Info

Shoot culture and

Bubble column

Alliin, coumarins

79,80

embryogenic

Aerated vessel

Differentiated biomass

81,82

culture

Airlift reactor

Coumarins

75,83

the flooding point situated on the top, plant cell and tissue culture bioreactors operate below this point. Suitable impellers are radial flow impellers, axial flow impellers, and impellers that distribute the power input over a large fraction of the total reactor volume (Fig. 7).

Radial flow impellers such as the Rushton impeller achieve high energy inputs. Therefore this impeller type is suitable for only limited duties in plant cell suspension cultivation procedures. Provided that a Rushton impeller should be applied, improved designs such as the Rushton impeller with concave blades should be preferred. This improved impeller type has a more uniform energy input and produces lower shear forces.

Large, slow-moving axial flow impellers providing good mixing at relatively low tip speeds up to 2.5 m sec 1 (e.g., marine impeller or special pitched blade impeller with rounded blades) are superior for plant cell and tissue cultures. Axial flow impellers are commonly used for more sensitive cultures requiring lower gas dispersion. There is a tendency to develop shear-sensitive improved axial flow impellers characterized by kinked, torsioned, and/or profiled large stirring paddles. Newly published design criteria for bioreactors that are really fashionable show tall vessels with multiple axial as well as radial flow impellers (e.g., multiple cross-arm paddle mixer, In-termig impeller and Sigma impeller). These impeller types also ensure intense and turbulent mixing in the top of the bioreactor. Improved asymmetrical impellers with the power input distributed over a large fraction of the total reactor volume and an axial displacement flow with superposed three-dimensional motions occurring additionally (e.g., multiple Seba impeller and Hint impeller) could be favorable for plant cell suspension cultures in tall bioreactors. It is commonly postulated that such improved asymmetrical impellers are suitable for fluids with medium to high viscosity. Furthermore, in those asymmetrical impellers, mixing zones with minimized mass transport as well as uncontrollable liquid swirling inside the impeller can be prevented (86) (H. Schindler, MAVAG Ltd., Switzerland, personal communication, 2000).

As demonstrated in the literature, impellers positioned near the vessel wall, for example, the spiral stirrer, helical ribbon impeller, and anchor impeller, are suitable impeller types for different plant cell lines (25,46-49), especially for cell broths with high viscosity. Major drawbacks are high costs of spiral and helical ribbon impellers resulting from the fabrication process and problematic cleanability. Alternatives are cut opened multiple helical ribbon impellers offered by various manufacturers. In addition, alternative systems were developed to avoid typical stirring movement, for example, the cell-lift fermenter. It is designed so that its cell-lift impeller (Fig. 8) acts as a fluid pump and aerator. Macrocirculation mixing is generated by a hollow central shaft with three rotating horizontal jet tubes in a low-shear

Figure 7 Suitable impeller types for plant cell suspensions: (a) Rushton impeller, (b) Rushton impeller (concave blades), (c) marine impeller, (d) pitched blade impeller (rounded blades), (e) intermig stirrer (Ekato, Germany), (f) Hint stirrer (Chema Balcke Durr, Germany), (g) spiral impeller, and (h) anchor impeller.

Figure 8 Cell-lift fermenter (New Brunswick Scientific, USA, with kind permission of IG Instrumentengesellschaft, Zurich), (a) Cell-lift impeller and (b) reactor principle (88).

bulk movement of cells and nutrient medium. This reactor type was used successfully for cultivation of Santalum album L. cells producing phenolic compounds (87).

Airlift reactors and bubble columns have also been applied by a number of workers for plant cell suspension cultures (see Table 4). Although the airlift reactor is the system of choice for newly developed cell lines, it has been suggested by Doran (63) and Tanaka (89) that this reactor type can provide inadequate mixing related to low aeration rates and/or suspension broth viscosity. Another potential disadvantage of airlift technology is that it may not be possible to achieve cell growth as high as obtained in stirred reactors regularly (about 20 g dry weight L 1 (26)). One exception is the cultivation of Berberis wilsonae suspension cells in an 20-L airlift reactor, where Breuling et al. (51) described the highest growth rate reported to date for plant suspension cells (40 g dry weight L-1). Tanaka et al. (37) developed a special rotating drum reactor that guarantees suspension homogeneity, a low-shear environment, and reduced wall growth. A few authors (42,58-60) recommend the application of fluidized bed reactors for suspension cultures, which also ensures cell immobilization. In conclusion, airlift reactors and stirred reactors are most often used in commercial as well as semicommer-cial plant cell cultivation.

B. Reactors for Hairy Root Cultures

Suitable bioreactor performance for cultivation of hairy roots should consider the structural roots' integrity including physiology, morphology, and their rheological properties (Table 5). The growth behavior of the roots causes essential difficulties concerning the inoculation, harvesting, and sampling procedure in the course of the cultivation process. A special and enlarged inoculation assembly in the form of special inoculation vessels, cell bags, and inoculation tubes became necessary (72,77). A nonuniform biomass distribution in the reactor and insufficient mass transfer in the densely packed mass of growing roots result in cell necrosis and autolysis as well as loss of biosynthetic capability. It became clear that standard reactors are generally not suitable for hairy root cultures. In the only exception described in the literature, hairy roots of Catharanthus trichophyllus L. were successfully cultivated in a simple stirred reactor with a 14-L working volume (61) at low agitation rates ( 100 rpm) and a high aeration rate of 1 vvm. All other reported configurations (43,62-73) are modified standard reactors.

Usually, stainless steel or nylon meshes (basket) and polyurethane foam are introduced into the chosen standard reactor for roots to protect against mechanical and hydrodynamic shear (43). The basket makes it pos-

Table5 Characteristics of Hairy Root Cultures (HRCs)

Advantages

Stable production of pharmaceutical active compounds Comparable or even higher growth rates than cell suspensions Simplified cultivation process Less expensive and less complicated medium Emerged growth possible (reduced medium volume and forced aeration)

Disadvantages

Morphology and growth behavior of HRCs Root development Degree of branching Organized growth Pressure sensitivity Shear sensitivity sible to separate the growth space of the roots from the aeration and mixing area and to support the self-immobilization of the roots. Figure 9 illustrates such hairy root support systems applied for scopolamine- and hyoscyamine-producing hairy root cultures of Hyoscyamus muticus L. Laboratory rotating drum reactors were also adapted for hairy root cultivations. Such a modified rotating drum reactor named Inversina (Fig. 9c) produces an oloid movement with coupling of three basic movements (rotation, translation, inversion). The consequence is that the rhythmic movement and low shear stress did not inhibit the growth and biosynthetic capability of the cultivated roots. Biomass growth of about 45-fold was achieved in 28 days (77).

It could also be demonstrated that working trickle bed reactors in which the nutrient medium is sprayed or dispersed offer more ideal conditions for growth and productivity of hairy roots than ordinary submerged bioreactors. Although moisture is required by root tissue, if the medium film is too thick it limits nutrient and gas transfer to the tissue. The solution is a droplet or mist application cycle, which decreases the development of thick medium films on the roots. Laboratory trickle bed reactors with a support matrix usually with working volumes of 1 to 14 L have been suitable for mass propagations of Hyoscyamus muticus (77,90), Nicotiana tabacum (70), Beta vulgaris (73), and Carthamus tinctorius hairy roots (91,92).

Wilson (72) described the only designed large growth droplet bioreac-tor system with a volume of 500 L. The Wilson reactor consists of a sparge ring, a number of sample as well as addition ports, sterile filters for the air, a central inoculation port, two concentric rings of spray nozzles, a drain port for recycle of growth medium, a pressure relief valve, and the immobilization matrix. This immobilization assembly, consisting of a number of barbs forming chains, is located within the droplet reactor. A high-pressure diaphragm pump guarantees nutrient medium feeding. Measurable control parameters are temperature, pH value, pressure, oxygen, and weight of the growth vessel. The last parameter allows indirect root growth control. Wilson and co-workers were able to cultivate hairy roots of Datura stramonium in this bioreactor type. They showed the suitability and advantages of the designed immobilization matrix for even distribution of the roots and an easy harvesting procedure. A total biomass of 39.8 kg fresh weight was harvested after 40 days of cultivation (72).

C. Reactors for Embryogenic and Shoot Cultures

The characteristics of embryogenic cultures are quite different from those of shoot cultures, but they often seem to be similar to cell suspension cultures. In this case, the same bioreactor types suitable for suspension cultures can be applied for embryogenic cultures.

Figure 9 Tested baskets for immobilization of Hyoscyamus muticus hairy roots, (a and b) Portable stainless steel basket in a 3-L droplet phase bioreactor (homemade, University of Applied Sciences Wadenswil, Switzerland), (c) Flexible multiple inoculation sieve plates in a 1-L modified drum reactor (Type Inversina, Bioengineering Ltd., Switzerland, with kind permission of reactor manufacturer), (d) Fixed multiple inoculation sieve plates in a 14-L droplet phase reactor (homemade).

Figure 9 Tested baskets for immobilization of Hyoscyamus muticus hairy roots, (a and b) Portable stainless steel basket in a 3-L droplet phase bioreactor (homemade, University of Applied Sciences Wadenswil, Switzerland), (c) Flexible multiple inoculation sieve plates in a 1-L modified drum reactor (Type Inversina, Bioengineering Ltd., Switzerland, with kind permission of reactor manufacturer), (d) Fixed multiple inoculation sieve plates in a 14-L droplet phase reactor (homemade).

Introduction of light into the bioreactor becomes necessary for some embryogenic cultures as well as shoot cultures. The literature describes not only external illumination tubes that are installed around the reactor but also internal systems made from tubes and hollow fibers (77,79,93-95). Figure 10 shows the illumination cage of the 15-L reactor with an eccentric motion stirrer that has been used for cultivation of an embryogenic culture of Allium sativum (78). Another advantage of this reactor, in terms of oxygen transfer and the stirring principle, is that the motion of the multifunctional stirring system protects shear-sensitive cultures against damage and prevents the sedimentation of cell aggregates in the medium. The air enters the reactor through the cell-free interior of the cylindrical stirrer. The medium passes the metallic membrane of the stirrer and oxygenates without cells. Thus, the problems with flotation that arise with standard reactors are prevented.

Similar systems with bubble-free aeration achieved by silicone tubing inserted into the reactor and vibrating stirrers (vibromixers) instead of rotating stirrers have been proposed by Preil et al. and others (84-96) and Luttmann et al. (85).

Aerated vessels are also suitable large-scale reactors for shoot mass propagation. Akita et al. (82) reported their observations concerning mass propagation of shoots of Stevia rebaudiana L. in a 500-L reactor of this type (64.6 kg fresh weight after 4 weeks of cultivation).

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