Biochemical Product Recovery--First Steps

Why is product recovery challenging?

•  recovery costs for the products are sometimes considerably greater than costs of the bioprocess steps. For one antibiotics plant, the investment for the recovery facilities was about four times that for the fermenter vessels and their auxiliary equipment. As much as 60 per cent of the fixed costs of fermentation plants for organic acids or amino acids is attributable to the recovery section.
• difficult challenges because of very dilute solutions.
• must separate them from byproducts with very similar properties.

Although most of the purification equipment for biotechnology is the same as that used throughout the chemical process industries, there are distinct differences.

• many biochemicals are not stable enough for hot operations.
• methods such as chromatography and electrophoresis that have little practical importance for the chemical process industries can be crucial for certain biochemical isolations.

Some tried-and-true chemical engineering unit processes and unit operations, e.g., adsorption and filtration, have been used routinely for years by biochemical engineers. Other steps are presently undergoing extensive research because they are needed for the difficult isolations of the exciting new protein products.

Dual goals: concentration and purification for the early steps in recovery that deal with dilute, impure materials.

Very large vessels are required when the solutions are so dilute. It is essential to reduce the amount of water.

An example is the solvent extraction of penicillin from acidified fermentation broth. The purity of penicillin at the end of fermentation may be a few per cent. Extraction into an immiscible solvent such as MIBK (methylisobutylketone) leaves behind the salts and polar organic molecules, e.g., sugars. In older processes, the ratio of MIBK to broth was about 1/5, and the distribution coefficient so favors penicillin that step yields were excellent. At this point the penicillin purity on a dry basis was already 70 to 80 per cent. Extraction back into water had a ratio of MIBK to water of 40/1, and the pH was between 7 and 8 where the distribution coefficient greatly favors water. The concentration factor increased by 5 times 40 or 200, and the purity was over 90 per cent. Modern penicillin fermentation achieves a concentration of roughly four per cent by weight of product in the broth, and the above ratios have been adjusted to suit the solubilities in organic solvent and water. It is relatively easy to crystallize a final, very pure product from concentrated aqueous solution.

Early purification by ion exchange has roughly similar performance. A large volume of broth is passed over the ion exchange resin, and the volume of eluant is relatively small. Again there is a highly favorable reduction in the volumes for subsequent handling and a significant increase in purity. 

Recovery of Ethanol

The method for recovery of ethanol from fermentation broth is shown in this sketch: 
 
Other options have had a great deal of attention in the research laboratory, but steam stripping is the mainstay of the industry. One very important advance in the last decade or so has been recycle of the waste aqueous stream from stripping back to the fermenter. This puts back a small amount of ethanol but more importantly reuses nutrients. The fermentation uses almost all of the carbohydrates but only part of the nitrogenous compounds. There is also a significant saving because less liquid goes to waste treatment. Recycle has the restraint of buildup of non-volatile components, however, glycerol reaches concentrations that make its recovery for sale practical.

Distillation of Ethanol

At atmospheric pressure, ethanol and water form an azeotrope that has about 95 % ethanol and 5 % water. An azeotrope is a liquid in equilibrium with vapor of exactly the same composition; this means that distillation of an azeotrope and condensation of the vapor gives the same liquid with which you started. Other solutions of ethanol and water are in equilibrium with vapor that is richer in ethanol. This is why distillation works. In fact, dilute solutions of ethanol vaporize to gas that is much richer in ethanol. 

The black line for the composition of the liquid just goes from one corner to the other. The red curve (left axis) shows the fraction of ethanol in vapor in dependence of the fraction of ethanol in liquid. For small concentrations of ethanol in the liquid, the concentration of ethanol in the vapor is higher than that in the liquid. But the lines pinch together at higher concentrations. Multiple distillations to purify ethanol work extremely well until the lines pinch. The lines intersect at the azeotrope and are so close together from there on up that the gap can hardly be seen.

The composition of the azeotrope changes with pressure, and distillation at a different pressure is one way to get above the azeotrope. Another is to add another organic liquid such as benzene to completely alter the liquid-vapor behavior. In any event, the pinching of the lines causes distillation to switch from a great process for starting the recovery of ethanol to a poor process for finishing the job. Vapor-phase drying has become a practical alternative for getting past the azeotrope.

Modern distillation technology saves heat energy by using the vapor from one stage to heat another. The enthalpy of the vapor is fine, but its temperature is too low to provide much driving force for heat transfer. There can be stages at different pressures to change boiling points to get more driving force, but a better solution is to compress the vapor. This causes some liquid to condense but at a higher temperature and thus creates more driving force.

Direct precipitation

This is very uncommon, but there are two examples. The early method for purification of the antibiotic cycloserine was to add silver nitrate to the clarified fermentation broth to precipitate its insoluble silver salt. This was far too expensive, and alternate methods were soon discovered. Furthermore, the dry silver salt could explode.

Example #2 is the recovery of proteolytic enzymes that are used in detergents. It seems crazy to add acetone directly to the fermentation broth. Some yield is lost because of enzyme instability in acetone, and the volumes of solvent are enormous. However, other methods of recovery tend to be even worse in terms of cost.

Many substances that are soluble in water are much less soluble in liquids that are miscible with water. Common solvents that are miscible with water are methanol, ethanol, propanol, and acetone. A good method of precipitating the substances is to add the solvent to an aqueous solution. Best practice is to concentrate the starting aqueous solution. Otherwise the decreased solubility after adding the solvent may still be too high to produce an acceptable yield.

One puzzling observation is that you can add too much solvent. The following applet should clarify this for you. Move the scrollbar and note that the yield goes through a maximum. You probably would not benefit from trying to reach this maximum exactly because the cost of so much solvent is an important consideration.

A little reflection leads to understanding. Note that the amount of total solvent is increasing. Even though the solubility decreases, the amount of substance in solution is the solubility times the volume. Too great a volume allows too much to remain in solution. Also, it takes an infinite volume of solvent to approach 100 % solvent.

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