solubility through ion pair or complex or adduct formation

Solvent recovery after extraction process is essential one which is usually done by distillation
The distillation is performed in three stages: 1. Evaporation of solvent into vapour phase 2. Vapour-liquid separation & 3. Condensation to collect solvent
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Aqueous two phase system (ATPS)

The basic principle involves differential partitioning of solute in two immiscible phases Phase separation occurs when hydrophilic polymers are added to an aqueous solution At low concentration of polymers, homogenous solution is formed At discrete concentration rise, two immiscible phases are formed using two aqueous phases with incompatible polymers such as PEG and dextran
Eg. PEG water / dextran water and PEG water/Kphosphate water, PEG phosphates

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Aqueous two phase system (ATPS)

Homogenates are prepared with two incompatible polymers Mixerphase separation is done by keeping idle Bottom phase and top phase separated The soluble and non-soluble substances are separated by ultra filtration and product recovered The retentate may be recycled for further recovery
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Membrane filtration
Involves the use of a semi-permeable membrane that selectively retains the particles / molecules bigger than the pore size, while smaller molecules pass through the membrane pores Membranes used in filtration made of polymeric materials such as polyethersulfone and polyvinyl difluoride Membrane filters difficult to sterilize Recently, microfilters & ultrafilters composed of ceramics and steel- cleaning and sterilization easy

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Membrane filtration
3 major types of filtration based on size of particles separated
Type Sizes of particles separated Compound/ particle separated

1. Microfiltration 2. Ultrafiltration

0.1 - 10 m 0.001 0.1 m

Cells or cell fractions, viruses Compounds with mol wt.s greater than 1000 (e.g. enzymes) Compounds with mol wt.s less than 1000 (e.g. lactose)

3. Reverse osmosis (hyperfiltration)

0.0001 0.001 m

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Other membrane filtration techniques:

Membrane adsorbers: - Micro- or macroporous membranes with ion exchange groups and / or affinity ligands - Membrane adsorbers can bind to proteins and retain them - Such proteins can be eluted by chromatography

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Other membrane filtration techniques:

Pervaporation: - A technique in which volatile products can be separated by a process of membrane permeation coupled with evaporation - Quite useful for extraction, recovery and concentration of volatile products - Cannot be used in large scale due to cost factor

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Other membrane filtration techniques:

Perstraction: - Advanced technique on the principle of membrane filtration coupled with solvent extraction - Hydrophobic compounds can be recovered / concentrated by this method

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Precipitation
by decreasing the solubility of the solutes the solute can be separated by precipitation

Solubility of the particle can be changed by, 1. Salting out by increasing ionic strength by adding salts
as ammonium sulphate, disodium sulphate

2. Solubility reduction at low temperature by adding

organic solvents at low temperature

3. Solvent precipitation adding salt, pH adjustment and

low temperature

4. Isoelectric precipitation by the changing the pH to

isoelectric pH (no charge in proteins)

5. Use of electrolytes ionic polymers (ionic

polysaccharides), non ionic polymer (dextrans)
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Adsorption
Biological products of fermentation can be concentrated using solid adsorbent particles Earlier activated charcoal used Now, cellulose-based adsorbents employed for protein concentration For concentration of low molecular wt compounds (vitamins, antibiotics, peptides), polystyrene, methacrylate and acrylate based matrices used Process of adsorption carried out by making a bed of adsorbent column and passing culture broth through it Desired product held by adsorbent can be eluted
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