Biocatalysts in organic chemical synthesis 2.1 Introduction 2.2 Micro-organisms as catalysts of organic synthesis 2.3 Enzyme preparations versus whole cell processes 2.4 Scale of production 2.5 Modes of operation of bioprocesses 2.6 Biotechnological processes verses chemical synthetic processes 2.7 Bioprocess development Summary and obiectives
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10 Chapter 2 Biocatalysts in organic chemical synthesis 1 Introduction traditonal and Biotechnology is often divided into two categories called'traditional biotechnology new and'new biotechnology The major products of the traditional biotechnology industry toachnoogy 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 The market for products of traditional biotechnology is currently worth around 250 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 major 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
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 Esterase enzymes- cleavage of various ester bonds to yield an acid and an alcohol, ie RI COoR2+H20 R1COOH+R2OH LIpase enzymes(a subgroup of esterase enzymes)-hydrolyse fats into glycerol and fatty acids, eg H2C-, H,COH R1 OOH 2+929 HCOH R2OOH H2C- OR H2CO fatty acids Proteolytic enzymes-hydrolyse proteins selectively, either on terminal groups (exopeptidases)or intemal linkages(endopeptidases),eg A Oxldoreductases-catalyse oxidation-reduction reactions, eg dehydrogenase H.C. CH,OH →H2C.CHo NAD+ NADH +H+ Oxygenases- add one(monooxygenases)or both(dioxygenases)atoms of molecular oxygen to molecules, eg NADH NAD+ cis-dihydrodiol Table 2.1 Some reactions catalysed by microbial enzymes. In principle each enzyme catalyses the reverse process as well
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
Chapter 2 Lyases-catalyse the breakage of c-c bonds, eg socitrate HC- COOH ase CH,COOH H Hoc一cooH CH2COOH + C-COOH Isocitric acid Succinic acid Glyoxylic acid Transferases-catalyse the transfer of functional groups, eg CHOl CH2 OPO3H2 Glucokinase D-Glucose D-Glucose-6-phosphoric acid es-catalyse the inter-conversion of isomers, eg CH2OH Triosephosphate C=O somerase HCOH CH2OPO3H2 CH2oPO3H2 Dihydroxyacetone phosotnyde-3 Tabe2.1… Continued rather than plant and animal cells, are generally preferred for the production of organic chemicals. There are several reasons for ∏ Make a list of at least five commonly found features of micro-organisms that would benefit their use as catalysts for organic synthesis Your list could have included the following commonly found features high growth rates which allows the generation of large amounts of catalyst advantages of substrates for growth are often cheap and include waste materials from other ing microbial industrial processes they are generally more robust and less fastideous than plant and animal cell cultures
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 are many different types of microbes, each with unique nutritional and ological features, which may be desirable for process development; collectively, micro-organisms have a broad complement of enzymes capable of a of chemical production plants involving micro-organisms are generally independent of climatic conditions and require little space( compared to crop plant production) for some microbes, such as Escherichia coli, the genome is well known and relatively easy to manipulate genetically many microbes are single celled organisms that grow well in stirred tank bioreactors ]though 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 tion 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 precursor for further chemical modification 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 pros 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 ydrolytic Hydrolytic enzymes such as proteases, esterases and lipases(Table 2.1)account for nzymes more than half of all reported biotransformations. These enzymes are particularly easy to use because: 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 dehydrogenase enzymes are still carried out using whole cells? dehydrogenases 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
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 bornsforination 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 Similarly, many oxygenation reactions (Table 2.1), 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 of industrially applied biotransformations. 2. 3. 1 Enzyme catalysed processes Enzymes isolated from micro-organisms have many desirable properties as catalysts for he synthesis of industrial chemicals, but there are associated problems associated the provision of enzyme cofactors can be expensi 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 preparation of a crude or purified cell-free enzyme preparation is necessary Advances in genetic and chemical enzyme modifications, enzyme immobilisation and immobilisation 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 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 Biocatalyst enzyme Enzyme immobilisation allows the construction of enzyme reactors in which the bioreactor enzyme can be reused. Furthermore, the process operates continuously and can be sign readily controlled Enzyme reactors currently in use include those illustrated in Figure
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 b .. Hproduct immobilised immobilised substrat product column substrate substrate immobilised substrat ( fluidised) ultrafiltra enzyme Figure 2. 1 Examples of enzyme bioreactor design Bioreactors: a)batch stirred tank; b )continuous stirred tank; c)continuous packed-bed i) downward flow, i)upward flow and ii recycle; d) continuous fluidised-bed; e)continuous ultrafiltration. Redrawn from Katchalski-Katzir E(1993)Trends in Biotechnology 1,471-477. advant Some of the potential advantages of enzyme immobilisation are: · high enzyme loads enzyme can often be regenerated by suitable ment and used again, the ability to recycle products · 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
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 process e can also use microbial cells(fermentation) containing the desired catalytic activity without isolating the enzymes responsibl Advantages of whole cell processes include: cell disruption not necessary enzyme isolation not necessary; cofactor regeneration not a problem reduced catalyst preparation costs Among the disadvantages of whole cell processes are system not fully understood(black box situation) product contamination by cellular enzymes or other end products of metabolism reduced catalytic specific activity 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 ells), immobilised growing cells, immobilised resting cells or immobilised dead cells immobilised Immobilised cells have all the advantages of immobilised enzymes. cell immobilisation cell 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 SAQ 2.1 Identify which of the following statements are true for immobilised biocatalysts hen compared to free enzyme or free cell system 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 SAQ 2.2 Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodio Give two reasons why this biotransformation should be carried out using whole cells as opposed to using enzyme preparations
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 2.4 Scale of production The decision as to which approach -free enzyme, immobilised enzyme, fermentation, immobilised cells-is mainly dictated by economIcs ommercial aspects of bioprocess development 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 entists engineers and marketing personnel. For products derived from micro-organisms, process design can be based mainly on biochemical engineering considerations or on microbial physiology considerations When compared to the improvements achieved bulk chemicals manufacture and in petroleum refining, the application of communication biochemical engineering principles to microbial proo 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) production range examples of blotechnology sIngle plant products ne chemicals 100 kg/annum- 100 vitamins. vaccines, nucleotides onnes/annum amino acids) usually batch reactors antibiotics ntermediate volume 0,000 tonnes/annum h or continuous reactors citric acid food industry bionics fo enzymes industr ermented foods and bulk chemicals > 20.000 tonnes/annum single cell protein, ethanol for usually continuous flow industry biopolymers for enhanced oil recovery), biogas (methane), sewage and wastewater treatment plants Table 2.2 Product categories in chemical manufacture The design of production plants for the manufacture of the three categories of product a lly produced in batch olume and also be used for the production of a variety of similar products. Fine chemicals usually bulk chemicals have demanding product quality specifications and uently, 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 usually manufactured in product-specific-plants, either as batch or continuous flow processes. Bulk chemical production plants usually operate continuous flow processes
Biocatalysts in organic chemical synthesis 17 k.4 Scale of production The decision as to which approach - free enzyme, immobilised enzyme, fermentation, P obilised cells - is mainly dictated by economics. Commercial aspects of bioprocess 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 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 batch mode 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 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 life cultures are considered further in Section 2.1 res and thus product yields; fed-batch fetime of batch cul continuous The alternative to batch mode operation is continuous operation. In the continuous mode 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) ∏I 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 aretime,, plug flowand'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 flexibi 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; e 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
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