Fermentation Pilot Plant

Yujiro Harada, Kuniaki Sakata, Seiji Sato and Shinsaku Takayama

The rapid development of biotechnology has impacted diverse sectors of the economy. Many industries are affected, including agricultural, bio-based chemicals, food processing, biological medicines, nutraceuticals, and biofuels. 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.

Keywords

Fermentation; cultivation; scale-up; bioreactor; pilot plant

Prologue

Yujiro Harada

The rapid development of biotechnology has impacted diverse sectors of the economy. Many industries are affected, including agricultural, bio-based chemicals, food processing, biological medicines, nutraceuticals, and bio-fuels. 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 data must 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 commonplace 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. (now Kyowa Hakko Bio Co. Ltd.) have addressed the problems of experimentation and pilot scale-up for 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

Kuniaki Sakato

Chemical engineers are still faced with problems regarding scale-up and microbial contamination in the fermentation of 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:

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 of the pilot plant is, however, not limited to steps 3–7. The pilot plant also provides the cultured broths needed for downstream 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 are grouped into either culture vessels, or reactors using biocatalysts (e.g., immobilized enzymes/microorganisms) or plant and animal tissues.

Table 1.1 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 1.2). 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 of growth 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 1.2.

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 of fed-batch operation are summarized in Table 1.3. 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.

The continuous operations of Table 1.2 are elaborated in Table 1.4 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. 1.1 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 concentration 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. Figure 1.1 is an example of chemostat equipment that we call a single-stage continuous culture. Typical homogeneous continuous culture systems are shown in Fig. 1.2.