For the experts

A recovery process typically consists of a pretreatment followed by a primary separation of the enzyme from the biomass. Afterwards, the enzyme is concentrated by removal of water, and unwanted impurities are removed in a purification step.

Pretreatment

The fermentation broth obtained is often pretreated in order to improve its separation properties. For extracellularly produced enzymes the complexity of the pretreatment varies depending on the type of microorganism and the type of product to be produced. 

The pretreatment of a fungal broth is simple and usually consists of dilution combined with pH adjustment. The dilution is carried out to reduce the viscosity and thereby enhance the separation. As the surface of the cells is negatively charged, the enzyme might bind to the surface if the pH of the process is higher than the isoelectric point of the enzyme. In these cases a pH adjustment is needed in order to release the product from the cells. Salts can also be added to neutralize the surface charge of the microorganisms. Furthermore, the salts can be used as precipitation agents, for example for removal of metabolites such as organic acids that have been formed during the fermentation.

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For bacteria the pretreatment of the broth typically consists of coagulation followed by flocculation. In the coagulation process salts (e.g., calcium, sodium, and aluminum) are added to neutralize the surface charges of the microorganism and other colloid materials. Next, a flocculating agent such as a polyelectrolyte (e.g., a cationic polyacrylamide-based polymer) is added. Depending on the conditions and dosage, flocs (clusters) with different morphologies are obtained in this process. The optimal dosage and conditions are characterized by the production of dense flocs and a clear supernatant, but the exact conditions also depend on the separation devices used in the primary separation. 

Primary separation

In the primary separation the pretreated liquid containing the enzyme is separated from the biomass and other insoluble materials by filtration or centrifugation. The two technologies use different driving forces; pressure in the case of filtration, and gravity in the case of centrifugation, but they can both be used for both bacteria and fungi.

Rotary drum vacuum filters are a widely used piece of equipment for this filtration step due to the simple operation and low investment costs. The drum is typically precoated (e.g., with diatomaceous earth, silica, perlite) to improve the process performance. Additional filter aid is often added to improve the filtrate quality; hence the amount of sludge generated from this unit operation is relatively large. Several other filtration devices such as filter machines and cross-flow filtration devices are also used for filtration. One of the benefits of the latter is that they can be operated without the addition of filter aids, and consequently the amount of generated sludge can be reduced.

The centrifugation step is an attractive, high-capacity unit operation that is available in several different configurations, for example disc stack centrifuges, decanters, and basket centrifuges. The centrifugation step often results in a liquid containing small amounts of sludge, which means that a fine centrifugation or a filtration step is needed to ensure complete removal of the microorganisms and to produce a liquid with a satisfactorily low turbidity.

The two main technologies mentioned above, drum filtration and centrifugation, can also be combined. For example, the centrifuges can be used for the first separation and the drums to remove the residual turbidity.
 

Concentration

The liquid obtained from the primary separation is a clear liquid containing the enzyme of interest in a diluted form. The next step is therefore to increase the concentration of the enzyme. Two technologies are widely used for this purpose, evaporation and ultrafiltration.

Evaporation is performed under vacuum, where water can be removed in conditions where the enzyme is stable (30–40 °C). Evaporation removes water and other volatile compounds, while enzymes, salts, and other dry matter are retained. The liquid is typically concentrated up to 20–40% dry matter during the evaporation. When using evaporation for concentration of enzymes, it is important to ensure that the temperature of the heating surface is as low as possible to avoid denaturation of the enzyme. The advantage of evaporation is that the dry matter, which typically stabilizes the enzymes, is retained. Savings on the formulation costs can therefore be realized, but of course these savings should be weighed against the costs associated with the higher energy input.
Concentration using membranes is another widely used technology. Membranes are available in a range of pore sizes, from nanofiltration membranes that only allow passage of water molecules to ultrafiltration membranes that allow passage of salts and lower-molecular materials. For concentration of enzymes between 10 and 100 kDa ultrafiltration is preferred. The ability of the membranes to retain the high-molecular molecules is described by the membrane’s cutoff value. For example, a membrane with a cutoff value of 10 kDa will allow passage of molecules with a molecular weight less than 10 kDa. The selected membranes typically only allow less than 1% passage of the enzyme to the permeate. Ultrafiltration can be used to concentrate liquids to 20–25% dry matter. Concentration to higher dry matter is possible, but the viscosity of the liquid often becomes a limiting factor, and the flux (flow per membrane area) decreases significantly.

Ultrafiltration is attractive compared to evaporation due to the lower energy consumption, and compared to nanofiltration the capacity (flow per membrane area) is significantly higher.
An ultrafiltration setup can also be used to wash out lower-molecular impurities by diafiltration. In this mode of operation water (tap water or deionized water) is added to the ultrafiltration concentrate, and the liquid is concentrated to compensate for the dilution. With this approach the content of low-molecular impurities can be reduced.

Other concentration methods include precipitation with salts, polymers, or organic solvents, but they are less attractive due to the high environmental impact (disposal or regeneration of salts and/or organic solvents). Finally, concentration can be achieved by spray-drying or lyophilization (freeze-drying). Handling issues are observed for both methods due to the formation of enzyme dust, but the latter in particular is potentially beneficial for heat-sensitive enzymes.
 

Purification 

The purification steps are aimed at addressing issues related to the undesirable effects of impurities in the product or in the application. The technology used depends on the type of impurity to be removed. Some of the methods available are:

• Precipitation
• Adsorption
• Inactivation
• Crystallization
• Chromatography
• Two-phase extraction


When precipitation is used for purification, the enzyme is precipitated using salts (e.g., Na2SO4), an organic solvent (acetone), or isoprecipitation. This technique is primarily useful if the impurity is of nonprotein origin as the precipitation is usually unspecific.

Adsorption, for example using activated carbon, is known to be useful for process-related impurities like color, odor, or antifoam used in fermentation. A range of activated carbon types is available, and a screening is typically needed to find the best carbon for the specific problem. The main concern about including a carbon adsorption step in a recovery process is the handling issues, especially related to poor filterability of activated carbon. Filter plates/cassettes impregnated with activated carbon have overcome some of these problems.

Inactivation is applicable when the impurity is another enzyme activity, for example a protease, that is labile under conditions where the main enzyme activity is stable. With this approach the enzyme impurity is denatured at a specific pH and temperature and will subsequently be removable by filtration.
Protein crystallization is a very complex process, much more complex than for small molecules (e.g., salts) that crystallize when the liquid is supersaturated. The crystallization process for proteins and enzymes in particular consists of at least two steps: 1) nucleation; and 2) crystal growth. When developing a crystallization process, the conditions for both stages need to be optimized. Addition of salts, adjustment of pH, addition of polymers, and addition of organic solvents are some of the techniques available for generating a supersaturated liquid and inducing crystal formation (nucleation). Crystal growth is often optimized by the selection of process conditions. If conditions can be found where crystals are formed at a sufficiently high yield, the process is a very simple and cost-effective protein separation technique.

Chromatography is a widely used method for protein separation in the biopharmaceutical industry where very high purity is required. For industrial enzyme production the technology is generally regarded as too expensive, except for a few high-value products. Aqueous two-phase extraction is an alternative recovery technology that has been investigated for many years in the industry and at universities. The standard two-phase system consists of PEG and salts (e.g., potassium phosphate). The polymer is added to the enzyme concentrate, and after mixing the enzyme is isolated from the PEG-containing phase. One of the main hurdles for application of this type of process is the cost of disposal or regeneration of the PEG and/or salts.