GB2168721A - Improvements relating to biotransformation reactions - Google Patents

Abstract

A method of effecting biotransformations comprises, as a first step, the fixation of biological material from suspension by passing suspension through a liquid permeable member which is capable of filtering the biological material. The liquid permeable member is an integral body or a composite body whose components are maintained in a substantially fixed relationship to each other. The biological material may comprise living or biochemically active cells and/or tissues and/or particles which themselves comprise pre-immobilised biocatalysts. After fixation, the biochemical transformation is effected by contacting the biological material supported on the member with a liquid and/or gaseous medium to form a product and/or biomass. Finally, the product is recovered and/or the biomass is harvested. Various apparatus for effecting the fixation of the biological material are described and generally incorporate means for establishing a relative velocity between the liquid permeable body and the suspension so as to effect the filtration. Also described are reactor systems comprising biological material which has been entrapped by fixation on a liquid permeable support body. The invention may be applied to the fixation of plant cells for growth into "plantlets" (i.e. micropropagation).

Description

SPECIFICATION Improvements relating to biotransformation reactions This invention relates to biotransformation reactions as well as the fixation of biological material for use in such transformations.

There is growing interest in the use of plant and animal cell culturesforthe production of high cost iow volume products. 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.

Plant and animal cells are fragile and have very low tolerance to shear. They prefer two stay in contact with each other. There is recentevidencethatcell-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.

Various techniques are used for immobilising the biological material on a support. Thus, for example, GB-A-2 006 181 describes the use of support bodies of substantial internal viodagewhich are agitated in a liquid in a vessel in the presence of a biological population some ofwhich 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.

However, this method has two disadvantages. Firstly, the liquid elements carrying the microorganisms move at almost the same speed as the foam particles resulting in a relative velocity value of almost zero between the microorganisms and the foam particles.

Therefore whatever immobilisation occurs is left to chance by random uncontrolled collision ofthe microorganisms with the foam particles. The second disadvantage is due to the entrapment of sparged air bubbles in the foam particles which leads to the floatation of the foam particles to the liquid surface.

When this happens, the mixing is disrupted and no effective immobilisation can take place.

In another method of immobilising biological material on a support, support material (e.g. small cubes of a reticular plastics foam) are simply introduced into a liquid containing the biolgical population. A proportion ofthe biolgical population migrates onto the support and becomes immobilised thereon. Once again, however, the proportion of material which is immobilised is not high, even after the system has been left to stand for several weeks.

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 ofthe 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 ofthe biotransformation reaction.

It is an object ofthe present invention to obviate or mitigate the abovementioned disadvantages.

According to a first aspect ofthe present invention there is provided 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/ortissues and/or particles comprising pre-immobilised biocatalysts in a liquid medium through a liquid permeable member capable of filtering said biological mate rial to filter said biological material from said liquid medium, said member being an integral body or a composite body whose components are main tained in a substantially fixed spatial relationship to each other; (b) Effecting a biotransformation reaction by con tacting said biological material supported on said memberwith a liquidand/orgaseousmediumto form a product and/or biomass; and (c) recovering said product and/or harvesting said biomass.

According to a second aspect ofthe present invention there is provided apparatus forthe fixation of biological material in the form of biochemically active cells and/or tissues and/or particles comprising preimmobilised biocatalystsfrom a suspension thereof in a liquid medium, the apparatus comprising a vessel, a liquid permeable member within the vessel capable offiltering the biological material, said member being an integral ora 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 membersuch that biological material in suspension in the liquid is filtered therefrom and retained on the support member.

An importantfeature ofthe 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 substantiallyfixed relationship to each other, e.g. by means of a liquid permeable frame structure. It is the maintenance of the component parts ofthe compo- site body in substantiallyfixed spatial relationship which allows the composite body to efficient filtration ofthe biological material.

In the method ofthe invention, the biological material is filtered from suspension by passing the suspending liquid ata sufficiently high relative velocity through the liquid permeable body. The biological material is entrapped by fixation on the body (so that is can be said to be immobilised with respect thereto) and may be used for biotransformation reactions, as detailed more fully later.

The fixation technique used in the present invention may be distinguished from the prior art immobilisation techniques in that, in these lattertechniques, the supportforthe immobilised material is free to move at random in a liquid whereas, in the present technique, the liquid permeable support body will either be stationarywithin avessel or mounted on a rotor or the like which provides predetermined motion ofthe body.

Theterm 'pre-immobilised' as used herein with reference to biocatalysts should be distinguished from the fixation by filtration described in the preceding paragraph. The pre-immobilised biocatalysts are produced by immobilising the biocatalyst in oron a particulate material (for example a polymeric gel bead or an ion exchange resin), e.g. by the method described in EP-A-22434. Asuspe sion of these particles may be used in the method of tne invention and held by fixation on the liquid permeable body.

Thefixationstepofthepresentinvention is very much quickerthan conventional immobilisation tech niques due to the fast rate offiltration ofthe biological material from the bulk liquid. This leads to reduced risk of contamination and almost no damage to the bioligical 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 offixation achieved by the method of the invention is good forthe "well-being" ofthe material resulting in less time being requiredforthe material to growto a particular "concentration" on the support. Furthermore, the viability ofthe fixed material is longerthan that obtained with other immobilisation techniques. This is due to the higher amount of biological material immobilised on the supportfacilitating transport of substances essential for growth ofthe material.

An additional advantage ofthe present invention overthe method described above in which small reticulate foam particles are moved in a bulk liquid is that, in this lattertechnique, a maximum of only 30% by volume ofthe reactor may be occupied by the foam particles. Amounts of foam particles greater than 30% result in loss of circulation ofthe particles. A much hig her volume percentage ofthe reactor may be occupied bythe support bodies by using the method ofthe 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 byfixation. However the liquid permeable member may be of a variety of otherconstructions. For example, the member may comprise a network of one-dimensional ortwodimensional materials orthree-dimensional structures. One-dimensional materials can be straight or curled laths,fibres,strands,orthreadsofsuitable material arranged to define a network structure with a plurality of pores. Two-dimensional structures can be flat, undulating orcurled 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 materialsforproducing such one-, two- orthree-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 suspended biological material may be made to flow through the support media by using suitable agitators, pumps or gas lifttechniques. Ifthe support media are moved mechanically, then the fluid does nothaveto be agitated as the movementofthe support media will give sufficent 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 effectfiltered out of the fluid by the support media and is physically entrapped in or between the support media. This fixation step can be considered similarto filtration mechanisms of particulate matter.

The physical entrapment process will be continued by circulating the liquid medium which contains the rest ofthe biological material which is not yet entrapped until a satisfactory level of entrapment is achieved.

The size ofthe holes in two-dimensional support media, the size ofthe pores in three-dimensional support media and the space between the onedimensional 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 togetherto create structures with uniformly or randomlyvarying pore sizes.

Ifthe size of the pores, orthe space in or between the structures available for fixation is chosen according to the size distribution and the amount of the biological material to be entrapped, then almost all of the original freely suspended biological material can be immobilised without any wasted inoculum. This yields a very clear bulk liquid after immobilisation which is not achieved easily in other methods of immobilisation.

Owing to the clear bulk liquid it is now possible to illuminate the biological material if necessary using internal or external light sources or glass-fibre material.

Having a clear bulk liquid leads to many other advantageous possibilities. There is no blockage of the pipes and openings in the apparatus with freely suspended biological material. Hence, the products of a biotransformation reaction effected with the fixed material can be continuously extracted from the apparatusanda new batch can befilledortheliquid medium can be made to flow continuouslythrough the apparatus. Mixing and if necessary aeration can be achieved more easily and effectively in the clear bulk liquid.

This last point is significant because it is possible to choose the degree of agitation and aeration without being restricted by the effect of the fluid dynamics on the movement ofthe support media since they are either stationary or moved mechanically, indepen dently ofthefluid mechanics.

We can also make better use ofthe available reactor space in our apparatus when compared with other immobilisation techniques.

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 restrictthe growth ofthe 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 compoundsfound in the system.

The physical entrapment itself may be strong enough for fixation ofthe 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 the biological material, electromagneticfields, and various bridging or binding materials.

After the physical entrapment (but before effecting the biotransformation reaction) the biological material may be encouragedtogrow on the liquid permeable body so as a result in stronger anchorage ofthe biological material.

Alternatively, the subsequent growth of the entraped biological material may be prevented or slowed down if necessary, by using chemical, biochemical and physiological methods, such as the withdrawal of hormones, vitamins, phosphorous or nitrogen source or addition ofgrowth inhibitory enzymes or other compounds.

If necessary, the support media can be taken out of the reactoraftera suitabletime, all orsomeofthe 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 ofthe 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 they are not than they can be immobilised in oron a suitable material in such a way that they can be handled like particlesforthe purposes of immobilisation.

The suspension of biological material may have been prepared by homogenisation of,forexample, 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.

Once the biological material has been fixed on the liquid permeable member, it may be used to effect a biotransformation reaction (e.g.forthe 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 orsolid form or in the form of cells, organelles or other biological material such as viruses, plasmids, antigens, enzymes, co-factors etc. Illumination may also be necessaryforthe process.

The biotransformation reaction may be one which is carried outforthe 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 material as nutrient medium.

Examples of biotransformation reaction to which the invention is applicable are: (1 ) the production of chemical compounds and/or biological materials by endogeneous enzymic transformations within cells; biosynthesis from precursors supplied to cells ; and de novo synthesis of complex materials or compounds from simple subsrates supplied to cells; (2) the production of chemical compounds and/or biological material by cells co-immobilised with pre-immobilised biocatalysts. These biocatalysts may, for example, be enzymes within cells (living, dead or resting) co-immobilised on the support.

Alternatively, 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.

(3) the production of biomass, e.g. the multiplica tion of plant cells or animal cells; Examples of(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. 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 biotransformations include steroids, terpenoids, and alka- loids, but the substrates need not necessarily be endogeneous to the plant species employed forthe transformation.

This opens the possibility forthe production of novel compounds.

Examples of biotransformation using plant cells.

(a) Hydroxylate the allylic position ofthe C=C bond of linalool by Nicotiana tabacum; (b) Glucosylation of phenolic compounds by Lithosperum erythrorhizon Perilla frutescens var. crispa Gardenia jasminoides Mallotusjaponicus Catharamthus roseus Datura innoxia (c) Hydroxylate digitoxin to digoxin by Digitalis lanata (d) Reduction of double bond to transform then mine to Ajmalicine-- isomers by Catharanthus rosens (e) Hydroxylation Digitoxigenin or Gitoxigenen to 5-ss hydroxygitoxigenin by Daucuscarota; Examples of biotransformation reactions which may be effected by using enzymes entrapped with the biological material byfixation on the supports are:: (f) In case of multienzyme systems, the deficient enzymes could be co-entrapped by fixation with plant cells; (g) If primary substrate cannot enter plant cells it could be treated by on enzyme co-entrapped by fixation with the plant cells and then changed to the secondary state of substratewhich which could enter the plant cells; (h) If primary substrate cannot be used by plant cells, it could be transformed by an enzyme co-entrapped by fixation with the plant cells to secondary substrate which can be used by plant cells.

A specific example of (3) to which the inventin has significant importance isthe cultivation of immobilised, undifferentiated cells (e.g. callus) to produce a plurality of plantlets, i.e. small plants which may then be grown in normal manner. The cells are in fact supplied with special media which induces differentiation and shoot growth. This micro-propagation ofso-called "mass propagation via micropropagation of plants. The technique has the advantage over conventional agricultural methods of cultivation because virus and other disease free, genetically similar, high yielding plants may be produced.

Consequently, plants of a particular gender may be produced exclusively since only one genderofthe species may yield a desired product It is possible to plant cells which have been produced bygenelic manipulationtechniquesto produce new species.

Various constructions of reactor may be used for effecting the method ofthe invention.

The support media can be held stationary in reactor vessels like baffles and the bulkfluid can be madeto 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 mayflow over and through these by the action of gravity,gas/airliftprovided bysparginggas/airinto 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 andthe liquid can flowoverandthrough 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 similar to wet-sieve devices. there may be side arms to add or remove nutrients or products orto supply dissolved gases.

A preferred unit in accordance with the invention comprises a plurality of liquid permeable bodies in the form of screens, trays orthe like arranged one above the other and being such thatthe average size of biological material which anyone 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 maythen 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 reactorvessels. The bulk fluid mayflowthrough and overthesesupportmedia by using pumps. The supply of 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 likea paddle, andthewhole 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 ofthe support media due to excess growth. The design ofthis 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 ofthis invention.

Ifthe size ofthe aggregates ofthe biological material which is cut off from the support media is too largeto pass through the exit ports, then their size can be reduced using a high shear device preferably placed at the bottom ofthe reactor.

If 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 exam- plethe agitators orthe walls ofthevessei. Asthe bulk liquid will be clear it will allowforthetransmission of the necessary illumination. lfthe light source has to come into contact with any liquid, then it can be rendered waterproof. The light sources can also be located outsidethereactorvessel andtheycan transmit lightthrough glass windows ofthe reactor.

alternatively, the support media can be made from glass fibre the cut ends of which can be used to illuminateusinga lightsourcewhich issuppliedto the main core of the branching glass fibre structure.

The invention will befurtherdescribed byway of exa m pl e on Iy with reference to the accompanying drawings, in which: Fig. lisa 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 reactorforeffecting the method of the invention; Fig. 3 is a top plan view ofthe reactor shown in Fig.

2; Fig. 4 shows the cutting device used in the reactor ofFig.3; Fig. 5 is similarto Fig. 3 but shows a modified 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 view of a further embodiment of reactor; Fig. 8 is a top plan view ofthe reactor shown in fig.

7; Fig. 9 is a schematic view of a further embodiment of reactor; Fig. loins 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 endview ofthe reactorshown in fig. 11; Fig. 13 is a side view of a first embodiment of plant propagation apparatus in accordance 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 ofthe results of experiments (see later); and Fig. 22 is a schematic view of the experimental reactor used in experiment 2 (see later).

Sincefermenters are well known, the present description does not include other equipment, units such as hold-up tanks, sterilizers, etc., and instruction and design details which are familiarto anybody skilled in the art offermenter design and operation, and will be directed in particularto the elements forming part of or cooperating directly with the apparatus in accordance with the present invention.

Referring to Fig. 1, there is shown a general schematic diagram of a complete system for perform ing 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 ofthe biological material, a wet-sieve column (3), product separation unit (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 ofthe 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).

In orderto preventthe entrapment of air bubbles in the support media during fixation, the aeration of the nutrient solution can be achieved by continuous recycling of the nutrient solution through a separate aeration vessel (5).

If the increased products concentration starts to inhibit the reaction or biological activity, then the products can be removed by continuous recycling of the liquid through the products separation unit (4).

As the biological material is held by fixation, the culture broth becomes progressively clearer and only the very large aggregates ofthe 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.

Referring nowto Figure 2 ofthe drawings, it will be seen that 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). As can be seen clearly in Figure 3 which is a top view of Figure 2, the sheets of polymer foam (8) can be oblong cross-section and are arranged in circum ferentially 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. Basically, as shown in top view of Figure 4the cutting device may consist of several blades (1 3a) 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 viewsimilarto Figure 2 but showing the use of support media (8a) of wedge shaped cross-section. Figure 6 is similarto Figure4 but shows a modified shearing device (1 3b).

In another preferred reactor configuration, as shown in Figure (7) and inthetopview in Figure (8), the support media (14) may be used in the form of stationary concentric cylindrical shells which are held vertically in the reactor vessel (15). In this embodi ment,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).

In the modified reactor shown in Figures 9 and 10, the support media in the form of concentric cylindric al shells (14a) is rotatably supported about a horizon tal axis.The assembly ofthesupport media (14a) is partially immersed belowthe liquid level Land is rotated about a horizontal axis in a reactor vessel (18) which incorporates a cutting device (19) comprised of several concentric fixed blades. When the assembly ofthe support media (14a) rotates, the cutting device (19) will shearthe plant cells outgrown on the external surfaces ofthe support media (14a).

In the embodiment of reactor shown in Figures (11) and (12) 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.

The embodiments of Figures 9-12 allows the support media to be moved intermittently into contactwith air, which is important if the biological material requires oxygen.

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 1800 apart on a shaft 24 driven by a motor25.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. In use, vessel 22 is substantiallyfilledwith a suspension of plant cells and motor25 is operated to sweep the support bodies arounclvessel thereby filtering the plant cells and entrapping then on these bodies. At the end ofthis operation, the support bodies 23 are brought to the horizontal position illustrated in Fig. 14.The liquid wlthinvessel 22 maythen be drained through ports 27 and replaced by a special media to induce differentiation and shootformation to a level approx imating'L0 line30 in Fig. 14. The cells maythen be cultivated under lsnown conditions to produce "plant lets" (i.e.small plants) which may be harvested from the propagator=,orgrowth in soil orthe like.

An second embodiment of propagator is shown in Figs. 15 andi 16. This propagatorcomprises a cyZin- drical 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.

In operation ofthis embodiment of plant propagator, 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 mannerindicatedforthe propagator shown in figs. 13 and4.

Theplantpropagatorshown 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.

As will be seen from the diagrammatic plan view of Fig. 118,vessel 39 is of circular cross-section. The support bodies 40 are each identical size major segments of a circle ofthe 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 angularlystaggered relationship such thatthe spaces 43 are in the staggered relationship clearly illustrated in Fig. 18.

To effectfixation, a suspension of plant cells is introduced into vessel 39 and motor 42 is operated to stir the suspension and thus effect filtration ofthe biological material on the bodies 40. During this immobilisation operation 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.

Thepropagatorshown in Fig. lSissimilartothat 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 orthe like 46 on the inside of vessel 39.

Afterfixation of plant cells and the supplythereto of media to effect differentiation and growth, the 'plantlets' may be cultivated with the aid of the lights 46 as well as liquid sprayed onto the bodies 40 by spray heads 45.

The method of the invention will be further illustrated by way of example only with reference to the following experiments.

In order to establish the difference in the efficiency of physical entrapment between the static support medium and the moving particulate medium the following experiments have been performed: Exrperiment 1: Relative velocity between foam parti cles andplantcell aggragates.

The relative velocity between two particulate materials which are moving in a liquid can be reflected bythe difference in their individual terminal free settling velocities in the same liquid. The terminal free settling velocities were measured by droppingthefoam particles and cell aggregates of various sizes into water and measuring the time they tookto fall a certain distance. The results are shown in Figures 20 and 21.

According to these figures, the free fall velocity varies with the size of the foam particles and with the size ofthe 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 velocityforthe 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). However, the average terminal velocity of cell aggragates of interest (i.e., 0.5 mm - 1.5 mm) is about 0.5cm/sec.

Therefore, when the foam particles and cell aggra- gates are stirred together in a reactor, the relative velocity between them is (2.5 - 0.5) = 2.0cm/sec.

When theterminal free settling velocity is less than 0.5 cm/sec, the particles tend to follow the circulation pattern ofthe liquid (W.L. McCabe and J. C. Smith, (1967) "Unit Operations of Chemical Engineering", p, 267, McGraw-Hill.). The average velocity in a liquid agitated with an impellervariesfrom 0.7 to 0.1 ofthe impellertip velocity (W.L. McCabe and J.C. Smith (1967). "Unit Operations in Chemical Engineering", p252, McGraw-Hill). Therefore, in an agitated vessel the plant cell aggregates of size range upto 1.5mm, which have an average terminal settling velocity of 0.5 cm/sec, will have velocities of 0.7 - 0.1 of the tip velocity ofthe impeller.When the foam matrix is held stationary as in the case ofthe baffle-reactor, the relative velocity between the cell aggregates and the foam matrix will be 0.7 - 0.1 ofthe tip velocity of the impeller. Hence, in such a reactor, the relative velocity can be controlled at a desired value by simply changingtheimpellerspeed.Asan example, ifthetip 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 higherthan the average relative velocity between moving foam particles and cell aggregates, which is 2.0 cm/sec.

Experiment2: Comparison ofphysical entrapment between a baffle-reactoranda circulating-foamparticle-reactor.

In this experiment, the same vessel and impeller (as illustrated in Figure 22) are used first with foam sheets held stationary like baffles and then with circulating foam particles. The impeller speed and position,the liquid volume, the initial amount and size of cell aggregates, the total volume of the foam matrix, and the duration ofthe experiment are kept the same for both experiments. Apart from whether ornotthefoam matrix moves in the vessel, the only other difference is the total external surface area of the foam matrix.

The vesselandimpeller(Fig. 20) Total liquid volume: 1000cm3.

Height of liquid level: 13cm.

Diameterofvessel: 10cm.

Width of impeller: 2.5cm.

Length ofimpeller: 7.6cm.

The distance of the impellerfrom the bottom ofthe vessel: 0.5cm.

Impeller speed: 90rum.

Baffle-reactor: Total volume offoam matrix = 187.5cm3.

Total external surface area offoam matrix = 387.5cm2.

Dimensions of each foam baffle = 2.5 x 2.0 x 7.5cm.

Numberoffoam baffles = 5.

Initial amount of freely suspended cell aggregates = 730mg (dry weight).

Amount entrapped after 30 minutes = 285mg (dry weight).

Percent entrapment = (285/730) 100 = 39%.

The average relative velocity between foam matrix and cell aggragates = 25-3.6cm/sec.

Circulating-foam -particle-reactor: Total volume offoam matrix = 190cm3.

Total external surface area of foam matrix = 1140cm2.

Dimensions offoam particles = 1 x 1 x 1 cm.

Numberoffoam particles = 190.

Initial amuntoffreelysuspended cell aggregates = 730mg (dry weight).

Amount entrapped after30 minutes = 13 mg (dry weight).

Percent entrapment = (13/730)100 = 1.8%.

The average relative velocity between foam matrix and cell aggregates = less than 2.0cm/sec.

The results show that, despite the advantageous factthatthe circulating foam particles have much largerexternal surface area availablefor contact and penetration of cell aggregates, the total entrapment within the same time period is much less than that which can be achieved in a baffle-reactor. This increased efficiency on entrapment in the baffle -reactor is due to the higher relative velocities between thefoam matrix and cell aggregates than those in circulating-foam-particle-reactor.

Thefollowing data relatingtotheviabilityofthe culture entrapped by fixation and production rate of products also illustrate the advantages ofthe invention.

I. Comparison of viability (a) Entrapped by fixation in foam sheets (Invention) ; viable over 24 months when the nutrient medium is changed every4weeks using eitherfull-growth or production medium.

(Afterthe 15th month, no medium was changed but cells were still viable atthe end of the 24th month) (b) Suspension culture (in 250ml flask) viable upto 2 months without addition of fresh nutrients.

(c) Immobilised culture (in 250m I flask) viable up to 4 months without addition offresh nutrients.

(d) Immobilised in foam particles ; viable over 6 months, when the nutrient medium is changed every 4 weeks using full-growth medium butwhen it was changed to production medium, viability sharply dropped to 40% and eventually all the cells were dead after six months from the start of immobilisation.

II. Comparison of production ofcapsaicin a) cells entrapped by fixation in foam sheets (3 x 8 x 1 cm) using the method ofthe invention : capsaicin concentration was 25,ug/ml of medium with total reactor working volume being 700ml. (total mass of plant cells was 1 75g-wet weight). Therefore, pro duction was 1001lg/g-wetcell weight.

b) cells immobilised in foam particles (1 x 1 x 1cm) capsaicin concentration was less than 1 pg/ml of medium with total reactor working volume being 5000 ml. (Total mass of plant cells was 500g wet weight). Therefore, production was 1 0ug of cap- saicin/g-wetcell weight.

The following example illustrate the application of the invention to 'micropropagation' of plants.

Plant Regeneration from Tissue Cultures ofCapsicum Frutescens using Micropropagator The callus or suspension culture initiated from stem explants of Capsicum frustescens (chilli pepper) was homogenised according to the method described in U.K. Patent Application No.8519180 and then aseptically introduced into the reactor illustrated in Fig. l5whichcontainedSchenkand Hildebrandt (Sh) medium. when the support material was held horizontally the liquid covered it completely.

The reactor was rotated around its horizontal axis in orderto filterthe cells into the support material.

This was continued until the cells completely filled the structure. Then, the medium was drained com pletely and the reactor was filled with a differentiation inducing medium unto such a level thatwhen the support structure was held horizontally the liquid medium just touched the bottom surface of the support structure. This new medium was a modification ofthe Murashige and Skoog's (MS) basal medium. The MS medium was supplemented with indoleaceticacid (1 mg/l) and benzyladenine (2mg/l).

Plant cells treated in the above way would yield shoot buds and buds with roots after 6 weeks from the addition of differentiation including medium and fully developed plants after 10 weeks.

Claims (30)

1. 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/ortissues and/or particles comprising pre-immobilised biocatalysts in a liquid medium through a liquid permeable member capable of filtering said biological mate rial to filter said biological material from said liquid medium, said member being an integral body or a composite body whose components are main tained in a substantially fixed spatial relationship to each other; (b) effecting a biotransformation reaction by con tacting said biological material supported on said memberwith a liquid and/or gaseous medium to form a productand/orbiomass; and (c) recovering said product and/or harvesting said biomass.

2. A method as claimed in claim 1 whereinthe biological material comprises plant cells.

3. A method as claimed in claim 1 wherein the biological material comprises animal cells.

4. A method as claimed in claim 1 wherein the biological material comprises organelles.

5. A method as claimed in claim 1 wherein the biological material comprises plant tissue.

6. A method as claimed in claim 1 wherein the biological material comprises animal tissue.

7. A method as claimed in claim 1 whereinthe biological material comprises microorganisms.

8. A method as claimed in claim 1 wherein the biological material comprises a biocatalyst in oron a polymeric gel bead,on an ion exchange resin oron an inorganic salt.

9. A method as claimed in claim 1 wherein the biotransformation reaction is effected to produce a secondary metabolite.

10. A method as claimed in claim 1 wherein the cells of the suspension of biological material are and/ortissues and/or particles ofthe suspension of biological material are of different sizes and said suspension is filtered through a plurality of said liquid permeable members which areintheformof screens,traysorthe like arranged one above the other and being such thatthe average size of biological material which any one of said members is capable of filtering is less than that which may be filtered by the nextmost upper body.

11. A method as claimed in claim 1 wherein the liquid permeable member comprises a plastics foam material.

12. A method as claimed in claim 1 whereinthe liquid permeable member comprises a network of optical fibres with exposed ends within the member, and light is transmitted along said optical fibres during the biotransformation reaction.

13. Apparatusforthefixation of biological material in the form of biochemically active cells and/or tissues and/or particles comprising partially immobil ised biocatalystsfrom a suspension therof 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 ora 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 ofthe apparatus, in the vessel and the support member such that biological material in suspension in the liquid is filtered therefrom and retained on the support member.

14. Apparatus as claimed in claim 13 wherein the support member is relativelyfixed within the vessel and means are provided for moving liquid contained, in use ofthe apparatus, in the vessel through the liquid permeable member so as to effect said filtration.

15. Apparatus as claimed in claim 13 wherein the liquid permeable member is moveablewithin the vessel and is associated with drive means for moving the support memberthrough liquid contained, in use ofthe apparatus, in the vessel so as to effect said filtration.

16. Apparatus as claimed in any one of claims 13 to 15 provided with means for shearing excess biological material from the support member.

17. Apparatusforthefixation of biological material in the form of biochemically active cells and/or tissues and/or particles comprising partially immobilised biocatalysts from the suspension thereof in a liquid medium, the apparatus comprising a vessel, a plurality of liquid permeable members in the form of screens, trays orthe like arranged one above the other and being capable of filtering said biological material, said members each being an integral body ora composite body whose components are maintained in a substantially fixed spatial relationship to each other and the bodies being arranged such that the average size of biological material which any one of said members is capable of filtering is less than that which may be filtered by the nextmost upper body.

18. Abiochemical reactorsystemcomprisinga biological material colonised on a supportwherein -the biological material comprises living or biochemically active cells and/ortissues and/or particles comprising pre-immobilised biocatalysts, -the support is a liquid permeable member capable offiltering said biological material from a suspension thereof in a liquid medium, said member being an integral body or a composite body whose components are maintained in a substantially constantfixed relationship to each other, and -the reactor system has been produced by filtering a suspension of said biological material in a liquid medium through said support.

19. A reactorsystem as claimed in claim 18 wherein the biological material comprises plant cells.

20. Areactorsystem as claimed in claim 18 wherein the biological material comprises animal cells.

21. Areactorsystem as claimed in claim 18 wherein the biological material comprises organelles.

22. Areactorsystem as claimed in claim 18 wherein the biological material comprises plant tissue.

23. A reactor system as claimed in claim 18 wherein the biological material comprises animal tissue.

24. Areactorsystem as claimed in claim 18 wherein the biological material comprises microorganisms.

25. A method as claimed in claim 1 wherein the biological material comprises a biocatalyst in oron a polymeric gel bead, on an ion exchange resin oron an inorganic salt.

26. A reactor system as claimed in claim 18 wherein said support comprises a network of optical fibres having ends exposed within the support, and the system is further provided with a light source arranged for transmission of lightfrom said source into the support.

27. A method of effecting a biotransformation reaction comprising contacting a reactor system as claimed in claim 18 with a liquid and/or gaseous mediumtoform a product and/or biomass.

28. A method as claimed in claim 27 which is effected to produce a secondary metabolite.

29. Plant propagation apparatus comprising a vessel, a liquid permeable support member within the vessel capable offiltering plant cells from a suspension thereof in a liquid medium, said support member being on integral ora 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 ofthe propagator, inthe vessel andthe support member such that plant cells in suspension in the liquid is filtered therefrom and retained on the support member.

30. Plant propagation apparatus as claimed in claim 29 wherein said support member is in or is capable of being positioned in a substantially horizontal disposition and said vessel has a liquid outlet below the horizontally disposed support memberto allow liquid to be drained from the vessel to a level suitable for plant propagation.