Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture

Ralf Pörtner and Rebecca Faschian

#### Abstract

Fixed-bed processes operated in perfusion, where cells are immobilized within macroporous carriers, are a promising alternative to processes with suspended microbial or mammalian cells. Their potential has been demonstrated for many purposes. Nevertheless, the number of industrial fixed-bed processes is quite small. To some extent, this is due to the lack of process development tools for fixed-bed processes. To fill this gap, a strategy was developed for the design and evaluation of relevant process parameters of fixed-bed processes. A scale-up concept is presented in order to evaluate the performance as part of process design of fixed-bed processes. This comprises fixed-bed reactors on three different scales, the smallest being the downscaled Multiferm with 10 mL fixed-bed units, the second a 100 mL fixed-bed reactor, and the third a pilot-scale reactor with 1 L fixed-bed volume. The performance of this concept will be discussed for fixed-bed cultures of lactic acid bacteria. Furthermore, a reaction kinetic model for the design of fixed-bed reactors will be presented.

Keywords: fixed-bed, bacterial culture, scale-up, modeling, lactic acid bacteria

#### 1. Introduction

Technologies for immobilization of biocatalyst, e.g., microbial or mammalian cells, are increasingly being considered for biotechnological processes due to many advantages compared to cell suspension culture such as continuous operation, accelerated reaction rates, high volumetric productivity, retention of plasmidbearing cells, prevention of interfacial inactivation, stimulation of production and excretion of secondary metabolites, and protection against turbulent high-shear environment, reduced susceptibility of cells to contamination, improved production efficiency, and reduced risk of washout [1–3]. Especially the increased importance of productivity for industrial processes due to restriction of production time and final product volume has drawn the attention to immobilization techniques in recent years, as they allow overcoming most of the limitations of commonly applied suspension cultures [2]. A summary of advantages and disadvantages for suspension cultures (stirred tank reactors) and immobilized cultures (fixed-bed reactors) is given in Table 1.

Various immobilization techniques such as the entrapment of cells in stable porous gels (e.g., alginate, agarose, collagen, chitosan, cellulose, κ-carrageenan, or


microorganisms [6]. Consequently, both the operational stability of the

Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture

Despite the obvious advantages of fixed-bed bioreactor systems, the number of industrial fixed-bed processes is quite small [2–4]. To some extent, this is due to the lack of process development tools for fixed-bed processes and meaningful concepts for design and operation of fixed-bed reactors on a large scale. To fill this gap, strategies for the design and evaluation of relevant process parameters of fixed-bed

In the following, the characteristics of fixed-bed bioreactors as well as a design concept for layout and scale-up will be introduced. Examples for macroporous carriers will be given. The design strategy for fixed-bed reactors will be discussed in detail for immobilized cultures of lactic acid-producing bacteria (LABs). Finally, a reaction kinetic model is introduced which allows evaluation of the culture perfor-

Fixed-bed bioreactors consist of a mostly cylindrical column containing macroporous carriers, wherein cells are immobilized (Figure 1A). The column is permanently perfused with fresh medium [4, 7]. If required, the medium can be circulated in a loop (Figure 1B). This might be useful if appropriate flow rates and

For small fixed-bed volumes with a height of approximately 10 cm, the medium can be pumped axially through the bed. In this case, at the outlet the oxygen concentration in the case of aerobic cells, e.g., mammalian cells [8], or the pH in the case of acid-producing anaerobic cells, e.g., LABs [6], should remain in a physiological range. A further increase of the length would result in too low oxygen or pH values in the upper zones of the bed. This can be overcome by applying a radial medium flow as shown in Figure 1C, where the radius determines the length of the oxygen or pH gradient, not the height of the column. This concept was successfully applied for mammalian cell culture [8] and lactic acid bacteria, as discussed in the following.

Examples for design concepts of fixed-bed reactors. (A) Axial-flow fixed bed with plug flow. (B) Axial-flow fixed bed with external conditioning vessel. (C) Fixed bed with radial flow integrated in conditioning vessel,

immobilized organisms and the productivity are improved.

processes are required, and a scale-up concept is introduced.

mance. Conclusions complete the text.

DOI: http://dx.doi.org/10.5772/intechopen.87944

medium supply rates vary significantly.

2. Fixed-bed reactor systems

2.1 Principle

Figure 1.

plug flow.

83

#### Table 1.

Summary of advantages and disadvantages of stirred tank (suspended cells) and fixed-bed reactors (immobilized cells).


#### Table 2.

Characteristics of fixed-bed reactors used for cultivation of microorganisms or mammalian/tissue cells (adapted from Pörtner and Märkl [5]).

gel-matrix polymers such as polyacrylamide-hydrazide) or hydrogels or immobilization in solid macroporous carriers have been developed and are applied in both laboratory and industrial scales for different purposes, e.g., food, dairy, and beverage industry, production of drugs, wastewater treatment, agricultural industry, and biodiesel production [2–4].

Bioreactors for immobilized biocatalyst are mostly operated continuously in perfusion mode. Here continuous stirred tank reactors with cell retention and fixedbed (packed bed) or fluidized-bed bioreactor systems can be applied. The following remarks focus on fixed-bed reactors, which consist of a packed column of macroporous carriers wherein cells are immobilized, as they have been used very successfully for a wide range of applications [4]. The advantages of fixed-bed reactors with immobilized cells (Table 2) are mainly with respect to general productivity and operational flexibility [6]. The volumetric productivity of immobilized cells is generally higher than the corresponding free cell fermentations [6]. This higher productivity can be explained by the fact that the microenvironments offered by the carrier are more stabilizing for the organisms, which generally show optimal activity only in a narrow range of physical conditions. Due to cell retention, it is possible to run fixed-bed bioreactors in a perfusion mode at a steady state with dilution rates higher than the maximum specific growth rate of the used strain. By this, very high volume-specific productivities can be reached and maintained for long periods of time and greatly facilitate recycling or reuse of

#### Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture DOI: http://dx.doi.org/10.5772/intechopen.87944

microorganisms [6]. Consequently, both the operational stability of the immobilized organisms and the productivity are improved.

Despite the obvious advantages of fixed-bed bioreactor systems, the number of industrial fixed-bed processes is quite small [2–4]. To some extent, this is due to the lack of process development tools for fixed-bed processes and meaningful concepts for design and operation of fixed-bed reactors on a large scale. To fill this gap, strategies for the design and evaluation of relevant process parameters of fixed-bed processes are required, and a scale-up concept is introduced.

In the following, the characteristics of fixed-bed bioreactors as well as a design concept for layout and scale-up will be introduced. Examples for macroporous carriers will be given. The design strategy for fixed-bed reactors will be discussed in detail for immobilized cultures of lactic acid-producing bacteria (LABs). Finally, a reaction kinetic model is introduced which allows evaluation of the culture performance. Conclusions complete the text.

#### 2. Fixed-bed reactor systems

#### 2.1 Principle

Fixed-bed bioreactors consist of a mostly cylindrical column containing macroporous carriers, wherein cells are immobilized (Figure 1A). The column is permanently perfused with fresh medium [4, 7]. If required, the medium can be circulated in a loop (Figure 1B). This might be useful if appropriate flow rates and medium supply rates vary significantly.

For small fixed-bed volumes with a height of approximately 10 cm, the medium can be pumped axially through the bed. In this case, at the outlet the oxygen concentration in the case of aerobic cells, e.g., mammalian cells [8], or the pH in the case of acid-producing anaerobic cells, e.g., LABs [6], should remain in a physiological range. A further increase of the length would result in too low oxygen or pH values in the upper zones of the bed. This can be overcome by applying a radial medium flow as shown in Figure 1C, where the radius determines the length of the oxygen or pH gradient, not the height of the column. This concept was successfully applied for mammalian cell culture [8] and lactic acid bacteria, as discussed in the following.

#### Figure 1.

Examples for design concepts of fixed-bed reactors. (A) Axial-flow fixed bed with plug flow. (B) Axial-flow fixed bed with external conditioning vessel. (C) Fixed bed with radial flow integrated in conditioning vessel, plug flow.

gel-matrix polymers such as polyacrylamide-hydrazide) or hydrogels or immobilization in solid macroporous carriers have been developed and are applied in both laboratory and industrial scales for different purposes, e.g., food, dairy, and beverage industry, production of drugs, wastewater treatment, agricultural industry, and

• Easy medium exchange and separation of cells and product simplifying downstream processing

Characteristics of fixed-bed reactors used for cultivation of microorganisms or mammalian/tissue cells

Advantages Disadvantages

Easy exchange of medium Nonhomogeneous

Summary of advantages and disadvantages of stirred tank (suspended cells) and fixed-bed reactors

High potential for scale-up

High productivity over long

Low-shear rates (relevant for

High cell density and productivity per unit

periods of time

mammalian cells)

• High volumetric cell density and high productivity

• Low-shear stress environment for mammalian and tissue cells

Known technology Aeration difficult at high cell densities

Good mass transfer Cell damage by shear and aeration (e.g.,

Cell count possible Low cell density and volumetric productivity

Good mixing Foaming (relevant for aerobic cells)

(relevant for aerobic cells)

Concentration gradients

Cell count impossible

Cell retention required for perfusion culture, techniques insufficient for long-term culture

mammalian cells)

Bioreactors for immobilized biocatalyst are mostly operated continuously in perfusion mode. Here continuous stirred tank reactors with cell retention and fixedbed (packed bed) or fluidized-bed bioreactor systems can be applied. The following

immobilized cells is generally higher than the corresponding free cell fermentations [6]. This higher productivity can be explained by the fact that the microenvironments offered by the carrier are more stabilizing for the organisms, which generally show optimal activity only in a narrow range of physical conditions. Due to cell retention, it is possible to run fixed-bed bioreactors in a perfusion mode at a steady state with dilution rates higher than the maximum specific growth rate of the used strain. By this, very high volume-specific productivities can be reached and maintained for long periods of time and greatly facilitate recycling or reuse of

remarks focus on fixed-bed reactors, which consist of a packed column of macroporous carriers wherein cells are immobilized, as they have been used very successfully for a wide range of applications [4]. The advantages of fixed-bed reactors with immobilized cells (Table 2) are mainly with respect to general pro-

ductivity and operational flexibility [6]. The volumetric productivity of

biodiesel production [2–4].

(adapted from Pörtner and Märkl [5]).

Stirred tank/ suspension

Growing and Handling of Bacterial Cultures

Fixed-bed/ immobilized cells

Table 1.

Table 2.

82

(immobilized cells).

Cell immobilization in fixed-bed reactors with macroporous carriers is fairly simple compared to other methods such as entrapping in gels (e.g., alginate). Cell loading is often carried out by simply pumping a cell suspension through the bed of carriers, and cells are kept under same physiological conditions for the immobilization. As only the natural properties of the surface and cells interact, there are no toxic effects arising from activating reagents compared to cell entrapment within polymers. Additionally, high load of cells can be avoided by desorption of cells from the solid surface to the cell suspension.

3. Carriers

In cell immobilization, properties of the carrier materials play an important role. This type of immobilization on solid synthetic materials firstly has the advantage that the microorganisms attach independently to the carrier (interaction with the surface) and thus no additional process steps and reagents are required for immobilization. At this point, carrier materials have to demonstrate several certain characteristics. Atkinson et al. [12] and Pörtner and Märkl [5] summarized these properties for cell immobilization such as simple and nontoxic material, high cell loading capacity, mechanical stability, stable at appropriate operational pH values, autoclavable, resistant to microbial degradation, cost appropriate to the application, density appropriate to reactor type used, as well as reusable, if possible. Examples are given in [4, 6]. In our own studies, carriers made of glass [Siran (QVF, Mainz, Germany), VitraPOR® (ROBU® Glasfilter-Geräte GmbH)] or ceramics [(CERAMTEC EO 19/30 (CeramTec, Marktredwitz, Germany) (Figure 3) or Sponceram (Zellwerk, Oberkrämer, Germany)] were applied. All carriers showed similar results with respect to immobilized

Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture

DOI: http://dx.doi.org/10.5772/intechopen.87944

cell density and lactic acid productivity for immobilized LAB strains [6].

4. Case study: fixed-bed cultivation of LAB strains

volumes go up to 100 m<sup>3</sup>

Figure 3.

carrier).

85

4.1 Overview on immobilization techniques used for LAB strains

Lactic acid bacteria are commonly used in the production of fermented dairy products as well as for production of lactic acid, antimicrobial substances (bacteriocins), and biodegradable polymers, among others [13–15]. Industrial processes use mostly conventional batch or fed-batch fermentation with suspended cells. Reactor

Examples for carriers applied in fixed-bed cultures: (A) CERAMTEC EO 19/30 (α-aluminum, ring, diameter 3–8 mm, height 8 mm, porosity 65%; manufacturer, CeramTec; up-right, carrier; up-left, carrier in fixed-bed cultivation of Lactococcus lactis; down, SEM of carrier); (B) VitraPOR® (glass, sphere, diameter 4 mm; manufacturer, ROBU; up-left, carrier; up-right, carrier in fixed-bed cultivation of L. lactis; down, SEM of

depending on the strain and the process strategy [14]. Even if high cell and product

, and process time varies between several hours and days

#### 2.2 Concept for design and operation

Process parameters that have to be optimized during process development comprise selection of carriers, medium selection, appropriate flow velocity, and longterm performance, among others. All these information are required to evaluate the overall performance, e.g., productivity, and to layout the scale-up strategy. In the following, a platform for development of processes for immobilized cells is introduced (Figure 2). As a start, suitability of different carriers can be compared in a small-scale multi-well system. After this, bioreactor systems of different sizes can be used to work out the required process parameters. The first, very small scale of 10 mL working volume is the multi-fixed-bed bioreactor "Multiferm" [9]. The next step is an axial-flow 100 mL fixed-bed system, which can be operated continuously with reasonable effort to investigate the performance and long-term stability of the culture [6]. As a first approach for scale-up, a radial-flow 1 L fixed-bed reactor is applied [6, 10]. Even if this is probably not the final industrial scale, the reactor system has already been the main characteristics of a large-scale system, mainly the radius. For further increase of the volume, just the height has to be increased [11]. For all three systems, a "proof of concept" has been shown before [10]. In Chapter 4 the performance of these three fixed-bed systems is compared for fixed-bed cultures of LABs.

#### Figure 2.

Platform for development of processes for immobilized cells. From left to right: multi-well plates with special inserts for the first evaluation of appropriate carriers under high-throughput, static conditions; multi-well flow chamber for evaluation of carriers under flow conditions; multi-fixed-bed reactor "Multiferm" (max. 12 small fixed-bed units containing approx. 10 mL carriers for evaluation of carriers under different process parameters (type of carrier, flow rate, medium, oxygen concentration, pH, etc.)); 100 mL axial-flow fixed-bed for longterm continuous culture under steady-state conditions; and 1 L radial-flow fixed-bed representative for a pilot scale for long-term continuous cultures under steady-state conditions.

Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture DOI: http://dx.doi.org/10.5772/intechopen.87944
