9 10 Chapter 2 Biocatalysts in organic chemical synthesis 2.1 Introduction tradHionaiand new bboe*mb!JY Biotechnology is often divided into two categories called 'traditional biotechnology' and 'new biotechnology'. The major products of the traditional biotechnology industry are industrial alcohol, food and flavour ingredients, antibiotics and citric acid. The new biotechnology involves the newer techniques of genetic engineering and cell fusion to produce organisms capable of making useful products. Products of the new biotechnology are extremely diverse and include steroid derivatives, antibiotics and special proteins for therapeutic use (eg human growth hormone, interferons and interleukins). The market for products of traditional biotechnology is cmntly worth around w) times more than those of the new biotechnology, although it is predicted that the new biotechnology will account for an increasingly larger fraction of the total biotechnology industry. The purpose of this chapter is to compare and contrast various production strategies of the biotechnology industry and to consider some of the mapr decisions that have to be made during bioprocess development. Many of the areas touched upon will be developed in greater detail in other chapters of this book. The book is limited to the use of micro-organisms and enzymes as bioprocess catalysts and does not consider catalysis by plant and animal cells. As you will see later in this chapter, industrial microbiology is the major foundation of biotechnology and there are many reasons why micro-organisms dominate as production organisms in both traditional and new biotechnological processes. 2.2 Micro-organisms as catalysts of organic synthesis Microbial cells are very attractive as a source of catalysts for the production of organic chemicals because of their broad range of enzymes capable of a wide variety of chemical reactions, some of which are illustrated in Table 2.1. Biocatalysts in organic chemical synthesis 11 Table 2.1 Some reactions catalysed by microbial enzymes. In principle each enzyme catalyses the reverse process as well. 12 Chapter 2 Table 2.1 ...... Continued Microbial cells, rather than plant and animal cells, are generally preferred for the production of organic chemicals. There are several reasons for this. n would benefit their use as catalysts for organic synthesis. Your list could have included the following coINl[lonly found features: high growth rates which allows the generation of large amounts of catalyst (microorganism); substrates for growth are often cheap and include waste materials from other 0 they are generally more robust and less fastideous than plant and animal cell cultures; Make a list of at least five commonly found features of mi<3.o-organisms that advantagesof -9 mia*u industrial processes; Cells Biocatalysts in organic chemical synthesis 13 there are many different types of microbes, each with unique nutritional and physiological features, which may be desirable for process development; 0 collectively, micro-organisms have a broad complement of enzymes capable of a wide variety of chemical reactions; production plants involving micro-organisms are generally independent of climatic conditions and require little space (compared to crop plant produdion); 0 for some microbes, such as Esc/~m-ichia coli, the genome is well known and relatively easy to manipulate genetically; 0 many microbes are single celled organisms that grow well in stirred tank bioreactors (fermentors). Although it is possible to obtain cells from whole animals or plants and to cultivate them in suitable nutrient solutions, in general they are not as easy to handle as microbes. Nevertheless, plant and animal cells are a valuable genetic resource for biotechnology and many newly developed bioprocesses rely on transfer of their genes to micro-organisms. Microbial enzymes can be applied as catalysts for chemical synthesis in biosynthetic processes or in biotransformations (bioconversions). In a biosynthetic process the product is formed de novo by the microbial cell from substrates, such as monosaccharides, molasses, soybean and corn steep liquor. In a biotransformation, however, a precursor that is usually chemically synthesised is converted in one or several enzyme catalysed steps into the desired chemical. This chemical may be the end product or may serve as a pmrsor for further chemical modification. biosynthetic Pr-wsand bornsforin- ation 2.3 Enzyme preparations versus whole cell processes In designing a process we have the choice of using the whole organism or specific enzymes isolated from it. As always both options have pro's and cons. Broadly speaking we could say that biosynthetic processes mostly rely on whole cells, whereas biotransformations can be catalysed by whole cells and by enzyme preparations. Hydrolytic enzymes such as proteases, esterases and lipases (Table 2.1) account for more than half of all reported biotransformations. These enzymes are particularly easy to use because: hydrolytic 0 they are available in large amounts from industrial sources; they are stable in non-aqueous solvent; they do not have cofactor requirements. Why do you think many processes based on redox reactions involving n dehydrogenase enzymes are still carried out using whole cells? Dehydrogenase enzymes generally require NADH or NADPH, and although methods for recycling these cofactors are now available on a laboratory scale, little progress has been made in the scale-up to industrial level. dehydrogenases 14 Chapter 2 oxygenases associated problems enzyme immobilisation enzyme boreactor design Similarly, many oxygenation reactions (Table Z.l), which also require cofactors, are usually performed using whole micro-organisms. Collectively, oxidoreductases and oxygenases account for around 30% of all reported biotransformations. Enzymes such as lyases, transferases and isomerases (Table 2.1) account for most of the remainder of industrially applied biotransformations. 2.3.1 Enzyme catalysed processes Enzymes isolated from micro-organisms have many desirable properties as catalysts for the synthesis of industrial chemicals, but there are associated problems: their protein structure may not be stable under non physiological conditions which may be detrimental to their long term use, especially at elevated temperatures; the provision of enzyme cofactors can be expensive; 0 most enzyme reactions are carried out in water and the enzymes must be separated from the product stream; the product stream is often very dilute, presenting problems of product concentration and recovery; 0 preparation of a crude or purified cell-free enzyme preparation is necessary. Advances in genetic and chemical enzyme modifications, enzyme immobilisation and enzymatic reactions in organic solvents, have increased the actual use and potential of enzymes in the production of industrial chemicals. Enzyme immobilisation, in particular, has proved to be a valuable approach to the use of enzymes in chemical synthesis. The term denotes enzymes that are physically confined or localised in a defined region in space with retention of their catalytic activities. A detailed consideration of immobilisation techniques is beyond the scope of this chapter; the subject is covered adequately in the BIOTOL text entitled 'Technological Applications of Biocatalysts'. Enzyme immobilisation allows the construction of enzyme reactors in which the enzyme can be reused. Furthermore, the process operates continuously and can be readily controlled. Enzyme reactors currently in use include those illustrated in Figure 2.1. Biocatalysts in organic chemical synthesis 15 Figure 2.1 Examples of enzyme bioreactor design. Bioreactors: a) batch stirred tank; b) continuous stirred tank; c) continuous packed-bed i) downward flow, ii) upward flow and iii) recycle; d) continuous fluidised-bed; e) continuous ultrafittration. Redrawn from Katchalski - Katzir E. (1993) Trends in Biotechnology I/. 471 -477. Some of the potential advantages of enzyme immobilisation are: 0 high enzyme loads; 0 prolonged enzyme activity; 0 enzyme can often be regenerated by suitable treatment and used again; the ability to recycle products; 0 high flow rates; reduction in cost (energy and labour), energy and waste products; easy to scale up to large systems; high yields of pure materials; higher substrate concentrations can be used. advantages of enzyme irnmobilisation 16 Chapter 2 2.3.2 Whole cell processes We can also use microbial cells (fermentation) containing the desired catalytic activity without isolating the enzymes responsible. Advantages of whole cell processes include: cell disruption not necessary; enzyme isolation not necessary; advantages 0 more suited to multiple step processes; 0 cofactor regeneration not a problem; 0 increased enzyme stability; reduced catalyst preparation costs. Among the disadvantages of whole cell processes are: 0 system not fully understood (black box situation); 0 product contamination by cellular enzymes or other end products of metabolism; 0 cell structures acting as diffusion barriers; contamination by other micro-organisms may be a problem. Synthesis of industrial chemicals by microbial cells may be by fermentation (free, living cells), immobilised growing cells, immobilised resting cells or immobilised dead cells. Immobilised cells have all the advantages of immobilised enzymes. Cell immobilisation is preferred for reactions catalysed by intracellular enzymes because it avoids tedious and expensive extraction and purification procedures, which often result in preparations of low yield and stability. disadvantages 0 reduced catalytic specific activity; immobilii cells Idenbfy which of the following statements are true for immobilised biocatalysts, when compared to free enzyme or free cell systems. 1) Conversions carried out by immobilised cells give higher yields than those carried out by growing and dividing cells. 2) Downstream processing can be much easier. 3) Smaller reactor volumes achieve similar rates of product formation. 4) The volume of effluent can be reduced. Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodiol. Give two reasons why this biotransformation should be canied out using whole cells as opposed to using enzyme preparations. Biocatalysts in organic chemical synthesis 17 k.4 Scale of production The decision as to which approach - free enzyme, immobilised enzyme, fermentation, obilised cells - is mainly dictated by economics. Commercial aspects of bioprocess P evelopment are considered in Section 2.6. The analysis of the various factors involved is a critical part of the decision-making process and involves inputs from scientists, engineers and marketing personnel. For products derived from micrmrganisms, process design can be based mainly on biochemical engineering considerations or on microbial physiology considerations. When compared to the improvements achieved in bulk chemicals manufacture and in petroleum refining, the application of biochemical engineering principles to microbial processes has not been as successful over the past thirty years. It is now accepted that individual factors affecting overall optimisation of microbial processes are best handled by individual specialists - microbial biotechnologists and chemical engineers. The biotechnologist, in addition to having an in-depth knowledge in a particular field, must also have the appropriate skills and knowledge to communicate and interact effectively with chemical engineers. Such interaction is thought to be essential for technological innovation and commercial success of microbial processes of the future. The necessity for interaction between biotechnologists and chemical engineers increases with the scale of production. In chemical manufacture three categories of product can be defined according to the scale of production (Table 2.2). -ww needfor communication production range examples of biotechnology single piant products fine chemicals 100 kg/annum-100 vitamins, vaccines, nucletides, tonneslannum amino acids) usually batch reactors antibiotics ) some enzymes ) intermediate volume 100-20,000 tonnes/annum glutamic acid) chemicals batch or continuous reactors citric acid ) food industry lactic acid ) antibiotics for agriculture, enzymes for industry, many fermented foods and beverages industry, biopolymers (for enhanced oil recovery), biogas wastewater treatment plants bulk chemicals > 20,000 tonnes/annum single cell protein, ethanol for usually continuous flow I (methane), sewage and I Table 2.2 Product categories in chemical manufacture. The design of production plants for the manufacture of the three categories of product varies considerably. Fine chemicals are usually produced in batch reactors, which may also be used for the production of a variety of similar products. Fine chemicals usually have demanding product quality specifications and, consequently, a significant fraction of the production costs are involved in product purification and testing. Intermediate volume chemicals have less rigorous quality specifications than fine chemicals and are usually manufactured in product-spe4c-plants, either as batch or continuous flow processes. Bulk chemical production plants usually operate continuous flow processes fine, volume and bulk demimls Chapter 2 batch mode continuous mode advantages of batch mode advantages of continuous mode and the products do not have rigid quality specifications; rather they are marketed on the basis of overall product performance criteria. 2.5 Modes of operation of bioprocesses As you might have already gathered, the majority of industrial fermentations are batch processes. In closed batch systems, the growth medium is inoculated with cells and growth and product formation is allowed to proceed until the required amount of conversion has taken place. After harvesting the culture the vessel is cleaned, sterilised and filled with fresh medium prior to inoculation. For some processes, addition of all the feedstock prior to inoculation, as is done in closed batch fermentations, is undesirable and it is preferable to incrementally add the carbon source as the fermentation proceeds. Such a process is known as fed-batch culture and the approach is often used to extend the lifetime of batch cultures and thus product yields; fed-batch cultures are considered further in Section 2.7.4. The alternative to batch mode operation is continuous operation. In the continuous mode there is a continuous flow of medium into the fermentor and of product stream out of the fermentor. Continuous bioprocesses often use homogenously mixed whole cell suspensions. However, immobilised cell or enzyme processes generally operate in continuous plug flow reactors, without mixing (see Figure 2.1, packed-bed reactors). n Complete the following statements by filling in the missing words. Conditions change with in batch fermentations but with distance along the reactor in reactors. conditions are maintained in continuous suspended fermentations. The missing words are 'time', 'plug flow' and 'constant'. Advantages of the batch mode over the continuous mode of operation include: several different products can be made using the same bioreactor, thus providing operational flexibility; genetic stability of the process organisms is not as great a problem; the risk of contamination is relatively small; it is possible to identify all the material involved in making a particular batch of product, which may be important in quality control. Advantages of the continuous mode over the batch mode of operation include: the proportion of the down-time (time spent preparing the bioreactor for a new run) to fermentation time is relatively small; they are relatively easy to operate and control when in steady state; the demand on services are relatively constant during operation; the product streams have a relatively constant composition (it may be difficult to prevent batch-to-batch variability); waste products of metabolism are unlikely to accumulate to inhibitory levels. Biocatalysts in organic chemical synthesis 19 2.5.1 Productivity in batch and continuous systems Productivity is an important parameter in evaluating the cost-effectiveness of a fermentation. It is defined as: productivity = product concentration/fermentation time Typical units for productivity are kg m-3 h-'. Factors that influence productivity include the production time of the fermentation, the time required to clean and set up the reactor, the sterilisation time and the length of the lag phase of growth. Figure 2.2 shows how total productivity and maximal productivity can be calculated for a batch fermentation. The decision as to when the fermentation is terminated (maximum or total productivity) depends on the operating costs, which include the capacity of the fermentation vessel, energy costs and labour costs. total and maximal pmductivitY Figure 2.2 Types of productivities for batch fermentation. n affect productivity. Your list could be extensive since any factor that influences the rate and/or amount of product formed, ie the fermentation conditions or the characteristics of the process organism could influence productivity, eg pH, temperature, solubility of substrate. In continuous fermentation: Make a list of factors, other than those mentioned already, that could directly productivity = D . X where - D = dilution rate (flow rate/culture volume; units 9f h-*) x = steady state product concentration (units kg m- ). In continuous fermentation, maximum productivity equals total productivity since the preparation time and the time in lag phase of growth are small relative to the total fermentation time. 20 Chapter 2 n Explain what is meant by a ’steady state’ for a continuous fermentation. A steady state is when the conditions in the bioreactor (biomass concentration, residual substrate concentration etc) remain constant over time. In a continuous fermentation the preparation time is 20 hours and the fermentation is run at two dilution rates: 0.1 h-’ for 48 hours and 0.2 h-’ for 96 hours. What is the total productivity of the fermentation if the steady sta$ product concentrations at the two dilution rates are 05 kg m” and 2.1 kg m- respectively? (Try to do this before looking at our solution below). n - Time period Time D X D.Y (h) (h) (h-’) (kg m-7 (kg m-3 h-’) 0-20 20 20-68 48 0.1 0.5 0.05 68-1 64 96 0.2 1.1 0.22 ( 48 x 0.05 ) i- ( 96 x 0.22 ) = o.14 kg m-3 h-l 164 Total productivity = Which of the following statements apply to ’batch mode’ and which to ’continuous mode’ of operation of fermentation? In each case give a reason for your decision. 1 ) Provides operational flexibility. 2) Genetic instability of the process organism is more likely to be a problem. 3) Loss of product as a result of microbial contamination is very much greater. 4) The ’down time‘ of the bioreactor is a significant portion of the total fermentation time. 5) It is possible that substances inhibitory to the fermentation may accumulate 6) More suitable for fermentations in which the product is synthesised after 7) The source of variation occurring in the product may be difficult to locate. 8) Relatively easy to operate and control. throughout the fermentation. growth. Biocatalysts in organic chemical synthesis 21 2.6 Biotechnological processes verses chemical synthetic processes Biotechnology has attracted enormous interest and high expectations over the past decade. However, the implementation of new technologies into industrial processes has been slower than initially predicted. Although biocatalytic methods hold great industrial potential, there are relatively few commercial applications of biocatalysts in organic chemical synthesis. The main factors that limit the application of biocatalysts are: 0 the inherent disadvantages of biocatalysts; the existence of well-developed traditional technologies (economic considerations). The disadvantages of biocatalysts will now be considered, followed by their advantages. You should note that the pros and cons of biotechnology versus chemical synthesis are very general and that exceptions may exist. 2.6.1 Disadvantages of biocatalysts From an economic perspective, it is important for bioprocesses to be integrated into existing organic chemical infrastructure. Typical production sites for bulk chemicals manufacture, such as those of the petrochemical industry, are unlikely to provide all of the requirements of industrial microbiological processes. The requirements of industrial microbiological processes include: economic P~sPe~v* high quality process water; 0 low temperature cooling water; 0 cleanair; 0 cleansteam; hygienic surroundings; waste water treatment facilities; storage facilities for feedstock and product; sophisticated quality control laboratories. Satisfying these requirements invariably requires capital investment that can significantly affect overall economics of microbiological processes. As far as fine chemicals are concerned, many biotransformations and some fermentations can be carried out in existing fine chemical reactors. In the absence of suitable existing facilities, significant investment in a state-of-the-art fermentation/biotransformation plant is required. However, existing chemical technology is well known and, in many cases, the investment in a factory plant has been paid for, so there is little economic incentive for implementing new processes. It is, therefore, unlikely that existing chemical technology will automatically be replaced by biotechnology. 22 Chapter 2 m ixed chemical/ biochemical processes metabolic control mild conditions organic solvents At present most bioprocesses in the organic chemical industry are actually mixed chemical/biochemical processes. In such processes, chemically synthesised educts (chemical precursors) are biotransformed and then re-enter chemical synthesis. The main reason for this approach is that, in general, higher volumetric productivities can be achieved with chemical catalysts. Apart from economic considerations, the inherent disadvantages of biocatalysts have also limited the transformation of new technologies into industrial processes. Table 2.3 lists the mapr drawbacks of bioprocesses. Educt, substrate or product inhibition of reaction. Poor understanding of reaction kinetics. Educt or substrate poorly soluble in water. High temperatures damage enzymes or cells (cooling required). Contamination by unwanted micro-organisms. Genetic instability of process micro-organism. Need for purified substrates and water to avoid poisoning of biocatalysts. Low concentration product stream. Poor reliability and reproducibility of process. High cost of state-of-the-art fermentationhiotransformation plant. Table 2.3 Possible drawbacks of bioprocesses and mixed chemicalhiochernical processes compared to purely chemical synthetic processes. Several of the problems associated with whole cell bioprocesses are related to the highly effective metabolic control of microbial cells. Because cells are so well regulated, substrate or product inhibition often limits the concentration of desired product that can be achieved. This problem is often difficult to solve because of a poor understanding of the kinetic characteristics of the metabolic pathway leading to the desired product. When compared to purely chemical synthesis, bioprocesses are operated under relatively mild conditions and in aqueous solvents: they are essentially low temperature processes with operating temperatures usually below 40°C. The pH of most bioprocesses is between 6 and 8 and the pressure is usually one atmosphere. Under these conditions, substrates (eg oxygen) can be poorly soluble in water, which may limit productivity. Since reactions can generate considerable amounts of heat, waste heat generated during bioprocesses often has to be adequately dissipated to ensure high temperatures do not damage enzymes or cells. Many substrates currently produced in the chemical industry are immiscible with water, but are readily miscible with organic solvents. Most enzymes, however, will not operate efficiently, or not operate at all, in non-aqueous media. Some exceptions do exist, such as lipases and esterases, which can operate in non-aqueous environments. Currently, there is considerable interest in extending the range of enzymes that do work in organic solvents. n a bioprocess? Organic solvents may improve substrate solubility, specificity of reaction and equilibrium reaction. The last two benefits are considered further in Section 2.6.2. What possible advantages might enzymes operating in organic solvents have for Biocatalysts in organic chemical synthesis 23 Contamination genetic inStdbili chemical con tam inalion product purification reproducibility Since most bioprocesses are monoseptic (one type of organism) operations where the production strain has been genetically modified (crippled) to overproduce the desired product, contamination by unwanted micro-organisms is a constant threat. The contamination may arise externally and aseptic procedures must be used to reduce the risk - such sterile engineering can be complex and costly. Genetic instability of the producing organism can give rise to contamination from within, where some change in the producing organism (reversion of a mutation; loss of a plasmid) affords a growth rate advantage that enables the unwanted organism to outcompete the producing strain. Cells and isolated enzymes are often susceptible to poisoning at low levels of chemical contamination. It is, therefore, necessary to carry out expensive purification of substrates (feedstocks) and water used in bioprocesses. In whole cell bioprocesses, extracellular products are preferable because this removes the requirement for cell disruption and this reduces the level of impurities in the product solution. Nevertheless, product isolation and purification can be prohibitively expensive particularly for low concentration product streams, which is a feature of many bioprocesses. When compand to traditional chemical synthesis, processes based on biocatalysts are generally less reliable. This is due, in part, to the fact that biological systems are inherently complex. In biopmsses involving whole cells, it is essential to use the same strain from the same culture collection to minimise problems of reproducibility. If cell free enzymes are used the reliability can depend on the purity of the enzyme preparation, for example iso-enzyme composition or the presence of other proteins. It is, therefore, important to consider the commercial source of the enzyme and the precise specifications of the biocatalyst employed. Give a possible drawback of bioprocesses, when compared to purely chemical synthesis, under each of the following headings (refer to Table 2.3 if you are unable to do this). n Reaction kinetics: Economics: Product stream: Reliability: Temperature: Genetics: Inhibition: Solubility: 2.6.2 Advantages of biocatalysts Despite the inherent disadvantages of bioprocesses, there are many advantages which can make a biotechnological approach to chemical synthesis the sole or desirable approach. The possible advantages of bioprocesses are shown in Table 2.4. 24 Chapter 2 St ereospedfcity Regiospecificity Reaction specificity Reduced disposal costs Waste products likely to be less environmentally damaging Reduced number of synthesis steps Reduced temperature and pressure costs Mild reaction conditions and aqueous solvents Mild reaction conditions give slower destruction of reaction vessel Mild reaction conditions allow reactions with labile molecules Safer reaction technology and working environment Renewable feedstocks Non-toxic, biodegradable and non flammable educts and substrates Table 2.4 Potential advantages of mixed chemicakiochemical processes compared to purely chemical synthetic processes. One of the most important advantages of biocatalysts is their stereospecificity. An example of a stereospecific biotransformation is given in Figure 2.3. In 1992 the Food and Drug Administration (FDA) in the United States addressed the issue of whether it mattered, for drug approval purposes, that a preparation of a synthetic chiral molecule (one or more asymmetric centers) contains not one but two compounds (racemates) - the two mirror-image stereoisomers (individual enantiomers). stereospecific^ Figure 2.3 An example of a stereospecific biotransformation: resolution of bicyclic lactam. A and AI are an enantiomer pair as are B and BI. The FDA gave drug companies the choice of developing chiral drugs as rammatf3 or as single enantiomers. In effect, this means that although the development of rammates is not prohibited, such drugs have to undergo rigorous justification before approval by the FDA. This justification includes putting each of the two foxms of the molecule FDA gU*hs Biocatalysts in organic chemical synthesis 25 separately into animals and, possibly, humans. Consequently, the maprity of drug companies have decided to develop single enantiomers, if feasible, in preference to racemates; some have decided to avoid the problem by switching to nonchiral molecules. The FDA's policy on chiral compounds has created, in effect, many opportunities for chemists in 'chirotechnology' and biotechnology is proving to be a powerful tool. The annual sales of the top ten best selling single enantiomeric drugs has been estimated to be around US $10 billion. These molecules include several penicillin and cephalosporin based antibiotics and market research has indicated that chiral molecules are likely to take an increasing share of the pharmaceutical drug market in the 1990's. The specificity of biocatalysts also extends to site specificity (regiospecificity). This means that if several functional groups of one type are present on the molecule, only one specific position will be affected. An example of this is the microbial oxidation of D-sorbitol to L-sorbose, a key step in the synthesis of vitamin C (Figure 2.4). regiospecifbty Figure 2.4 Microbial oxidation of D-sok)iiol to L-sorbose. The spechcity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic cosolvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and The high specifity of biocatalysts also has the advantage of reducing disposal costs (pollution control costs) because relatively few useless and potentially harmful byproducts are generated. In addition, waste products that might be produced are, by their very nature, likely to be biodegradable and, therefore, less environmentally damaging compared to those produced in purely chemical synthetic processes. In processes involving whole cells the required product can often be formed in a single step, although the cells essentially cany out a multistep synthesis. This means that only a single product purification is necessary. Conversely, in chemical synthesis of compounds, each step in the synthesis is usually carried out separately. Thus the product of one reaction must often be purified before it can be used in the next step in the synthetic sequence. This multistep approach is expensive, time consuming and can require a complex process plant to handle the individual steps on an industrial scale. medum engineering Selectivity. polldon amw multi-s* syn*esis 26 Chapter 2 non-mrrosive conditions The almost noncorrosive conditions (low temperature, neutral pH, atmospheric pressure) of most bioprocesses have the advantage that there is much slower destruction of the reaction vessels, which means financial depreciation over a much longer period of time. Indeed, it has been estimated that biotechnological reactors have a life span three times that of chemical reactors. The relatively mild conditions of bioprocesses are also favourable compared with chemical synthesis, where the high temperatures and pressures can incur considerable costs. In bioprocesses, the feedstocks required to grow the catalysts and produce the chemical are generally renewable resources, such as sugar from crops. Conversely, purely chemical synthesis relies largely on non-renewable resources such as oil, coal and natural gas. It follows that as non-renewable resources dwindle, it is likely that biotechnology will become increasingly important to the chemical industry. renewable Make a list of possible advantages of bioprocesses, when compared to purely chemical synthetic processes, under each of the following headings (refer to Table 2.4 if you are unable to do this). n Specificity: Reaction conditions: Educt/ substrate/ products: Others: Briefly compare and contrast downstream processing for bioprocesses and for purely chemical synthetic processes, from an economic perspective. SAQ 2.4 2.7 Bioprocess development Micro-organisms have produced chemicals for industry for many years and their potential for the future in this respect is enormous. However, the development of new and improved bioprocesses depends on the realities of the commercial world the product must generate a profit; the bioprocess must have a significant advantage over existing or potential chemical processes; the industry must be willing to risk substantial capital expenditure. Other factors that are important for the development of bioprocesses include: 0 the identification of new large-market products and/or high-profit margin products; 0 the ability to protect patents or the confidentiality of new technical advances; 0 the need to minimise the use of expensive capital equipment and labour-intensive processes; the need to minimise the requirements that new products have to meet to attain regulatory approval (because of the high costs, time-delay and risks involved). um~ercid Biocatatysts in organic chemical synthesis 27 research and develapment flexibility shear resistance resistance to environmental stresses Even when a successful process has been established, continuous improvement is required to meet the challenges of competitors. Ongoing commercial success will rely, therefore, on optimisation of the many diverse stages in the bioprocess through research and development. For a typical biopnxess, the stages may include: growth and improvement of cells for catalytic use; immobilisation of biocatalyst; preparation of chemical feedstocks to be modified or purification of growth substrates; design of bioreactor to allow contact with biocatalyst and feedstock; optimisation of conditions in bioreactor; separation of product from byproducts and waste products; recycling or disposal of byproducts and waste products; 0 product isolation from dilute aqueous solution; 0 product purification and formulation; scale-up of a laboratory scale process to a new and large scale, ie design, construction and operation. Next, some of the decisions involved in bioprocess development will be considered. 2.7.1 Choice of process micro-organism Increased flexibility into the choice of organism will rely on knowledge of the genetics of species other than those presently used in genetic engineering. Bioreactors nowadays can be designed and constructed in such a way that simple modifications permit the growth of prokaryotes, filamentous fungi, plant cells and mammalian cells in the same reactor. Obviously genetic stability of the organism is extremely important but physical, chemical and nutritional factors also have to be considered. In aerobic processes, mechanical agitation by rotating impellers is often used to disperse air in the form of fine bubbles throughout the growth medium and to promote adequate mixing. However, the shearing action of the impellers also tends to damage the cells employed. Such damage is particularly pronounced in the case of filamentous fungi, actinomycetes and bacteria with appendages (flagella and fimbriae). Fungal morphology can be important with respect to the formation of certain products, for example citric acid. For these products, culture conditions must be adapted or strains selected to minimise mycelium injury or alterations in morphology. As far as damage to bacterial cells is concerned, the main hydrodynamic effects on appendages, particularly flagella that can have lengths many times that of the cells, can lead to stripping of such appendages with subsequent release of cell contents. Selection of a more robust fungus or bacterium may enhance the commercial success of the process. Micro-organisms with resistance to environmental stresses such as solvents, extremes of pH, high salt concentration, and having broad temperature and dissolved oxygen optima are more suited to process applications. Improved process instrumentation and 28 Chapter 2 control reduces the disadvantages of fastidious microbes in this respect. However, some commeraalde bioreactors, for example column and pressure-cycle system, have rapidly changing physical and chemical conditions and robust micro-oxganisms are essential if good overall process economics are to be achieved. Process organisms also need to be resistant to inhibition by substrates and products and to inactivation by proteases. In addition, the bioprocess must use micrmrganisms that are acceptable to the regulatory authorities, that can be grown easily on media, and that possess constitutive enzyme activities. n unable to do this reread Won 2.7.1. Make a list of desirable characteristics for a process micro-organism. If you are 2.7.2 Strategies for improvement of process micro-organisms Natural isolates usually produce commercially important products in very low concentrations and the potential productivity of the organism is controlled by the genome. In practice, the process of strain improvement involves the continual genetic modification of the culture, followed by reappraisals of its cultural requirements. Genetic modification may be achieved by: selecting natural variants (eg enrichment culture of soil organisms); 0 selecting induced mutants (eg produced by use of W light or chemical mutagens); selecting recombinants (eg produced by protoplast fusion or by in Vitro genetic engineering). genetic mdifimtim An array of strategies and techniques is available for each approach. A detailed consideration of these is beyond the scope of this chapter, although you will encounter several strategies elsewhere in this BIOTOL text. There is a small probability of a genetic change occurring each time a cell divides. Therefore, selection of natural variants may result in increased yields but it is not possible to rely on such improvement, and techniques must be employed to increase the chances of improving the culture. In its broadest sense recombination can be defined as any process which helps to generate new combinations of genes. The use of recombination mechanisms for improvement of industrial micro-organisms has been limited by the lack of basic knowledge of the genetics of industrial micro-orgaNsms. The ease of applying mutation and selection techniques and the spectacular success of these approaches have also limited the use of recombination techniques for industrial micro-organisms. 2.7.3 Composition of the medium The growth medium for a bioprocess should ideally provide all the requirements of the process organism in such a way that the organism grows rapidly, produces large amount of the desired product, does not degrade the final product, and is able to survive under harsh environmental conditions (such as shear forces, oxygen limitation etc). Whenever possible the individual components of a medium should not be provided in excess because if they are not consumed or built into the product, they must be removed by costly purification processes. genetic recombination ided *ara**h Biocatalysts in organic chemical synthesis 29 Make a list of characteristics of an ideal medium for biotransformations using n whole ells. (Try this on a piece of paper before reading on). Your list could have included the following characteristics: allows fast growth and high yields of process micro-organism; does not allow growth of contaminants; 0 no excess of any component (all substrates utilised by end of biotransformation); no colour, odour or byproduct at the end of the biotransformation; substrates cheap, stable, soluble and of constant quality; product stable; 0 protects cells from adverse environmental conditions; prevents foaming. 2.7.4 Technical options In many cases, problems cannot be overcome by biological means. This is especially true for those related to inhibition by substrate or product. There may, however, be technical solutions to these problems. Nowadays, complicated feed strategies with different substrates can be achieved through the use of flow injection analysis, on-line sensors, mass flow meters and sophisticated computer control. Such control coupled to a fed-batch mode of operation (Figure 25) can often eleviate problems caused by substrate inhibition. For some processes, continuous product removal can avoid the problems associated with product inhibition; the various options include: vacuum fermentation (volatile products); solvent extraction; conh~~~us product ~Wal dialysis; 0 ion exchange; 0 cell recycling. Some of the most important technological approaches to bioprocess problems are shown in Figure 2.5. 30 Chapter 2 L Figure 2.5 Possible technological solutions to bioprocess problems: a) Fed-batch culture; b) Continuous product removal (eg dialysis, vacuum fermentation, solvent extraction, ion exchange etc); c) Two-phase system combined with extractive fermentation (liquid-impelled loop reactor); d) Continuous culture, internal multi-stage reactor; e) Continuous culture, dual-stream multi-stage reactor; f) Continuous culture with biomass feedback (cell recycling). (See text for further details). We have already seen that the term 'fed-batch culture' is used for batch cultures which are fed, continuously or intermittently, with fresh medium without removal of culture fluid. Benefits of such systems include: 0 extending the lifetime of the product formation phase beyond that possible with batch culture; 0 maintaining very low concentrations of the growth limiting nutrient. feb-batch ~~ltum In what circumstances would the maintenance of very low concentrations of the n growth limiting nutrient be an important process design criterion? When excess substrate interferes with growth and/or product formation. One example is the production of baker's yeast. It is known that relatively low concentrations of certain sugars repress respiration and this will make the yeast cells switch to fermentative metabolism, even under aerobic conditions. This, of course, has a negative effect on biomass yield. When maximum biomass production is aimed at, fed batch cultures are the best choice, since the concentration of limiting sugar remains low enough to avoid repression of respiration. Biocatalysts in organic chemical synthesis 31 two-phase systems multi-stage continuous PrOCeSSeS singlestream multi-stage mu Iti-stream multi-stage biomass feedback In a two-phase system (Figure 25c), the organic (water immiscible) solvent may be used as product extractant. In addition, recirculation of the organic phase can serve to transfer oxygen and to mix the aqueous phase. The advantages of the continuous mode of operation of fermentations were considered in Section 2.5. One of the main drawbacks is that in aerated well mixed continuous processes, high substrate concentrations can remain. This problem may be overcome using a continuous cascade (multi-stage) reactor, in which substrate concentration near the outflow of the bioreactor is reduced to the same low levels characteristic of fed-batch processes. In multi-stage continuous processes, conditions for growth and/or product formation may vary considerably between stages, which may have considerable benefits for the process. For example, in secondary product processes, where product formation takes place after growth has ceased, the first stage of the multi-stage process may be optimised for growth with subsequent stages optimised for product formation, in the absence of growth. With complex media (eg with more than one source of carbon) a singlestream multi-stage process may be necessary to achieve utilisation of all substrate. For example, such an approach is necessary if utilisation of one carbon source represses the expression of genes for utilisation of another. In the singlestream multi-stage process, utilisation of one substrate in the first stage allows utilisation of the other in the second stage. In general, the system provides a series of different environments. The multi-stream multi-stage system is a valuable means for obtaining steady-state growth when, in a simple chemostat, the steady-state is unstable eg when the growth-limiting substrate is also a growth inhibitor. This system can also be used to achieve stable conditions with maximum growth rate, an achievement that is impossible in a simple chemostat (substrate-limited continuous culture). Biomass feedback refers to increasing the concentration of biomass in the culture vessel. This is achieved by fitting some device, either internally or externally, to the continuous culture which retains or returns biomass to the vessel. The main advantage of biomass feedback is that the maximum output rate of biomass (and products) in the vessel with a given medium can be increased. This is particularly useful when the growth-limiting substrate is unavoidably dilute, for example if substrate has low solubility or has to be limited because of the formation of an inhibitory product. Combinations of different technological approaches are, of course, possible. Several methods have not been mentioned in this chapter, but you will encounter these in a specific context elsewhere in the text. 32 Chapter 2 Select an appropriate combination of process design factors for each of the p'ocesSeS. Process problems Design fadon Process A: Mode of operation: Genetic instability Batch Substrate repression Fed-batch Multi-step synthesis Continuous Product (volatile) inhibition Genetic instability Free enzyme Poor enzyme stability Free cells Cofactor requirement Immobilised enzyme Product (non-polar) inhibition lmmobilised cells Product purification complicated Vacuum fermentation by presence of low molecular components Single-step biotransformation; Two phase system Enzyme stable and readily available; No cofactor requirement; Low molecular weight product Multi-stage Process B: Biocatalyst: Process C: Reactor technology: Biomass feedback (cell recycle) Ultrafiltration with enzyme recycling Single-stream multi-stage dual-stream Solvent extraction Biocatalysts in organic chemical synthesis 33 Summary and objectives Biotechnology has the potential to become the most important tool in organic chemical industry in the early part of the next century. However, this will rely on successful integration of biology, chemistry and engineering. Micro-organisms have tremendous potential as biocatalysts because of their flexibility, but they can be unpredictable. All bioprocesses quire active biocatalysis, which may require improvement by mutagenic procedures or by recombinant DNA technology. Downstream processing is invariably a decisive cost factor and its minimisation is an important prerequisite of a successful bioprocess in organic chemical synthesis. Rapid progress currently being made in reactor design and process control strategies, together with advances in enzyme and recombinant DNA technologies, will ensure that biotransformations of increasing complexity and that novel products will continue to be realised. Now that you have completed this chapter you should be able to: list advantages of using micro-organisms as catalysts of organic synthesis; compare and contrast the use of enzyme preparations and whole cells as catalysts of organic synthesis; categorise products of the chemical industry according to scale of production; compare and contrast batch and continuous fermentations; list advantages and disadvantages of bioprocesses and mixed chemical /biochemical processes compared to purely chemical synthetic processes; list factors that are of major importance for the development of bioprocesses; describe broadly the mapr decisions that have to be made in bioprocess development.