product recovery

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Wohlgemuth R (2011) Downstream Processing and Product Recovery | ProductRecovery. In: Murray Moo-Young (ed.), Comprehensive Biotechnology, Second

Edition, volume 2, pp. 591–601. Elsevier.

© 2011 Elsevier B.V. All rights reserved.

Author's personal copy

2.42 Product Recovery R Wohlgemuth, Sigma-Aldrich, Buchs, Switzerland

© 2011 Elsevier B.V. All rights reserved.

2.42.1 Introduction 591 2.42.2 Historical Background 592 2.42.3 Modular Unit Operations in Downstream Processing 592 2.42.3.1 Recovery of Solids and Liquids 593 2.42.3.2 Cell Treatment 594 2.42.3.3 Solvent Extraction 594 2.42.3.4 Liquid–Liquid Phase Separation 594 2.42.3.5 Crystallization and Precipitation 594 2.42.3.6 Adsorption 595 2.42.3.7 Distillation 595 2.42.3.8 Chromatography 595 2.42.3.9 Membrane Filtration 595 2.42.3.10 Other Unit Operations 596 2.42.4 Integrated Unit Operations in Downstream Processing 596 2.42.4.1 Solid–Liquid Separation and Product Recovery 596 2.42.4.2 Reaction and Solvent Extraction 596 2.42.4.3 Reaction and Liquid–Liquid Phase Separation 597 2.42.4.4 Reaction and Crystallization/Precipitation 597 2.42.4.5 Reaction and Adsorption 597 2.42.4.6 Reaction and Distillation 597 2.42.4.7 Reaction and Chromatography 597 2.42.4.8 Reaction and Membrane Filtration 598 2.42.5 Product Purification 598 2.42.5.1 Metabolite Purification 598 2.42.5.2 Lipid Purification 599 2.42.5.3 Protein Purification 599 2.42.5.4 Nucleic Acid Purification 599 2.42.5.5 Carbohydrate Biopolymer Purification 599 2.42.6 Product Formulation and Stabilization 600 2.42.7 Conclusion 600 References 601

Glossary adsorption Favored product binding to an adsorber/resin compared to the aqueous bulk phase as a way of concentrating the product. chromatography Workhorse unit operation for product separation from byproducts by passing a mixture over a suitable column and collecting the separated pure product fraction. crystallization/precipitation Popular and often used downstream processing step making use of decreased product solubility by changing the medium/solvent composition and/or conditions.

distillation Separation of more volatile products from less volatile products by evaporation and subsequent condensation. phase separation Primary recovery step making use of the separation of nonmiscible phases such as solid– liquid, organic–aqueous, and aqueous–aqueous phases. membrane filtration Spatial separation of different molecules or particles by polymeric or ceramic membranes of defined pore sizes, which retain the products larger than the pore size and pass the impurities or vice versa.

2.42.1 Introduction

The knowledge of how to obtain products after biotechnological processes from a mixture of many ingredients has not only been essential in the historical development of biotechnology from small manual procedures to large industrial technologies, but is also a key factor for industrial biotechnology and biotransformations today [1, 2]. The significant amount of work, energy, and equipment

591 Comprehensive Biotechnology, Second Edition, 2011, Vol. 2, 591-601, DOI: 10.1016/B978-0-08-088504-9.00135-5

Author's personal copy592 Downstream Processing and Product Recovery

that is needed for the separation of products, reusable materials, and wastes depend to a large extent on the type of product (product mixture or highly purified product, low-molecular-weight product or high-molecular-weight product, and stable product or highly labile product), and also on the type of biological source. Whether the source of the biomass, crude extract, broth, or fluid comes from biotransformation, microbial fermentation or cell culture, plant or animal tissue or body fluids, processes of separation, refinement, purification, or transformation are required to recover the desired product. The product recovery operations, generally, require the isolation and purification of the product from dilute aqueous media and often constitute the major cost contribution of a production process. It is therefore important to develop high-yield processes with a reduced amount of work and energy, with a minimum number of steps. Each operation needed depends both on the preceding bioprocess design and on the form and concentration of the final stable product required. The variety of product types has led to different approaches and methodologies for these processes, and the number of publications on product recovery has steadily increased over the last century (Figure 1).

2.42.2 Historical Background

Product recovery operations from complex mixtures, obtained after bioprocesses, have accompanied human history since the time of producing beer, wine, vinegar, and other food preparations to the present industrial processes for making a variety of products such as organic acids and other chemicals, antibiotics, amino acids, vitamins, proteins and enzymes, solvents, and liquid fuels. As waste has accumulated with the industrial scale-up of processes, the waste treatment and utilization aspects of such bioprocesses have become more important.

The increasing know-how in the selection of the best product recovery operations [3–5] has been a key to the industrial large-scale production of these products, which have increased the quality of life over the last two centuries.

2.42.3 Modular Unit Operations in Downstream Processing

The great number of individual downstream processes in biotechnology can be perceived as an entity of steps or unit operations, which are based on common technologies and the same fundamental sciences as the unit operations in chemical engineering [6]. Each unit operation is based on exploiting unique physico-chemical properties of the product of interest compared with the other species in the mixture. The challenges in the downstream processes of biotechnology are not diminished compared with chemical engineering, but are different and are an important research area of biochemical engineering. The great product diversity makes separate considerations of low-molecular-weight and high-molecular-weight products useful.

In the area of low-molecular-weight products, many unit operations in biotechnology such as extraction, phase separation, crystallization, adsorption, or distillation have the same physicochemical principles as in chemistry (Figure 2). Important physicochemical properties that can be utilized for the recovery of small-molecular-weight compounds are the charge, molecular weight, hydrophobicity, volatility, and solubility behavior. The fundamental chemical and biochemical engineering of the underlying complex processes are essential for industrial production processes. Purification steps such as the separation of regioisomers,

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Figure 1 Publications in product recovery over the last century.

Comprehensive Biotechnology, Second Edition, 2011, Vol. 2, 591-601, DOI: 10.1016/B978-0-08-088504-9.00135-5

Author's personal copyProduct Recovery 593

Adsorption

Distillation

Biomass, crude extract, or broth from bioprocess

Crystallization and

precipitation

Liquid–liquid phase

separation

Solvent extraction

Cell treatment

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and liquidsMembrane

filtration

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Figure 2 Overview of unit operations in product recovery.

cis/trans-isomers, diastereomers, or enantiomers, and protection of labile functional groups and their final deprotection also benefit from the interaction between chemistry and biotechnology. Unit operations such as cell harvesting and cell separation, cell preservation and cell immobilization, product secretion, or cell homogenization and cell lysis can build on the vast biochemical experience and are specific for biotechnology operations (Figure 2). This is also true for large-molecular-weight products such as proteins, nucleic acids, polysaccharides, and other biopolymers, where additional factors like increased viscosity, degradation by enzymes, stability under process conditions, and folding play a role.

The development of the optimal product recovery process may require substantial work, and the final engineering of this process is often having a big influence on cost, equipment size, and waste. It is therefore of much interest to use conventional unit operations and to employ currently established and available techniques and materials like resins and membranes. This reduces the risks in scale-up, process robustness, technology transfer, and Good Manufacturing Practice (GMP) issues for equipment, methods, and processes.

2.42.3.1 Recovery of Solids and Liquids

The separation of solid particles, such as adsorbers, cells, inclusion bodies, virus-like particles, or crystals, from a solution depends both on particle properties such as size and density and on the properties of liquid media such as viscosity, density, and gel, emulsion, or foam formation tendency. The selection of a suitable separation method is mainly determined by size, size differences, and density. Particle–particle separation of coarse particles from fine particles may be useful to separate first macroscopic particles of nonutilized solid substrates or high concentrations of products on crystals or on solid adsorbers. Larger polymeric adsorber particles can be well separated from smaller biological cells by a high-performance sieving step prior to solid–liquid separation. Initial harvesting, medium exchange, or clarification requires suspensions of solids to be separated from their liquid media. Primary recovery processing steps such as decanting, sedimentation or settling, centrifugation, depth filtration, or tangential flow micro-filtration [7] are in widespread use. Different types of centrifugal separators and continuous centrifuges provide robust and broadly applicable equipment for handling various suspensions with different methods of discharge. The pore size of the filtration unit can thereby be adjusted to the size of the particles to be separated. The advantage of using microfiltration membranes with 0.2 μm pore size compared with sedimentation or centrifugation is the generation of a particle-free solution, which can be directly used in the next downstream processing steps and does not need further clarification [8]. Depending on whether the product is contained in the solids, which can consist of cells and intracellular products, cell debris, inclusion bodies, or crystals, or is dissolved in the liquid medium, the best-suited techniques for collecting or removing the solids are selected. The size and size distribution, morphology, and concentration of the solid particles have a key influence on the technique that is selected for the recovery of the solid particles. Therefore, it is already in the bioprocess design phase that these key parameters of the solid particles need to be considered in order to minimize the effort going into solid–liquid separations.

The separation of liquids from a solution can be straightforward if the liquid is nonmiscible, the formation of foams, soaps, and emulsions can be avoided, and liquid droplets coalesce fast to form a separate phase. Since the presence of biological cells and nonmiscible product in the liquid aqueous medium can lead to complex phase behavior, the phase separation time can in certain cases be very long. Addition of antifoam reagents, salts, buffers, or other reagents to break emulsions can be used to reduce phase separation times to reasonable values.



2.42.3.2 Cell Treatment

The nature of the product (whole cell, extracellular product, or intracellular product) also determines the type of cell treatment. From the gentle recovery of viable whole cells, mechanical dewatering or water removal by drying or lyophilization to the complete homogenization and lysis of cells by enzymatic, chemical, and physical methods, each cell treatment procedure needs to be optimized for the specific target products. Various methods of flocculation can be used to aggregate the cells and therefore make sedimentation, centrifugation, or filtration easier. For intracellular products, selective product release strategies, considering key factors for manufacturability such as viscosity reduction, removal of product-related contaminants, and elimination of enzymes that reduce product quality, are crucial for successful downstream processing [9]. One particular interesting selective product release strategy is product secretion, making use of natural transport mechanisms or engineering cells capable of secreting the product into the medium. This is essential in large-scale protein production, where the work-up is simplified significantly if the product can be recovered from the medium. In the case where the product of interest cannot be secreted, cell disruption is the key unit operation, which liberates intracellular products. A case-by-case assessment is required to find whether mechanical, physical, chemical, or biochemical method is the best disruption technology.

2.42.3.3 Solvent Extraction

Organic solvents that are nearly immiscible with water and rapidly form two liquid phases have been the classic way of extraction. High solubility of products in organic solvents or components of an extractant phase can be utilized to extract the product from the aqueous phase, if the distribution coefficient of the product between the aqueous and the organic phase is favorable [10]. The product solubility of ionic molecules in the organic solvent may be increased by neutralization. The extraction method depends on the use of final product. This liquid–liquid extraction is a product concentration step and requires a good and fast separation of the organic and aqueous phases. If products are localized in cells or cellular compartments, medium and water removal can be useful for high-yield solvent extraction of the products. The liquid–liquid extraction of organic acids [11–13] and alcohols [14, 15] has focused on the extractive recovery of neutral undissociated molecules and has improved the process technologies based on extraction. Phase separations of aqueous and organic phases into two phases with clear phase boundaries are scalable low-cost unit operations, which can be developed rapidly and are therefore used extensively in industry. As the time for phase separation can vary depending on the influence of additional components in the reaction mixture, small additions of antifoam compounds or salts help to accelerate phase separation processes. Although solvent extraction has been widely adopted by industry, some final products may preclude the use of solvent extraction and the large solvent consumption is a disadvantage.

2.42.3.4 Liquid–Liquid Phase Separation

While solvent extraction with organic–aqueous two-phase systems has been mainly the domain of small molecules product recovery, aqueous–aqueous two-phase system has been advantageous for the recovery of proteins and enzymes without any damage [16]. The situation is different for the practical applications of aqueous two-phase partitioning and phase separations, where, in general, the costs of phase-forming polymers and process development times have limited this operation in industrial processes [17]. A direct comparison of ion-exchange chromatography and aqueous two-phase separation (ATPS) has shown the superior overall process yield of ATPS at lower costs than ion-exchange chromatography.

2.42.3.5 Crystallization and Precipitation

The simplicity and low cost of these liquid–solid phase separations have made crystallization and precipitation one of the most often used and popular downstream processing steps. The ideal case for this product recovery operation is a preceding bioprocess whereby the product is formed at a concentration well above the product solubility in the reaction medium. If the product is soluble even at high concentrations in the reaction medium, changes in pH, temperature, solvent composition, and ionic strength can offer opportunities for product crystallization or precipitation. The development of new product precipitation and crystallization procedures is still challenging for both small and large molecules, but rewarding if successful. Even if the best conditions for the crystallization of pure molecules have been developed, their extension to the crystallization of the same molecules in their more complex media is not trivial. Despite its extensive applications, crystallization at production scale can be difficult to characterize and improve by process analytical technology [18]. Precipitation and crystallization of products as insoluble barium or calcium salts have been commonly employed for the recovery of organic acids. This technique has been used for a long time in the small molecule field as well as in the protein field, although in the latter field mostly at small scale. Ammonium sulfate is among the commonly used precipitation reagents due to its high solubility and low cost and the availability of extensive data on its saturation concentrations under various conditions. Crystallization and precipitation are both separation and purification processes and do not need expensive equipment. Therefore, they are experiencing a renaissance as simple and low-cost purification methods for small biomolecules such as metabolites and large biomolecules such as proteins.



2.42.3.6 Adsorption

The distribution of products between the bulk aqueous phase and an adsorber is a good tool for product recovery, if organic solvents have damaging effects. The use of polymeric adsorbers for the recovery of small molecules from dilute media is well established [19]. A variety of hydrophobic polymers, ion exchangers, and other functionalized polymers, which are available at large scale, can be used for designing the best product distribution between the adsorber and liquid medium. As the adsorption characteristics such as capacity, selectivity, or kinetics are not known, in general, for the product to be recovered, these data have to be determined experimentally. The assessment of adsorbent resins for their capabilities of product recovery has been useful for selecting the most suitable adsorbers for small molecule recovery (antibiotics and chiral building blocks). After separation of the product-loaded adsorber from the reaction mixture, the product can be easily recovered from the adsorber by solvent elution or extraction, and the adsorber can be recycled.

2.42.3.7 Distillation

Volatile products can be separated from nonvolatile products by evaporation, gas stripping, and subsequent condensation or adsorption. Besides traditional alcoholic drinks and biofuels, a variety of products such as solvents, flavors and fragrances, terpenes, and oils from bio-based processes are preferentially recovered by distillation. Fractionation according to boiling points depends on the boiling point differences of product and other volatile components of the reaction mixture. If simple distillations do not achieve the required product purity, advanced fine distillation techniques have proven useful in the purification of terpenes from challenging natural oil mixtures with components of similar boiling points. The effect of small impurities on possible reactions of the product during distillation at increased temperatures needs to be checked.

2.42.3.8 Chromatography

Passing a product mixture over a chromatography column is not only an important standard unit operation, but also a workhorse of downstream processes for product recovery. Differences in the partitioning of the individual components between the solvent and the chromatographic material in the column and the selection of the best conditions for enlarging these differences enable high-resolution bioseparations. Highly selective chromatographic capture steps under mild product recovery conditions are key elements of a flexible, but generic downstream process platform. Adsorption chromatography, affinity chromatography, hydrophobic and hydrophilic interaction chromatography, ion-exchange chromatography, and gel filtration chromatography can provide unit operation platforms for certain product classes with biochemical similarities. These major chromatographic methods use charge, affinity, polarity, and size as the basis of separation. Although the best purifications can be obtained with affinity chromatography, the most common chromatographic method is ion-exchange chromatography. The optimization of chromatographic separations relies heavily on experimental data, and the combination of high-throughput screening with genetic algorithms has provided powerful tools for rapid process development [20]. Different small molecule product classes such as metabolites and lipids and large molecule classes such as proteins, nucleic acids, and polysaccharides can be separated by a wealth of specialized chromatographic experience accumulated over the years. Although chromatographic separations have a high resolving power for many mixtures, there are limitations from the industrial and large-scale perspective. Alternatives such as membrane chromatography can increase throughput and overcome traditional bottlenecks in column chromatography [21].

2.42.3.9 Membrane Filtration

Nature provides the role model processes for product recovery by highly selective as well as nonselective membrane filtrations through the natural lipid membranes of biological cells. As the macroscopic engineering counterpart of the natural cell membranes, polymeric membranes for the spatial separation of different molecules have not yet reached their high performance, but are nevertheless a key tool for the spatial separation of different molecules in biotechnological downstream processes. Most of the membrane separations are performed in an aqueous environment and are based on size differences of the components to be separated. The pore sizes of the membranes vary from micrometers (microfiltration) over pore sizes characterized by the molecular weight cutoff (500 000–1000) of the molecules which no longer pass to the filtrate (ultrafiltration) to pore sizes with a molecular weight cutoff below 1000 (nanofiltration). Tangential flow membrane filtration is a state-of-the-art operation with membranes of selected molecular weight cutoffs, and is employed almost everywhere. It is used in batch or continuous mode as a major technique for both product concentration and product purification. Microfiltration is applied, for example, for the concentration of cells, crystals, and precipitates, whereas ultrafiltration is applied for the concentration of high-molecular-weight products and the separation of high-molecular-weight biopolymers from unwanted low-molecular-weight byproducts, media components, and salts [8, 22]. Nanofiltration is used in the downstream processing for the desalination and concentration of small-molecular-weight compounds [23] and reverse osmosis membranes can be used for a subsequent concentration step. A variety of micro-, ultra-, and nanofiltration membranes made of different polymers and pore sizes are in widespread use. Other membrane materials of interest include highly porous, acid- and solvent-resistant ceramic membranes with pores size from 1 to >1000 nm. Membrane filtration equipment from small to large scale is available for the selection of the best product recovery yields and minimal adsorption.



2.42.3.10 Other Unit Operations

Electroseparation processes such as electrophoresis and electrodialysis are based on charge and mobility differences in an electric field and are orthogonal to the molecular weight-based separation methods; however, they are used in more specialized preparative applications. Free-flow electrophoresis equipment has been applied for a variety of high-performance separations of cells, proteins, and small molecules over the last decades. Electrodialysis has been a suitable separation method with respect to energy requirements in the large-scale production of amino acids. The use of ion-exchange membranes in electrodialysis-based separation technologies is of much interest for process integration and scale-up [24].

The removal of buffers from small molecules at the end of chromatographic separations can be achieved by lyophilization if volatile buffers are used in the preceding chromatographic steps. The best volatile buffer can be selected from various compounds according to the completeness of removal at the desired pH value, temperature, and pressure.

2.42.4 Integrated Unit Operations in Downstream Processing

The scalability, yield per step, and number of unit operations in downstream processing are key factors to the economics of product recovery. The replacement of multiple downstream processing steps by single-stage processes can improve overall operational efficiency by optimizing product quality, space–time yield, timing, and cost issues. In situ product removal (ISPR) techniques remove product from the vicinity of the reaction space thereby preventing its interference [25, 26]. The integration of product removal unit operations with bioreactions has been a successful way of process intensification for making the success of the bioprocess clearly visible from Pasteur’s biocatalytic tartaric acid resolution process in 1858 until the large-scale industrial bioprocesses today (Figure 3).

2.42.4.1 Solid–Liquid Separation and Product Recovery

The integration of these two unit operations can principally be done in one of two different ways, depending on whether the product is in the solid or liquid phase. In the first case, the solid particles can be the pure product in the form of crystals or the product in association with other components such as inclusion bodies, adsorbers or cell organelles, viruses, and virus-like particles. The liquid phase contains the rest of the impurities, side products, and additional reagents, which have to be separated from the product. In the second case, the liquid can be the pure product itself or the product can be dissolved in the liquid phase; the solid phase contains the rest of the impurities.

Expanded-bed adsorption is such an integrated primary downstream process where the solid phase usually contains the product. This technique combines solid–liquid separation with product recovery into a single operation [27].

2.42.4.2 Reaction and Solvent Extraction

A simultaneous reaction and extraction system involves organic–aqueous two-phase systems that require the organic solvents used to be compatible with the bioprocess. Such organic–aqueous two-phase systems are attractive for increasing the yield of hydrophobic products toxic to the biocatalysts or cells and that are highly soluble in the organic phase. A scheme that integrates a homogeneous phase consisting of organic–aqueous tunable solvents with a carbon dioxide-induced phase separation allows simultaneous product recovery and recycling of the biocatalyst [28].

Adsorption Solvent

extraction

Crystal or precipitate formation

Bioreaction Bioprocess Biocatalysis

Membrane filtration

Figure 3 Examples of integrated operations in product recovery.



2.42.4.3 Reaction and Liquid–Liquid Phase Separation

The applications of ATPSs for extractive bioconversions have not yet resulted in wide industrial applications, despite attractive scientific exploitations for many years [17]. Further work on reducing the costs of the phase-forming polymers and on reducing the complexity of the ATPSs involved will stimulate industrial applications of this research area. The use of two aqueous phases in extractive fermentation has been an attractive approach for overcoming low product yield or for obtaining the product in a cell-free phase. The increases in productivity have been achieved by overcoming existing bottlenecks such as product inhibition and degradation through phase separation of the product as it is formed in the reaction. Both small-molecular-weight and largemolecular-weight extracellular products have been recovered by this methodology. In the case of intracellular products, cell disintegration or cell treatment is required and therefore different integration strategies for product recovery need to be developed. Despite the current lack of translation of research applications into industrial processes, environmentally friendly ATPSs in view of the organic solvent waste contribution of solvent extraction processes have tremendous potential for new integrated bioprocesses.

2.42.4.4 Reaction and Crystallization/Precipitation

The combination of these two unit operations is useful for crystalline product formation by bioprocesses where the product is inhibiting the bioprocess or degrading under process conditions. The removal of the product by crystallization as soon as it is formed in the bioprocess can circumvent these limitations of product inhibition and degradation. As the thermodynamics of the integrated bioprocess can be changed compared with the nonintegrated process, unfavorable equilibriums in the nonintegrated bioprocess can be pulled to completion by the integration of the equilibrium bioprocess with the product crystallization. An example of such an integrated process is the fermentation and enzymatic deacetylation of adipoyl-7-amino-deacetoxy-cephalosporanic acid in one reactor [29]. By using the liberated adipic acid again in the fermentation, avoiding the use of acids and bases for pH shifts, a reduced number of downstream processing units, and a reduction in waste salts production, the integrated process leads to economic advantages such as lower manufacturing costs and lower capital investments. The integration of product formation and crystallization has been shown to lead to significant advantages over the nonintegrated case [30].

2.42.4.5 Reaction and Adsorption

The integration of the reaction part (biocatalytic reaction, fermentation, or cell culture) with an adsorption operation can serve the purpose of easier product recovery or of removing reaction components that inhibit the reaction at higher concentrations. Such inhibitions prevent the process to be run at higher concentrations and can be caused by substrates, products, and byproducts. Preferential adsorption of fermentation inhibitors from biomass hydrolysates can be used to improve the ethanol yield of the process [31]. Adsorbent resins are gaining significant applications in antibiotic and natural product recovery, and the characterization of different adsorbers under the given conditions is important [32].

If both substrate and product inhibit the reaction, the adsorption of both substrate and product is a way of keeping the free substrate and product concentration below the inhibitory concentrations, and this substrate feed and product recovery (SFPR) technique has been useful for improving space–time yields. The most suitable adsorbers can thereby be selected by a straightforward experimental determination of substrate and product adsorption as a function of concentration [33]. The scale-up and further product recovery of a reaction in an SFPR mode benefit from complete conversion and scalable simple operations at large scale.

2.42.4.6 Reaction and Distillation

Alcoholic beverages with higher ethanol contents such as whiskey, gin, rum, brandy, and vodka have been traditionally recovered by distillation for centuries following fermentation of various grains, fruits, or molasses. The production of biofuels and solvents by fermentation and traditional distillation has been of varying interest since the nineteenth century and the improvement of the thermal product recovery techniques after the fermentation with respect to cost reduction, energy reduction, and simple integration is a key factor for the whole production processes. Simple product recovery methods such as gas stripping, condensing the volatile product, and recycling the stripped gas to the fermentation are efficient alternatives. The combination of fermentation or a biocatalytic reaction with the removal of the volatile product via the gas phase can improve conversion yields by reducing product inhibition in the liquid phase. Selective removal of volatile products such as flavors and fragrances is highly interesting; however, the specialized fine distillation technology makes a division between the fermentation/reaction step and the distillation step necessary.

2.42.4.7 Reaction and Chromatography

This coupling is of interest for nonvolatile and temperature-sensitive components and for intensifying bioprocesses with unfavorable equilibriums by product removal and feeding back unreacted substrate to the bioreaction. Annular reactive chromatography has been found less efficient, but convenient for collecting multiple products, whereas simulated moving bed (SMB) reactors are more efficient, but allow the separation of only two products [34]. A high-fructose syrup process using immobilized glucose



isomerase has been improved by simulating continuous countercurrent contact of the liquid stream with the solid adsorbent. Adsorption columns have been advanced against the fixed inlets and outlet of liquid streams without actual movement of the solid adsorbent, while the immobilized enzyme reactors have been stationary [35]. Since the coupling adds constraints to the chromatographic separation, a robust product–substrate separation in the presence of additional components from the bioreaction is of key importance. In the biocatalytic condensation of glycine and acetaldehyde to L-allo-threonine, the separation performance with respect to the two amino acids has been shown to be only slightly reduced by coupling the SMB separation to a continuously operated enzyme membrane reactor (EMR) whose efflux contained, in addition to the amino acids, acetaldehyde and the cofactor pyridoxal-5-phosphate [36].

2.42.4.8 Reaction and Membrane Filtration

The combination of a bioreactor with a membrane filtration system can serve different purposes of either mass recycling into the bioreactor or product recovery from the bioreactor [3]. A large range of microfiltration membranes for the recycling of biological cells and ultrafiltration membranes for the recycling of large-molecular-weight compounds such as enzymes or large coenzymes are available. The membranes can have a symmetric or asymmetric structure and can thereby have a dual function of immobilizing the biocatalyst and of product separation or purely a product recovery function. In the first case, where the membrane serves as biocatalyst support and separation unit, the biocatalyst can be immobilized within the porous membrane or at the membrane surface by different methods such as covalent attachment, ionic interactions, adsorption, entrapment, gel formation, or cross-linking. A variety of different geometries such as flat-sheet, spiral-wound, and tubular structures are available for the membranes, which can then be assembled into the appropriate membrane modules and units for standardized couplings to the feed, retentate, and permeate lines.

The concept of the EMR has been developed and successfully applied by Kula and Wandrey [37] and others. The classical work on the enzymatic reductive amination of α-ketoisocaproate to L-leucine with L-leucine dehydrogenase and simultaneous cofactor regeneration with formate dehydrogenase in a continuously operated membrane reactor [38] has pioneered this integration mode for a large number of industrial bioprocesses. The enzymatic production of amino acids in membrane reactors with simultaneous regeneration of β-Nitotinamide adenine dinucleotide reduced (NADH) has been developed to industrial scale [39] at Evonik in Germany. The EMR has been successfully applied in numerou routine productions by other industries (e.g., Tanabe Seiyaku, Sepracor, and Sigma-Aldrich), demonstrating the industrial relevance of this integration of reaction and membrane filtration [1]. The membrane reactors have the advantage of using soluble components (enzymes, substrates, and products), which can be easily replenished, for example, for substrate supply or if more enzyme is needed to keep the bioconversion rate constant because of enzyme deactivation effects. Therefore, attention has to be given to the preparation of enzyme so that the operational stability of the free enzyme is good enough for its use in the EMR.

Extensions of the classical EMR concept include the charged ultrafiltration membrane enzyme reactor, where negatively charged ultrafiltration membranes are used to retain the native cofactor in the reactor, or the use of nanofiltration membranes. For the bioconversion of poorly soluble substrates, an emulsion membrane reactor consisting of a separate chamber with a hydrophilic ultrafiltration membrane for emulsification, an EMR loop with a normal ultrafiltration module, and a circulation pump can be used [1].

Other examples include enantiomerically pure intermediates, anticancer drugs, vitamins, anti-inflammatory compounds, cyclodextrins, and antibiotics [40].

2.42.5 Product Purification

Although the ideal downstream processing and purification scheme would be the direct separation, for example, by solid–liquid or another phase separation of the highly pure product, the reality at the end of the biocatalytic process is similar to the work-up in organic chemistry. Many downstream processes and purification operations are unit operations which are robust, well established, and scalable. Therefore, available methods are often chosen in the development of purification methods. Since the selectivity of biocatalytic reactions is high, less side products and auxiliary reagents have to be removed in general, but depending on the number of main products formed and the type of educts and auxiliary compounds used, advanced isolation and purification technologies may be of use. In the case of two very similar products, such as regioisomers, formed in an equimolar ratio, the SMB chromatography is a useful purification method [41], which is also available at large scale.

2.42.5.1 Metabolite Purification

Biological cells represent a valuable source for metabolites, because the chemical synthesis in many cases is not possible or economically viable. As metabolites are small-molecular-weight compounds, many product purification processes from chemistry and biochemistry can be transferred to biotechnology. The wide structural diversity of metabolites requires a large series of purification procedures, which can vary from simple one-step purifications, utilizing one or more unit operations described previously, to the most challenging separations of homologues with very similar molecular properties [10, 42]. The diversity of closely related metabolites created by biological cells in biocatalytic pathways can be both an opportunity for multiproduct bioprocesses and a challenge for the purification of a single product. Upstream and bioprocess developments can narrow down



the metabolite diversity and therefore simplify the purification work of such metabolite mixtures to highly defined and enriched single metabolite products. The production of industrially important metabolites such as organic acids, amino acids, vitamins, and chiral intermediates at large scale focuses on the selection of high-yield mutant strains and avoids the design of extensive separation processes. Rapid and simple purification methods such as pH adjustments, extraction, adsorption, crystallization, and chromatographic separations are in high demand, with crystallization/precipitation and chromatographic separation being the most commonly used techniques. In addition, special cases of liquid metabolite mixtures can be checked for purification opportunities by fine distillation.

2.42.5.2 Lipid Purification

The large-scale processing of fats, oils, and lipids involves rendering, screw-pressing, expelling, or solvent extraction-based methods compatible with the applications. Separation and isolation procedures for lipids include crystallization, urea fractionation, distillation, enzymatic procedures, and liquid chromatography [43]. Adsorption, partition, ion-exchange, and supercritical fluid chromatography are versatile and useful tools for fractionating lipid mixtures on a preparative scale. Sephadex LH-20 and Sephadex G-25 have been commonly used for chromatography. Preparative high performance liquid chromatography (HPLC) and super-critical fluid chromatography (SFC) have also been successfully established lipid purification techniques.

2.42.5.3 Protein Purification

Although the isolation and purification of proteins has been established long ago, today’s challenges require faster ways to purify more proteins. More than a century of practice and know-how in protein purification has resulted in a great variety of methods and techniques [44], each with its own advantages, disadvantages, and limitations. The number of unit operations involved depends on the demands on purity and safety of the final product. The development of efficient and simple protein purification sequences is a major bottleneck and makes use of the best methods. Miniaturized systems for the major unit operations and for screening relevant downstream process parameters have been developed in order to reduce the development time significantly. The fundamental investigation of basic purification processes is crucial and can lead to extremely novel applications reducing this major part of production costs. As the preservation of fully functional proteins during protein purification and high yields are the goals, methods for avoiding protein degradation or modification have become important. However, in certain cases, the purification of inclusion bodies and the subsequent in vitro refolding into fully functional proteins can be the purification method of choice.

2.42.5.4 Nucleic Acid Purification

A variety of well-known laboratory methods for DNA and RNA purification are traditional molecular biology procedures. The transfer to large scale of many laboratory methods for DNA purification such as density gradient centrifugation with cesium salts, ion-exchange chromatography, and reversed-phase chromatography can, however, be difficult due to the use of toxic and mutagenic reagents, kits, or time-consuming procedures [45]. Large-scale processes using only generally recognized as safe reagents and scalable methodologies such as precipitation, salting-out, and chromatography (ion exchange, hydrophobic interaction, and size exclusion) are available for the large-scale purification of DNA. Nonchromatographic procedures based on selective precipitation with cetyltrimethylammonium bromide [46] or aqueous two-phase systems containing polyethylene glycol with potassium citrate or potassium phosphate have allowed the fast and simple recovery of plasmid DNA. The complete sequence of lysis, precipitation, clarification, and extraction can be performed in a single vessel [47]. Suitable large-scale methods for RNA employ overproduction and purification of recombinant RNA [48] and polyacrylamide-free size-exclusion chromatography [49].

2.42.5.5 Carbohydrate Biopolymer Purification

The carbohydrate biopolymers are widely distributed in animals, plants, seaweeds, algae, mushrooms, fungi, yeasts, and bacteria, where they have different functions such as nutritional reserve or structure-forming compounds. Many polysaccharides are formed extracellularly in larger amounts, are water-soluble, and have a long history of applications in industry, medicine, and our daily life. As the carbohydrate biopolymers show the greatest structural diversity of all biopolymers, it is also clear that the purification and analysis pose the biggest challenges. Depending on the localization in the biological cells, the carbohydrate biopolymers may have to be deproteinized without destroying the carbohydrate structure. Carbohydrate biopolymers with cross-links, consisting of glycosidic bonds or peptide units, are insoluble in aqueous media and require selective removal of byproducts. Economical production of polysaccharides is usually achieved at very large scale. Soluble carbohydrate biopolymers have the advantage of being extractable, but a variety of related products are usually extracted as well. Fractionation is based on selective precipitation and solubilization, but in contrast to small molecules the increased viscosity even at low product concentrations is one of the main factors influencing not only purification but also formation and isolation of polysaccharides above a certain molecular weight. Different physicochemical parameters such as ionic strength, pH, cations, and viscosity, even at very low levels, can have a substantial influence on the purification scheme. The fractionation of structurally diverse carbohydrate polymers as well as the



separation of different molecular weight fractions consisting of the same building blocks (homopolysaccharide) or of different building blocks (heteropolysaccharide) requires the whole toolbox of unit operations and case-specific troubleshooting, until finally purified carbohydrate biopolymers are obtained [50].

2.42.6 Product Formulation and Stabilization

In our interdependent economy, it is crucial that bioproducts can be transported and stored worldwide in a biologically active state from the production site to the application site. The properties of the product and its application, on the one hand, and the minimum shelf-life requirements for products, which are kept permanently under controlled storage or transportation conditions, on the other hand, set the boundary conditions for the formulation and stabilization of the final product.

The storage stability of products is important for research, biomedical, and industrial applications and is influenced by many of the preceding process steps in production. The last operations are, however, often the most important ones [5], because these are determining the form, standardized quality, and impurities of the product. A pure powder or liquid is the preferred form of the product; however, criteria such as ease of application, production costs, or stability issues often make other formulations such as dried powders as products, immobilized or stabilized solids, and buffered and stabilized suspensions or solutions the formulation of choice. The actual best practices for the formulation of stable products focus on preventing the relevant molecular mechanisms of the product degradation pathway. When the molecular structure of the product is given, the formulation design of the product can protect against degradation by microbial, chemical, osmotic, pH, and oxidative stress. Changes in molecular product integrity as well as more physical changes in solubility, adsorption, and aggregation can be prevented by the addition of protecting agents such as buffers, anti-oxidants, cryo- or lyoprotectants, and osmolytes. Although much experience has been gained for determining the best formulation and method to maintain and store, stabilize and homogenize, and apply and deliver bioproducts under given conditions in an active state, predictions have to be experimentally tested for each new product. Process analytical technologies that are capable of recognizing changes in the activity and molecular integrity of the product are thereby of prime importance. Biocompatible additives such as glycerol, polyethylene glycol, carbohydrates, or amino acids can be useful for optimizing stability and production issues of the product, but have to be checked for their compatibility with the intended applications. Since pH or oxygen can influence product stability tremendously, the addition of pH or redox buffers is often required in the quality design in order to have a robust production process.

The operational stability of products under the conditions of research, biomedical or industrial applications, is of equal importance, and strategies for product stabilization are not restricted to the ones already mentioned for storage stability, but can aim at a tailor-made product design.

2.42.7 Conclusion

The future directions of product recovery technology are influenced by numerous scientific and technological, industrial, economic and commercial as well as environmental and social developments. Nevertheless, it is quite clear that process improvements in product recovery with respect to waste minimization, volatile organic solvent reduction, energy conservation, safety, health, and environment issues are beneficial not only for the ecoefficiency of a product recovery scheme, but also for economical aspects such as production cost reductions, because the costs of product recovery are substantial determinants of the total production costs. In addition, these process improvements can lead to additional macro- and microeconomic benefits on the local and global levels.

The waste minimization in the recovery of a particular bioproduct depends both on technology changes, for example, replacing an extraction step with organic solvents by crystallization, and on finding new opportunities for useful applications of byproducts and thereby turning these from waste into products. Besides the engineering aspects of waste minimization, there is also the key molecular aspect of selectivity and the parameters influencing it in all product recovery operations, which are important for optimizing the recovery scheme to a defined purity of the product of interest. Therefore, innovative downstream processing taking into account both the molecular and engineering aspects continues to be of great importance for the whole bioprocess.

With the changing costs of energy and the limited resources of certain fossil energy, it becomes not only a cost-saving exercise to minimize the energy input into product recovery bioprocesses, but also an opportunity to develop new efficient product recovery processes for the economic production of biofuels for cars and jet planes.

The introduction of renewable resources into product recovery processes can occur in various contexts and offer, in addition, great opportunities as raw materials in bioprocesses. Many renewable resources can be utilized for the production of auxiliaries for product recovery such as cellulose and dextran, which have traditionally been used in the form of functionalized cellulose ion exchangers (e.g., DEAE-cellulose), cross-linked agarose and dextran (e.g., Sepharose and Sephadex) for chromatography, or as dextran components in ATPSs.

As the carbon dioxide and volatile organic carbon compounds in the Earth’s atmosphere need to be reduced, product recovery processes can be transformed into more sustainable processes that reduce the use of volatile organic solvents or reduce carbon waste burned to carbon dioxide [51–53].



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