SYSTEM AND APPARATUS FOR FERMENTATION IN SOFT CONTAINERS

Abstract: Fermentation or culture of microorganisms takes place in soft receptacles which are sterile when the fermentation starts. The soft receptacle contains the medium which is to be fermented. The receptacle, which has a flexible casing, is immersed in a liquid with desired qualities, for example with reference to the density of the liquid. The medium within the receptacle may be kept in motion by an outer influence of the flexible casing.

Description: System and Apparatus for Fermentation in soft containers The present invention relates to a system for fermenta¬ tion of initially sterile liquids by microorganisms in pure culture under conditons avoiding all infection of other microorganisms, the fermentation being either conti- nuous or not, the method using one or several soft contai¬ ners arranged in series or parallel to each other and being initially sterile or completely sterilized, as well as cultivation of microorganisms. The invention also relates to refermentation of liquids not being recontaminated by undesired microorganisms as well as cultivation of microorganisms. The soft container(s) consist of soft, flexible envelo¬ pes of a any general form, but especially cylindrical ones and of containers with se ispherical ends or other end sections submersed in a liquid possibly being water in which possibly are solved or dispersed solids, liquids or salts to provide the liquid with the desired features. In its simplest execution the system and the apparatus for enabling the function of the system comprise only one soft container for fermentation. Figure 1 shows: 1 soft container 2 liquid, into which the container is immersed 3 gaseous atmosphere at atmospheric pressure, in be¬ tween the liquid 2 and walls of the confinement covering the basin 4. 4 basin containing the liquid 2 5 rigid or soft roof (at choice, if the atmosphere 3 is air) aseptic circulating pump inlet for the sterile liquid to be fermented, aseptic inlet for sterile air or oxygen and rigously pure culture of the microorganism^s) to be used 9: aseptic outlet for fermented liquid. 10: outlet for gas being produced by the fermentation. 11: apparatus avoiding all contamination of the content in the soft container 1 of other microorganisms an those initially been admitted at 8. 12: apparatus maintaining the desired conditions in liquid 2 (temperature, content of oxygen <£tc.) 13: nets, false bottoms or conducting nets fδr distribu¬ ting of the immersion liquid serving to achieve local deformations of the soft diaphragm of the con¬ tainer. The whole system is sterilized before it is used and, if no accident occurs, the sterilization does not need to be redone during the lifetime of the system under the con¬ dition that the liquid to be fermented as well as the air or the oxygen are perfectly sterilized by methods known in the art before they are admitted into the system, that the culture is rigorously pure and asepticly admitted into the system, that the withdrawal of fermented and stored liqu¬ ids is aseptically performed and that the content in the soft container is kept in circulation, this being achieved by pumping or by a mixer, and also that the evacuation of the fermentation gases does not allow any microorganism others than the ones being used to enter the liquid, this being the object of a controlled fermentation of a selec¬ ted microorganism. The soft container at the start being empty and steri¬ le, is aseptically fed with sterile liquid and with pure culture and oxygen or sterile air according to the requi¬ rements considering the type of microorganism and thus the fermentation being used, the sterile liquid having a low content of gas. The liquid is mixed e.g. by pumping in a closed circuit. It is kept at the desired temperature and is completely protected against parasitic fermentation, the possibly occuring fermentation gases being evacuated from the upper part of the soft container to the exteriors or to a recuperating device passing through an apparatus eliminating all hazards of reinfection of the content in the soft container. The microorganisms are kept dispersed in the soft con¬ tainer, the content of which is kept in circulation, the deposits with a significant thickness on the containers soft diaphragm being regularly separated due to the exte¬ rior action on these soft diaphragms by localized forces causing deformations of the diaphragm; these localized forces are provided mechanically or hydraulically by loca¬ lized beams using the liquid in which the soft container is immersed. The circulating pump guaranteeing the circulation of the liquid during the process of fermentation is of a type with large capacity and low pressure or of a type with low capacity and high pressure, if the circulation of the con¬ tent in the soft container is maintained indirectly by a hydroejector, but this pump is preferably an aseptic pump of a new type to be described below (figure 2) or an ana¬ logous pump. Figure 2 shows: 1 motor 2 pump shaft 3 pump packing 4 pump casing 5 inlet for the fluid to be pumped 6 outlet for the fluid to be pumped 7 container with aseptic fluid 8 aseptic fluid 9 movable platform 7'and 9' Position for 7 and 9 when the pump is not im¬ mersed in the aseptic fluid, during repair and sterilization by heat. The elements 3. , 5, 6 of the pump are sterilized in a classical manner, amongst others with water or steam pos¬ sibly containing a product guaranteeing the distruction of micro-organisms, the container then, containing a surplus of stable and cold fluid 8, is lifted by the platform 9 in a way that the sterile elements of the pump, with which a reinfection could be possible within a few days," months or years is immersed in a considerable amount of cold anti¬ septic fluid. This fluid is chosen amongst a variety of possible com¬ positions so that it cannot damage the organoleptic pro¬ perties of the liquid transported by the pump or the health of the user if traces thereof are penetrating into the liquid. The aseptic pump described in figure 2 is badly suited for the purpose, if the quantity per hour is very large, of the order of 500-1000 m or more. For very large quantities the diameter of the pipes are lying between 0,5 and 1 m and even more and, moreover, it is preferred to design the pump in accordance with the figures of the drawing. In these cases the movable container 7 is not conve¬ nient. It is replaced by a chamber or a tank with large dimensions. The pump or the pipe including a screw, its shaft and the packing of the pump and also the flanges for connec¬ tion to the net of rigid piping are sterilized. The antiseptic liquid is then directly poured over the normally very warm and sterile parts, through which a re- infection could be possible if there is a microleakage, whereafter the tank is quickly filled with antiseptic li¬ quid, thus being equivalent to the container 7 in figure 2 Tight valves with very large diameters are not necessa¬ ry when, in case of a repair, it is important to isolate the very large pump of the soft containers , because, in case the pressure In the liquid piping does not surmount 0,5 kg/cm2, it is sufficient to flatten two soft sections of the piping for transmission of the liquid, one down¬ stream and the other upstream of the circulating pump, to be able to isolate the latter one. It then remains in a proper way to resterilize the part of the device which has been Infected and to reinstall the circulating pump for operation guaranteeing the total and permanent aseptic condition due to the antiseptic liquid. At request the exterior pressure on the soft envelopes can be maintained hydraulically for obtaining pressure balance between the inner and the outer parts of the enve¬ lope. The simultaneous use of soft envelopes and the sterili¬ zation with streaming steam nessicitates that the pump includes a steam inlet, an outlet for condensate and a device for feeding in sterile air or inert gas. The simplest antiseptic liquids suitable are concentra¬ ted solutions of caustic soda or potassium hydroxide as well as strong acids such as sulphuric acid, but some ten other compositions can also achieve the aseptic condition in simpler apparatus which not have been especially desig- nad for aseptic operation with one or several possibili¬ ties for microleakage which allow for reinfection of the product circulating in the apparatus. The latter can be a pump, a dispersing device, a colloidal grinding device, a continuous centrifugal separator and even a simple connec¬ tion consisting of dis ountable flanges or pipe fittings. A remarkable property of this new technology is that the preservation of the aseptic condition is guaranteed during extremely long periods of time in a very simple, reliable and cheap manner, the elimination of hazards for reinfection being total as long as, from time to time, a survey is made of the volume of the bactericidal immersion liquid and its composition. This new technology being necessary for apparatus not being available in an aseptic form presents a significant advantage over the aseptic forms known in the art, as the aseptic condition is completely guaranteed and only requi¬ res a minimum of survey, whereas the device responsible for the aseptic condition for aseptic forms known in the art requires a maintenance followed by a sterilization, the frequency of which can vary between once a day and once every 50-100 days, the frequencies creating hazards and downtimes which are not allowable for the fermenta¬ tions in question. The use of soft containers allows to avoid all gas movements during the successive stages of filling and emp¬ tying. To those skilled in the art it is evident how dif- ficult and uncertain it is to sterilize considerable amounts of gas. This problem has been eliminated. The immersion of the soft containers in a liquid results in the following advantages: - Reduction of stresses the envelope is facing and, consi- dering this, the possibility to create soft immersion con¬ tainers for liquids. - Protection against pillage by own will or by unfortunate accidents, by mankind or by animals. - Easy control of the temperature. - In conjunction with a suitable form of the surface, on which the container is placed to guarantee an almost cons¬ tant pressure which facilitates the emptying of the con¬ tained liquids. - Reduction or suppression of oxidation of the content in the containers. - Easiness to transform deposits attached to the soft walls into suspension which Is hydraulically achieved by localised stream of immersion liquid or by mechanical inf¬ luence. As for the oxidation the diaphragm forming the soft containers wall can be chosen as a function of what the liquid requires and the fermentation it is submitted to. The penetration of oxygen through the diaphragm in the soft containers and the soft or rigid envelopes or pipings which are connected thereto and in which the liquid can circulate is the only source of an involuntary oxidation of the liquid of fermentation. Depending on the type of fermentation the liquid requi¬ res extremely varying amounts of oxygen, ranging from zero to large amounts. 'Zero' corresponds to strictly anaerobic microorganisms and 'large amounts' to strictly aerobic microorganisms, whereas the necessary amounts for fermen¬ tation of alcohol, being of great economic importance, are small. In the latter case it is generally convenient to perform any admission of dry air or oxygen in carefully decided quantities during the periods in which the fer en- ted liquid is not withdrawn from the container in connec¬ tion with its packaging before a final sale. This rule however is not an absolute one, as even for finished beer, for which air is a horrible enemy, certain kinds of " dark beer such as 'Stout' from top fermentation can be improved by the presence of traces of oxygen. When It is necessary to reduce or to suppress oxidation the method of the present invention is using one or seve¬ ral of the methods below: - Complete or partial reduction of the content of - oxygen in the immersion liquid by physical, chemical or biologi¬ cal processes. - Protection or covering of the surface of the liquid, the speed of dilution of gases in the immersion liquid being suppressed or reduced. - Exchange of air at the surface of this liquid with gas containing little or no oxygen. - The influence of the immersion liquid on the pressure in the diaphragm is very important. For a 300m container with a diameter of 5 m and a height in filled condition of 3 (filling degree 80?) the density of the liquid to be fermented is 1,08. - When the container is situated in free air the pressure to be obtained is as follows: 2 o 2 , , 2 DH = 1,08 x 300 x 1 = 24,3 kg/cm 4 4 1000 In the container immersed in the liquid we get: 2 - n , 2 DH = 0.08 x 300 x 1 » 1,8 kg/cm 4 4 1000 When the container is immersed in water with a specific weight being brought up to 1,07 we get: DH = 0.01 x 300 x 1 = 0,225 kg/cm 4 4 1000 If the densities are identical inside and outside of the container it will retain at the most its initial form in the shown example being cylindrical, and thef softness in its walls is at the top due to the lack of pressure. Moreover, the container can under these conditions been filled up to 100?. The process allows the performing of discontinous fer¬ mentations, the container being filled, the liquid being fermented and the fermented liquid then been transferred to other containers, equal or unequal. This way of opera- tion is necessary for certain fermentations. In breweries a continuous or almost continuous fermen¬ tation is generally preferred, the fermented beer in cir¬ culation having a density of the order of 1,015 with an initial, already augmented density of 1,072 by way of ex- ample. - Pressure across the diaphragm: 0,3375 kg/mc . - CaC12 for addition to the water in the basin to equalize the densities +/- 2% . The choice of diaphragm and of the treatment for dega- sing the immersion liquid depends on the grade of anaero- bicity to be preserved in the liquid during fermentation. With a diaphragm made of high density polyethylene with a thickness of 2 mm and water in circulation, saturated with oxygen, the permeabiblity of the diaphragm is P = 3,2 x 0.2 = 3,2 cm oxygen/day/m . 0,2 3 3 If a container of 300 m under 24 hours receives 60 m dense wort saturated with oxygen, this would correspond to about 540 g or 336,000 cm oxygen, the diaphragm only being able to let about 1181 cm pass. In the same way gaseous oxygen must be injected into the beer flow to enable a sufficient large reproduction of yeast. However, if the container is used for performing a very slow secondary fermentation, it is obvious that a dia¬ phragm with very low permeability should be chosen. With a diaphragm with reduced permeability of the type 2 mm:s high density polyethylene with a barrier layer 'Clarene L' (Solvay) or of polyvinylidenchlorlde a minimum of permea¬ bility is obtained in the diaphragm. As the permeability of the barrier layer 'Clarene L' is 6 x 10 cm /cm/day/atmosphere/m , it is then for a film of 'Clarδne' with a thickness of 0,01 mm equal to 6 x 10 3 3 2 x 10 x 0,2 = 0,12 cm /day/m and the same figure can be calculated for the total sheet according to: I = 1 + 1 = 8,646 which lead ox- ygen per d neg- lectible figure. For an even better improvement of the anaerobicity the thickness of the barrier layer can be augmented or the content of oxygen in the immersion liquid be reduced. In practice it is better to reduce the leakage of oxy¬ gen in through the diaphragm as much as possible, as the same apparatus then also can be adapted to fermentations demanding anaerobicity or for aseptic storage of to oxygen sensible products such as fruit juices. Soft containers for fermentation are easily transformed to aseptic storage containers for sterile liquids, whereas the reverse proce¬ dure is more difficult. When it is important that the amounts of oxygen passing through the soft diaphragm should be absolutely negligib¬ le, the immersion liquid is degassed and is covered with a barrier film or a gas very poor of oxygen or these precau¬ tions are accumulated. For a diaphragm out of 2mm:s high density polyethylene barrier layer 'Clarene L' 0,01 mm; and degassed water in balance with a gas containing 1% oxygen, one gets: Permeability of the polyethylene layer: 3 2 3,2 x 0,01 = 0,16 cm oxygen/day/m . 0,2 Permeability of the 'Clarδne'-layer: 6 x 10 x lO x 0,01 = 0,006 cm oxygen/day/m Permeability of water: not calculated, as the water is in circulation. Permeability for the whole: you have I » 1 + 1 = 173, P 0,16 0,006 3 2 and therefore P = 0,00578 cm oxygen/day/m , and for a 3 3 300m container about 2,13 cm oxygen/day. The oxidation is zero. It is easy to eliminate the oxygen in the circulating water when it is cooled under vacuum or by replacing this gas with a neutral gas such as anhydride of carbon acid. It is preferred to feed the rigid containers from be¬ neath with deaerated and/or cooled water. These problems originate from the usual techniques as also the problems with the isolation of the containers, with receiving an atmosphere with low contents of oxygen and the design of a mattress or a film floating on the surface of the immersion liquid and limiting the contact between this liquid and the atmosphere. The soft buffert containers with the only task to asep- ticly store sterile fermentable products can in may cases be sterilic-d before they are put into place. The same is valid for certain soft containers, pipings and apparatus being used within the scope of the present invention, whereas other devices preferably are designed to be steri- lized before they are put into place and to be able to be resterilized each time it is appropiate to use e.g. a new pure culture of the microorganism in question or even another type of microorganism. It is always of interest to be able to sterilize at o rather high temperatures, over 100 C, the interior of such pipings and soft container being able to withstand this treatment, while it is necessary for the soft containers and envelopes in plastics being freely deformable not to pass the atmospheric pressure, the maximum temperature o then being 99-100 C. For these rare operations one can of- fer the time needed and nevertheless it is of interest, o rather than keeping e.g. 100 C during 20 hours to destroy most of the thermoresistant spores to increase the letha¬ lity of the steam or the humid heat due to one or several classical chemical products such as strong acids, alkali, formaldehyde, hydroperoxide, halogen and derivats of chlo¬ rine or iodine, quaternary ammonia compounds, nitrites, sulphuric acid. Germicides are added in gaseous form or in form of a mist in the steam flow or in a mixture of 'sterile warm gas and mist of warm water' , if there is a need for opera- o ting below 100 C. Then the bactericidal products are fully eliminated. The easiest way is to use pure steam for the diaphragm, the envelopes and the accessories out of plastics or elas- o tomeres re- sisting 100 C. In this case a final elimina¬ tion of a strong antiseptic medium is obtained, such as formaldehyde by rinsing carried out with pure steam. This eliminates the antiseptic gas phase while the clean steam condensate is rinsing the surfaces and removes the unclean residues in liquid state. The effectiveness of the treatment requires that a small regular inclination exists between the bassin of the first soft container and the opening for the final dis¬ charge equipped with a tight aseptic valve. A new technique for sterilization of a combination of pipes, soft envelope - rigid pipe or apparatus - soft en¬ velope consists of a sterilization of the rigid element in an autoclave and under pressure. Figure 3 shows the necessary sterilization device: 1 and 4: soft envelope 2: rigid pipe or rigid device 3: valve and possibly air filter autoclave lock for the autoclave inlet for sterile gas clamps for plugging the soft envelope If the envelope Is sterile from the beginning the clamps 8 are adapted and then the rigid element 2 is ele¬ vated between the reinfected parts. When the rinsing valve 3, terminated by a filter 9 being tight for microorganisms and permeable for gas, is open the autoclave is closed and the envelopes 1 and 2 are flattened between this part and the lock of the autoclave and then sterilized under pressure with pure steam. The pressure is slowly reduced and air or a neutral gas is admitted at 7 depending on the application. An analogue technique makes it possible to sterilize or to keep sterile such a paratus such as valves and pipings connected thereto, but then the autoclave is of a type shown in figure 4, where: 1-2-3: tight valves, operated automatically or by hand 4: body of the autoclave 5 : lock of the autoclave 6: packings, flanges, welded joints, etc. 7: packings allowing 5 to be moved towards 4. This apparatus is especially interesting for steriliza¬ tion units and for aseptic cooling independent of the fluid to be treated, as It allows to eliminate the hazards for reinfection and even to maintain at an elevated tempe¬ rature the interior of the tight valves equipped e.g. with diaphragms. They are a potential source for reinfection as soon as the part downstream of the diaphragm ceases to be sterile. o .When the product is sterilized over 100 C and the appa- o ratus over 120 C the bactoriologlsts know that by keeping o the part downstream of the diaphragm-valve at 100 C the main hazards are already avoided for a reinfection via the valve. All the more, if a higher temperature is maintained the hazards are reduced and thereby the hazard is zero at the sterilization temperature for the product, being a function of the pH-value. It is necessary to inhibit an infection of the content in the soft containers and their pipes, an infec€ion ente¬ ring by the gas exit as well as breakdowns due to too high inside pressure. Besides, a slight overpressure is desi¬ rable up to the soft containers and in the soft emvelopes to evacuate the gases. One of the devices ascertaining these three conditions is shown in figure 5 - It has the advantage to be able to function even during power failures. Figure 5 shows: 1 Inlet for gas arriving from the soft containers. 2 Entering of the gases into the gas holder 3- 3 Gas holder or rigid container open in the lower part. 4 Filling material or Raschig rings ascertaining a maxi¬ mal contact with the gas penetrating the material. 5 Perforated bottom supporting 4. 6 Tank 7 Antiseptic liquid 8 Pump 9 Distributor for the liquid 7 over the filling material 10 Inlet for filtered gas, air or a non condensable gas depending on the cicrcumstances. The. interior of the unit in figure 5 can be sterilized independent of the soft containers and their pipes, the most common case being a simultaneous sterilization of the entire system. The gases (steam and antiseptic agent or a mixture of sterile, non condensable gas, steam and antiseptic agent) being used to sterilize the soft containers and their ac¬ cessories and pipes are entering from 1 in at 2 and steri- lize the contents in the gas holder 3, being already par¬ tially disinfected by the circulation of the antiseptic liquid 7 by the pump 8 and the distributor 9. The liquid is heated up. When one Is sure that the sterilization is completed and that the pure steam or the mixture 'warm sterile water mist /sterile and warm non condensable gas' has been emp¬ tying the antiseptic residαes from the containers, the ac¬ cessories and the pipes, the warm or cooking liquid 7 is concentrated with antiseptic agent and the pump 8 is star¬ ted. The introduction of steam, or steam and sterile gas, is interrupted upstream of 1 and also in the liquid . The envelope 1 is sealed or an aseptic valve situated at 2 is closed and air or a filtered neutral gas Is lead in at 10. This is then completely rinsed and sterilized by the flow of antiseptic liquid sprinkling down on the agent 4. When the gases arrive at 2 the pump operates continu¬ ously, but the elements between 1 and 4 in figure 5 remain sterile, even if the pump is at rest during more than one day. It is easy to avoid a shortage of liquid in the tank 6. Also, the only important control -"unction is directed towards the content of antiseptic agent and the grade of impurities in the liquid 7. The choice of antiseptic agent is varying depending on, if the gas arriving in the gas holder is clean or polluted with organic agent. The antiseptic agent being scarcely sensitive to orga- nic agents is e.g. formaldehyde, sulfltes in a slightly acid environment andhalogen derivats of acetic acid. o Acidified water being kept above 70 C is also suitable, but it is preferred to reduce its pH-value to 1 by a non volatile acid such as sulphuric acid and/or adding an an- tiseptic agent as the aseptic condition must be maintained during power failures. Many fermentations and productions of microorganisms require an insertion into a pilot plant, in which the soft containers have a lower capacity per unit in the order of 1 m or less. The stirring of the contents in the small containers can be effected without a pump by exposing these contai¬ ners walls to deformations. The rotating, centrifugal or volumetric pumps being made aseptic are not in all cases suitable in cases of in- dustrial plants, especially pilot plants. The aseptic transfer of a sterile fermentable liquid, stored in a sterile soft container, to a fermentation con¬ tainer requests not only an aseptic metering pump but also that It does not allow the microorganisms to contaminate the sterile liquid upstream of the pump. Aseptic extraction of the deposits of organic material or of sediments of microorganisms in the conical bottom in a static decanting device does not permit foreign microor¬ ganisms comtaminating the outlet into the open air to penetrate into the decanting device, one microorganism being sufficient. The quantites per hour to be considered can vary extre¬ mely, the maximum lying in the order of 10.000 litres and the minimum at 0,0003 litres. This minimum corresponds to the sedimentary volume coming from 100 litres refermented material in one year. An aseptic pump of a new type with the required pre- standa is represented in figure 6 showing: 1: Part upstream of the rigid cofferdam of the pump. 2: Activating device or driving unit, normally shut-off by the action of a spring, for the suction valve of the socket valve type Stationary support on which the soft envelope 4 is flattened by the activating device 2. Soft envelope. Opening allowing the valve 2/3 to be opened. Flange or other tight connection between 1 and 7. 7: Body of the rigid cofferdam. 8: Inlet for liquid filling the rigid cofferdam and ac¬ ting upon the soft envelope 4 and upon the aseptic valve motors. 9: Tight valve 9': Possible container for surplus of liquid to fill the rigid cofferdam. 10: Equal to 1 11: Equal to 2, but this is an outlet valve or pressure valve, normally closed. 12 Equal to 3- 13 Equal to • 14 Rigid pipe. 15 Semi-rigid pipe. 16 Envelope. 17 Packing. 18 Equal to 14. 19 Equal to 15. 20 Equal to 16. 21 Equal to 17. The length of the pumps 7 body and the diameter of the envelope 16-4-20 as well as the form of the envelope bet¬ ween the valves 2/3 and 11/12 are functions of the desired quantity per cycle. An possibly sterile cylindrical envelope can be posi¬ tioned after that the valves 2/3 and 11/12 have been ope¬ ned with the openings 5 and 13. The cofferdam has been tightened with the packings 17 and 21, pressing the envelope 16-20 together against the semi-rigid pipes 15-19, supported by the rigid walls in the pipes 14-18. The semi-rigid pipes 15-19 serve as seals and also to support the soft envelope at the end of the rigid pipes 14-18 at the side of the cofferdam. They prevent the soft envelope to deform In a dangerous way. When the cofferdam is tight and the interior of the soft envelope is sterile or sterilized, the cofferdam is filled completely through the pipe 8 with cold water or other liquid with low boiling point. The surplus of liquid is led to the eventual container 9'. The valve 9 is closed. The pump is ready to operate as follows: A vacuum at 8 acts on the soft envelope at 4 and on the immersed motor of the activating device 2, withdrawing from the stationary support 3« The pump sucks up the li¬ quid at 22. When the envelope 4 is filled the vacuum is abolished at 8. The activating device for the suction 2 is closed. When a pressure at 8 has been transmitted to the interior of the cofferdam, the activating device 11 is opened, the pressure rising around the envelope which is flattened and discharges its content at 23. The valves 2/3 and 11/12 include one or several clamps compressing the envelope against the stationary supports 3 and 12 and, if necessary to avoid the presence of microor¬ ganisms downstream of the nonreturn valve situated in the interior of the cofferdam, at least one valve c the type shown in figure 7 must be installed, downstream of the discharge seal 21. Figure 7 shows: 23 Soft envelope 24 Rotating shaft 25 Flexible plate 26 Driving device for the movement of plate 25. 27 Rigid support on which the envelope rests. 25' The position of the plate 25 under control of dri¬ ving device 26 completely flattening envelope 23 against the support 27. 26' Driving device 26 in lowered position. This valve, supplied with a spring, is of a type nor¬ mally closed. The liquid filling the pipe 8 and the cof¬ ferdam 1-7-10 in figure 6 are entering the hydraulic motor acting on the driving device 26 and the valve in figure 7 opens even when the pressure in the cofferdam is dischar¬ ging the products in the envelope of the cofferdam through the valve 11/12. In figure 7 the compression of the envelope between 25 and 27 quickly discharges the contents thereof and also the interior of the end of the envelope is not infected, considering the fequency of cleaning and the fact that the soft envelope Is completely flattened over a considerable length between two parallel surfaces, i.e. the surface of the support 27 and the surface of the plate. Figure 7 is schematic and relates only to the principle of this valve, its plate possibly having another profile and evidently with a decreasing thickness at the hinge 24 at the end where the driving device 26 is acting. The security is increased by placing more than one val¬ ve of the type shown in figure■ 7 in series, downstream of the pump, and one can even exchange the nonreturn valve 11/12 in it with a valve of the type shown in figure 1. This valve can as a sealing device, instead of the spring 25, include a spring with double driving devices, being one the driving device 26 and the other a driving device parallel with the first one acting on the other end of the plate. Another device comprises a compressor for the soft, un¬ der pressure inflated envelope, which normally flattens the envelope but rises when this discharge valve has to be opened. In all cases the principle remains the same: The envelope is first flattened and from this moment sealed. Then the part situated downstream of this envelope is progressively plugged up. There remain only neglectible traces of pumped product in the flattened part of the en¬ velope and the quick emptying of the envelope in the downstream direction combined with the quick deformation of the walls of the envelope expels all parts of the pro¬ duct which can create bacteriological problems and the system also prevents an infection originating adjacent to the valve 11/12 and all the more returning to the valve 2/3 and, even worse, upstream of this valve. In figure 7, upstream and downstream of the aseptic pump, there is no rigid pipe in the soft envelope. For certain applications however, this is not the case. If one wishes to use a rigid piping downstream of the pump the valv(s) of the type shown in figure 7 can be re¬ placed by a socket valve or a diaphragm, normally closed, which is opened and closed at the same time as the valve 11/12 in figure 6. The ideal is to have at least two final valves in series and that the compression of the envelope or the diaphragm is less in the final valve. In pumps with a large capacity the diameter and the length of the cofferdam in figure 6 are substantial and thus the diameter of the envelope can ly near the diameter of the cofferdam. In this case it can be advantageous to have a disk at 6 and at 10 also comprising a packing of the type 17-21. There are then three cofferdams, i.e. 1-7 and 10. The cofferdam 1 is In connection with the source for vacuum and the cofferdam 10 with the source for pressure. Alternatively can vacuum and pressure been produced e.g. by a piston pump the suction side of which is connected to the inlet cofferdam and the pressure side with the dis¬ charge cofferdam. Pressure variations in the central cof¬ ferdam can be obtained thanks to interior and exterior pipes each of them comprising a valve for quantity control and even a spring device. This allows to reduce the load on the diaphragm due to the feeding to the opening of the valves. When the cross section of the emvelope is substan¬ tial between the valves it will be impossible to pass the envelope through the first packing, the sterilization of the interior of the envelope being achieved after the ins- tallation of the pump, with the consequence that especial¬ ly interesting envelopes have a relatively constant diame¬ ter. A partial elimination of material ih suspension in the liquid upstream and downstream or in the soft envelopes can be achieved by static decanting, possibly followed by cooling and even combined with an addition of a floccula- ting agent. If the absence of all foreign microorganisms is indispensable, a decanting device is convenient, with soft walls initially sterile and possibly having a conduit for evacuating the gas, if it is supplied with an aseptic pump acoording to figures 6 and 7. At least the bottom of the decanting device is conical or pyramidal. Its soft walls are deformed slowly but regularly with the purpose to loosen the deposits. The disposition of the conduits for liquids and gas in series of soft containers is shown in figures 8 and 10: 1 Soft container. 2 Circulating pump 3 Conduits for liquid. 4 Conduits for gas. 5 Conduits for gas. The conduits for gas are not shown in figure 10, being a side view. When a single pump makes the liquid circulate in a series of soft containers the loss of added material, the consequences thereof and of a pump failure must be limi¬ ted. The diameter of the conduits can also be augmented due to that these conduits permit workers and even certain ap- paratus to enter. This fact simplifies and improves the production and the installation of the units. In figures 8 and 9 possible domes are not shown impro¬ ving the collecting of gas. They are situated on the con¬ tainers 1 in figure 8 and on the conduits 4 in figure 9. These are details the importance of which is a function of the type of fermentation or cultivation to be performed. Figure 11 represents a cut through a building compri¬ sing a single soft container: 1-13 : See figure 1 . 14: Thermal isolation. 15: Gas sealing layer. 16: Exterior protection of the unit. The wall of the soft containers with large capacity facing their discharge openings or liquid outlets " is rein¬ forced and supplied with tension bolts or exterior cables limiting their displacement. They inhibit that the dia- phragm obstructs the opening and stops, if needed, the discharge and acts on an indi .tor for the discharge de¬ gree. Another safety device interrupts the filling before an excessive pressure is developed In the diaphragm. At least one part of the soft containers containing the liquid is Immersed. The proportion between the height of liquid and the height of gas can be any one. This depends on the type of fermentation or cultivation. The part of the container filled with gas can also been kept together with any mechanical means whatsoever to par¬ tially stabilize the form of the soft containers and to reduce the faible overpressure to be kept therein, this being achieved by the sterilization washing device descri¬ bed in figure • Example 1 Beer from top fermentation is significantly improved by refermentation in the bottle. However, the hazards for infection by foreign, i.e. wild fermentations and by bacteria, especially bacteria of lactic acid are significant, especially in cases of beer with low contents of alcohol. All the more as the beer, tapped at low temperature, must be reheated for a suffi¬ ciently quick refermentation. This concerns a scracely industrial technology with ac¬ companying hazards. -The curing - in the bottle requires at least 15 days, rather a month. With good conditions the quality is rising during about 3 months, even longer for strong beers. The beer, refermented in the bottle, is expensive as the storage applies to the packaged product. It has consi¬ derable hazards and many comsumers and retailers do not appreciate the presence of deposits on the bottom of the bottle. Therefore only small quantities of luxurious beer are recommended. The aforementioned example is using wort suitable for a top fermentation beer. The sterile wort is asepticly coo¬ led and Is stored asepticly in immersed soft containers. The sterile wort is kept in circulation in the aseptic storage container as a buffer and some part with internal haze or deposits in suspension is eliminated asepticly during its aseptic transfer to the soft container for main. fermentation containing yeast for top fermentation, lead in asepticly at the time of the start of the device. The o fermentation between 15 and 30 C takes 3-7 days under asepticly performed mixing. The continously fermented beer contains maximum 0,595-1,38% not fermented wort, and also either sterile wort or a sterile sugar sirup is lead in in an aseptic way at the time for the transfer to the soft container(s) for secondary fermentation. The surplus of yeast, mainly lumps and large cells are separated from this flow of green beer by a separator being accordingly regulated. The other fermentation performed during mixing _ o lasts 15-60 days at a temperature of between 15 and 30 C. The mature stale beer is then treated according to well known rules common for the brewery trade by centrifugation or filtration, carbonizing and is tapped on barrels, bott¬ les or cans. As the secodary fermentation allows to transform all fermentable extract into alcohol and eliminates the total amount of oxygen, the stabilization of the beer becomes rather higher than that of top fermented, not refermented beer. For the preservation of the special taste and smell properties from the refermentation the packaged beer re¬ quires very small contents of oxygen. This is achieved with new commercial tapping devices (less than 0,1 ppm oxygen after packaging) . If the production of wort is X litres per day the capa¬ city of the container(s) is varying for the main"fermenta¬ tion between 3X and 7X and that for the secondary fermen¬ tation unit from 15X to 60X and it thus is sufficient to adapt the capacity and/or the number of soft containers and their accessories to cover all needs from a pilot plant to a very large brewery. Example 2 This example concerns low fermentation beer. The slower o main fermentation is run rather below 10 C and the secon- o dary fermentation between 1 and 5 c « Continuous fermentation is once again not suitable and therefore it is performed in cylindrical-conical tanks without tryirg to achieve a carbonisation of the fermenta¬ tion with carbonic acid. The beer can be sold but it is purified by continuous refermentation. This requires a degassing of the beer, its filtration on fuller's earth or its centrifugation and then pasteurization. The sterile and cooled stale beer is then asepticly lead into the refermentation unit. This latter and the sequence of ope¬ rations are analogues to those described in example 1 with the exception of the temperature. The use of an aseptic buffer container is possibly jus- tified as a function of the discharge frequency of the tanks for the main fermentation. Example 3 The conditions are the same as for example 1, only that one fraction of the green beer after clarifying, pasteuri¬ zation and cooling is subjected to to a fermentation in a soft container with the assistance of a selected lactic acid bacterium. The proportions of products been treated with yeast or with a lactic acid depend on the requested beer type and even on the amount of extract which can be fermented by the lactic acid bacteria. The sour beer is filtrated, pasteurized, cooled and then asepticly mixed with the 'mature* beer. The mixture is subjected to a complementar " y maturing improving especially the colloidal stability. The stabilized mixture is then packed as in example 1 according to the rules in this technique. Example 4 A pilot plant has been erected for a quick start of a fermentation process. This device is using a number of soft containers, whose uniform capacity is less than lm . Except the apparatus and the devices of the type shown in figures 1-10 the unit comprises a big soft container, in which the quantity of sterile and cooled liquid to be fer¬ mented is stored protected from all oxidization and the aseptic accessories such as decanting devices, small hyd- rcyclones, sterilizable and movable filters and continuous mini-pasteurizers. The content of certain soft containers is mixed by pum¬ ping. That in other containers is only mixed by mechanical action deforming the diaphragm of the container which for¬ ces the liquid to circulate. Each soft container is immersed in deaerated water. The water serves as a control for the temperature and of the oxidization of the products via the soft containers, pipes and accessories. The parameters varying during the tests are several: the temperatures, types of microorganisms, times and quan¬ tities, contents of microorganisms, mixtures of products been fermented by the different microorganisms, elimina- tion of a surplus of microorganisms, decantation, centri¬ fugation, filtration. The tests have been carried out parallelly, each test using containers connected in series. The bad results usually obtained in classical pilot plants using rigid fermentation tanks and the high costs of this classical equipment impose the adaption of a method according the present invention. Moreover, only this method can be adapted to a unit in industrial scale.

Claims: Claims A system for fermentation or cultivation of microorga¬ nisms in a liquid, being from the beginning sterile or not, containing any unwanted microorganism and an appara¬ tus for the performance of the system, characterized by the following points, separately or in combination: A) 1. The system is using an initially sterile apparatus comprising at least one soft container immersed in a liqu- id with suitable properties, at least up to a level higher than the product contained; a liquid being kept in circu¬ lation, where either a fermentation or a cultivation is taking place in the absence of any foreign unwanted micro¬ organism, the soft conduits and other eventual units with soft walls containing a liquid are immersed, while the possible units and conduits with soft walls containing a gas, are not. These conduits can float on the surface of the immersion liquid. 2. The system of claim 1 comprising several containers connected in series. 3. The system of claims 1 and 2 comprising one or seve¬ ral decanting devices with soft walls initially sterile, immersed like the soft container(s) . 4. The Immersion liquid is contained in one or several containers of concrete, brickwork, metal, plastics or of earth and embankments possibly sealed by a plastic film. 5. The immersion liquid is cooled or heated. 6. The immersion liquid Is deaerated by a physical, chemical or biological process. 7- The immersion liquid is water or a mixture of water and solved salts or salts in suspension, increasing its density and/or its viscosity. 8. The transfer of liquids and gas outwards, upstream or downstream of the soft containers, is performed with the assistance of an apparatus avoiding all introduction of unwanted microorganisms into the pure culture or even all contamination with those of the content in the possib¬ le soft container and in the aseptic stock for the aseptic liquid to be treated. 9. A slight constant overpressure is maintained in the gas phase in the soft containers and in the conduits lea¬ ding the gases from there. I) An apparatus for performing the system characterized by the following points, separately or in combination: 1. The soft containers, 'reactors' or 'fermentation devices' are designed starting from soft diaphragm of thermoplastic or elastic material, possibly reinforced by textile and possibly beeing sterilized in the factory by common means before installation or sterilization after installation and before usage, all parts of the unit which could be contaminated are sterilizable and especially the connecting parts med devices for feeding in the primary materials and dicharging the obtained products. 2. The reactors according to Bl are immersed in a li¬ quid, preferably water possibly blended with salts or pro¬ ducts in suspension to regulate Its density with the pur- pose to reduce the mechanical load on the diaphragm and also to control the temperature in the interior of the reactor. 3. Pipe flanges are arranged in the bassin and in the close vicinity of the reactors, governing the pulsating immersion liquid against the walls of the reactors to tem¬ porarily deform the soft walls and thereby loosen the deposits having a tendency to build up inspite of the cir¬ culation been kept in the contents of the reactors, the circulation creating decomposed deposits. 4. The surface of the immersion liquid in the apparatus according to claim B2 can be covered by a floating film to reduce evaporation, heat transfer or transfer of gaseous products (e.g. oxygen). 5. The complete apparatus or at least the reactors and the bassins containing the immersion liquid are placed in premises with an atmosphere of controlled composition. 6. The diaphragm out of which the apparatus Is composed are chosen to limit the leaking in of oxygen into the mix¬ tures during the process of fermentation or stocking. 7. The composition of these diaphragms is 'chosen to avoid migration towards the liquid during the process of production of all harmful material and penetration of aro- ma to the exterior. 8. The diaphragm consists, as a consequence of the re¬ quirements expressed in claim 7, of one or several of the materials below: - Polyethylene of low, medium or high density - Ionomeres of polyethylene - Polypropylene and its copolymers - Polyvinyl chloride and its copolymers, softened or not - Polyvinyllden fluoride and its copolymers - Copolymers of ethylene and polyvinyl alcohol as that, or modified - Polyamides - Linear polyesters - Derivats of cellulose - Thin metal foils such as aluminum These materials are used in form of films either stre- ched or monoaxially or biaxially stretched. If the mecha¬ nical stresses require the diaphragm will comprise a tex¬ tile reinforcement on the base of natural or synthetic fibres, including glass fibres. For complex diaphragms (in several layers) the adhesion between the different components is achieved by thermal welding or by a glue or 'adequate means'. The diaphragms used can also be tissue made Impermeable with the assistance of elastomeres, e.g. polyisobutylene. 9« The diaphragms are assembled to form the 'reactors* for the pipes and the connecting parts between the reac¬ tors and the soft pipes or between the soft pipes and the rigid pipes out of plastics or metal. They are assembled by welding or gluing and, if necessary, with seams sealed by coating, gluing or welding. 10. The circulation and the homogenization of the II- quids in the apparatus is preferably achieved by circula¬ ting pumps, not excluding the use of classical mixers or deformations obtained on the soft diaphragms. 11. All connecting pieces and sealing devices for rota¬ ting shafts and other places which in short or long terms could permit the introduction of microorganisms or spores of these into the apparatus are immersed in an antiseptic liquid or are placed in hermetically sealed and sterili- zable containers, the immersion, if so desired, is precee- ded by an intensive irrigation with this antiseptic medi- urn, if it is indispensably necessary to impede all rein¬ fection during the short period which is needed to immerse the apparatus which originates hazards for reinfection by microleakage and which has been sterilized. 12. A gas washing apparatus, sterilized, in which the gases penetrate a contact mass irrigated in a closed cir¬ cuit with a cold and antiseptic solution or is kept at at o least 70 C and also antiseptic before it is spread by splashing into the antiseptic liquid for holding a con¬ stant overpressure in the gas washing unit, protects the interior of the gas pipes against all hazards for infec¬ tion by unwanted microorganisms, avoids too high inner pressure and maintains an adjustable and constant over¬ pressure in the soft conduits leading the gas as well as in the part of the reactors which are filled with gas. 13. An aseptic suction and pressure pump, sterllizable, with adjustable dimensions and uniform capacity allowing to avoid all passage of microorganisms in the direction of downstream-upstrea of the pump, which pump consists of a soft envelope being sterile in the interior before it is inserted in the pump body or sterilizable after been in¬ serted into place, a cofferdam and at least three aseptic valves of the socket valve type, normally closed by springs, of which at least two, one being a suction valve and the other a pressure valve, are placed in the coffer¬ dam and at least one, a suction valve, outside the coffer¬ dam. The rigid cofferdam comprises two or rather three sec¬ tions. The first one includes an opening with a packing as inlet to the envelope, at least one suction valve and at least one opening which can be sealed by a plug allowing a corresponding valve to be opened to the outside, for in- sertion Into the envelope. The other section of the cof¬ ferdam constitutes the chamber with variable dimensions, in which the envelope is situated, either with constant diameter or not, between the suction valve(s) and- the pressure valves, an envelope in which the deformations achieve the tranfer of liquids or gas. This central cof¬ ferdam can also comprise inlets for driving fluid, a li¬ quid with low boiling point such as cold water as well as a spillway equipped with a valve and if so required con¬ nected to a container receiving the surplus of the driving fluid. The third section of the cofferdam is identical with the first section but comprises at least one pressure valve. The packings compress the wall of the soft envelope against an interior semirigid pipe, in which a rigid pipe is situated. The semirigid pipe extends over the rigid pipe towards the valve alongside the cofferdam and avoids being progressively deformed, that the envelope is defor¬ med too much alongside the rigid pipe when it is flatte¬ ned. The fluid with pressure modifications in the coffer- dam, acting upon the envelope and the valve driving devi¬ ces, acts with the assistance of a pipe conduit on the driving device of the pressure valve(s) situated down¬ stream of the cofferdam, at least the latter one of these being of a new type, which is self-purifying (according to the description of the application, pages 18-19 and figure 7), characterized by a plug action in the envelope. This flattens first the envelope and increases simultaneously the flattening pressure as well as the length of the flat¬ tening zone downstream, which zone is completely empty of liquid. 14. The pretension of the springs holding the normally closed valves is regulated so that a pressure valve beco¬ mes a safety valve for the mini-container, securing its exit. 15. The soft envelope for the transfer of the pumped liquid can be protected externally by an indipendent enve- lope out of plastics, elastomeres or textiles, its role being merely mechanical.

Inventors: COLINET EDOUARD, LEFEBVRE HENRI J L, LEFEBVRE PAUL HENRI, COLINET, EDOUARD, LEFEBVRE, HENRI, J., L., LEFEBVRE, PAUL, HENRI
Applicants: ALFA LAVAL FOOD ENG AB, ALFA-LAVAL FOOD ENGINEERING AB
Application year: 1986
Main classification: C12M1
IPC codes: C12M1