Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/14—Rotation or movement of the cells support, e.g. rotated hollow fibers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/02—Photobioreactors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/02—Membranes; Filters
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/16—Particles; Beads; Granular material; Encapsulation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M31/00—Means for providing, directing, scattering or concentrating light
  • C12M31/08—Means for providing, directing, scattering or concentrating light by conducting or reflecting elements located inside the reactor or in its structure

Definitions

• This invention relates to biotransformation reactions as well as the fixation of biological material for use in such transformations.
• Examples are pharmaceuticals (for example, alkaloids, vitamins and anti-cancer agents), flavours, perfumes, pigments, hormones, enzymes and genetic engineering materials from plant cell cultures. Further examples are monoclonal antibodies, vaccines, enzymes, hormones and interferon from animal cell cultures.
• pharmaceuticals for example, alkaloids, vitamins and anti-cancer agents
• flavours for example, alkaloids, vitamins and anti-cancer agents
• perfumes for example, flavours, perfumes, pigments, hormones, enzymes and genetic engineering materials from plant cell cultures.
• Further examples are monoclonal antibodies, vaccines, enzymes, hormones and interferon from animal cell cultures.
• Plant and animal cells are fragile and have very low tolerance to shear. They prefer to stay in contact with each other. There is recent evidence that cell-to-cell contact can ' improve secondary metabolite formation.
• One way of providing these requirements of low shear environment and cell-to-cell contact is immobilisation.
• GB-A-2 006 181 describes the use of support bodies of substantial internal viodage which are agitated in a liquid in a vessel in the presence of a biological population some of which becomes immobilised on the support bodies. More particularly, small reticulate foam particles are moved in a bulk liquid, which carries freely suspended microorganisms, by using sparged air.
• this method has two disadvantages. Firstly, the liquid elements carrying the microorganisms move at almost the same speed as the .. .
• support material e.g. small cubes of a reticular plastics foam
• a proportion of the biological population migrates onto the support and becomes immobilised thereon.
• the comparatively low degree of immobilisation achieved by the above method has the disadvantage that a biotransformation reaction effected with the immobilised material may not produce a satisfactory yield of the desired product. Additionally, the low degree of cell-to-cell contact may means that the immobilised material does not remain viable over a sufficiently long period of time for economic operation of the biotransformation reaction.
• a method of effecting biotransformations which comprises: (a) fixation of biological material by passing a suspension of biological material comprising living or biochemically active cells and/or tissues and/or particles comprising pre-im obilised biocatalysts in a liquid medium through a liquid permeable member capable of filtering said biological material to filter said biological material from said liquid medium, said member being an integral body or a composite body whose components are maintained in a substantially fixed spatial relationship to each other;
• apparatus for the fixation of biological material in the form of biochemically active cells and/or tissues and/or particles comprising pre-immobilised biocatalysts from a suspension thereof in a liquid medium
• the apparatus comprising a vessel, a liquid permeable member within the vessel capable of filtering the biological material, said member being an integral or a composite body whose components are maintained in a substantially constant fixed relationship to each other, and means for establishing a relative velocity between liquid held, in use of the apparatus, in the vessel and the support member such that biological material in suspension in the liquid i'S filtered therefrom and retained on the support member.
• An important feature of the above method and apparatus is the liquid permeable body which effects said filtration.
• This body may either be of integral construction or may be a composite body which comprises a plurality of individual elements which are either liquid permeable and/or arranged as a liquid permeable unit and which are maintained in substantially fixed relationship to each other, e.g. by means of a liquid permeable frame structure. It is the maintenance of the component parts of the composite body in substantially fixed spatial relationship which allows the composite body to efficient filtration of the biological material.
• the biological material is filtered from suspension by passing the suspending liquid at a sufficiently high relative velocity through the liquid permeable body.
• the biological material is entrapped by fixation on the body (so that it can be said to be immobilised with respect thereto) and may be used for biotransformation reactions, as detailed more fully later.
• fixation technique used in the present invention may be distinguished from the prior art immobilisation techniques in that, in these latter techniques, the support for the immobilised material is free to move at random in a liquid whereas, in the present technique, the liquid permeable support body will either be stationary within a vessel or mounted on a rotor or the like which provides predetermined motion of the body.
• pre-immo.bilised biocatalysts are produced by immobilising the biocatalyst in or on a particulate material (for example a polymeric gel bead or an ion exchange resin), e.g. by the method described in EP-A-22434.
• a suspension of these particles may be used in the method of the invention and held by fixation on the liquid permeable body.
• the fixation step of the present invention is very much quicker than conventional immobilisation techniques due to the fast rate of filtration of the biological material from the bulk liquid. This leads to reduced risk of contamination and almost no damage to the biological material. In some of our experiments, the whole physical entrapment took less than ten minutes. In addition to the rapid fixation, the comparatively high degree of fixation achieved by the method of the invention is good for the "well-being" of the material resulting in less time being required for the material to grow to a particular "concentration" on the support. Furthermore, the viability of the fixed material is longer than that obtained with other immobilisation techniques. This is due to the higher amount of biological material immobilised on the support facilitating transport of substances essential for growth of the material.
• An additional advantage of the present invention over the method described above in which small reticulate foam particles are moved in a bulk liquid is that, in this latter technique, a maximum of only 30% by volume of the reactor may be occupied by the foam particles. Amounts of foam particles greater than 30% result in loss of circulation of the particles. A much higher volume percentage of the reactor may be occupied by the support bodies by using the method of the invention.
• the liquid permeable member is preferably of a non-toxic (to the biological material), non-corroding, sterilizable, and optionally combustible material.
• a preferred member is in the form of an integral body (e.g. a sheet) of plastics (e.g. polyurethane) foam material with substantial internal voidage providing a plurality of interconnecting pores in which the biological material may be held by fixation.
• the liquid permeable member may be of a variety of other constructions.
• the member may comprise a network of one-dimensional or two-dimensional materials or three-dimensional structures.
• One-dimensional materials can be straight or curled laths, fibres, strands, or threads of suitable material arranged to define a network structure with a plurality of pores.
• Two-dimensional structures can be flat, undulating or curled screens, sheets, plates etc. Three-dimensional structures can be made in such a way that they would have continuous interconnecting networks of voids., of random or uniform size and geometry. Suitable materials for producing such one-, two- or three-dimensional support materials are stainless steel, glass, glass-fibres, carbon-fibre, ceramic, synthetic and natural polymers, cellulose, cotton, linen, wool, wood or other natural or synthetic materials.
• the liquid permeable member may be held stationary or moved mechanically in the suspension. If it is held stationary, then the fluid which carries the supended biological material may be made to flow through the support media by using suitable agitators, pumps or gas lift techniques. If the support media are moved mechanically, then the fluid does not have to be agitated as the movement of the support media will give sufficient mixing and flow. However, it is possible to employ both the agitation of the fluid and the mechanical movement of the support media simultaneously. A suitable relative velocity between the biological and the support media can easily be achieved and controlled. In this way, the biological material is in effect filtered out of the fluid by the support media and is physically entrapped in or between the support media. This fixation step can be considered similar to filtration mechanisms of particulate matter.
• the physical entrapment process will be continued by circulating the liquid medium which contains the rest of the biological material which is not yet entrapped until a satisfactory level of entrapment is achieved.
• the size of the holes in two-dimensional support media, the size of the pores in three-dimensional support media and the space between the one-dimensional support media will be determined by the size distribution of the biological material in such a way that most of the biological material will be physically entrapped. It may be necessary to pack the support media together to create structures with uniformly or randomly varying pore sizes.
• the support media do not necessarily have an extensive area of access from their outer surface to the interior. They can be covered with a suitable material such as polymers, gels or semi-permeable membranes after the entrapment of biological material, to restrict the growth of the biological material to the interior and also to eliminate contamination from the exterior. These coating materials will however have the necessary permeability to certain chemical compounds found in the system.
• the physical entrapment itself may be strong enough for fixation of the the biological material in and/or between the support media. Alternatively the physical entrapment can be enhanced by utilising electrostatic forces between the support media and 5 the biological material, electromagnetic fields, and various bridging or binding materials.
• the biological material may be encouraged to grow on the 0 liquid permeable body so as to result in stronger anchorage of the biological material.
• the subsequent growth of the entraped biological material may be prevented or slowed down if necessary, by using chemical, 5 biochemical and physiological methods, such as the withdrawal of hormones, vitamins, phosphorous or nitrogen source or addition of growth inhibitory enzymes or other compounds.
• the support media can be taken out o of the reactor after a suitable time, all or some of the biological material can be removed and then the support media can be introduced back into the reactor vessel.
• the biological material used in the method of 5 the invention may, for example, be plant cells, animal cells, tissue cultures, cell organelles, micro-organisms and other materials of biological origin. These biological materials may be of particulate or mycelial form by their nature. If 0 they are not than they can be immobilised in or on a suitable material in such a way that they can be handled like particles for the purposes of immobilisation.
• the suspension of biological material may have 5 been prepared by homogenisation of, for example, plant material, callus culture or suspension culture to produce a suspension which is sufficiently 'fine' for use in the invention. Any suitable method of homogenisation may be used, for example that described in U.K. patent application no. 85 19180.
• the biological material can be alive, growing or at a stationary phase of growth or it can be dead. Nevertheless the biological material will, in all cases, be active from the process point of view.
• the biological material may be used to effect a biotransformation reaction (e.g. for the production of secondary metabolites) although the exact nature of the reaction will be course depend on the particular biological material. It is usually necessary to supply nutrients, precursors, inducers and/or other reactants to the biological material. These may be supplied to the biological material in liquid, gaseous or solid form or in the form of cells, organelles or other biological mate'rial such as viruses, plas ids, antigens, enzymes, co-factors etc. Illumination may also be necessary for the process.
• the biotransformation reaction may be one which is carried out for the production of particular chemical or biological products, e.g. new cells, antibodies, nucleic acids etc.
• the products may be released or excreted by the biological material into the medium in which it is cultured (and from which the products will be recovered). Alternatively the products may be retained by the biological material which will need to be harvested for recovery of products therefrom.
• the invention is also applicable to biotransformation reactions which involve the breakdown of unwanted products (as in waste water or sewage treatment) supplied to the biological mateiral as nutrient medium.
• biotransformation reaction examples of biotransformation reaction to which the invention is applicable are:
• biocatalysts may, for example, be enzymes within cells (living, dead or resting) co-immobilised on the support.
• the biocatalysts may be enzymes or co-factors pre-immobilised in or on particulate material (e.g. polymeric gel beads or ion exchange resin beads, chlorides, carbonates or phosphates) which is then entrapped by fixation on the support with the cells.
• (1) and (2) as applied to plant cells are the production of pharmaceuticals (e.g. alkaloids, vitamins, and anti-cancer agents), flavours, perfumes, pigments, plant-hormones, enzymes, and genetic engineering material.
• pharmaceuticals e.g. alkaloids, vitamins, and anti-cancer agents
• flavours e.g. alkaloids, vitamins, and anti-cancer agents
• perfumes e.g. pigments, plant-hormones, enzymes, and genetic engineering material.
• Specific examples of (1) and (2) as applied to animal cells are the- production of genetic material and proteins, e.g. monoclonal antibodies, vacines, enzymes, hormones, and interferon.
• Specific examples of endogeneous enzymic transformations involve such reactions as oxidation, hydroxylation, (de) methylation, gly- cosylation, esterification, epoxidation and isomerization.
• Chemically variable substrates (reactants) used in biotransfor ations include steroids, terpen
• plants of a particular gender may be produced exclusively since only one gender of the species may yield a desired product. It is possible to plant cells which have been produced by genelic manipulation techniques to produce new species.
• reactor may be used for effecting the method of the invention.
• the support media can be held stationary in reactor vessels like baffles and the bulk fluid can be made to flow over and through these by means of agitators, pumps, or air/gas lift action.
• the support media may also be used in horizontal or slanting covered ducts in the form of vertical or slanting baffles and the bulk fluid may flow over and through these by the action of gravity, gas/air lift provided by sparging gas/air into the liquid occupying the compartments between the baffles, or by agitators in each compartment, or by liquid circulation using pumps.
• the support media can be placed horizontally in a reactor vessel like the trays in a distillation column and the liquid can flow over and through these by gravity and pump circulation.
• the space between each "tray" can be either totally or partially filled with liquid.
• This arrangement can also be viewed as simlar to wet-sieve devices.
• a preferred unit in accordance with the invention comprises a plurality of liquid permeable bodies in the form of screens, trays or the like arranged one above the other and being such that the average size of biological material which any one body is capable of filtering is less than that which may be filtered by the nextmost upper body.
• Filtration of a suspension comprising biological material of different sizes through the unit from top to bottom allows the material to be held by fixation on the bodies in fractions of different size range, the largest biological material being retained on the uppermost body and the smallest being retained on the lowermost body.
• Each of the fractions of biological material may then be cultivated under conditions which are optimal for that particular size fraction.
• the support media can be rolled sheets with space or another material between each layer and placed like cartridges in cylindrical reactor vessels.
• the bulk fluid may flow through and over these support media by using pumps.
• the supply of 15 The supply of 15 ,
• nutrients and removal of products can be achieved through the bulk liquid occupying the space between the layers.
• the support media can be used in the form of concentric cylindrical shells placed vertically or horizontally in the reactor vessel.
• the support media can also be attached to a rotating axle like a paddle, and the whole assembly paced vertically or horizontally in a reactor vessel dipping completely or partially in the bulk fluid.
• the reactors may contain a suitable cutting device to shear biomass which protrudes out of the support media due to excess growth.
• the design of this cutting device and its operation may vary depending on the individual reactor configuration. Some examples of the cutting device are given in the relevant sections on individual reactor descriptions of this invention.
• the size of the aggregates ' of the biological material which is cut off from the support media ' is too large to pass through the exit ports, then their size can be reduced using a high shear device preferably placed at the bottom of the reactor.
• illumination is required by the biological material such as algae, plant cells, etc.
• this can be achieved by fixing light sources into suitable positions, for example the agitators or the walls of the vessel.
• the bulk liquid will be clear it will allow for the transmission of the necessary illumination.
• the light source has to come into contact with any liquid, then it can be rendered waterproof.
• the light sources can also be located outside the reactor vessel and they can transmit light through glass windows of the reactor.
• the support media can be made from glass fibre the cut ends of which can be used to illuminate using a light source which is supplied to the main core of the branching glass fibre structure.
• Fig. 1 is a general schematic diagram of a complete reactor system which may be used in the method of the invention
• Fig. 2 is a schematic side view of one embodiment of reactor for effecting the method of the invention
• Fig. 3 is a top plan view of the reactor shown in Fig. 2;
• Fig. 4 shows the cutting device used in the reactor of Fig. 3;
• Fig. 5 is similar to Fig. 3 but shows a modifed form of support media
• Fig. 6 is similar to Fig. 4 but shows a modified cutting device used in the reactor of Fig. 5;
• Fig. 7 is a schematic side view of a further embodiment of reactor.
• Fig. 8 is a top plan view of the reactor shown in Fig. 7;
• Fig. 9 is a schematic view of a further embodiment of reactor;
• Fig. 10 is an end view of the react shown in Fig. 9;
• Fig. 11 is a view of a further embodiment of reactor.
• Fig. 12 is an end view of the reactor shown in Fig. 11;
• Fig. 13 is a side view of a first embodiment of plant propagation apparatus in accorda.nce with the invention
• Fig. 14 is an end view of the propagation apparatus shown in Fig. 13;
• Fig. 15 is a side view of a second embodiment of plant propagation apparatus
• Fig. 16 is an end view of the propagation apparatus shown in Fig. 15;
• Fig. 17 is a side view of a third embodiment of plant propagation apparatus
• Fig. 18 is a schematic plan view of the propagation apparatus shown in Fig. 17;
• Fig. 19 is a side view of a fourth embodiment of plant propagation apparatus.
• Figs. 20 and 21 are graphic representations of the results of experiments (see later); and Fig. 22 is a schematic view of the experimental reactor used in experiment 2 (see later).
• fermenters are well known, the present description does not include other equipment, units such as hold-up tanks, sterilizers, etc., and instrumentation and design details which are familiar to anybody skilled in the art of fer enter design and operation, and will be directed in particular to the elements forming part of or cooperating directly with the apparatus in accordance with the present invention.
• FIG. 1 there is shown a general schematic diagram of a complete system for performing the method of the invention.
• the system shown in Fig. 1 includes a reactor (1) which can be of various configurations as described hereinafter, a device (2) which will reduce the size of the aggregates of the biological material, a wet-sieve column (3), product separation u?it (4), and an aeration vessel (5). in operation, it is first necessary in running a reactor (1) to prepare an inoculum which can be cultured in a separate fermenter (6). If necessary, before the fixation, the size of the aggregates of the biological material in the inoculum can be ' reduced for entrapment using a suitable device (2) and then the suitable size fraction can be- collected through the wet-sieve column (3).
• the aeration of the nutrient solution can be achieved by continuous recycling of the nutrient solution through a separate aeration vessel (5).
• the products can be removed by continuous recycling of the liquid through the products separation unit (4).
• the culture broth becomes progressively clearer and only the very large aggregates of the biological material which cannot be entrapped in a given pore size and/or excess biological material remain in the nutrient solution.
• This excluded or excess biological material can be removed and reused as an inoculum after being passed through the size reduction device, if necessary, for another reactor or the inoculum fermenter itself.
• the illustrated reactor vessel (7) includes baffle-like sheets of vertically held supported media (8).
• This reactor vessel (7) also includes two-blade impellers (9,10); one (9) at the bottom of the vessel and the other (10) beneath the liquid surface (11).
• Figure 3 which is a top view of Figure 2
• the sheets of polymer foam (8) can be of oblong cross-section and are arranged in circumferentially spaced relationship.
• An air-sparger (12) is arranged in such a way that the air bubbles rise in the compartments inbetween the foam sheets, thus helping with the mixing.
• the reactor also includes a special cutting device (13) for controlling the excess growth out of the foam sheets.
• the cutting device may consist of several blades (13a) so that when they are moved up and down, the cutting device (13) will shear the plant cells outgrown on the surface of the foam sheets.
• Figure 5 is a view similar to Figure 2 but showing the use of support media (8a) of wedge shaped cross-section.
• Figure 6 is similar to Figure 4 but shows a modified shearing device (13b).
• the support media (14) may be used in the form of stationary concentric cylindrical shells which are held vertically in the reactor vessel (15).
• the reactor vessel (15) includes a special rotating device (16) as shown in the figure.
• This special rotating device (16) which consists of several blades attached to the impeller (17) shaft acts as both an agitator and a cutting device to control the excess growth out of the support media (14).
• FIGs 9 and 10 are modified reactors shown in Figures 9 and
• the support media in the form of concentric cylindrical shells (14a) is rotatably supported about a horizontal axis.
• the assembly of the support media (14a) is partially .immersed below the liquid level L and is rotated about a horizontal axis in a reactor vessel (18) which incorporates a cutting device (19) comprised of several concentric fixed blades.
• the cutting device (19) will shear the plant cells outgrown on the external surfaces of the support media (14a).
• the support media (20) is fixed on a rotatable horizontal axle (21) which allows certain of the support media to dip into the bulk liquid.
• FIGs. 13 and 14 illustrate a first embodiment of plant propagation apparatus in accordance with the invention.
• the propagator comprises a vessel 22 in which are located two liquid permeable support bodies 23 which are mounted 180° apart on a shaft 24 driven by a motor 25.
• Vessel 22 includes two upper liquid inlet ports 26, two lower liquid outlet ports 27 a gas inlet port 28, and a gas outlet port 29.
• vessel 22 is substantially filled with a suspension of plant cells and motor 25 is operated to sweep the support bodies around vessel 22 thereby filtering the plant cells and entrapping them on these bodies.
• the support bodies 23 are brought to the horizontal position illustrated in Fig. 14.
• the liquid within vessel 22 may then be drained through ports 27 and replaced by a special media to induce differentiation and shoot formation to a level approximating to line 30 in Fig. 14.
• the cells may then be cultivated under known conditions to produce "plantlets" (i.e. small plants) which may 21 ,
• FIG. 15 and 16 An second embodiment of propagator is shown in Figs. 15 and 16.
• This propagator comprises a 5 cylindrical vessel 31 in which is fixed, across a diametral plane of vessel, a liquid permeable support material 32.
• Vessel 31 is rotatably mounted on a stand 33 and may be rotated by motor 34.
• the vessel includes liquid inlet ports 35, liquid outlet ports 36, a gas inlet port 37, and a gas outlet port 38.
• a suspension of plant cells is introduced into vessel 31 which is then rotated by motor 33.
• Support body 32 rotates with the vessel 31 and filters the plant cells from suspension. Once this fixation operation is complete, the plant cells may be cultivated in the manner indicated for the propagator shown in Figs. 13 and 14.
• the plant propagator shown in Figs. 17 and 18 comprises a vessel 39 in which are provided a plurality of vertically spaced, liquid permeable support bodies 40.
• An agitator arrangement comprising upper and lower blades 41 and a motor 42 is also provided.
• vessel 39 is of circular cross-section.
• the support bodies 40 are each identical size major segments of a circle of the same diameter as vessel 39. There is thus a space 43 between the plane edge of each body 40 and the wall of vessel 39.
• the support bodies 40 are in angularly staggered relationship such that the spaces 43 are in the staggered relationship clearly illustrated in Fig. 18.
• a suspension of plant cells is introduced into vessel 39 and motor 42 is operated to stir the suspension and thus effect filtration of the biological material on the bodies 40.
• the biological material remaining in suspension can pass through the openings 43 and this ensures that the biological material is evenly distributed among the supports 40.
• the propagator shown in Fig. 19 is similar to that shown in Figs. 17 and 18 (including angularly staggered, segmental support bodies) and like parts are designated by the same reference numerals. However, this propagator additionally comprises an irrigation system 44 including a plurality of spray heads 45 arranged to spray onto the bodies 40, a well as a lighting system including bulbs or the like 46 on the inside of vessel 39.
• irrigation system 44 including a plurality of spray heads 45 arranged to spray onto the bodies 40, a well as a lighting system including bulbs or the like 46 on the inside of vessel 39.
• the 'plantlets' may be cultiviated with the aid of the lights 46 as well as liquid sprayed onto the bodies '40 by spray heads 45.
• Experiment 1 Relative velocity between foam particles and plant cell aggregates.
• the relative velocity between two particulate materials which are moving in a liquid can be reflected by the difference in their individual terminal free settling velocities in the same- liquid.
• the terminal free settling velocities were measured by dropping the foam particles and cell aggregates of various sizes into water and measuring the time they took to fall a certain distance. The results are shown in Figures 20 and 21.
• the free fall velocity varies with the size of the foam particles and with the size of the cell aggregates.
• the foam particle used for this experiment had 30 pores per inch. This gives an average pore size of 0.8mm.
• Our previous experimental results indicate that the cell aggregates up to 1.5mm in size can be entrapped in 30 ppi foam matrix.
• An average terminal velocity for the foam particles can be taken as 2.5cm/sec ( Figure 20).
• the terminal velocity for cell aggregates varies from 0.5 to 3.2cm/sec depending on the size (Fig. 21).
• the average terminal velocity of cell aggregates of interest i.e., 0.5 mm - 1.5 mm
• the relative velocity between the cell aggregates and the foam matrix will be 0.7 - 0.1 of the tip velocity of the impeller.
• the relative velocity can be controlled at a desired value by simply changing the impeller speed.
• the tip speed of the impeller is about 36 cm/sec as in the case of experiment 2 below
• the average relative velocity between the foam matrix and cell aggregates is 25 - 3.6 cm/sec in a baffle-reactor. This value is much higher than the average relative velocity between moving foam particles and cell aggregates, which is 2.0 cm/sec.
• Experiment 2 Comparison of physical entrapment between a baffle-reactor and a circulating-foam- particle-reactor.
• each foam baffle 2.5 x 2.0 x 7.5cm.
• Number of foam baffles 5.
• Amount entrapped after 30 minutes 285mg (dry weight) .
• the average relative velocity between foam matrix and cell aggregates 25 - 3.6cm/sec.
• Circulating-foam-particle-reactor
• Total volume of foam matrix 190cm 3 .
• Total external surface area of foam matrix 1140cm 2 .
• the average relative velocity between foam matrix and cell aggregates less than 2.0cm/sec.
• capsaicin concentration was 25 ⁇ g/ml of medium with total reactor working volume being 700ml. (total mass of plant cells was 175g-wet weight). Therefore, production was lOOug/g-wet cell weight.
• cells immobilised in foam particles (1 x 1 x lcm) capsaicin concentration was less than l g/ml of medium with total reactor working volume being 5000 ml. (Total mass of plant cells was 500g wet weight). Therefore, production was lOug of capsaicin/g-wet cell weight.
• the reactor was rotated around its horizontal axis in order to filter the cells into the support material. This was continued until the cells completely filled the structure. Then, the medium was drained completely and the reactor was filled with a differentiation inducing medium upto such a level that when the support structure was held horizontally the liquid medium just touched the bottom surface of the support structure.
• This new medium was a modification of the Murashige and

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• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Saccharide Compounds (AREA)

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• 1984
  • 1984-11-07 GB GB848428085A patent/GB8428085D0/en active Pending
• 1985
  • 1985-11-07 JP JP60505087A patent/JPS62500701A/ja active Pending
  • 1985-11-07 ES ES548668A patent/ES8704003A1/es not_active Expired
  • 1985-11-07 AU AU50967/85A patent/AU5096785A/en not_active Abandoned
  • 1985-11-07 ZA ZA858584A patent/ZA858584B/xx unknown
  • 1985-11-07 GB GB8527452A patent/GB2168721B/en not_active Expired
  • 1985-11-07 WO PCT/GB1985/000508 patent/WO1986002944A1/en not_active Application Discontinuation
  • 1985-11-07 EP EP85905851A patent/EP0231193A1/de not_active Withdrawn
• 1986
  • 1986-07-03 NO NO862698A patent/NO862698L/no unknown
  • 1986-07-04 DK DK318686A patent/DK318686D0/da not_active Application Discontinuation
  • 1986-07-07 KR KR1019860700429A patent/KR880700058A/ko not_active Application Discontinuation

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