Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/001—Culture apparatus for tissue culture

Definitions

• FERMENTER The present invention relates to fermenters Many types of organism are grown (fermented) in closed vessels to provide, directly or indirectly, valuable substances.
• hairy root cell cultures are obtained by infecting plants (usually dicotyledons, although the technique may be applicable to monocotyledons) with Agrobacterium rhizogenes.
• the resulting proliferation of roots can be excised from the plant and maintained indefinitely in axenic culture in order to produce substances native to the parent plant or substances encoded by genes inserted artificially into the plant.
• Such cultures have many advantages over cell suspension cultures (i.e. suspensions of free cells) but they also have disadvantages, particularly because root organisation must be maintained, otherwise a callus forms which generally reduces the yield of the desired product.
• the present invention seeks to provide an improved apparatus, and provides a fermenter for plant growth, comprising a vessel defining a fermentation chamber and a space-filling lattice of inoculation points adapted to be located within the chamber.
• each inoculation point comprises an inoculant-trapping means comprising means to define a gap suitable for accommodating and retaining plant material of a predetermined size.
• the lattice may comprise a plurality of wires so arranged as to constitute the said inoculant-trapping means or a plurality of barbs constituting the inoculant-trapping means.
• the gap is less than or equal to the "effective diameter" of the plant material.
• the "effective diameter" of hairy roots is illustrated in Figure 1 as parameter "D" .
• the lattice comprises a plurality of wires and the wires meet one another to form nodes constituting the said inoculation points.
• the average density of inoculation points in the lattice is one per X, where X is the volume which will be filled by the growing plant, under the intended conditions of operation of the fermenter, from a single inoculation point.
• the parameter X is species-dependent but is typically 10 -4 to 10 -2 m 3 .
• the fermenter will normally be operated in a substantially conventional manner, except that it can be advantageous to have one or more of the following features present: a system for delivery precipitation (for example a fine mist) of nutrient solution over the roots instead of immersing them in the nutrient solution; recycling the nutrient solution; and passing any recycled solution over a suitable absorbent (such as activated charcoal or Amberlite XAD-2, XAD-4 or XAD-7) to remove auxins, cytokinins and phenolics which may promote callus formation.
• a suitable absorbent such as activated charcoal or Amberlite XAD-2, XAD-4 or XAD-7
• the fermenters of the invention are suitable for the fermentation of hairy root cell cultures but may also be suitable for fermenting other cultures which grow from an inoculation point to form a multicellular mass, particularly a mass having a more-or-less predetermined shape.
• Figure 1 is a schematic view of a hairy root
• Figure 2 is an isometric view of a space-filling lattice of wires forming part of a fermenter in accordance with the invention
• Figures 3A to 3C show respective views (end, side and perspective) of the lattice of Figure 2, wherein the wires do not quite meet one another;
• Figures 4A to 4C show respective views (end, side and perspective) of an alternative form of the lattice of Figure 2, wherein the wires do meet one another;
• Figures 5 to 10 illustrate differing ways of forming a barb on a wire for use in forming an alternative lattice to that of Figure 2, wherein Figure 5 shows an inset peg construction, Figure 6 shows a welded construction, Figure 7 shows a screwed construction, Figure 8 shows a recessed construction, Figure 9 shows a collar construction and Figure 10 shows a one-piece moulded construction;
• Figures 11 to 13 illustrate differing ways of arranging the barbs of Figures 5 to 10 on the wires, wherein Figure 11 shows a multi-barb "crown" (Fig 11A is the top view and Fig. 11B the side view), Figure 12 shows single barbs arranged with a helical pitch (12A top view; 12B side view) and Figure 13 shows multiple barbs with a staggered pitch (13A top view; 13B side view); Figures 14 and 15 are top views of two alternative arrangements (in-line and staggered, respectively) of the barbed wires of Figures 5 to 13 to form a space-filling lattice;
• Figure 16 is an isometric view of the lattice of Figure 14;
• Figure 17 is a schematic vertical section through a fermenter in accordance with the invention, with the space-filling lattice indicated in outline only;
• Figure 18 is a process diagram for the operation of the fermenter of Figure 17;
• Figures 19 and 20 show respective configurations of barbs for use with the inter-barb link of Figure 21 to form a chain
• Figure 22 shows such a chain in tension
• Figure 23 shows such a chain being withdrawn from the fermenter.
• Figure 1 is a schematic view of a hairy root comprising the root itself 1 and a plurality of side branches 2 growing from the root.
• the root grows in the direction shown by arrow A in Figure 1, with the branches 2 growing laterally to reach an "effective diameter" D.
• the fermentation vessel is inoculated with suitable small lengths of roots which then grow to substantially fill the fermenter vessel. It has been found to be highly advantageous if the lengths of roots which form the inoculant are distributed throughout the fermentation vessel. To achieve this, a space-filling lattice of inoculation points is provided in the fermentation vessel and, in a manner to be described in more detail below, the inoculating lengths are suspended in a suitable medium which is then passed into the vessel. The inoculant lengths lodge at the inoculation points and thereafter grow in a substantially conventional fashion.
• Figure 2 illustrates one example of a space-filling lattice constructed from seventy five lengths of steel wire 3 (diameter 0.9 mm, grade 304) so arranged orthogonally to form a 40 ⁇ 40 ⁇ 40 cm cube with sixty four 10 ⁇ 10 ⁇ 10 cm sub-elements.
• the term "wire” is used herein generically to cover articles which, according to their diameter, might more usually be referred to as, for example, filaments or rods. There are two main ways in which the wires 3 can form inoculation points 4.
• X represents the volume which can be filled by the roots, starting from a single inoculation point, under the intended conditions of operation.
• X is from 10- 4 to 10 -2 m 3 .
• Figure 4 represents an alternative way in which the wires 3 of Figure 2 can be arranged, in that in this embodiment the inoculation points 4 are formed by actual connections between the wires 3 where they meet perpendicularly.
• the inoculation points 4 are constituted by the angle formed by the adjoining wires 3.
• the wires 3 may be formed of any suitable material which is strong enough to support the weight of the growing roots, which does not inhibit the growth or metabolism of the roots and which does not interfere with any subsequent downstream processing.
• suitable material which is strong enough to support the weight of the growing roots, which does not inhibit the growth or metabolism of the roots and which does not interfere with any subsequent downstream processing.
• stainless steel, platinum, carbon fibre composites, ceramics, nylon and other natural or synthetic polymers or combinations thereof are suitable.
• FIG. 5 An alternative way of forming an inoculation point 4 on a wire 3 is illustrated in Figure 5, with variants thereof illustrated in Figures 6 to 10.
• a peg 5 or similar means is attached to the wire 3 (or formed integrally therewith as in Figure 10) to form a barb defining an angle, the size of which can be arrived at by experimentation but will usually be 30 to 60°.
• the particular way in which the barb is formed can be selected from the possibilities illustrated in Figures 5 to 10 according to such criteria as cost and effectiveness and will in general depend upon the materials which are used for the wire 3 and the peg 5 and also on the root species and conditions of operation.
• the barbs 5 may be flexible in relation to the main wire 3.
• the barbs can be arranged in any convenient manner. Three possibilities are illustrated respectively in Figures 11 to 13, namely a multi-barb "crown", a single-barb helical pitch and a multi-barb staggered pitch.
• the wires used in the embodiment where barbs form the inoculation points are generally thicker than the wires used in the lattice of Figure 2, for example 5 mm.
• Figures 19 to 21 Further ways of forming an inoculation point are illustrated in Figures 19 to 21.
• the barbs 50 or 50' are formed on a link 51 or 51' which is part of a chain, supported within the fermentation chamber.
• the chain comprises "barbed links” and "inter-barb links".
• Figures 19 and 20 show examples of construction of the barbed links, either by bending wire into a suitable shape ( Figure 19) or by forming a link by fabrication ( Figure 20).
• Figure 21 shows an example of construction for an inter-barb link 52.
• the links may be formed of any suitable material, especially stainless steel, and may be formed of wire or flat strips.
• the links may be twisted in a tortional manner to allow orientation of the barbs to produce a desired pitch, as illustrated in Figures 12 and 13.
• the barb 50 or 50' should be long enough to trap the root, for example at least 2mm, and may be up to 10 cm long, to help guide the root pieces down to the inoculation point 53 during the stage of filling the fermenter with root inoculum.
• the chain is held in tension, which orients the barbs in the required position.
• Figure 22 is an illustration of the chain in this position. Removal of this support means from the biomass may be achieved by releasing the tension on the chain and withdrawing the assembly down through the biomass.
• the chain construction has the advantage of facilitating this removal, because the barbs can retract from the biomass by re-orientation of the barbed links as shown in Figure 23.
• the spatial density of the barbs can be chosen by manipulation of the length of the inter-barb links.
• the barbed wires may be arranged in any suitable manner within the fermenter, running vertically or horizontally, and need not be parallel to one another, but two convenient arrangements are illustrated in Figures 14 and 15, namely an in-line arrangement and a staggered arrangement respectively.
• the barbs are not shown in these Figures.
• An in-line arrangement similar to that of Figure 14 is shown isometrically in Figure 16, showing a typical distribution of barbs to give substantially the same arrangement of inoculation points as in the embodiment of Figure 2.
• a multi-stage process for growth of transformed root on a large scale has been designed which depends on the development of procedures for inoculation of the fermenter 10 and the development of a specially designed bioreactor which satisfies the nutritional, gaseous and environmental requirements for growth of the roots so that high density beds may be rapidly established.
• the desired biomass density is dependent upon the species of root cell and can be arrived at by routine experimentation. Typically, however, there is at least 50% partial volume of roots, 25-50g dry wt/litre.
• Establishing the static bed would usually involve inoculation with a low density of roots (for example about 6g wet wt/litre) and preferably an even distribution of this inoculum throughout the reactor volume.
• the fermentation is divided into three main stages, as follows:
• the culture medium may be passed over an adsorbent 10 such as activated charcoal, or Amberlite XAD-2, XAD-4 or XAD-7, to remove factors produced by damaged roots which may promote callusing.
• the resin may be included at a time when the desired metabolite is not being produced (i.e. during early growth)and removed later or left in place. The resin may or may not remove the metabolite and can be used when there is no need to provide for metabolite removal by way of a resin.
• the vessel is operated (a) with a continuous gas (air) phase with growth medium being continuously recycled as a thin film over the root mass via a separate recycle vessel 12, or (b) with the roots fully submerged in medium, with the facility for liquid recycle and/or mixing.
• phase iii A period of product formation and, depending on the species concerned, release, during which further growth can be reduced if required by application of growth-limiting media. In some cases, phase iii would not be applied, particularly with products whose synthesis is strictly growth dependent.
• the vessel 10 is constructed of a sturdy, sterilisable material (preferably stainless steel) which may be opened to allow removal of intact biomass at the end of the fermentation period. To facilitate this process, it is advisable to have a tapered configuration.
• the vessel should be capable of being sterilised in situ. The overall size of the vessel may be arrived at by experimentation; too large a volume may lead to uneven distribution of nutrients etc.
• the vessel is fitted with ports 13-16 to allow liquid addition, removal and recycle and also for the introduction of air.
• air is used to include any gaseous medium.
• a suitable inoculation port 17 is provided, to allow the introduction of root material without damage to the roots, or blockage of the port.
• Ports 18 are provided to house monitoring probes (for example for pH, dissolved oxygen and temperature).
• a sight glass 19 is provided to allow visual inspection of the vessel contents.
• the vessel is fitted with a jacket 20 for purposes of vessel sterilisation and temperature control.
• Air filtration units 21 are provided to allow sterile air to be introduced into the vessel.
• a condenser 22 and equipment 23 for removal of entrained liquid droplets on the air outlet 24 should be provided, should process conditions lead to unacceptable liquid losses.
• the interior of the vessel is smooth to prevent attachment of the roots to the walls.
• a stirrer 25 (or other liquid mixing system) is provided to distribute the root material as described above. The stirrer is so situated and designed as not to damage the root material.
• the vessel is fitted with a rigid or semi-rigid removable support 26 to allow harvesting of the root mass at the end of the fermentation.
• a further network of root inoculation sites 4 is provided to allow even distribution of the inoculum, as described above.
• the vessel is fitted with a means of introduction of air, either by direct gas injection 16 or by the supply of pre-aerated growth medium during the growth phase.
• the vessel will be provided with a means of illumination 27 to allow inspection of the root material.
• the vessel 10 is fitted with a liquid distribution device such as a spray 30, atomiser, sprinkler or weir discharge system. This is capable of distributing the medium over the root mass without causing mechanical damage or dislodging the biomass from its support.
• a liquid distribution device such as a spray 30, atomiser, sprinkler or weir discharge system.
• This is capable of distributing the medium over the root mass without causing mechanical damage or dislodging the biomass from its support.
• Two or more types of distribution system may be provided for different stages of biomass packing, for example to produce a fine mist during early stages of growth when the inoculation points are widely separated and to provide simple liquid distribution over the surface of the biomass when the roots have grown into a continuous mat.
• the liquid medium is recycled from the bottom to the top of the growth vessel via the recycle vessel 12.
• This vessel may be smaller than the main growth vessel 10, to allow better control over the liquid inventory, and may be fitted with a wide range of instrumentation (not shown) to allow accurate monitoring and control over the progress of the fermentation. Additions to the recycling medium, including air, can be made into this vessel, which is preferably stirred to ensure homogeneity and bubble break-up.
• Roots may be harvested from the vessel by stopping the liquid flow, allowing the remaining liquid to drain from the root surfaces and opening the vessel .
• the root mass may then be removed from the vessel by use of the support structure 26. If the barbs of, say, the Figure 6 embodiment are sufficiently flexible, or the chains of Figures 19 to 22 are used, then the wires or chains can be withdrawn individually through the biomass. This can facilitate subsequent processing of the biomass and/or the re-use of the array of inoculation support points.
• the process may be operated as a "fed-batch” process, with nutrients being added to a less than full vessel, or as a “continuous” process, where the vessel starts full and is supplied with further medium such that excess medium is led away.
• the inoculum is preferably cut into suitable lengths of roots (determined experimentally according to the species and fermentation conditions, but typically 1 to 10 cm) and pumped as a slurry into the main fermenter . These two processes can lead to damage, resulting in callusing and loss of productivity. The following procedure has been found to reduce or avoid this problem.
• Roots are inoculated into a 20-40 litre inoculum vessel 31 (preferably glass or stainless steel), the inoculation being carried out in a laminar flow hood (not shown).
• This vessel 31 is operated as an air sparged stirred tank so that the inoculum of 100g fresh wt is grown in free suspension to about 20-25% v/v roots (about 6 kg of roots suitable to inoculate a 1 m 3 reactor).
• the inoculum vessel 31 is provided with a rotary blade which is driven by an external motor and operated at intervals during the preparation of the first inoculum to chop the roots into small pieces.
• the medium may be recycled through an adsorbent 32 (for example activated charcoal, XAD-2, XAD-4 or XAD-7) to remove factors (for example auxins, cytokinins and phenolics) produced by the damaged roots which may promote callusing.
• an adsorbent 32 for example activated charcoal, XAD-2, XAD-4 or XAD-7 to remove factors (for example auxins, cytokinins and phenolics) produced by the damaged roots which may promote callusing.
• Pumping the inoculum slurry is achieved by means of a pumping system giving minimal damage such as over pressuring the inoculating vessel or by use of an air-lift pump.
• a recycling pump enables back flushing of the inoculating vessel to ensure complete transfer of the inoculum.
• the fermenter is generally suitable for fermenting hairy root cultures and other multicellular organisms. Specific examples include hairy root cultures of the plants shown in Table 1, and all those mentioned by Mugnier in "Plant Cell Reports", 1988, 7 , 9-12.
• plants of these and other species may be transformed with genes for particular compounds of interest.

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• 1988
  • 1988-05-14 GB GB888811478A patent/GB8811478D0/en active Pending
• 1989
  • 1989-05-11 WO PCT/GB1989/000510 patent/WO1989010958A2/en not_active Application Discontinuation
  • 1989-05-11 JP JP1505603A patent/JPH03505278A/ja active Pending
  • 1989-05-11 EP EP89905729A patent/EP0414766A1/de not_active Withdrawn
• 1990
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