Fermentation Pilot plant Yujiro Harada, Kuniaki Sakata, Seiji sato and Shinsaku Takayama PROLOGUE (by Yujiro Harada The rapid development of biotechnology has impacted diverse sectors of the economy over the last several years. The industries most affected are the agricultural, fine chemical, food processing, marine, and pharmaceuti cal. In order for current biotechnology research to continue revolutionize industries, new processes must be developed to transform current research into viable market products. Specifically, attention must be directed toward the industrial processes of cultivation of cells, tissues, and microorganisms Although several such processes already exist(e. g, r-DNA and cell fusion), more are needed and it is not even obvious which of the existing processes best To develop the most cost efficient process, sc data must be collected by repeating experiments at the bench and pilot scale level. These data must beextensive. Unfortunately, the collection is far more difficult than it would be in the chemical and petrochemical industries. The nature of working with living material makes contamination commonplace and repro- ducibility of data difficult to achieve. Such problems quickly distort the relevant scale-up factors In this chapter, three research scientists from Kyowa Kogyo Co have addressed the problems of experimentation and pilot scale-up
1 Fermentation Pilot Plant Yujiro Harada, Kuniaki Sakata, Seiji Sato and Shinsaku Takayama PROLOGUE (by Yujiro Harada) The rapid development of biotechnology has impacted diverse sectors of the economy over the last several years. The industries most affected are the agricultural, fine chemical, food processing, marine, and pharmaceutical. In order for current biotechnology research to continue revolutionizing industries, new processes must be developed to transform current research into viable market products. Specifically, attention must be directed toward the industrial processes of cultivation of cells, tissues, and microorganisms. Although several such processes already exist (e.g., r-DNA and cell fusion), more are needed and it is not even obvious which of the existing processes is best. To develop the most cost efficient process, scale-up data must be collected by repeating experiments at the bench and pilot scale level. These datamust be extensive. Unfortunately, the collection is far more difficult than it would be in the chemical and petrochemical industries. The nature of working with living material makes contamination cobonplace and reproducibility of data difficult to achieve. Such problems quickly distort the relevant scale-up factors. In this chapter, three research scientists from Kyowa Kogyo Co. Ltd. have addressed the problems of experimentation and pilot scale-up for I
2 Fermentation and Biochemical engineering Handbook microorganisms, mammalian cells, plant cells, and tissue. It is our sincere hope that the reader will find this chapter helpful in determining the best conditions for cultivation and the collection of scale-up data. Hopefully, this knowledge will, in turn, facilitate the transformation of worthwhile research programs into commercially viable processes 1.0 MICROBIAL FERMENTATION (by Kuniaki sakato) Chemical engineers are still faced with problems regarding scale- and microbial contamination in the fermentation by aerobic submerged cultures. Despite many advances in biochemical engineering to address these problems, the problems nevertheless persist. Recently, many advances have been made in the area of recombinant DNA, which themselves have spun off new and lucrative fields in the production of plant and animal pharmaceuti cals. A careful study of this technology is therefore necessary, not only for the implementation of efficient fermentation processes, but also for compli ance with official regulatory bodies There are several major topics to consider in scaling up laboratory processes to the industrial level. In general, scale-up is accomplished for a discrete system through laboratory and pilot scale operations. The steps involved can be broken down into seven topics that require some elaboration 1. Strain improvements 2. Optimization of medium composition and cultural condi tions such as pH and temperature 3. Oxygen supply required by cells to achieve the proper metabolic activities 4. Selection of an operative mode for culture process 5. Measurement of rheological properties of cultural broth 6. Modelling and formulation of process control strategies 7. Manufacturing sensors, bioreactors, and other peripheral Items l and 2 should be determined in the laboratory using shake flasks or small jar fermenters. Items 3-7 are usually determined in the pilot plant The importance of the pilot plant is, however, not limited to steps 3-7. The pilot plant also provides the cultured broths needed for downstream
2 Fermentation and Biochemical Engineering Handbook microorganisms, mammalian cells, plant cells, and tissue. It is our sincere hope that the reader will find this chapter helpful in determining the best conditions for cultivation and the collection of scale-up data. Hopehlly, this knowledge will, in turn, facilitate the transformation of worthwhle research programs into commercially viable processes. 1.0 MICROBIAL FERMENTATION (by Kuniaki Sakato) Chemical engineers are still faced with problems regarding scale-up and microbial contamination in the fermentation by aerobic submerged cultures. Despite many advances in biochemical engineering to address these problems, the problems nevertheless persist. Recently, many advances have been made in the area of recombinant DNA, which themselves have spun off new and lucrative fields in the production of plant and animal pharmaceuticals. A careful study of this technology is therefore necessary, not only for the implementation of efficient fermentation processes, but also for compliance with official regulatory bodies. There are several major topics to consider in scaling up laboratory processes to the industrial level. In general, scale-up is accomplished for a discrete system through laboratory and pilot scale operations. The steps involved can be broken down into seven topics that require some elaboration: 1. Strain improvements 2. Optimization of medium composition and cultural condi- 3. Oxygen supply required by cells to achieve the proper tions such as pH and temperature metabolic activities 4. Selection of an operative mode for culture process 5. Measurement of rheological properties of cultural broth 6. Modelling and formulation of process control strategies 7. Manufacturing sensors, bioreactors, and other peripheral equipment Items 1 and 2 should be determined in the laboratory using shake flasks or small jar fermenters. Items 3-7 are usually determined in the pilot plant. The importance ofthe pilot plant is, however, not limited to steps 3-7. The pilot plant also provides the cultured broths needed for downstream
Fermentation Pilot Plant 3 processing and can generate information to determine the optimal cost structure in manufacturing and energy consumption as well as the testing of various raw materials in the medium 1.1 Fermentation Pilot plant Microorganisms such as bacteria, yeast, fungi, or actinomycete have manufactured amino acids, nucleic acids, enzymes, organic acids, alcohols and physiologically active substances on an industrial scale. The" New Biotechnology"is making it increasingly possible to use recombinant DNA techniques to produce many kinds of physiologically active substances such as interferons, insulin, and salmon growth hormone which now only exist in small amounts in plants and animals This section will discuss the general problems that arise in pilot plant, rmentation and scale-up. The section will focus on three main topics: (i) bioreactors and culture techniques, (ii) the application of com sensing technologies to fermentation, and (iii) the scale-up itse bouter and 1. 2 Bioreactors and Culture Techniques for Microbial Processes Current bioreactors are grouped into either culture vessels and reactors using biocatalysts(e.g, immobilized enzymes/microorganisms)or plant and animal tissues. The latter is sometimes used to mean the bioreactor Table I shows a number of aerobic fermentation systems which are schematically classified into(i) internal mechanical agitation reactors, (i) extemal circulation reactors, and (iii) bubble column and air-lift loop reactors. This classification is based on both agitation and aeration as it relates to oxygen supply. In this table, reactor I is often used at the industrial level and reactors(a)2, (b )2, (c)2, and( c)3, can be fitted with draught tubes to improve both mixing and oxygen supply efficiencies Culture techniques can be classified into batch, fed-batch, and con inuous operation(Table 2). In batch processes, all the nutrients required for cell growth and product formation are present in the medium prior to cultivation. Oxygen is supplied by aeration. The cessation ofgrowth reflects the exhaustion of the limiting substrate in the medium. For fed-batch processes, the usual fed-batch and the repeated fed-batch operations are listed in Table 2 A fed-batch operation is that operation in which one or more nutrients are added continuously or intermittently to the initial medium after the start of cultivation or from the halfway point through the batch process. Details
Fermentation Pilot Plant 3 processing and can generate information to determine the optimal cost structure in manufacturing and energy consumption as well as the testing of various raw materials in the medium. 1.1 Fermentation Pilot Plant Microorganisms such as bacteria, yeast, fungi, or actinomycete have manufactured amino acids, nucleic acids, enzymes, organic acids, alcohols and physiologically active substances on an industrial scale. The “New Biotechnology” is making it increasingly possible to use recombinant DNA techniques to produce many kinds of physiologically active substances such as interferons, insulin, and salmon growth hormone which now only exist in small amounts in plants and animals. This section will discuss the general problems that arise in pilot plant, fermentation and scale-up. The section will focus on three main topics: (i) bioreactors and culture techniques, (ii) the application of computer and sensing technologies to fermentation, and (iii) the scale-up itself. 1.2 Bioreactors and Culture Techniques for Microbial Processes Current bioreactors aregrouped into either culture vessels and reactors using biocatalysts (e.g., immobilized enzymes/microorganisms) or plant and animal tissues. The latter is sometimes used to mean the bioreactor. Table 1 shows a number of aerobic fermentation systems which are schematically classified into (i) internal mechanical agitation reactors, (ii) external circulation reactors, and (iii) bubble column and air-lift loop reactors. This classification is based on both agitation and aeration as it relates to oxygen supply. In this table, reactor 1 is often used at the industrial level and reactors (a)2, (b)2, (c)2, and (c)3, can be fitted with draught tubes to improve both mixing and oxygen supply efficiencies. Culture techniques can be classified into batch, fed-batch, and continuous operation (Table 2). In batch processes, all the nutrients required for cell growth and product formation are present in the medium prior to cultivation. Oxygen is supplied by aeration. The cessation ofgrowth reflects the exhaustion of the limiting substrate in the medium. For fed-batch processes, the usual fed-batch and the repeated fed-batch operations are listed in Table 2. A fed-batch operation is that operation in which one or more nutrients are added continuously or intermittently to the initial medium after the start of cultivation or from the halfway point through the batch process. Details
4 Fermentation and Biochemical Engineering Handbook offed-batch operation are summarized in Table 3. In the table the fed-batch operation is divided into two basic models, one without feedback control and the other with feedback control. Fed-batch processes have been utilized to avoid substrate inhibition, glucose effect, and catabolite repression, as well as for auxotrophic mutants Table 1. Classification of Aerobic Fermentation Systems (a) Internal mechanical agitation reactors 1. Turbine-stirring installation 2. Stirred vessel with draft tube 3. Stirred vessel with suction tube (b) Extermal circulation reactors 1. Water jet aerator 2. Forced water jet aerator 3. Recycling aerator with fritted dis (c) Bubble column and air-loop reactors 1. Bubble column with fritted disc 2. Bubble column with a draft tube for gyration flow 3. Air lift reactor 4. Pressure cycle reactor 5. Sieve plate cascade system Table 2. Classification of Fermentation Processes 1. Batch process 2. Fed-batch process(semi-batch process) 3. Repeated fed-batch process(cyclic fed-batch process Repeated fed-batch process(semi-continuous process or cyclic 5. Continuous process
4 Fermentation and Biochemical Engineering Handbook of fed-batch operation are summarized in Table 3. In the table the fed-batch operation is divided into two basic models, one without feedback control and the other with feedback control. Fed-batch processes have been utilized to avoid substrate inhibition, glucose effect, and catabolite repression, as well as for auxotrophic mutants. Table 1. Classification of Aerobic Fermentation Systems (a) Internal mechanical agitation reactors 1. Turbine-stimng installation 2. Stirred vessel with draft tube 3. Stirred vessel with suction tube (b) External circulation reactors 1. Water jet aerator 2. Forced water jet aerator 3. Recycling aerator with fritted disc (c) Bubble column and air-loop reactors 1. Bubble column with fritted disc 2. Bubble column with a draft tube for gyration flow 3. Air lift reactor 4. Pressure cycle reactor 5. Sieve plate cascade system Table 2. Classification of Fermentation Processes 1. Batch process 2. Fed-batch process (semi-batch process) 3. 4. Repeated fed-batch process (cyclic fed-batch process) Repeated fed-batch process (semi-continuous process or cyclic batch process) 5. Continuous process
fermentation pilot plant 5 Table 3. Classification of Fed-Batch Processes in Fermentation 1. without feedback control a. Intermittent fed-batch b. Constant rate fed-batch C. Exponentially fed-batch d. Optimized fed-batch 2. With feedback control a. Indirect control b. Direct control Setpoint control (constant value control) Program Control Optimal control The continuous operations of Table 2 are elaborated in Table three types of operations. In a chemostat without feedback control, the medium containing all the nutrients is continuously fed at a constant rate (dilution rate)and the cultured broth is simultaneously removed from the fermenter at the same rate. A typical chemostat is shown in Fig. 1. The chemostat is quite useful in the optimization of media formulation and to investigate the physiological state of the microorganism. A turbidostat with feedback control is a continuous process to maintain the cell concentration at a constant level by controlling the medium feeding rate. A nutristat with feedback control is a cultivation technique to maintain a nutrient concentra- tion at a constant level. a phauxostat is an extended nutristat which maintains the ph value of the medium in the fermenter at a preset value
Fermentation Pilot Plant 5 Table 3. Classification of Fed-Batch Processes in Fermentation 1. Without feedback control a. Intermittent fed-batch b. Constant rate fed-batch c. Exponentially fed-batch d. Optimized fed-batch 2. With feedback control a. Indirect control b. Direct control Setpoint control (constant value control) Program Control Optimal control The continuous operations of Table 2 are elaborated in Table 3 as three types of operations. In a chemostat without feedback control, the feed medium containing all the nutrients is continuously fed at a constant rate (dilution rate) and the cultured broth is simultaneously removed from the fermenter at the same rate. A typical chemostat is shown in Fig. 1. The chemostat is quite useful in the optimization of media formulation and to investigate the physiological state of the microorganism. A turbidostat with feedback control is a continuous process to maintain the cell concentration at a constant level by controlling the medium feeding rate. A nutristut with feedback control is a cultivation technique to maintain a nutrient concentration at a constant level. A phuuxostut is an extended nutristat which maintains the pH value of the medium in the fermenter at a preset value
6 Fermentation and Biochemical Engineering Handbook Figure l is an example of chemostat equipment that we call a single-stage continuous culture. Typical homogeneous continuous culture systems are shown in Fig. 2 Table 4. Classification of continuous fermentation processes 1. without feedback control a. Turbidostat lIlm WIllI Vent Broth eservoir reservoir Broth (a)single-stage continuous culture b)Level controller system Figure 1. Chemostat System. V: Operation volume. F: Feed rate of medium
6 Fermentation and Biochemical Engineering Handbook Figure 1 is an example of chemostat equipment that we call a single-stage continuous culture. Typical homogeneous continuous culture systems are shown in Fig. 2. Table 4. Classification of continuous fermentation processes 1. Without feedback control a. Chemostat 2. With feedback control a. Turbidostat b. Nutristat c. Phauxostat i F Med i urn reservoir Air Motor 1 Broth re servo i r (a1Single-stage continuous culture system f (bltevel controller Figure 1. Chemostat System. V: Operation volume. F: Feedrate ofmedium. Sf: Concentration of limiting substrate
Fermentation Pilot plant (a)single-stage continuous operat ion (b)single-stage continuous operation with feedback (c)Multi-stage continuous operation: simple chain (d)Multi-stage continuous operation: Multiple substrate addition Figure 2. Homogeneous systems for continuous fermentation
Fermentation Pilot Plant L r I 11 1 I I mM - 0 7 - _N_v (a)Single-stage m c o n t i n u o u s o p e r a t i o n Figure 2. Homogeneous systems for continuous fermentation
8 Fermentation and Biochemical Engineering Handboot 1.3 Application of Computer Control and Sensing Technologies for Fermentation Process The application of direct digital control of fermentation processes began in the 1960s. Since then, many corporations have developed computer-aided fermentation in both pilot and commercial plants. Unfortu nately, these proprietary processes have almost never been published, dueto corporate secrecy. Nevertheless, recent advances in computer and sensing technologies do provide us with a great deal of information on fermentation This information can be used to design optimal and adaptive process controls In commercial plants, programmable logic controllers and process computers enable both process automation and labor-savings. The present and likely future uses of computer applications to fermentation processes in pilot and industrial plants are summarized in table 5. In the table, open circles indicate items that have already been discussed in other reports while the open triangles are those topics to be elaborated here Table 5. Computer Applications to Fermentation Plants Pilot scale Production scale Fut Sequence control Feedback control Data acquisition imation state variables Advanced control △ Modelling Scheduling
8 Fermentation and Biochemical Engineering Handbook 1.3 Application of Computer Control and Sensing Technologies for Fermentation Process The application of direct digital control of fermentation processes began in the 1960’s. Since then, many corporations have developed computer-aided fermentation in both pilot and commercial plants. Unfortunately, these proprietary processes have almost never been published, due to corporate secrecy. Nevertheless, recent advances in computer and sensing technologies do provide us with a great deal of information on fermentation. This information can be used to design optimal and adaptive process controls. In commercial plants, programmable logic controllers and process computers enable both process automation and labor-savings. The present and likely future uses of computer applications to fermentation processes in pilot and industrial plants are summarized in Table 5. In the table, open circles indicate items that have already been discussed in other reports while the open triangles are those topics to be elaborated here. Table 5. Computer Applications to Fermentation Plants Sequence control Feedback control Data acquisition Estimation of state variables Advanced control Optimized Control Modelling Scheduling Pilot Scale Present Future Production Scale Present Future A few cases A A
fermentation Pilot plant The acquisition of data and the estimation of state parameters on commercial scales will undoubtedly become increasingly significant Unfortunately, the advanced control involving adaptive and optimize controls have not yet been sufficiently investigated in either the pilot or industrial scale Adaptive control is of great importance for self-optimization of fermentation processes, even on a commercial scale, because in ordinary fermentation the process includes several variables regarding culture condi tions and raw materials. We are sometimes faced with difficulties in the mathematical modelling of fermentation processes because of the complex reaction kinetics involving cellular metabolism. The knowledge-based controls using fuzzy theory or neural networks have been found very uset for what we call the"black box processes. Although the complexity of the process and the number of control parameters make control problems in fermentation very difficult to solve, the solution of adaptive optimization strategies is worthwhile and can contribute greatly to total profits. In order to establish such investigations, many fermentation corporations have been building pilot fermentation systems that consist of highly instrumented fermenters coupled to a distributed hierarchical computer network for on-and off-line data acquisition, data analysis, control and modelling. An example of the hierarchical computer system that is shown in Fig. 3 has become as common in the installation of large fermentation plants as it is elsewhere in the chemical industry. Figure 4 shows the details of a computer communi- cation network and hardware As seen in Fig 3, the system is mainly divided into three different functional levels. The first level has the YEWPACK package instrumenta tion systems(Yokogawa Electric Corporation, Tokyo), which may consist of an operators console (UOPC or UOPS)and several field control units (UFCU or UFCH) which are used mainly for on-line measurement, alarm, sequence control, and various types of proportional-integral-derivative(PID) controls. Each of the field control units interfaces directly with input/output signals from the instruments of fermenters via program controlle conditioners. In the second level, YEWMAC line computer systems (Yokogawa Electric Corporation, Tokyo) are dedicated to the acquisiti storage, and analysis of data as well as to documentation, graphics, optimi zation, and advanced control. a line computer and several line controllers constitute a YEWMAC. The line controller also governs the local area network formed with some lower level process computers using the bsc multipoint system. On the third level, a mainframe computer is reserved for modelling, development of advanced control, and the building of a data base
Fermentation Pilot Plant 9 The acquisition of data and the estimation of state parameters on commercial scales will undoubtedly become increasingly significant. Unfortunately, the advanced control involving adaptive and optimized controls have not yet been sufficiently investigated in either the pilot or industrial scale. Adaptive control is of great importance for self-optimization of fermentation processes, even on a commercial scale, because in ordinary fermentation the process includes several variables regarding culture conditions and raw materials. We are sometimes faced with difficulties in the mathematical modelling of fermentation processes because of the complex reaction kinetics involving cellular metabolism. The knowledge-based controls using fuzzy theory or neural networks have been found very useful for what we call the “black box” processes. Although the complexity of the process and the number of control parameters make control problems in fermentation very difficult to solve, the solution of adaptive optimization strategies is worthwhile and can contribute greatly to total profits. In order to establish such investigations, many fermentation corporations have been building pilot fermentation systems that consist of highly instrumented fermenters coupled to a distributed hierarchical computer network for on-and off-line data acquisition, data analysis, control and modelling. An example of the hierarchical computer system that is shown in Fig. 3 has become as common in the installation of large fermentation plants as it is elsewhere in the chemical industry. Figure 4 shows the details of a computer communication network and hardware. As seen in Fig. 3, the system is mainly divided into three different functional levels. The first level has the YEWPACK package instrumentation systems (Yokogawa Electric Corporation, Tokyo), which may consist of an operator’s console (UOPC or UOPS) and several field control units (UFCU or UFCH) which are used mainly for on-line measurement, alarm, sequence control, and various types of proportional-integral-derivative (PID) controls. Each of the field control units interfaces directly with input/output signals from the instruments of fermenters via program controllers and signal conditioners. In the second level, YEWMAC line computer systems (Yokogawa Electric Corporation, Tokyo) are dedicated to the acquisition, storage, and analysis of data as well as to documentation, graphics, optimization, and advanced control. A line computer and several line controllers constitute a YEWMAC. The line controller also governs the local area network formed with some lower level process computers using the BSC multipoint system. On the third level, a mainframe computer is reserved for modelling, development of advanced control, and the building of a data base
10 Fermentation and Biochemical Engineering Handbook Finally, the mainframe computer communicates with a company computer via a data highway. This is used for decision-making, planning, and other managerial functions. The lower level computer, shown as the first level in Fig. 3, is directly interfaced to some highly-instrumented fermenters Figure 5 illustrates a brand new fermenter for fed-batch operation. Control is originally confined to pH, temperature, defoaming, air flow rate, agitation speed, back pressure, and medium feed rate. Analog signals from various sensors are sent to a multiplexer and A/D converters. After the computer stores the data and analyzes it on the basis of algorithms, the computer sends the control signals to the corresponding controllers to control the fermentation process MAINFRAME COMPUTER LINE COMPUTOR 3 quISiTiOn Graphy ic display LIME CONTROLLER Sophist icated contre ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■国■■■■■■■■■■■■薯■■ DATA HIGHWAY Level I OPERATOR OPERATOR CONSOLE CONSOLE PID cor UFCU: Field control unlt Figure 3. Configuration of distributed hierarchical computer system for fermentation
IO Fermentation and Biochemical Engineering Handbook DATA HIOHWAY Finally, the mainframe computer communicates with a company computer via a data highway. This is used for decision-making, planning, and other managerial functions. The lower level computer, shown as the first level in Fig. 3, is directly interfaced to some highly-instrumented fermenters. Figure 5 illustrates a brand new fermenter for fed-batch operation. Control is originally confined to pH, temperature, defoaming, air flow rate, agitation speed, back pressure, and medium feed rate, Analog signals from various sensors are sent to a multiplexer and ND converters. After the computer stores the data and analyzes it on the basis of algorithms, the computer sends the control signals to the corresponding controllers to control the fermentation process. level m MAINFRAME COMPUTER Figure 3. Configuration of distributed hierarchical computer system for fermentation pilot plant. Optimi zit ion VEWMAC 300 LINE COMPUTOR tevei II Data aquisition LINE CONTROLLER 3600-M$A Optimization Sophisticated control I ! UFCU
