GB2167085A - Method for the operation of a bio-reactor - Google Patents

Abstract

Operation of a Bio-Reactor (1) using porous flexible biomass support particles in a moving bed (2) is described. The particles containing biomass are removed via off-take (5) and are subjected to pressure e.g. by counter-rotating rollers (13) to remove the biomass from the pores of the particles. The particles may be recycled to the bed via intake (11) together with the separated biomass or the separated biomass may be removed from the system. In other described embodiments, the biomass is separated from the particles by impinging jets of liquid on the particles, by vibration or by magnetic means when the particles are magnetic. The particles are e.g. of plastic foam or of wire mesh. The process may be used in batch beer fermentations. <IMAGE>

Description

SPECIFICATION Method for the operation of a bio-reactor This invention relates to a method for the operation of a bio-reactor i.e., a process and apparatus for the utilisation of living cells particularly microorganisms in a liquid containing suitable nutrient material The original bio-reactors (fermenters) were self stirred by rising of gas bubbles and by thermal convection. Later they were mechanically stirred.

Bioreactors have been designed using air lift, fluidisation of the beds, micro beads to permit movement of the mass through the bed and various modifications of stirred tank and packed beds.

The most successful were found to be gas-lift bioreactors and fluidised bioreactors. Nevertheless, agitation by gas flow through the mass can be difficult and if the gas contains oxygen can interfere with the reaction. Agitators in the reaction vessel may harbour infection. The main aim behind all of these reactors is to provide for a circulation within the bed of particles i.e., the beds of particles were capable of fluid flow. The bed might be revitalised by removal of portions so as to purify support particles, some part of the separated material being recycled. Types of bioreactor systems are described in Bio-technology May 1984 page 477 onwards.

The use of free cells in bioreactor systems such as continuous, stir-tank or batch systems had the disadvantages of low cell concentration per unit reactor volume and low productivity. It was recognised as desirable that greater concentration be developed. Therefore, for industrial processes involving biological fermentation there were developed systems involving dense suspensions of cells for example cell densities of 10 million cells per millilitre.

Sometimes the cells in bioreactors are supported on carriers. A bed formed from this combination of cells and carriers is sometimes called a biomass.

This technique has been employed particularly in the treatment of sewage and industrial effluent (the so-called activated sludge process) but has also been used in brewing and other fermentation techniques. These systems utilised immobilised cell systems in which cells were affixed to base materials such as gels or inorganic supports.

Immobilisation can be passive or active in nature. In passive immobilisation the bio-particles are introduced into the support merely by mechanical means while in active techniques a chemical process can be used to immobilise the cells. By polymerisation of monomer in the presence of the cells, polymeric support systems could be created.

For example, a gelling agent could be added to a concentrated suspension of bio mass and the cells then be entrapped into the gelling particles as these formed. These particules could then be loaded into a reactor to form a concentrated bed of material.

In Specification EP-A-0 025 309 the liquid passes downward through a bio-reactor in which the carrier particles have a specific gravity less than that of the liquid. Particles with excess biological material are removed from the base of the bed which, because of the gravity of the particles, is in the upper portion of the container and the particles are subjected to abrasion to remove excess biota from the particles. Particles suggested are cork, wood or plastic particles, hollow glass beads or other lightweight material, most of which would be rigid in nature.

In Specification GB-B-2 066 843 a continuous fermentation process for production of alcohol involves an agitated liquid biomass mixture. From the upper portion of the vessel the mixture is removed and allowed to settle and a separated biomass is returned by a simple mechanical recycle.

The use of bodies of plastic mesh on which to grow microorganisms is described in Specification GB-B-2 006 181. The bodies used in this patent are described as bodies having a substantial voidage in the internal structure, for example sponge-like or reticular constructions of stainless steel, plastics, glass or other non-corrodable materials.

In Biotechnology and Bioengineering Volume XXI, pp 193-200 (1979) there is described the production of biomass particles of given size, shape and density using the appropriate mesh support particles.

In Monograph-IX of the European Brewery Convention on Biotechnology held at Nutfield, Britain November 1983 in a paper number 13 by G.M.

Black there are described biomass support particles made from flexible, reticulated polyurethane foam or knitted, crushed stainless steel wire. These are indicated as being particularly suitable for yeasts which will readily occupy such porous particles. The desirability of simple mechanical introduction of the cells is that the production is much less expensive and one can perhaps more readily replace the cells by fresh cells compared to situations where the cells are chemically interlinked to the support mass. Chemically immobilised cells have been found to be subject to destabilisation which is not readily recoverable.

The use of biomass support particles, for example polyester foams, is also described in Biotechnology and Bioengineering volume XXVI pages 134-141 (1984). A polyester foam could be a material with a porosity of 0.97 and a variety of pore sizes from 10 to 80 pores per 2.54 cm (linear inch ppi). This paper refers to cleaning the cells by squeezing the foam to remove entrapped cells. The fermenters described are simple fermenters containing the biomass support particles. A circulating bed fermenter is described employing foam particles wherein the circulation is within the mass and does not involve removal of particles from the bed.

The density therefore of the bed has to be relatively low to allow for movement of the particles in the liquid.

Initially in the development of supported cell systems the biomass was in a tower fermenter in which the cells and support particles moved relatively freely. There was then developed the plug fermenter or reactor. The original type of plug fer menter was a solid bed of microbial cells and filterair earth, through which the liquid was made to flow by pressure or suction. In this type of reactor the cells and earth (which can be regarded as a form of support) were restrained so that the bed remained static and not free to move.

If the fermentation is allowed to continue in a mass of cellular particles supporting micro-organism without agitation, then cells of the micro-organism, particularly those well into the foam particles, will tend to die off and moreover will fill up the pores of the particles so blocking the bed and providing a less efficient fermentation system since they are not spread over a large surface area.

Moreover, additional flavours and undersirable flavours may be generated because of the presence of dead or dying cells, which, if not removed, will secrete abnormal amounts of metabolite into the mechanism. Particularly in fermentations for beverages such as beer, abnormality of yeast metabolites will result in unsatisfactory flavours.

In the present invention there is provided a method for utilisation of living cells, particularly yeast-based and other microbial fermentations, in which a container is substantially filled with densely packed particles of an open pore flexible porous material. At one point in the container there is an inlet for feed, e.g., fermentable, liquid which passes through the container. At a point in the container within the bed portion, there is provided an off-take for bed particles which are subjected to pressure to remove at least some of the cells from the pores of the particles. The particles can then be recycled to another point in the bed adjacent to the liquid inlet. The separated cells can be returned to the container and can be separated with the fermentation fluid at the outlet for of the vessel, or can be removed completely from the system.

The operation of this system can be continuous or cyclic depending on the condition of the bed at any given time.

The operation of the system provides in the dense structure of the bio-reactor bed, which is similar to the plug-reactor, a condition of plastic flow. Thus the invention permits an immobilisedcell bio-reactor in which there is co-current flow (as opposed to countercurrent or random mixing) of biomass and reactant solution. The speed of flow of the biomass is less than that of the reactant; the relative speeds (together with conditions such as temperature) can be varied to control the conversion rate and (to some degree) the composition of the product.

The present invention (a) permits higher bio-conversion than other bio-reactors per unit volume of vessel in unit time, and (b) simulates (to a greater or lesser degree) the course of bio-conversion in a batch vessel. Thus in a batch beer fermentation: fresh wort meets freshly growing yeast; half-fermented wort is in contact with yeast cells that are no longer growing, and whose assimilation and secretion of chemical compounds has changed; and fully fermented wort (i.e., beer) is in contact with yeast cells that have virtually stopped working.

This pattern is simulated in the passage of wort through the bed of the present invention.

In a preferred embodiment the inlet will be at the base of the container and the off-take in an upper portion. Such upward flow is appropriate for bioconversions which reduce the density of the liquid (e.g., for alcoholic fermentations) which probably constitute the overwhelming majority of fermentations. However, there is a possibility of producing temperature gradients by thermosetting different levels in the column at different temperatures. An example could be lager brewing, where chilling towards the exit could be necessary to make the yeast carry-out flavour maturation. In certain instances (but not lager) a fermentation could reduce the liquid density but chilling reverses this, so that the column would need to be inverted. Therefore in certain cases, the column could advantageously be run inverted or at any angle to the vertical.

To provide for concurrent flow of feed liquid and particles, the point of entry of fresh feed liquid and pressurised particles should be reasonably adjacent, and distant from the exit of product liquid and off-take for particles for pressure treatment.

The fresh feed could be in the particle recycle line.

The upper portion of the container can be provided with a retaining plate or other means of diminishing the cross section of the bed towards the point of removal of the bed particles. In the case of fermentation of beer, the foam or froth can be broken above the bed and carbon dioxide can be removed separately from fermented liquid produced.

Where the cells are separated from the cellular particles and returned to the container they can leave together with the fermented liquid through the same off-line. At or near the input for fermentation liquid there can also be provided an input for a small amount of oxygen.

For fermentation involving low alcholic content products the container may not require the foam breaker and carbon dioxide separater since the fermented liquid will leave under slight pressure to retain the carbon dioxide in liquid. Thus for brewed soft drinks for example low alcohol beers and drinks such a kvass with 1.2% alcohol there will not be much foaming as only a small percentage of carbon dioxide will be produced for example two volumes of carbon dioxide per volume fluid.

The term foam as applied to the froth or foam above a fermenting liquid ie. gas entrapped in liquid is to be distinguished from polymer foam as used for cell supports.

By a densely packed bed of particles of cellular material is meant that the particles of material are held in contact with each other as distinct from moving a fluidised bed/or relatively freely in a mass of liquid. However, they must be sufficiently free to permit movement of the bed upwards in the container i.e. equivalent to plastic flow.

As cellular materials can be used compressible webs or particles of metallic material or open cell polymer foam i.e. in which the cells of the foam are interconnecting substantially throughout the mass of the foam. The foam must be of a poly meric material which is sufficiently flexible to permit compression of the foam under the pressure of the system. Thus cellular materials as described in GB-B-2 006 181 or Biotechnology and Bioengineering Vol XXVI pages 134-141 (1984) could be used.

A preferred polymer foam is a polyester foam. The particle size of the foam particles could be from 2 mm to 200 mm maximum dimension which could be diameter in the case of approximately spherical particles, although the shape need not be spherical. Volumes of particles could be from 0.5 to 500 ml.

The invention also provides a reactor for utilisation of living cells particularly yeast and other microbial fermentations comprising a container, an input in one position in said container for feed liquid, an off-take in an opposed portion of the container within the portion of the container where a bed of particles can be located, a pressuring device in or at the mouth of said off-take to apply pressure to cellular particles from said bed of particles in said container and said off-take leading to a position in the container adjacent or at the entry point of feed liquid to effect recycle of cellular particles. Preferably the container is supplied with an off-take line for liquid product of the fermentation.

The fermenter can be provided with a foam breaker in an upper portion of the container above the normal bed content line and a carbon dioxide removal line optionally with pressure regulator.

The container can be a simple cylinder or other conventional shape for a fermenter vessel and could be of varying diameter in different portions of the container as in the known CCV (cylindrical conical vessel) used in batch fermentation for beer.

The pressure can be applied to the off-take by a pair of rollers or by applying pressure to the material passing into the mouth of the off-take by a horizontal cylindrical rotor which could be driven by external power sources. However other devices for applying pressure to squeeze the particles can readily be visualised. Preferably such a pressure device will simultaneously urge the bed material taken in, through the off-take to the intake of the container.

The invention is particularly applicable to yeast fermentation with production of beer with higher content of alcohol.

The invention can be applied to a wide variety of biological processes using plant or animal cells, particularly microbiological processes employing organisms which include yeasts and a wide range of bacteria. The conversions include brewing and wine making, and the production of power alcohol, antibiotics, food additives, industrial chemicals and enzymes.

The invention will now be illustrated in the accompanying drawings in which: Figure 1 illustrates a cross section of a container according to the invention.

Figure 2 represents a modification in the positioning of the pressuring device.

In Figure 1 the container is an upright vertical, generally cylindrical, container 1 filled to a substantial degree by a mass of polyester foam particles 2. Particles at the top of the bed are prevented from leaving the bed by retaining plate 3. An offtake 4 is provided for liquid in the side of the container at the highest point in the container. In the off-take line there is provided a foam breaker 6 and an off-take line 7 for carbon dioxide controlled by a pressure regulator 8. In the base of the container there is provided an oxygen feed line 9. An off-take line 5 is provided below the level of the liquid offtake line and below the upper level of the bed. Positioned a short distance from the inlet in said off take line is provided a pair of contra-rotating rollers 13.These are plastic coated and contain magnetisable elements so that they can be rotated by an external magnetic motor (not shown). From these rollers there is a recycle line 14 which feeds into the container at outlet 11 which is distant from the off-take 5. In the recycle line there is provided a feed line 10 for fermentable liquid. Inclined walls 15 and 16 at the top and bottom of the container assist in directing flow of the bed.

The container is filled with the polyester foam particles in amount sufficient to maintain a dense packing and with initiating bacteria or yeast and after a certain period of operation to secure a degree of fermentation, the rollers 13 are operated to withdraw material from the bed. As the particles are pressed in the rollers, bacteria or yeast are driven out of the pores of the foam. The separated cells and accompanying liquor can be separated at the rollers or the cells allowed to pass through the rollers into line 14 for recycle into the base of the vessel at outlet 11 together with the support particles. The latter may be appropriate where all that is desired is to open up the pores of the particles so as to allow fresh growth of the organisms. The drawing off of the bed material and recycle of the support particles to a distant point induces a plastic flow on the mass of the bed.Fermented product is drawn off by the off take line 4. The retaining plate 3 prevents escape of particles from the foam bed and maintains the density of the bed. In the case of a fermentation for say beer where a considerable foam is created escape of carbon dioxide is through the off-take line 7 through pressure regulator 8 after passing through the foam breaker 6.

In Figure 2 is described a similar system but with a different positioning of rollers. Again a vertical cylinder 20 is provided with an inlet 21 for fermentable liquid and an oxygen inlet 22. In the container there is a bed 23 of densely packed open cell foam particles. An outlet 24 at the apex of the container is provided leading to line 25 for removal of fermented product with retaining plate 26. A foam breaker 27 is provided in line 25. A recycle off-take 28 is provided, the inlet to which is within the bed and the outlet of which is distant from the inlet.

Close to this recycle line outlet are provided rollers 29 similar to those of Figure 1. The rollers subject the particles from the bed to pressure to release the micro-organisms from the foam. The rollers also draw the bed particles through the line 28.

The inlet 21 for fresh feed is positioned adjacent to the outlet of the recycle line.

The container or vessel in any of these embodi ments may be thermally insulated and the walls may be fitted with one or more cooling zones.

The product outflow for example through lines 4 and 25 in Figures 1 and 2 can comprise fermented liquid product and surplus microbial cells and also gas if present.

This invention relates to a method for the operation of a bio-reactor, ie, a process and apparatus for utilisation of living cells particularly micro-organisms in a liquid containing suitable nutrient material.

In Patent Application 8428541 there is described a bio-reactor and a method for the utilization of living cells in which a container is substantially filled with particles of porous material which are densely packed. The flow within the bed of densely packed particles is co-current with the flow of nutrient fluid. Particles are separated from the bed and cycled through a means of applying pressure to the particles. This dislodges cells from the particles so avoiding blocking of the bed and inefficiency in the fermentation system. In that patent application the particles are subjected to pressure to remove these cells from the pores of the particles. In the present embodiment of the invention the particles can be formed from materials which are less resilient than those described in the earlier application.

Thus for example particles of steel fibres or polymeric or other material which have substantially inelastic properties can be employed. So long as the particle has a pore or mesh structure such that there can be penetration of fluid into the interior of the particle and cells in turn can be reasonably easily removed from the particle, then the particle structure will be satisfactory for the purpose of the invention.

The present embodiment of the invention relates to the dislodgement of the cells from the particles by techniques other than the simple application of mechanical pressure. These techniques essentially involve the application of a pressure or physical effect which has the same effect as pressure, ie dislodgement of cells from the particles. Thus for example there can be applied to the particles jets of fluid, particular liquid, which penetrate into the pores or interstices of the particles so as to dislodge the cells. High pressure jets or very fine jets will have this effect. The cells can also be subjected to turbulent flow by the surrounding fluid which will break off cells from the particles. The fluid used for such jets or turbulent flow can be fresh nutrient fluid or recycled fluid.A high pressure gas could also be used for the purpose of the jets which gas is preferably inert, for example carbon dioxide. Thus carbon dioxide can be employed which is given off from a fermentation process.

The particles could also be subjected to a rotation which is sufficiently rapid to induce a centrifugal effect upon the particles so again displacing or dislodging the cells within the particles. In the case of turbulent flow a venturi effect which draws out cells from within the open particles by reason of the relative pressures. Such centrifugal flow can be induced either mechanically directly by drive on the particles or preferably by adjustment of the jets impinging upon the particles so as to induce rapid rotation.

In a particular form of the invention the particles can be formed from a magnetic grade of a metal or alloy particularly a magnetic steel. These particles could be wire knitted or crumpled or compressed or one or more of these states. These could be similar to the non-magnetic bio-support particles described in Black et al (Biotechnology and Bioengineering 198426 134). Where such particles are to be used in a fermentation system it is necessary that the particle be inert to the fermentation. Such inertness can arise inherently from the nature of the steel or by passivating the surface of the steel or by applying an inert coating such as polytetrafluoroethylene. Other materials which could be used are foamed polymeric materials as described in Patent Application No. 8428541 but incorporating magnetic particles dispersed in the polymer.For example a PTFE coated mild steel could be employed.

The use of such a magnetic material for the particles can have two functions in the system. In the first instance the magnetic properties of the particles can be used to drive the particles up through the densely packed bed and then through the recycle system to the entry point of the bed. In addition the magnetic effects on the particles can be used to develop the necessary impact on the particles to effect dislodging of cells. In particular the particles could be subjected to a magnetic field which causes the particles to rotate at high speed so creating the necessary centrifugal effect mentioned above which will dislodge the cells.

According to the invention therefore there could be provided a system as described in the earlier patent application in which the particles are formed at least in part from a magnetic material and at least one magnetic system is employed to recycle the particles or to cause dislodgement of cells from the particles. The magnetic systems could be two systems one for effecting recycle and a second system for causing dislodgement, eg, by rotating of the particles or could be a single system which applies both effects simultaneously.

Thus the vessel described in the earlier application would have in the side recycle limb the one or more magnetic systems just described. The magnetic system for driving the particles could of course be combined with other systems for dislodging the cells from particles as described in the earlier application and in the present application.

The invention is more particularly described in the accompanying drawings in which: Figure 1 illustrates an embodiment utilising magnetic particles which are moved in the system by magnetic means and the cells are dislodged by magnetic action on the particles; Figure 2 illustrates a similar system in which cells are dislodged by jets of fluid impinging on the particles; Figure 3 illustrates a system applicable to nonmagnetic particles.

In Figure 1 a vessel 1 forms a bioreactor having an inlet for oxygen 2 and a recycle line 3 at the lower end of which is wort inlet 4. At the top of the vessel is an outlet 5 for liquid. In the case of beer fermentation this will be an exit for beer, carbon dioxide and surplus yeast cells. A screen 6 of coarse gauze prevents exit of bio-support particles.

The main body of the reactor is filled with a packed bed 7 of non-compressible magnetic but non-magnetised bio-support particles. The sizing of the particles is as described in patent application 8428541. The particles are packed to a density to enable flow of the bed.

The particles exiting from the bed to the recycle line are subjected to pulsing magnetic field by resonator 8 which vibrates the particles to dislodge cells. The particles descend the recycle line 3 and are subjected to the magnetic effect of moving magnetic belt 9 which urges the particles from the recycle line back into the vessel.

In the second embodiment of Figure 2 there is provided, instead of magnetic resonator 8 an offtake re-circulating pump 10 which recycles liquid to inlet jets 11A, 11B and 11C. The liquid from the jets impinges on the particles to dislodge cells from the particles either by simple mechanical impact or by causing the particles to rotate and dislodge cells by centrifugal force.

In the case of jet 11C the effect of the jet may be sufficient to urge particles into the recyle line. In addition or solely there can be provided a rotating magnet 12 (or a belt similar to Figure 1) to urge particles by magnetic effect into the reactor.

In Figure 3 there is a fermenting vessel 1 with a double wall or inner wall 2 the space 2A between the walls providing a recycle line. A central rotatable shaft 3 has an archlmedian screw 4 which passes through an opening 5 in the lower portion of the recycle space 24 to draw material from the recycle space or line back into the main body 6 of the bed. The shaft is massive enough to remove heat from the reactor to a cold cone 7 which acts as a shaft bearing. The cold cone is cooled by refrigeration line 8 and the shaft is rotated by a drive means (not shown). From the upper edges of the cone to the wall of the vessel extends a screen 9.

Immediately adjacent to the screen (possibly located between the screen and cone) is a mechanical vibrating ring 10. Liquid passes through the screen to an upper space in the vessel with vent cock 11 and outlet line to a cylindrical separator vessel 12 with outlets 13 for CO2 and 14 for beer or other product. Yeast separated from the particles can exit through outlet 15. An inlet for nutrient fluid wort is provided at 16 which can enter a recycle line from the separator for start up with recycle at start up being urged by pump 17. A finings inlet can be provided at 18. An oxygen inlet to the bed is provided at 19.

Particles leaving the bed are subjected to vibration by ring 10 separating cells which pass with product and gas through screen 9 while particles pass into the space 2A and to the bottom of the vessel where they are drawn from the space and urged back into the vessel by screw 4. The effect of escaping liquid and gas at screen 9 upon the particles may impact on the cells and provide sufficient agitation to separate cells without additional vibration from the ring 10.

Claims (1)

1. A method for utilisation of living cells, particularly yeast-based and other microbial fermentations, in which a container is substantially filled with densely packed particles of an open pore flexible porous material; at one point in the container there is an inlet for feed, e.g., fermentable, liquid which passes through the container; at a point in the container within the bed portion, there is provided an off-take for bed particles which are subjected to pressure to remove at least some of the cells from the pores of the particles; the particles can then be recycled to another point in the bed adjacent to the liquid inlet, the separated cells can be returned to the container and can be separated with the fermentation fluid at the outlet for the vessel, or can be removed completely from the system.