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

#### 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 volumes go up to 100 m<sup>3</sup> , and process time varies between several hours and days depending on the strain and the process strategy [14]. Even if high cell and product concentrations can be reached, the known drawbacks such as low productivity, product inhibition, and also the variation from batch to batch remain [16–18].

Since the immobilization of LAB has many advantages, it has been examined extensively, e.g., for the production of lactic acid; the production of starter cultures; the production of bacteriocins, e.g., nisin; and the formation of aromatic compounds (reviewed in [6]). Different methods have been used for immobilizing LAB: physical entrapment in polymeric networks, microencapsulation, attachment or adsorption to a carrier, and membrane entrapment [6]. The purpose of all these techniques is either to keep high cell concentrations within the bioreactor or to protect cells from a hostile environment. In many applications of cell entrapment, droplets of thermal (κ-carrageenan, gellan, agarose, gelatin) or ionotropic (alginate, chitosan) gels are used to produce spherical gel biocatalysts, and these controlledsize polymer droplets are produced using extrusion or emulsification, under mild conditions (reviewed in [6]). However, although promising on a laboratory scale, the large-scale production of beads under aseptic conditions still has difficulties [19].

Another immobilization technique is to immobilize LAB cells onto solid macroporous carriers and apply these in fixed-bed bioreactors. Examples are given in [6]. In the following, our recent work in this area will be discussed.

> The scale-up from 100 mL to 1 L fixed-bed (Figure 5) was successful, as similar productivities could be obtained in both systems (see below). As a conclusion, the continuous cultivation of immobilized LAB strains in fixed-bed reactors shows a high biological stability as well as cell and lactate production in long-term

> Long-term cultivation of Lactococcus lactis in an axial-flow fixed-bed (100 mL, Medorex) filled with macroporous carrier CERAMTEC EO/90 at different perfusion rates D in MRS medium (data from S. Zengen (TU Hamburg), not published). Cell concentration in harvest flow (left) and volume-specific lactate

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

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

Fixed-bed and suspension cultures of L. lactis were compared with respect to the volume-specific lactate productivity (Figure 6) [10]. Continuous suspension culture in chemostat mode showed the expected course [20]. At first the productivity increases with increasing dilution rate up to a maximum. When the dilution rate

Pilot plant fixed-bed bioreactor system consisting of 1 L radial-flow fixed-bed reactor (Medorex), feed and harvest pumps, feed tank (100 L), and control unit (temperature, pH, oxygen, depending on the type of

4.2.2 Comparison of suspension and fixed-bed systems on different scales

fermentation.

Figure 5.

87

microorganism (aerobic or anaerobic)).

productivity versus cultivation time.

Figure 4.

#### 4.2 Fixed-bed cultures of LAB strains

#### 4.2.1 Examples for fixed-bed cultivation on different scales

For all three fixed-bed bioreactor systems (Multiferm 10 mL, axial flow 100 mL, radial flow 1 L), a "proof of concept" has been shown before [6, 9, 10]. More infos on Materials and Methods can be found there. In the following, the main results are highlighted. Both Lactococcus lactis subsp. lactis and Lactobacillus delbrueckii subsp. bulgaricus could be cultivated successfully in the fixed-bed reactors. As expected, the lactate concentrations in the harvest flow were in a similar range or slightly lower as in the corresponding batch cultures. The yield of lactate depended on the type of strain and the used medium. The cell concentration in the harvest flow was considerably lower as in the corresponding batch culture, especially in case of L. bulgaricus. This is probably due to the short duration of most experiments (50–100 h per perfusion rate). In longer experiments considerably higher cell concentrations in the harvest flow were found.

The volume-specific lactic acid and cell productivity increased with increasing perfusion rate (see below).

Parallel cultivation in the Multiferm bioreactor system showed a very high reproducibility [9]. Standard deviation for lactate concentration from different parallel runs was below 5%, indicating a high reproducibility of the system. Therefore, the system is well suited for evaluation of process parameters in a very small scale with reduced effort.

The microbiological and mechanical stabilities of continuous cultivations during prolonged fermentations are critical properties of an immobilized cell process, and industrial applications are largely dependent on these properties. Therefore, we focused on the examination of long-term (52 days) continuous cultivation of L. lactis immobilized on ceramic carriers in an axial-flow 100 ml fixed-bed reactor (Figure 4) [6]. This proved that the continuous immobilized cell fermentation with L. lactis demonstrated a high biological stability longer than 50 days. The viability of cells in the harvest flow was usually around 90%, and the growth rate of cells re-cultivated as batch was similar to the corresponding batch. This indicates that functional cells can be harvested continuously from the fixed-bed.

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

#### Figure 4.

concentrations can be reached, the known drawbacks such as low productivity, product inhibition, and also the variation from batch to batch remain [16–18]. Since the immobilization of LAB has many advantages, it has been examined extensively, e.g., for the production of lactic acid; the production of starter cultures; the production of bacteriocins, e.g., nisin; and the formation of aromatic compounds (reviewed in [6]). Different methods have been used for immobilizing LAB: physical entrapment in polymeric networks, microencapsulation, attachment or adsorption to a carrier, and membrane entrapment [6]. The purpose of all these techniques is either to keep high cell concentrations within the bioreactor or to protect cells from a hostile environment. In many applications of cell entrapment, droplets of thermal (κ-carrageenan, gellan, agarose, gelatin) or ionotropic (alginate, chitosan) gels are used to produce spherical gel biocatalysts, and these controlledsize polymer droplets are produced using extrusion or emulsification, under mild conditions (reviewed in [6]). However, although promising on a laboratory scale, the large-scale production of beads under aseptic conditions still has

Another immobilization technique is to immobilize LAB cells onto solid macroporous carriers and apply these in fixed-bed bioreactors. Examples are given

For all three fixed-bed bioreactor systems (Multiferm 10 mL, axial flow 100 mL, radial flow 1 L), a "proof of concept" has been shown before [6, 9, 10]. More infos on Materials and Methods can be found there. In the following, the main results are highlighted. Both Lactococcus lactis subsp. lactis and Lactobacillus delbrueckii subsp. bulgaricus could be cultivated successfully in the fixed-bed reactors. As expected, the lactate concentrations in the harvest flow were in a similar range or slightly lower as in the corresponding batch cultures. The yield of lactate depended on the type of strain and the used medium. The cell concentration in the harvest flow was considerably lower as in the corresponding batch culture, especially in case of L. bulgaricus. This is probably due to the short duration of most experiments (50–100 h per perfusion rate). In longer experiments considerably higher cell

The volume-specific lactic acid and cell productivity increased with increasing

The microbiological and mechanical stabilities of continuous cultivations during prolonged fermentations are critical properties of an immobilized cell process, and industrial applications are largely dependent on these properties. Therefore, we focused on the examination of long-term (52 days) continuous cultivation of L. lactis immobilized on ceramic carriers in an axial-flow 100 ml fixed-bed reactor (Figure 4) [6]. This proved that the continuous immobilized cell fermentation with L. lactis demonstrated a high biological stability longer than 50 days. The viability of cells in the harvest flow was usually around 90%, and the growth rate of cells re-cultivated as batch was similar to the corresponding batch. This indicates that

Parallel cultivation in the Multiferm bioreactor system showed a very high reproducibility [9]. Standard deviation for lactate concentration from different parallel runs was below 5%, indicating a high reproducibility of the system. Therefore, the system is well suited for evaluation of process parameters in a very small

functional cells can be harvested continuously from the fixed-bed.

in [6]. In the following, our recent work in this area will be discussed.

difficulties [19].

4.2 Fixed-bed cultures of LAB strains

Growing and Handling of Bacterial Cultures

4.2.1 Examples for fixed-bed cultivation on different scales

concentrations in the harvest flow were found.

perfusion rate (see below).

scale with reduced effort.

86

Long-term cultivation of Lactococcus lactis in an axial-flow fixed-bed (100 mL, Medorex) filled with macroporous carrier CERAMTEC EO/90 at different perfusion rates D in MRS medium (data from S. Zengen (TU Hamburg), not published). Cell concentration in harvest flow (left) and volume-specific lactate productivity versus cultivation time.

The scale-up from 100 mL to 1 L fixed-bed (Figure 5) was successful, as similar productivities could be obtained in both systems (see below). As a conclusion, the continuous cultivation of immobilized LAB strains in fixed-bed reactors shows a high biological stability as well as cell and lactate production in long-term fermentation.

#### 4.2.2 Comparison of suspension and fixed-bed systems on different scales

Fixed-bed and suspension cultures of L. lactis were compared with respect to the volume-specific lactate productivity (Figure 6) [10]. Continuous suspension culture in chemostat mode showed the expected course [20]. At first the productivity increases with increasing dilution rate up to a maximum. When the dilution rate

#### Figure 5.

Pilot plant fixed-bed bioreactor system consisting of 1 L radial-flow fixed-bed reactor (Medorex), feed and harvest pumps, feed tank (100 L), and control unit (temperature, pH, oxygen, depending on the type of microorganism (aerobic or anaerobic)).

#### Figure 6.

Comparison of suspension and fixed-bed cultures for Lactococcus lactis. Volume-specific lactate productivity q\*Lac versus dilution rate D. M17 medium (Difco) with 5 g L<sup>1</sup> lactose; carrier, 10 mL Multiferm; 100 mL, VitraPOR® 4 mm; 1 L, VitraPOR® 8 mm. The red arrow indicates the maximum specific growth rate of the strain.

5. Conclusions

Figure 7.

geometry can be easily scaled up further.

Abbreviations and symbols

<sup>F</sup>in Inlet flow rate (L<sup>h</sup><sup>1</sup>

D Dilution rate (h<sup>1</sup>

<sup>F</sup> Flow rate (L<sup>h</sup><sup>1</sup>

89

The goal of the studies was to evaluate the performance of fixed-bed bioreactor systems on different scales compared to suspension culture. The suggested concept for development of fixed-bed processes could be confirmed. The multi-fixed-bed bioreactor Multiferm provides an ideal downscaled and economical system that can be used for basic studies with low requirements on medium and cells. Here, questions such as optimal carrier design, appropriate medium, and process parameters (e.g., technique for immobilization, initial cell density, flow rate, temperature, oxygen, pH) can be evaluated. Especially the start-up phase can be investigated. The next step, a 100 mL fixed-bed system, provides data on the performance and long-term stability of the culture. Problems that might not have been shown up in the Multiferm, e.g., insufficient long-term stability, can be detected here. The 1 L fixed-bed can be regarded as a pilot scale already because medium requirement was already at 27.6 L per day at the highest dilution rate. Additionally, the radial-flow

Reaction kinetic model for fixed-bed cultures with immobilized microorganisms. For details see text and [21].

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

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

As expected, fixed-bed bioreactors could be operated in a perfusion mode at a steady state with dilution rates higher than the maximum specific growth rate. By this, very high volume-specific productivity with respect to lactate can be reached and maintained for long periods of time. The fixed-bed processes with lactic acid bacteria on macroporous carriers could be transferred on a pilot scale without loss in productivity. Furthermore, the productivity could be described by a spline, indi-

Therefore, a process development tool for fixed-bed processes is now at hand that will pave the way for an industrial application of this promising technology.

cating that the maximum growth rate was not reached in this study.

)

)

)

gets close to the maximum specific growth rate μmax, the productivity decreases, as washout of cells occurs.

For the fixed-bed-cultures, the productivity increases further due to cell retention in the carriers. The highest value determined here is approx. 3–4 times higher than the maximum in chemostat cultivation. Obviously the maximum for fixed-bed cultures has not been reached so far.

All fixed-bed systems used here can be described by the same spline. This is very important with respect to scale-up, as obviously data from small-scale systems can be used to predict the performance on a larger scale (for more details on scale-up, see [20]).

#### 4.3 Reaction kinetic model for start-up of fixed-bed reactors

For establishment of mathematical process model, biomass formation, lactose consumption, and lactate production during start-up of fixed-bed cultures with immobilized L. lactis were investigated experimentally and described by a reaction kinetic model [21]. Appropriate modeling and simulation of fixed-bed processes require biomass data. Therefore, a low-volume multiple fixed-bed reactor system (Multiferm) was used to investigate biomass formation of a L. lactis strain during the start-up phase of fixed-bed cultivation. The generation of data in parallel experiments was fast and easily compared to larger single reactor systems. Biomass data obtained from both fractions, retained and free suspended biomass, was used for modeling and simulation, together with data for lactose and lactate. The underlying Luedeking-Piret-like model structure was developed based on the results from suspension cultivations with the same strain. The fixed-bed system was described as perfusion culture with cell retention (Figure 7). For this, merely four additional parameters had to be defined to extend the suspension model to fixed-bed cultures. Experimental trends and steady states of both biomass fractions besides substrate and product could be described very well. Thus, this model could be used for process layout during process development.

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

Figure 7. Reaction kinetic model for fixed-bed cultures with immobilized microorganisms. For details see text and [21].
