BIOREACTOR AND USE THEREOF

Conventional bioreactors consist of a large container, which is filled with a mixture of microbial, plant- or animal cells, or cell fragments or enzymes and a nutrient or other reactant, and which is supplied with a feed gas, as required, usually oxygen or carbon dioxide. Product gas, such as carbon dioxide, is blown off as required. The tank is stirred either mechanically or by means of the feed gas which is injected. Such bioreactors can be either of the batch-type or of the continuously-operating type, but have relatively low volumetric efficiency and usually require a large space to house them if production is on an industrial scale. The stirring or gas injection, which is used for mixing, can also damage cells as, particularly plant and animal cells are very delicate. However, in order to minimize the culture or reaction time, it is important that the feed gas supply is adequate and that the gas is quickly and thoroughly mixed with the other components in the reactor. In accordance with a first aspect of the present invention, in a method of culturing, or of utilizing in a reaction, cells or cell fragments or enzymes, biological material thereof is located within a first chamber separated from a second chamber by a hydrophobic (i.e. non-wettable) gas-transfer membrane which is shaped to present to the first chamber a repetitive array of hollows; a liquid medium, in which the biological material is immersed, is pulsed in the first chamber along the array of hollows so that the liquid forms vortices in the hollows; and a gas for promoting the culture or reaction passes from the second chamber into the

liquid medium through the membrane.

The use of the vortex-mixing promotes high rates of gas transfer, excellent mixing in the first chamber with low shear stress and hence gentle handling of the biological material and low fouling of the membranes. As a result a high volumetric efficiency, that is to say yield of product divided by a reactor volume, can be expected when used on a laboratory, pilot plant or an industrial scale, with a consequent reduction in reactor size and cost. "

The culture or reaction will often produce a waste product gas which can pass out of the liquid medium through the membrane into the second chamber.

The second chamber will contain the relevant gases at appropriate concentrations to cause the necessary gas transfer through the membrane, and the gases may be in a gaseous form, dissolved, or in a carrier, for example in the case of oxygen a perfluoro compound. The culture or reaction could be in either a batch process, or continuously operated. When it is operated continuously, any necessary nutrient or reactant, which is consumed during the culture or reaction, will need to be supplied to the liquid medium in the first chamber.

The biological material may be free-floating in the first chamber, or immobilised therein on a support, located within the first chamber, such as beads or other solid suspended within the chamber or a lining on the membrane or other wall of the first chamber and with a porous or other geometry allowing percolation of the liquid medium therethrough.

The pulsatile flow is preferably a reversing flow, possibly with a mean flow through the first chamber, and provided, e.g., by diaphragm, roller, or other pumps operating out of phase at opposite ends of the first chamber.

The hollows may be an array of transverse furrows, or dimples, and the manner in which these hollows cooperate with the pulsatile flow to provide vortices in the hollows is more fully described in GB-A-1442754, GB-A-2042926. and EP-A-0111423, and the apparatus described in those specifications may readily be adapted as a bioreactor for use in the new method.

In accordance with the second aspect of the invention, therefore, a bioreactor for carrying out the new method according to the first aspect of the invention, comprises a first chamber separated from a second chamber by a hydrophobic gas-transfer membrane, which is shaped to present to the first chamber a repetitive array of hollows; a biological material comprising cells or cell fragments or enzymes, or means, for immobilizing such biological ^' material, located within the first chamber; means for pulsing a liquid medium in the first chamber along the array of hollows so that the liquid forms vortices in the hollows; and means for supplying a gas or its precursor to the second chamber.

The advantages of the vortex mixing can be extended to the purification, concentration, and/or separation of the biological material and product.

Thus, in accordance with a third aspect of the invention, a liquid medium resulting from the culture, or use in a reaction, of biological material comprising cells or cell fragments or enzymes, e.g. from the first chamber, is filtered by disposing the liquid in a third chamber, which might be common with the first chamber or in series with it, the third chamber being separated from a fourth chamber by a hydrophilic (i.e. wettable) liquid-transfer membrane which is shaped to present to the third chamber a repetitive array of hollows; and pulsing the liquid in the third chamber across this array of hollows so

that the liquid forms vortices in the hollows; whereby one or more components of the liquid are filtered out through the liquid-transfer membrane.

The corresponding bioreactor would then involve a reactor according to the second aspect of the invention, but having both a hydrophobic gas-transfer membrane separating the first chamber from the second chamber, and a hydrophilic liquid-transfer membrane separating the first chamber, or a third chamber which can be put in communication with the first chamber, from the fourth chamber.

Some examples of bioreactors constructed in accordance with the present invention, and their use, are illustrated in the accompanying drawings, in which:-

Figure 1 is a diagrammatic perspective view of one reactor;

Figure" 2 is a section taken on the line II-II in Figure 1; Figure 3 is a section taken on the III-III in Figure 1;

Figure 4 is a section taken on the IV-IV in Figure 1;

Figure 5 is a plan of the profiled surface of one plate of the reactor;

Figure 6 and 7 are sections through the two plates of the reactor juxtaposed with two membranes between them, and taken respectively on the lines VI-VI, and VII-VII, in Figure 5; Figures 8 to 11 show various uses of the reactor of Figures 1 to 7; and.

Figures 12 to 17 are graphs associated with various reactions.

As shown in Figure 1, a reactor 13 is carried face to face by an upright wall 14 and consists of similar opposed side plates 15 and similar pairs of end plates 16 and 17. The side plates 15 are

rectangular and elongate and the facing adjacent surface of these plates are profiled. Positioned between the two profiled surfaces of the side plates 15 are a pair of membranes 18. Along the longer sides of the plates 15, the membranes 18 are sealed to one another and to the plates 15 by clamping bolts 19, which draw the plates together, and pairs of sealing beads 20, which are seated in grooves in the plates 15, and abut the membranes 18. There is thus formed between the membranes 18, a central ^* primary chamber 22 and, between each membrane 19 and the adjacent profiled surface of the adjacent plate 15, an outer secondary chamber 23. At intervals along the longer dimension of the plates 15, their profiled surfaces are intersected by transverse channels 24 which ensure complete irrigation of the secondary chambers 23 between the membranes and profiled faces of the plates 15.

At each of the ends of the plates 15, the two plates 16 and 17 are bolted to them by bolts 25 and the ends of the membranes 18 are clamped between the ends of the plates 15 and the end plates 16. Clamped between each of the plates 16 and the adjacent plate 17, is it an outwardly extending flange 26 of a flexible diaphragm 27. A manifold 28, in communication with the adjacent end of the primary chamber 22, is formed within an open interior of the plate 16 and each of these manifolds 28 is connected through a bore 21 with an external nipple and hose 29, 29'. The diaphragms 27 are accommodated within openings within the respective plates 17 and are acted upon by respective pushers 30, 30' carried by arms 31, 31', which work through elongate slots 32, 32' in the board 14, and carried on respective ends of a member 33. This member is reciprocable in a linear bearing 34 by means of a motor 35 acting through a crank 36. As the member 33 is moved to and

fro liquid is flushed to and fro through the primary chamber 22. However, if mean flow through the chamber 22 is required, the stroke of the pusher 30 extends further into the respective plate 17 than does the pusher 30', as a result of which there is superimposed upon the reciprocating flow in the primary conduit 22, a component which provides a net mean flow from the inlet hose 29 to the outlet hose 29'. At each end of the plates 15, each of the secondary chambers 23 and one of the channels 24 communicate through a port 37 in the respective plate via a bore 38 in the respective plate, with a nipple and hose 39, 39*. As suggested in Figures 2, 3 and 4, and shown more clearly in the enlarged Figures 5, 6 and 7, the profiled face of each plate 15 is provided with a close packed array of substantially hemispherical recesses 40 which are arranged in rows extending parallel to the longer dimension of the plate, the recesses in each row being offset half way between those in adjacent rows, to provide the close packing. Adjacent recesses in each row are interconnected by grooves 41. The membranes 18 are each preformed with an array of dimples 42, corresponding in maximum diameter and centres to the recesses in the plates such that when the membranes are assembled between the plates, the dimples 42 nest in the recesses, as shown in Figures 6 and 7. The primary chamber 22 is thus formed in the continual spacing between the membranes 18, and the secondary chambers 23 are formed by the spaces between the bottoms of the dimples and the bottoms of the corresponding recesses, together with the grooves 41. Typically the maximum diameter of the recesses 40 and dimples 42 is 1.5 mm, the dimples having a maximum depth of 0.5 mm and the recesses a maximum

depth of 1 mm. The grooves 41 have a width of 0.5 mm and a depth, the same as that of the depressions, i.e. 1 mm. The channels 24, which intersect the grooves 41, have a greater depth by a factor of up to 3 or 4. To appreciate what this means in the context of the reactor, each of the plates 15 is substantially 150 mm long and 100 mm wide.

In use when liquid in the central chamber 22 is flushed to and fro between the manifolds 28, by the out of phase action of the pushers 30, vortices in the liquid are set up in the dimples 42. This brings a greater quantity of the liquid into intimate contact with the membranes and hence enhances the transfer of gas or other material through the membranes to or from the liquid.

Figures 8 to 10 shows various ways of using the reactor shown in Figures 1 to 7. Thus Figure 8 shows one module 13 using one of the reactors, i.e. a single stage reactor, and only the essential parts are numbered. The central chamber 22 contains the biological material comprising cells or cell fragments or enzymes. The biological material may be free-floating in this chamber, or immobilized therein on a support, located within the chamber, possibly as a lining on at least one of the membranes 18A, and with a porous or other geometry allowing percolation of the liquid medium therethrough, or in suspension, for example on or in beads. The membranes 18A separating the central chamber 22 from the outer chambers 23 are hydrophobic gas-transfer membranes. These may be made of polypropylene with a pore size in the order of 0.02μ, such as that sold under the name Celguard and provided with the dimples facing into the chamber 22. The diaphragm pumps, operated by the pushers 30, reciprocate the material along the chamber 22 so that vortices are set up in the biological material within the dimples, promoting

gas-transfer with the biological material through the membranes.

This reactor configuration may be used to pass biological material continuously through it, from the inlet 29 to the outlet 29' ,- provided that the mean flow is superimposed upon the reciprocatorary flow by appropriate setting of the pushers 30. Alternatively batch processing may be carried out, in which case valves 44 in the hoses 29, 29' will be closed. Figure 9 shows another single stage reactor but with a combined arrangement in which the chamber 22 is separated on one side by a hydrophobic gas-transfer membrane 18A from a first outer chamber 23A having its own inlet and outlet 39A and 39'A, while the other side of the chamber 22 is separated by a hydrophilic liquid-transfer filter membrane 18B, from the second outer chamber 23B, which has its own inlet and outlet 39B and 39'B. The hydrophilic membrane may be a polysulphone microfiltration membrane with a pore size of the order of 0.2μ. This arrangement could be used for culturing biological material, for providing gas to and removing gas from the biological material in the chamber 22, and also for nutrients supplied to and or product removal from the biological material in the chamber 22 via the second outer chamber 23B. The inlet and outlet 29 and 29' to the central chamber 22 may then be sealed by valves 44.

Figure 10 shows a reactor having two stages 13A and 13B in series. The membranes 18A of the reactor 13A are hydrophobic membranes and the membranes 18B of the reactor 13B are hydrophilic membranes. A connection 29A interconnects the outlet 29' of the first stage 13A to the inlet 29 of the second stage 13B. The first stage may be used for gas-transfer to promote growth of the biological material, and the second stage may be used for liquid-transfer, either

for nutrient into, or a product out of, the central chamber 22. If the second stage is only used for product filtration, the inlets 39 to the outer chambers may be sealed or omitted as all that is necessary is for the filtrate to be drawn off through the outlets 39* .

Figure 11 shows three stages 13A, 13B and 13C in a stacked arrangement, with their central chambers

22 interconnected by connections 29A and 29B. Each stage has a different function. Thus the first stage

13A may provide gas-transfer via hydrophobic membranes 18A. The second stage 13B may be provided with hydrophilic membranes 18B for the supply of nutrients to the biological material and/or removal of product which could, if required, be returned to the first stage. The third stage 13C would also be provided with hydrophilic membranes 18B and could be used for the further separation of the product into its constituent parts. All the pushers 30 could be operated by a common pair of bars 43.

The various reactors for supplying and/or removing gas, supplying nutrient and/or removing product to and/or from a culture medium could be arranged as a "bolt-on" or ancillary unit to another reactor, such as a standard fer entor.

Some examples of the use of these reactors will now be described.

Example 1

A reactor as illustated in Figure 8, containing two hydrophobic polypropylene (0.02μm, pore size) membranes was used for the batch growth of the strictly aerobic microorganism . Pseudomonas testosteronii. The microorganism was inoculated into growth medium, in this case nutrient broth, and the suspension was pumped aseptically into the central compartment 22 of the bioreactor, the inlet 29 and outlet 29' of which were then closed, completely

filling the area between the two hydrophobic membranes 18. Air moistened to limit evaporation was sucked across the membranes, in the outer chambes 23, at a flow rate of 2.5 litres by applying suction to the outlets 39'. This drew oxygen-containing air into the chambers 23, and withdrew from these chambers oxygen-lean, carbon dioxide-rich residue gas, resulting from transfer of oxygen through the membranes 18A into, and the transfer of carbon dioxide through the membranes 18A out of, the chamber 22. The inlet and outlet 29, 29' of the chamber 22 were sealed such that any oxygen entering the chamber and contacting the cells has to pass across the membranes 18A. The bacterial suspension in the chamber 22 was oscillated at 3 Hertz via the diaphragm pumps at the ends of the reactor. The system was operated at room temperature 20-25°C- Samples were removed over a time course to monitor growth by measuring increased absorbance at 600 nm, the loss in volume'in the chamber 22 was replenished from a syringe containing sterile medium attached to the inlet 29.

The rate of growth of the microorganism in this bioreactor system was equivalent to that observed under standard microbial growth conditions in shake flask cultures at the same temperature, indicating that the cell culture within the bioreactor was adequately oxygenated and not limited by the rate of gas transfer (see Figure 12) . To ensure that cell growth that has occurred within the bioreactor was due to adequate aeration, the experiment was repeated using identical conditions and the same microorganism but the air flow in the outer chambers 23 was substituted by a constant flow of nitrogen. As can be seen in Figure 12 very limited growth was observed under these conditions indicating severe oxygen limitation.

Example 2

The bioreactor configuration shown in Figure 9, that is having one gas-transfer hydrophobic membrane and one liquid-transfer hydrophilic membrane, was used. In this particular case the orientation of the reactor was with the upper membrane 18B being hydrophilic and the lower membrane 18A hydrophobic.

The test microorganism was a Pseudomonas sp. , a strictly aerobic bacterium. A simple defined growth medium was used with succinonitrile (20 mM) being the sole source of carbon and nitrogen, all other conditions were as used in Example 1 except that air was drawn solely through the chamber 23A, the chamber 22 containing the microorganism plus growth medium and the chamber 23B growth medium only.

The rate of growth of the microorganism within the bioreactor was monitored and compared to an ^' identical stationary culture. Microbial growth was slow in the stationary culture, only having limited oxygen available, but the rate of growth in the bioreactor was significantly faster being equivalent to that obtained under standard microbial growth conditions (see Figure 13).

In Example 1 it was demonstrated there was no oxygen limitation to growth in a bioreactor having two hydrophobic membranes and in Example 2 that again there is no oxygen limitation using only a single hydrophobic membrane.

Example 3 A bioreactor configuration as used in Example 2 was in this example used to monitor the accumulation of a product produced during growth of a microorganism. The chamber 22 of the reactor was filled with nutrient broth media inoculated with a species of the bacteria Pseudomonas aeruσinosa known to produce pyocyanine during growth on nutrient broth. Pyocyanine is a blue green pigment produced

as a secondary metabolite by this microogranism that accumulates towards the later stages of batch growth. Air was passed across the gas-transfer membrane 18A, i.e. through the chamber 23A and the chamber 23A was connected to a 300 ml sterile reservoir of nutrient broth. This medium was continually circulated through the chamber 23A at 2 l/min. The production of pyocyanine was monitored by the increase in absorbance of the fluid passing through the chamber 22. Pyocyanine produced by the microorganisms contained within the chamber 22 was able to permeate the hydrophilic liquid-transfer membrane 18B, accumulating within the external loop (see Figure 14) . The bioreactor can therefore be used to accumulate products of cell metabolism produced during growth. The product is separated from the cellular material which is retained by the liquid-transfer membrane 18B. At the same time oxygen is supplied by gas-transfer across the hydrophobic membrane 18A. ^■ Example 4

The bioreactor configuration used in Examples 2 and 3 was again utilized. Conditions were as previously described. The microorganism Nocardia rhodochrous LL100-21 was grown in batch in chamber 22 of the bioreactor, the growth substrate supplied as sole source of carbon and nitrogen being acetonitrile (20 mM) . The chamber 23B of the bioreactor, above the liquid-transfer membrane 18B, was sealed off during batch growth of the microorganism within the chamber 22.

Growth was monitored as previously described. Nocardia rhodochrous LL100-21 is known to be able to utilise acetonitrile as a growth substrate as a sole source of carbon and nitrogen. The

microoganism can be hydrolyse the vinylic nitrile acrylonitrile converting it to acrylic acid but is unable to utilise this acid product. Thus the 300ml volume external loop was charged with acrylonitrile 20 mM and the release of ammonia and formation of acrylic acid monitored. Growth of the microorganism on acetonitrile was monitored until an absorbance of 0.6 was reached. The external loop containing acrylonitrile (20 mM) was then connected and circulated at 3.5 ml/min with an induced back pressure of 5-10 mm Hg to prevent collapse of the bioreactor membranes by maintaining a positive pressure in the chamber 22.

Within eight hours all the acrylonitrile had been biotransfor ed to acrylic acid (see Figures 15 and 16) . The bioreactor can therefore be used to perform biotransformations, in this particular case a 100% conversion was achieved of a 300 ml volume of acrylonitrile (20 mM) to a ^' 300 ml volume of acrylic acid (20 M) with no requirement for cell separation.

The addition of a maintenance medium or initiation of further growth in the chamber 22 may well prolong the ability of the cells to perform biotransformations enabling continuous operation of the system. This is of course dependant upon the cell culture system under study. Example 5

The bioreactor configuration as used in Examples 2-4 was utilised as a small scale culture vessel.

A strictly aerobic microorganism Pseudomonas sp. was cultured in the chamber 22, the inlet 29 and outlet 29' being initially sealed. The growth substrate was succinonitrile 20 mM as sole source of carbon and nitrogen. On entering the stationary phase of growth, fresh medium containing succinonitrile (20 mM) was pumped continuously into

the chamber 22 via the inlet 29 and cell-containing effluent was removed via the outlet 29', at a flow rate of 2.5 ml/min giving a dilution rate of 0.125/hr (see Figure 17) .

A continuous culture was readily achieved operating in this case for 50 hours demonstrating that the bioreactor can be used as small scale continuous culture system.

The invention has many possible uses including:-

1. The culture of microbial cells (including bacteria, yeast, fungi, algae and viruses) or the utilization of such cells under non-growing conditions.

2. The culture of plant cells, or their utilization under non-growing conditions.

3. The culture of animal cells (including the production of vaccines or monoclonal antibodies or other products) , or their utilization under non-growing conditions.

4. As an enzyme reactor or a reactor using other components derived from living cells.

5. For the purification, concentration (e.g. de-salting, growth medium removal, removal of metabolites) and recovery of biological material and product.

6. For the recovering of product, such as proteins, peptides, nucleotides, antibodies or other biological cell constituents, or products produced or excreted by biological cells such as amino acids, antibiotics or extracellular enzymes or other products.