Crystallization & Whole broth processing

y Crystallization is important as an industrial process

because of the number of materials that are and can be marketed are in the form of crystals. y Crystallization may be carried out from a vapor, from a melt, or from a solution. y More than 80% of the substances used in pharmaceuticals, fine chemicals, agrochemicals, food and cosmetics are isolated or formulated in their solid form. y Crystallization is in general the last chemical purification step in the production of ingredients.

y Crystallization is the (natural or artificial) process of

formation of solid crystals precipitating from a solution, melt or more rarely deposited directly from a gas. y Crystallization is also a chemical solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. y Its extensive use is based on the fact that this single operation is both a separation and a purification process whereby a solid crystalline product can be isolated with high purity and with relatively low capital and operating costs.

Crystalsy A crystal may be defined as a solid composed of atoms arranged y y y y

y

in an orderly, repetitive array. Crystals are grown in many shapes, which are dependent upon downstream processing or final product requirements Crystal shapes can include cubic, tetragonal, orthorhombic, hexagonal, monoclinic, triclinic, and trigonal. In order for crystallization to take place a solution must be "supersaturated". Supersaturation refers to a state in which the liquid (solvent) contains more dissolved solids (solute) than can ordinarily be accomodated at that temperature The crystallization process consists of two major events, nucleation and crystal growth.

Nucleationy Nucleation is the step where the solute molecules

dispersed in the solvent start to gather into clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes stable under the current operating conditions. y the clusters reach a critical size in order to become stable nuclei. This is dictated by the operating conditions (temperature, supersaturation, etc) y Total nucleation is the sum effect of two categories of nucleation - primary and secondary.

Primary nucleationy Primary nucleation is the initial formation of a crystal

where there are no other crystals present or where, if there are crystals present in the system, they do not have any influence on the process. y This can occur in two conditions 1. homogeneous nucleation 2. heterogeneous nucleation

Secondary nucleationy Secondary nucleation is the formation of nuclei

attributable to the influence of the existing microscopic crystals in the magma. y first type of known secondary crystallization is attributable to fluid shear, the other due to collisions between already existing crystals with either a solid surface of the crystallizer or with other crystals themselves

Crystal growthy Once the first small crystal, the nucleus, forms it

acts as a convergence point for molecules of solute touching - or adjacent to - the crystal so that it increases its own dimension in successive layers. y Growth rate is influenced by several physical factors, such as surface tension of solution, pressure, temperature, relative crystal velocity in the solution

Artificial methodsy For crystallization to occur from a solution it must be

supersaturated y This can be achieved by various methods, 1. solution cooling, 2. addition of a second solvent to reduce the solubility of the solute 3. chemical reaction 4. solvent evaporation

Applicationsy There are two major groups of applications for the

artificial crystallization process: 1. crystal production and 2. purification.

Equipment for crystallizationy Tank Crystallizers y Forced circulation crystallizer y Scraped surface crystallizers y Circulating-magma vacuum crystallizer y Oslo crystallizer

Tank Crystallizersy This is probably the oldest and most basic method of y y y y

crystallization Hot, saturated solutions are allowed to cool in open tanks. After crystallization, the mother liquor is drained and the crystals are collected. Controlling nucleation and the size of the crystals is difficult. The crystallization is essentially just "allowed to happen"

Scraped surface crystallizersy One type of scraped surface crystallizer is the

Swenson-Walker crystallizer, y it consists of an open trough 0.6 m wide with a semicircular bottom having a cooling jacket outside. y A slow-speed spiral agitator rotates and suspends the growing crystals on turning. y The blades pass close to the wall and break off any deposits of crystals on the cooled wall.

The Forced Circulation ("FC") crystallizery the most common type of crystallizer in the industry y The average FC crystallizer evaporates solvent, thus

increasing the supersaturation in the process liquor, and causing crystallization to occur. y Most conventional FC units operate under vacuum, or at slight super atmospheric pressure. y The FC consists of four basic components: the crystallizer vessel, heat exchanger, the circulating pump and the vacuum equipment y Slurry from the crystallizer vessel is circulated, in plugflow fashion, through the heat exchanger, and returned to the crystallizer vessel again, where its supersaturation is relieved by deposition of material on the crystals present in the slurry.

Circulating-magma vacuum crystallizery The magma or suspension of crystals is circulated out of y y y

y y

the main body through a circulating pipe The magma flows though a heater, where its temperature is raised. The heated liquor then mixes with body slurry and boiling occurs at the liquid surface. This causes supersaturation in the swirling liquid near the surface, which deposits in the swirling suspended crystals until they leave again via the circulating pipe. The vapors leave through the top. A steam-jet ejector provides vacuum. DTB crystallizer is an example of this type of crystalliser

DTB (Draft Tube and Baffle) crystallizer

Oslo-Krystal Cooling crystallizersy A small quantity of warm

concentrated feed solution enters the crystallizer vessel at point A, located directly above the inlet to the circulation pipe B. y Saturated solution from the upper regions of the vessel together with The small amount of feed liquor, is circulated by pump C through the tubes of heat exchanger D, which is cooled rapidly by a forced circulation of water or brine. y On cooling the solution becomes supersaturated, but not sufficiently for spontaneous nucleation to occur (metastable). y The supersaturated solution flows down pipe E and emerges from the outlet F, directly into a mass of crystals growing in the vessel.

Whole broth processingy The concept of recovering a metabolite directly from

an unfiltered fermentation broth is of considerable interest because of its simplicity, the reduction in process stages and the potential cost savings. y It may also be possible to remove the desired fermentation product continuously from a broth during fermentation so that inhibitory effects due to product formation and product degradation can be minimized throughout the production phase.

Methodsy Ion exchange resins y Reciprocating-plate extraction column y Dialysis y Expanded-Bed Adsorption y Resin method.

Ion exchange resinsy Ion exchange resins are polymers that are capable of exchanging

particular ions within the polymer with ions in a solution that is passed through them y The resins are prepared as spherical beads 0.5 to 1.0 mm in diameter. y These appear solid even under the microscope, but on a molecular scale the structure is quite open. y This means that a solution passed down a resin bed can flow through the crosslinked polymer, bringing it into intimate contact with the exchange sites.

Extraction of Stretomycin y A process for adsorption of streptomycin on to a series of cationic ion-exchange resin columns was developed directly from the fermentation broth, which had only been screened to remove large particles so that the columns would not become blocked This procedure could only be used as a batch process. Extraction of Novobiocin y The harvested broth was first filtered through a vibrating screen to remove large particles. y The broth was fed into a continuous series of well mixed resin columns fitted with screens to retain the resin particles, plus the adsorbed novobiocin, but allow the streptomycete filaments plus other small particulate matter to pass through. y The first resin column was removed from the extraction line after a predetermined time and eluted with methanolic ammonium chloride to recover the novobiocin.

Reciprocating-plate extraction columny The Karr Reciprocating Plate Column consists of a series of

y

y y

y

perforated plates with large diameter holes and high free cross sectional area mounted on a reciprocated central shaft The reciprocating motion imparts energy to the liquids, creating droplets and thus providing interfacial area so that mass transfer can take place Along with the perforated plates are periodic baffle plates that suppress axial mixing. The entire series of perforated and baffle plates is assemble in a single cartridge, which is suspended from the drive motor on top There are no internal bearings or guide bushings, and all maintenance is performed outside of the column

Dialysisy Removal of soluble impurities from solution by the use y y

y y

of semipermeable membrane is known as dialysis Solutes present in a solution(broth) can pass through a semipermeable membrane. Cycloheximide was extracted using methylene chloride. Methylene chloride was circulated in a dialysis tubing loop which passed through a fermentor. Cycloheximide was extracted into methylene chloride. The product yield increased by almost double by this dialysis-solvent extraction method.

Resin Methody Sterile beads of an acrylic resin, as dispersed beads

or beads wrapped in ultrafiltration method, were put in fermentors 48 hours after inoculation. y Some of the cycloheximide formed in broth was absorbed in resin. y Recovery of antibiotic from resin is achieved by solvents or by changing temperature.

Electrodialysis(ED)y Electrodialysis(ED) is a well known separation

process where ionized compounds are separated from non ionized compounds in aqueous solutions based on transport through ion exchange membranes in an electric field. y Since in a fermentation broth the lactate salt is ionized, whereas the carbohydrates and proteins and amino acids are either non ionized or weakly ionized, recovery and purification of lactate salts from a fermentation broth by electrodialysis is feasible.

Expanded-Bed Adsorption Theory

Expanded-Bed Adsorption Theory

When the resin has packed in the column, the beads are close together (1). As the column is fluidized, the resin beads establish a concentration gradient (2). The sample feedlot is injected, and particulates and cell debris (green dots) move past the resin and out of the column, while the compound of interest (red dots) interacts with the beads (3). The column is then repacked, the flow is reversed, and the compound is eluted from the beads (4).