DE1642653A1 - Fermentation Process and Apparatus - Google Patents

Description

Fermentation process and apparatus

The present invention relates to a fermentation process using a multi-stage "3-stage retention device, which comprises the stage of aerobic or anaerobic cultivation of microorganisms," wherein the device has a set of vertically superposed chambers, which are separated by horizontally arranged "plates with one or more holes are separated. The invention also relates to the presentation device.

The present invention relates to a fermentation process using a multi-stage fermentation device and in particular a fermentation process / according to which Microorganisms are cultured aerobically or anaerobically in a perforation device with one or more plates

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is equipped, the oich horizontally by dleae hinduroh extend, each of these plates having one or more openings.

In industry, microbiological fermentation is usually carried out in a batch process. Continuous processing techniques have been sought, using either a single fermenter or several such fermenters arranged in series »but no technique has been developed so completely that it is suitable for technical purposes. Generally, a continuous single fermenter process is carried out in the case of growth associated fermentation in which the formation of a desired product takes place along with the growth of the microorganism in the culture medium. It is also carried out with more than two fermenters in the case of non-growth fermentation, in which the formation of a desired product does not occur when the microorganism is growing but occurs after the microbial growth ψ has ceased . In the latter case, the continuous pigmentation must be carried out with more than two fermenters, each of which has a particular cultivation phase, different from the others, so that microbial growth and product formation take place separately during the process. However, this requires additional equipment, such as a ventilation system, a device for moving, a metering pump, etc. with a Ka pa-

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zit & t »which is proportional to the number of permers used 1st. When used on a technical scale, let the procedure be carried out on the basis of a stationary state required system very expensive.

The present invention relates to a fermentation process "in which" a permenter is used which has several chambers and has novel operating characteristics. These characteristics can provide better and more effective results be achieved. With this device, not only can discontinuous and continuous processes be carried out successfully, but also synchronous breeding and Obtaining the cells can be done very effectively. In addition, the above-mentioned disadvantages of the known continuous processes, both economically and functionally, can be completely eliminated due to the following advantageous features: (1) The Device has a simple construction j (2) it does not necessarily require a device for moving, da the liquid is stirred well by aeration (2) it does not require a pump to transfer the broth from a vessel in another (4) it is easy * to maintain the steady state; (5) the operation is simple and easy to automate; (6) The cooling and heating takes place with high efficiency due to the large heat transfer surface.

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That is, the internal volume j (7) a solid substrate material is used in the fermentation (hereinafter referred to as "solid fermentation"), bo the dispersed solid particles are kept in the column by the operation of the flow stage device, which usually results in a good yield in relation to the The raw material used is achieved «since there is little material loss due to the overflow of the liquid auitrittj (8) during solid fermentation, it is neither necessary to pulverize the material nor to use a solvent which can be harmful to the microorganism. Another important feature in continuous operation is that in the method according to the invention the cell age in the fermented broth is kept more closely related to the average residence time than in a single stirred tank in which the cell age in the fermented broth is between zero and is infinite.

The multi-stage used for the process according to the invention Fermentation device essentially consists of a column forming the housing of the fermenter and one or more columns Plates, each of which is provided with one or more holes, the plate or plates being horizontally transverse to the Fermenter is or are arranged so that it is divided into sections iefc. Structurally speaking, the housing absenteeism wex of the fermenter, each of which is open and closed at both ends

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flanges is «placed on top of one another * with a plate provided with a loophole or holes being inserted between two sections / so that a series of chambers is formed » which are closed by covering the top and bottom. (When the first chamber is referred to as "second chamber, etc." in the following, each chamber is numbered from the bottom upwards.) The device has inlet and outlet openings at both ends for both air and medium, as well as heating and refrigeration equipment (such as a jacket), a thermometer * tube (or faucet) for sampling, and other ancillary equipment that may be useful.

The fermenter will now be described in more detail with reference to the drawing. Show it:

Fig. 1 shows an axial section through the for the invention Method used fermenter;

Fig. 2 is a plan view of a perforated plate;

3 shows an axial length through a different embodiment of the fermenter i used for the method according to the invention

. 4 a graphiäsoft « Baimt ^ ltimg dar Hesishung between the ation ιηχά der äureh ^ ohtilttilJksii Vsfweil & äifc In Km-tm? In the Verglaiuh to a WaeiHöt '* £ äii & vi2 »tfe bsi d: la» ko »^ llQhu & s, gmuäss attempt h-, al® aaigt, uA & n die

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Cell concentration in a continuous process below Use of the multi-level ferrule according to the present one Invention reached a steady state j

Fig. 5 shows the operating status within each canine of the Permentere " the culture medium is in fermentation;

Fig. 6 shows the overall flow characteristics of the ferment or s in With reference to various combinations of hole diameters one Plate with the ratio of the opening transverse son of each plate to the overall cross-section of this plate using ordinary water according to experiment tj and

Figure 7 also shows the overall flow characteristics of a fermenter in relation to different combinations of the hole diameter a plate with your ratio of the opening cross-section Using each plate to the total cross-section of the plate an actual fermented bread according to experiment 2.

Bs Referring now to Fig. 1, reference "a cylindrical multi-stage fermenter in vertical section showing the _s are arranged as Trennviandungen consists of four chambers (1)» the superposition with the plates (2). (Fig. 2 shows the surface of an oil plate.) Each chamber can be equipped with a temperature regulating device, such as, for example, a jacket (5) and an electric heater, and the headgear (5) and the bottom (6) are equipped with a pair of Pipes (

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connected to the inflow and removal of air or Enable medium. These pipes can be connected directly to the chambers * and in this case end parts (5) and (6) not required.

The fermenter usually has a number of chambers of uniform shape and size arranged one above the other, but it can also have chambers which are different in shape and size. The main thing is that the fermenter is divided by a plate or plates, each of which has at least one opening. The size, height and width of the fermenter can therefore be freely selected, and the shape of the chamber can be partially spherical, as in Flg. 3, or oval or cubic. It is better that the overall structure of such a fen entere, when all the chambers are connected, be elongated. In a special embodiment of the fermenter, as it was used in the experiments described in some of the following examples, each chamber has a height of 12 cm and a diameter of 7 mm, each plate has 100 holes 2 mm in diameter, and "the total cross-sectional area > occupied by the holes is approximately 8-10 # of the total cross-sectional area of the plate. The values given here are only an example of the relationship between the hole diameter and the opening ratio of a plate to the total plate cross-sectional area. The combination of these sizes varies, as will be discussed below, in a wide range.

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The housing parts can be made of metal, glass or plastic. A transparent material is useful, however, because the inside of the fermenter is then visible during operation. The material from which the panels are made is 1st likewise freely selectable, but the constituents of the medium and the product produced as a result of the fermentation should contain the act on the plate material and this can corrode »be considered. Sometimes wire mesh panels are used or porous or perforated plates are used. The size of each hole and the number of holes (opening cross-sectional area) are caused by such factors as oroese of the fermenter and type the desired microbial response. The number of Chambers is determined by the best conditions from the standpoint of such factors as desired fermentation type, height and volume of the fermenter, ventilation system, pressure drop and the like. certainly. Each inserted plate can be screwed to the flanges of the housing parts with seals in between. Sampling, filling and / or discharge lines (or taps) (7), which are mounted on individual chambers, are also included useful for the control and regulation of the fermentation as they allow the exit or entry of liquid contents. An air filter is provided as in the usual fermenters. Ultimately, this multi-stage Ferraenter can either do it alone or used in a group.

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Special features of this fermentation device, as described above can be seen from the following experiments:

Versuoh 1

The effect of the plate quality, ie the diameter of the holes and the ratio of the perforated cross-sectional area of each plate to the total cross-sectional area of this plate * on the flow characteristics of the fermentation variant used for the process according to the invention was investigated. The device was as shown in Fig. 1, divided by perforated plates into four chambers "each having a DUI * © Haesser of 7 cm and a height of 12 cm had. Water was pumped into this device from the bottom at a rate of 6 liters per hour and flowed off from the uppermost chamber. On the other hand, air was drawn in from the floor through the nozzle attached to this at a rate of 10 liters per minute.

When the air and liquid flowed in steady state, a tracer solution was passed through the water inlet £ -functional injection introduced. The change in tracer concentration was determined at certain time intervals at the outlet of this device, and a sensitivity curve was i.e., susceptibility to disturbance. The overall flow characteristics were expressed by the corresponding numbers of the containers arranged in a row, as shown in Fig. 6 is shown. When each chamber of the device is

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dependent on the. other located in the steady state with ventilation »so would be the corresponding number of in series arranged containers four. If, however, any looking back takes place under the chambers, the number would be less than four. This means that a multi-stage fermenter according to of the present invention behaves in the same way as the corresponding number of containers arranged in series P with the same volume.

Using the plates with holes 2 mm in diameter and a ratio of the total hole cross-sectional area in each plate to the total plate cross-sectional area in the range of 1 to 30% , the behavior of the heel was not affected. With a hole diameter of more than 3 am, however, the opening ratio should be smaller in order to maintain the heterogeneity in the fermenter. From this experiment it can be concluded that very complicated relationships exist between the hole diameter and the ratio of the perforated cross-sectional area of a plate to the oesarat cross-sectional area of this plate in order to achieve an optimal condition for the combination of the two. The above experiment was based on the pure water-air system and the situation is therefore largely influenced by the system used.

Attempt 2

The effect of the plate properties, as described in Experiment 1, on the flow characteristics of an invented

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according to the fermenter was examined, this time an actual fermentation broth was used *

Xanthomonas eampestoris E-J24 was cultured at JK) ⁰ C for two days in a medium containing 3 £ »u2.ur.ose> Ö» 2 & Pt on, 0.2 % fumaric acid * 0.2 % KH ₂ PO ₂ ^ 0 * 2 # Ns ₃ CO ₃ and 0 * 05 % MgSO ^. 7HgO and adjusted to pH 6.0 with ITaOH. The viscous culture broth obtained above was used to determine the flow characteristics of this ferment in the same manner as described in Experiment 1. The results are shown in FIG.

Using the plates with holes 5 mm in diameter and an opening ratio in each plate in the range of 1 to 40 % , the heterogeneity in the fermenter was fully maintained. With a hole diameter of more than 8 mm, on the other hand, the aperture ratio should be smaller in order to avoid the heterogeneous! did maintain.

From the above experiments 1 and 2 it can be seen that such a Relationship is a complicated function of fluid properties such as density, viscosity and surface tension Aeration rate »the liquid flow rate * the distance from one plate to another, deε the diameter of a plate (the size of the fermenter), etc. is. The choice of plate regarding the above coordination of hole diameter and off-

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The ratio of the fermentation also depends on the fermentation object. Taking into account the factors described above, the optimal properties of a plate should be determined individually for each experiment before the Percenters are actually used. Although it is very difficult to generalize such plate properties, the most frequently used combination is in the range between 1 and 20 mm for the diameter of the holes and 1 and 40 % for the opening ratio.

Attempt 3

A zuohtungsmedium (pH 7 * 0), each 1 1 g Oluooee, 0.5 g yeast extract, 5 * 0 gm ^ Cl, 1.0 g NH ₄ NO, 2.0 g Na ₃ SO ₄ , 0.1 g MgSC ₄ . 7H ₂ O, 3 * 0 g of K ₃ HPO ₄ and 1.0 g of KH ₂ PO ₄ contained »was introduced into a cylindrical fermenter with a height of 130 cm and a diameter of 7 cm, through five transversely arranged plates was divided equally into six chambers.

The inoculation was carried out with 5% of an inoculum of E. coil B-548 * which had been grown for 18 hours. the Supplied air flowed through the fermenter at a linear velocity of 7 cm / second. After the onset of microbial growth, the air in each chamber filled an empty résumé in the upper part of this chamber against the bs-

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sensitive plate. A certain amount of liquid that is in every single Cairo was held "became dependent on the amount of air supplied »the diameter of the holes * the focal ratio in each plate, the physical view Properties of the liquid and the like. Determined. This certain amount of liquid was kept in each chamber, whereby infrequent mixing with the liquid in the adjacent chamber occurred. In this way each chamber worked separately and individually as a single fermentation device.

The stages of bacterial growth in the culture in the respective Chambers were observed and recorded (Table I below). In summary, they differ Stages of growth from one chamber to another, and the differences between the values obtained lie »outside the range of statistical errors. The values in Table I give the optical density unit (hereinafter referred to as U.O.D .: optical density at 610 πΐμ χ number of dilutions) *

Table I.

chamber

(from the ground) U.O.P. to different breeding hours

0 1 2 3 4

No. 1 0.10 0.220 0.421 0.756 1.02

No. 2 0.10 0.210 0.412 0.74} 0.98

No. 3 x 0.11 0.207 0.410 0.735 0.95

No. 4 0.11 0.205 0.405 O _: 731 0.95

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Attempt 4

According to the above procedures, fermentation was carried out in another fermenter, which this time was divided into 10 chambers with equally spaced plates, each plate having holes with a diameter of 2 mm and an opening ratio of 11 % .

fe After culture initiation for 3 hours, a medium with the the same composition as in Experiment 3 is fed at a constant rate from the bottom of the permenter. The dilution rate, based on the total volume of the? Medium »was 1/5 per hour. In particular, the amount of zugefUhrtem mo medium was adjusted so that the Verdünnungerate did not exceed the maximum specific growth rate of the microorganism in the soil chamber, excess liquid which has been passed through the fermenter up "VOA flowed over the upper end.

ψ The differences in cell concentration from one chamber to the next became more apparent with the passage of time, and after 17 to 20 hours, the gradient in the cell concentration became steady. The cell concentration (U.Q.D.) at this condition and the nucleic acid content depending U.O.D. in the individual chambers are given in Table II. Also in Fig. 4 are the cell concentrations in some Chambers shown as a function of the average residence time, for energy calibration purposes together with the growth curve for discontinuous culture.

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• Table II

Chamber Cell Concentrate Nucleic Acid (from the bottom ) (U.0.D.) r-HNS / UOD

No. 3 0.310 15 * 0

Mr. 5 0.492 10.0

Mr. 7 0.698 8 * 3

Mr. 9 0.792 8.0

No. 10 0.810 7 * 9

The above two experiments J and k show * that the extensive fermentation device has the following features: When microorganisms are "grown" in such a fermenter as in the experiment, each chamber acts as an individual fermenter in which the air adjoins the upper part of the room fills the plate above and acts as a cushion here. With other fourths, “the medium within one chamber is thoroughly mixed by aeration , but it hardly mixes with the medium in the adjacent chambers. The content of ropes therefore varies between the individual chambers.

On the other hand, if the culture medium is introduced at a certain rate from the bottom of the percentile, ie the fermentation is carried out continuously, * the distribution of the cells is somewhat different. As the medium in each chamber is well stirred and mixed, an amount equal to the amount flowing in from the bottom moves upward from one chamber to the next through the holes in the top plate. Hiei ^ ei prevent Foktoran _r ι * ίη tfie Eei.tnang at the PlaV.to: V. £ 109882/0298

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the liquid housing, the aeration and the like. * The medium flows on and so ₅ that the air fills the upper part of a chamber under the upper plate ₉ as if all chambers are operated separately as described above. These conditions are shown in FIG. However, the upward flow and supply of the medium are carried out quantitatively. If the medium is thoroughly mixed in each chamber _s takes the flow of medium as a whole the significant form of a piston flow. The medium is thus pushed upwards and drawn off quantitatively at the upper end of the fermenter. The same principle can be applied to operation with a battery of fenenters * arranged in series «.

Experiment 4 shows that * if the growth is stationary State reached, each chamber holds cells at the average residence time. The cell concentration therefore varies with it the chambers. More specifically, the cultivation is continuous under such conditions * that the gels on the ground are always at the beginning of their logarithmic growth phase, those in the middle in the intermediate phase and those at the top in the final phase »by one appropriate coordination of the flow rate of the medium with the rate of cell growth. Becomes a desired product in cell growth or formed during a certain period of the growth phase, so the different distribution of the product concentration occurs in the same way on.

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The fact that the difference in the cell concentration in the respective chambers is based not only on physical factors was demonstrated by means of the biological activities of cells that were removed from the respective chambers “for example by the nucleic acid content of the cells. It is essential * that when each chamber is operated as a single fermenter device in a continuous process, the ratio of the volume of the chamber to the flow rate of the medium * ie the dilution rate * does not exceed the maximum specific growth rate of the microorganism. The cells obtained from the upper reaches therefore have a certain average residence time or a certain cell age under stationary cultivation conditions. The more the fermenter is subdivided, the more the overall process works according to a perfect piston flow pattern and the clearer the pattern of the cell concentration gradient becomes. This can be proven both theoretically and experimentally.

A continuous process has been described * with soft the medium is introduced from the bottom of the fermenter / however, the same basic procedure can also be used in reverse, introducing the medium from the upper end without Sinuses of the advantageous characteristics of that already described Procedure to be carried out. In this second case, the distribution of the cells naturally follows from the top to the ground. In the case of anaerobic fermentation, inert

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gas s> such as nitrogen or carbon dioxide and / or gas bubbles developed in the course of fermentation have the same effect as achieved with air or aeration in aerofcer fermentation.

The creation of the new fermentation device described above enables a new fermentation process to be used the characteristics described above in the field of microbiological reactions that differ from purely chemical Distinguish reactions. Some examples of fermentation processes using the device according to the invention and various types of materials are given below ί

(1) Ordinary fermentation

Many examinations and studies of the usual fermentation have already been carried out. " but none of them applied a method like the one according to the invention. This is particularly effectively applied to a continuous fermentation process. Initially, as in a batch process, the medium is introduced into a fermenter ₃ inoculated with the desired strain of microorganisms and cultured with aeration at a suitable temperature. No moving is required because the plates with the holes do the job. After a certain period of time, additional medium is added in a volume that depends on the capacity of the fermenter and the specific growth rate of the strain »

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depends, introduced? in order to obtain the desired final broth at the outlet of the fermenter »As shown in experiment ¹ I. -» the cell age varies from chamber to chamber, and the cell age distribution in the overflow is very similar to a piston path characteristic. It is easy to see that a continuous process is distinguished by its high productivity compared to a batch process.

(2) Solid fermentation

There have been some reports of fermentations * in which solid materials are used * but they have hardly been developed for industrial use with the exception of steroid fermentation. The fermentation of aromatic substances, such as naphthalene and Anfchracan * the use of solid paraffin in fermentation and the oxidation of steroids are some examples of solid fermentations « which to a certain extent have been successfully carried out on a laboratory scale. When performing on a technical scale however, there are various problems and adequate yields are not always obtained. A major problem relates focus on the treatment of solid materials. The solid materials mentioned are those which are insoluble or in water are sparingly soluble. A microorganism would only absorb the very small portion of the poorly soluble materials in the Water dissolves »assimilate and produce a desired product from it. It is therefore both theoretical and experimental

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Raentell verifiable * that the rate of dissolution of the material in Water is the limiting factor for the growth rate of microorganisms and the production rate of a undesirable product and that the productivity per unit of time in long-term operation is low. To increase the rate of dissolution of the solid Material, the material is liquefied * by first pulverizing it is used to create a large surface, and then a large amount is given in water or by putting it in one Solvent is dissolved in water before adding and then finally by vigorous agitation.

This method not only requires an extraordinary amount of time and labor and equipment, but also results in a considerable loss of material. The solvent often impaired the growth of microorganisms _s also having problems in the choice of materials with regard to the quantity and quality up, and finally the cost is certainly not negligible. In addition to the above problems, the powdered materials put in water sometimes tend to "go to the colloidal state." When used in large quantities, unused solid materials are also lost in the withdrawn culture broth. This problem occurs in both discontinuous and continuous processes, but especially in the latter, since a certain amount of medium flows out of the fermentation device.

The present invention enables all of the above problems to be overcome. In the discontinuous process

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the fermenter does not need to have a special facility for fulverization or emulsification of the solid material, and a greater amount of solid material than is used is usually used. This proportionally increases the surface area of the solid material per unit amount of the medium and does not limit the growth rate of the microorganism, thereby overcoming the limitations that are easily encountered in the ordinary solid-permeation method. Then a medium is introduced into the permenter ₉ which contains certain components essential for the growth of the microorganism, and the temperature is _{adjusted when the fermentation takes place 3} and air is sprayed in. With the discontinuous process, it is not necessary for each chamber to act as such * and there is a free choice with regard to the size and the perforated area of a plate. The holes can be large. Even a coarse wire mesh can be used. The choice is made depending on the microorganisms and the material used. The plates fulfill several tasks here, namely to keep the material in the medium, to break up the upward-flowing air introduced from the bottom, so that the air in the medium spreads in all directions * and so does the medium and keeping the air in close contact, resulting in a high rate of oxygen inflation.

After fermentation, the fermented broth is drawn off and subjected to the isolation process of the product. After peeling the liquid phase, solid material remains in the

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menter that did not react with the microorganism, and a further fermentation can be carried out by simply adding the medium. This can be repeated many times until the remaining solid material is completely consumed. So the process in this fermenter becomes free carried out by the disadvantages, the always ordinary fermentation process using solid materials.

The continuous process is started in the same way as the batch process. There is however much _s that each chamber separately and worked individually, and it may be necessary /. Using plates with smaller holes, but without making them so small? that a loss of air pressure is caused or foreign elements can clog the holes and prevent a smooth flow of the liquid; and the like. The size of the holes and the perforated area of a plate should be determined by such factors as the height of the column used, its cross-section , the ratio of column height to cross-section "the distance from one plate to another (height of a chamber) / the physical properties of the medium to be used, the amount of air supplied" the type of fermentation and the like. These are determined before the start of the process.

A certain time after the inoculation of the microorganism and after the start of fermentation, additional medium becomes available from the soil off quantitatively with ventilation at a controlled temperature

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fed * and the fermentation is carried out continuously for as long as desired. The aeration causes a thorough movement of the medium in each chamber, excess medium, which has been pressed up by the supply from the bottom, flows over ₃ at the upper end, the amount of which corresponds to the supplied amount. This creates a cell concentration gradient from the bottom chamber to the chamber at the top in the direction of the medium flow.

If a solid material is used as the sole source of carbon for the microorganism to be grown, it should be used in the individual chambers are introduced before the start of the proceedings. In this case the amount that goes into each chamber is to be brought in;, through the relationship of cell age to the solid material to be consumed is determined. If the material is not a direct fermentation source but a substrate, on which one by the microorganism during fermentation generated Enayni works {in the steroid oxidation), and the production of enzyme has a certain relationship to the cell age, the continuous fermentation proceeds with a multistage Fermenter even more complete. In other words, the action of an enzyme produced by a microorganism can be effectively induced by only introducing the material into the chambers, the cells of one certain ages that produce this enzyme »contain, as the cell age in the respective chambers varies. Da3 continuous Procedure can be continued until the material is completely covered.

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p) Fermentation in petroleum products

Problems arise with the use of petroleum products for fermentation on an industrial scale because of the insolubility of some materials. Microorganisms are vsrwenclet for desulphurisation or dewaxing of oil and petroleum products in _s bred and erfiridungsgemMsse irehrstufige fermenter Kaiin to advantage in such ^ learn be used

In the continuous fermentation in conventional fermenters, there is no complete assimilation of petroleum products ₂ and it is difficult to separate oil residues from the bacterial cells in the overflowing culture broth for oil residues «ircti so that the ropes in it are completely grown \ xnä the withdrawn culture broth consists mainly of water.

For desulfurization or dewaxing, int a continuous Method using his multi-stage fermenter advantageous around a final broth with the expected level of reaction to obtain. With at least one petroleum product that is easily emulsified in the medium and continuously from the bottom of the Fermenter was introduced from, - the fermentation with aeration and adjustment of the amount of medium supplied be carried out, then a "Bi'tthe will be received" the one

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has uniform reaction outcome. In this case, too, the distribution of the age of the pelts is determined , and the cells obtained have a certain determined quality. "

synchronous fermentation

As has already been mentioned repeatedly, * indicates when carrying out a continuous process in a multistage fermentation apparatus geraäse the invention, the medium in each Kammex * a culture phase, _s is different from those in other chambers. In some cases, fermentation requires the use of a synchronous method to achieve the optimum gellenvertellung _s. It was beispielsvieise determined that an extraction of RNA from Hefesellen effective at the end of the logarithmic growth phase of the Stamirs can be carried out, this linear meters to reach * were some devices and methods _g designed to cells of the optimal age to obtain with continuous fermentation "The inventive method and However, devices are new. The method of operation is the same as for the other fermentation, and the feed quantity and rate * the capacity of the respective chambers and number of plates are accordingly measured and determined _! that the fermented final broth is brought to the final state of the logarithmic growth phase.

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-'26 -

_S has already set out the vile erfindungsgeniSssa Fermentationsvorrichtimg can be very effective applied to various continuous fermentation process due iiirsr particular quality. In addition to the high effectiveness, the costs of the required system are low * and a high yield per unit is achieved in all cases.

It is also an essential feature of the fermentation furnace Teehnlk used the basic procedure, that the microbial cells be separated from the medium or the suspension in a medium or a suspension after the fermentation has ended have to. This is usually done by centrifugation and filtration. Neither centrifugation nor filtration can but lead to the isolation of pure bacterial cells · because solid substances other than the cells were also collected in the medium and it is difficult to separate such substances from the cells by washing. When a special form of Cells is desired or, for example, only one spore suspension is to be used for a technical purpose neither centrifugation nor filtration is used to separate spores from vegetative cells. If the cells as industrial Materials to be used for the production of enzymes * it is required only the cultured cells that have a special property of collecting, for example those with high nucleic acid content or highly activated after removal of autolyzed cellular materials

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l'ic-j · le «. Usual l5o3ie: inmjjsra © methods cannot meet these requirements. In addition, it is from the technical stability point, ■ for example from the physiological * immunological? From a cytolo-cisohen and microbiological point of view , often necessary, i'Girie bacterial ropes to gain. No perfect method of separating such cells has been known to date.

'& qX imUörsuohungen = those on the concentration, removal and / or isolation of bacterial lines with specific JBigensohaf-

i; oj ». were directed? it has been found that these purposes and requirements can be met through the use of a multi-stage fermenter _: into which a bacterial cell suspension or a suspension of sell fractions is introduced. This rüspenqion is aerated from the ground to form a foam. Cells or protein particles are adsorbed on the surface of the Schauras due to their charge. These bacterial cells are fractionated, concentrated and / or distributed upwards in the fermenter.

Ira individuals will use the Eaktex-ienaellen suspension or the Suspension of cell fractions in a multistage fermenter introduced, if necessary, a foaming agent is introduced and which is ventilated from the ground. These BaIcteriensielle or cell tractions are * when the suspension foams, depending on the surface of the foam at a certain rate

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according to the composition of the suspension, the arrangement of the foam, the type of foaming agent and the like. The concentration rate differs from that in a liquid medium * and the foam goes through the holes in the separating plate up into the one immediately above it Chamber _t in which it comes into contact with the medium * and further up through the medium in this and through another plate and so on until it reaches the upper end of the Fermenfe ters.

In the steady state, there is no change in the amount of liquid contained in each chamber, and the kedium flows out of the upper chamber as the foam moves up from one chamber to the next, in an amount equal to that of the liquid content of the foam . That is, the foam rises while the medium moves downwards, and an interaction of the medium solution takes place at each divider plate between the chambers involved. ψ At this junction, if the cell concentration on the foam is higher than that in the medium, the barks are pushed upwards. If the cell concentration in the medium is higher than that on the foam, an opposite effect occurs. The concentration of cells on the foam differs in degree according to such factors as the type of medium, bacterial cells or cell fractions used, and the type of foaming agent and pH. These conditions

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should be set beforehand by means of a preliminary test ©. In this way, cells with a specific property can be concentrated and / or fractionated. This procedure can be carried out more effectively by repeated cycles of the procedure described above. In contrast, if the medium or the liquid phase vjlrd used ₃ may be derived from the chambers in which the cell content 1st low, are removed.

The fermentation device according to the invention can thus be used for collecting and separating desired cell bodies are used "wherein generally more than 90 # of the total cells are separated and / or be fractionated * without any disadvantageous physical and chemical influence through heating, through larger ones Changes in pH and the like occur. In addition, the cells can then, if necessary, with a higher degree of efficiency be filtered or centrifuged than with the usual methods. Simple modes of operation in the implementation a continuous process are a further advantage of the present invention. In contrast to the inventive Process the cells in a conventional fermenter uniformly distributed and therefore cannot be concentrated and / or separated.

The type and consistency of the overspill «included in the proceeding can be used depends on the condition and type of inch to be used. Different types of surfactants, fatty acid amines> inorganic SaI

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zen and the like, can be used individually or in coordination will. The consistency can be determined by simple checks.

The above procedure can cause spores. " Frote granules, cell fragments and the like, as well as vegetative cells and microorganisms are separated, concentrated and / or fractionated. on the other hand a medium or a culture broth is obtained which is free from bacterial cells or other foreign elements.

By the inventive method, in which the special Characteristics of the multi-stage fermentation device can be used «bacterial cells can be separated from a culture medium, concentrated and / or fractionated. Such a method has not yet become known and it is extremely valuable for industrial applications.

The following examples explain the invention without adding to it restrict.

example 1

A cylindrical fermenter with an inner diameter of 6 cm and a height of 120 cm was divided into equal parts by four transversely arranged plates * each plate had several holes with a diameter of 2 mm and the opening ratio was 20 % (see Fig. * ;) ·

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⁸ ^ ORIGINAL

Crude technical naphthalene was introduced into the second chamber from below in an amount of 10% by weight of the total liquid, and a culture medium free from bacteria and containing 0 * 25 ammonium chloride * 2 % hangan chloride, 0.005 % ferrous sulphate and Of05 % calcium chloride., was poured, after which the chamber was inoculated with Pseudomonas aeruginosa Mr. B-3 ^.

When the fermentation was carried out at 50 ° C. with aeration at a linear speed of 3 cm / second, a certain amount of the solid naphthalene present in this chamber passed through the holes in the plates into the adjacent chambers. After 36 hours of fermentation, 10.6 g of salicylic acid were produced. This salicylic acid is not reduced or broken down by further fermentation.

When the fermentation was complete, the fermented broth was drained from the bottom of the fermenter and 85% of the initial solid Naphthalene was collected in the fermenter. Using this recovered naphthalene and cells * that remained in the fermenter "became another fermentation cycle by simply introducing those described above Mineral solution carried out. This operation was done six times repeated.

Example 2

A breeding medlura with a content of 10 56 glucose «1 % urea, 0.1 $ KH ₂ PO ₁ ^ OpO ^ % MgSO ₄ . 7HgO, 0.6 # soaked corn

tract * 2 parts j

109882/0298

water, 0.1 % kefe extract * 2 parts per million. Fe * " ⁸ ", 2 ¹ EeIIe each

BATH ORIGINAL

Million Mn ⁺ * and 2 ing / i biotin were introduced into the same fermenter as in Example 1 in the same manner. Mach sterilization the medium at 30 ⁰ C was cooled "ad with the seed culture in an amount wherein the inoculum gelatinosum by culturing Corynebacterium no. 718jJ was obtained in a medium 2 \ ^r ol .- # corresponded to the medium inoculated _f, the same composition as described above hattej with the only exception that the Gluoosogehalt was "8% Εε was then at 30 ^e C under aeration of 200 -. ecm 400 per cultured minute after 58 hours reached dor DL-alanine content. in the medium 40 g / l. The number of plates used can be chosen freely and does not even affect the result of the operation in the case of this discontinuous process.

A continuous process using the same ingredients and five equally spaced plates across the fermenter was then carried out. 1/12 of the total volume of the medium was brought in from the bottom of the fermenter per hour. The steady state was reached in about 24 hours. The content of cells and alanine in the medium was different in the respective canoes. As was found, both contents were highest in the top chamber, namely 34 g / l alanine and 9.2 UOD bacterial cells. For example, the alanine content in the respective chambers was from bottom to top * (1) 0.5 g / l _f (2) 5 g / l, (3) 17 g / l, (4) 29 g / l, (5 ) 34 g / l.

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Example 3

Following the procedure described in Example 1, the Medium prepared and with the same bacterial inooulum inoculates. Naphthalene was used as in Example 1.

After fermentation for 24 hours, the salicylic acid content in the medium reached 6.2 g / l. From this point onwards, a sterilized inorganic salt solution was introduced from the bottom by means of a metering pump. The proportion of this solution to the total liquid in the fermenter, or the fermentation rate, was 1/15 per hour. 40 hours awake from the beginning of the process, each chamber contained bacterial cells and salicylic acid, and the steady state was reached. The amount of salicylic acid formed from the bottom chamber upwards was: (1) 0.8 g / l, (2) 0.8 g / l, O) 6,} g / l and (4) 7.5 g / L The content in the fourth chamber was identical to the content in the fermented broth drawn off. The fermentation was carried out continuously for about 10 days until the solid naphthalene in the fermenter was completely consumed.

Example 4

A 5 liter columnar fermenter was divided into 6 chambers, the holes in each plate making up 20 % of its total area and the holes each having a diameter of 2 mm. The division was such that the first chamber (bottom chamber) had a volume of 1.5 liters and the remaining chambers each had a volume of 0 * 7 liters. Under the use of

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The fermentation of yeast (Candida 1-ypolitica) and the dewaxing were carried out with light oil.

100 parts of medium (pH 4.5), including 3.0 g of ammonium sulfate and 1.0 g of potassium phosphate «0.5 g magnesium sulfate, 0.1 g yeast extract and one small amount of trace elements each 1 contained, were with 13 Part of light oil and 0.02 part of a surfactant (Tween 40) mixed so that a stable emulsion was formed.

Before this step, Candida lypolitica was grown for 35 hours in a medium of the same composition as above. 5 vol. # Of this preculture of Candida lypolitica was added to the above emulsified medium, and cultivation was carried out at 28 ° C. with aeration at a rate of 15 1 / minute.

After fermentation for 24 hours, an enulsified medium, which was mixed with 15 parts of light oil per 100 parts of water, was introduced from the bottom at a rate of 200 ml / hour and a continuous fermentation was carried out. The fermented broth, which consisted of yeast cells, unused light oil and water and had a certain retention time, was gradually withdrawn from the top of the fermenter.

After the steady state was reached, a sample of the broth was taken from each chamber. The content of kefezelia and light oil was the following 1.6 g / l (dry weight) cell mass and 14.0 parts (parts by volume) oil layer (per 100 parts of initial water) in the first chamber, 2.5 g / l and 12, 8 parts in

109882/0298

the third and 2 _? 1 g / l and 11.5 parts in the fifth. The overflowing broth was separated into cell bodies, dewaxed light oil and water using known methods.

Example 5

In a fermenter with an inner diameter of 6 _× 5 cm, which was divided into 10 chambers, each of the plates having 100 holes with a diameter of 2 mm, the production of bacterial spores was carried out in a continuous process. (

As Xmpfkultur mounted Bacillus stearothermophilus ATCC was inoculated into glucose-Bouillionmedium 8005 and grown under aerobic conditions at 55 C on a ⁹ Bouillonschrägagar. The main pore product was initiated with 5% by volume of this culture. The total volume was 4.0 l.

The medium contained 6 g glucose * 12 g yeast extract, 1 g KHgPO ^, 0.1 g MnSO ₁ ^ .H ₂ Q * 0.01 g PeSO ^ .7H ₂ O, 0.1 g CaCl ₂ and 1 liter of distilled water . After 4 to 5 hours, additional medium was continuously added to the bottom of the fermenter in an amount of j $ 60 ml / hour. The fermented broth, which contained spores, overflowed at the top. The dipoleic acid content and the UOD of the cells in the jetted chambers reached the steady state. The cell concentration increases against the upper KaoMem . No dipicolinic acid was found in the lower chambers, only in the chambers above the sixth.

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The * diplcolic acid content was kept at 60 γ / ml. This enabled more than 80 % of the total cells to form spores under the growth conditions. The broth containing spores was centrifuged or the spores were collected from the spore suspension by the shower flotation method.

Example 6

Were 1253i-XAM the eyelet using a culture solution of Escherichia colii in a 2 # GIu- and 1% yeast extract-containing medium for 24 hours at 30 C ^a grown "BaIcterienzellen were concentrated and / or fractionated. 0.2 g / l of aluminum sulfate and 40 mg / l of lauric acid (ethanol solution) were added to the above culture solution. The mixture was adjusted to pH 7-2 and poured into a cylindrical multi-stage fermenter with a height of 120 cm and a diameter of 6 cm, which was divided into 11 chambers by 10 plates. in each paltte the holes of 5 mm diameter made up 11 % of the total surface area. 7 cm / sec was air in the above ψ Permenter after its filling with the mixture with a G ~ ^e speed of efcwa introduced. After 25 minutes of ventilation, a sample of the solution was taken from each chamber and the number of cells therein was determined. The Ex * results showed »that the number of cells in the top chamber exceeded that in the bottom chamber by about 10 times. The number of cells in the intervening chambers gradually differed from one chamber to the next from the bottom

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from ascending upwards. Although the cell bodies in the various chambers were not visible through a microscope, non-bacterial solids were visible in the lower chambers, their number increasing towards the bottom. The physiological Activities of the cell bodies removed from the respective chambers were measured in terms of the oxidative capacity of a cell purity determined with glucose as substrate. The following table shows the values obtained. The total U.0.D./ml was determined by Multiply the optical density at 610 μm of the diluted Sample obtained with the number of dilutions. ' It was 5.0 in all chambers before ventilation. Varying after ventilation however, they go from 1 * 0 at the bottom to 10.4 in the ninth chamber.

The table below also shows the number of cells in each chamber versus the total number of cells: 73 % of the total number of cells were found in chambers 7 to 10 and the remaining 27 % in the first to sixth chambers. Q-; ₂ in the table, the rate of oxygen absorption per cell unit, is given as a base value of 1.00 compared to the value in the first chamber. It can be seen that the activity is lowest in the first chamber and increases towards the upper chambers.

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Table IXI

Chamber U.0.D. (6tQ ναμ) Distribution of the (vqra soil made of) ropes, $

number 1
No. 3
No. »5
No. 7
9

1 1.97 1.00 2.1 4.13 1 ₉ 10 3.8 7.48 n3o 8.1 15.95 1.38 10.4 20.47 1.50

Example 7

A culture stage (pH 7.0) containing 0.5 # glucose, 1 ₃ 3 % yeast extract, 0.1 # KH ₂ IPO ₄ , 0.01 % MnSO ₄ -HgO, 0.001 % FeSO ₄ ^ H ₃ O and O , OO5S6 MgSO ₄ .7H ₂ O was MIFC Bacillus subtilis IAM / 1214 and inoculated with aeration movement 24 hours at 30 ^e C geattchteto After this period, 1.9 x lOVoom spores were formed from a total of 2.1 χ 10Vcera cells. 2 liters of the above solution, in which the spores and vegetative cells of the above strain were mixed, were placed in a multi-stage fermenter with a volume of 3.2 liters, which was filled by 8 plates (in which the holes of 4 mm in diameter each had 16 % their total area) was divided into 9 chambers. The air was introduced through an air vent on the floor at a rate of 4 liters / minute. At a pH of 7.3 and a temperature of 28 ®C, foaming began in the chambers and advanced foaming was observed immediately below each plate. The amount of air was increased by 0.5 l / minute every 5 minutes until it finally reached 19 l / minute. As a result, the volume of the foam layers was

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ORIGINAL

stepped places increased so that a certain amount of the Culture solution was kept in each chamber. Naoh 120 minutes Ventilation under the above conditions were the number of the spurs intended for the upper two and lower two canoes: 1.1 10 / ecm and 1.0 χ 10 / ecm for the two

7 7

upper chambers and 2 χ 10 '/ com and 1.2 χ 10 / ecm for the two lower chambers. In other words, the above results show that 90 % or more of the qesarate spores were collected in the upper two chambers. They also show that since the number of cells in the respective chambers did not differ much, the number of spores per number of inches in these upper chambers was considerably increased. The liquid in these chambers was drawn off and used as a spore suspension.

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Claims (8)

Claims 1. Process for discontinuous fermentation * thereby marked that an ear-stage fermentation preparation is used and air into the multi-stage fermentation device is introduced from the bottom and thus passed through the fermentation device. " that they are in the upper part of each divided section in this fermentation device forms a free space, the multi-stage fermentation device comprises a set of vertically one above the other arranged chimneys, which by one or more horizontally extending plates are separated, and each of the plates is provided with one or more openings. 2. A method for continuous fermentation, characterized in that a multi-stage fermentation device is used and a medium is supplied with aeration with air from the bottom of the fermentation device and passed up through the fermentation device ν.Ά at the upper end of the fermentation _{device P} overflows while the air in the upper Part of each subdivided section in the fermentation soldering device occupies a space and thus forms a free space or has several openings. 109882 / 02S8 Untei lay << vt 7 § ι At * ζ ν * »? Sat * sd ·· % r * im »mmm * BATH ORIGINAL 3 Process for continuous fermentation, characterized in that a multi-stage fermentation device is used and a medium from the top of this fermentation device is used and air in countercurrent from the lower end are fed, the multi-stage fermentation «device has a set of chambers arranged vertically one above the other, which are separated by one or more horizontally extending plates. and each of these plates has one or more has multiple openings. 4. Multi-stage fermentation device, characterized by a set of chambers arranged vertically one above the other, which are separated by one or more horizontally extending plates, each of these plates having one or more openings having. 5. Method for fermentation naoh one of claims 1 - 3 # characterized in that a solid substrate material in the respective chambers of the multi-stage fermentation device suspended and held. 6. The method for fermentation according to any one of claims 1-3 *, characterized in that at least one petroleum product is introduced as a medium component. 7 · Process for continuous fermentation according to one of the Claims 2, 2, 5 and 6, characterized in that a micro- 109882/0298 _{οιΛ | ΜΑΪ} ORIGINAL - 48 - Organism is bred in such a way * that a different distribution the cell concentration, the cell age or the concentration of product in the respective chambers in the multi-stage Fermentation device achieved under steady state will. 8. A method for fermentation according to any one of claims 1-3 _t . characterized. that the product, which consists of at least one type of microbial cells * a suspension of cell fractions and spores? removed from the appropriate chambers and separated at the end of the fermentation. 109882/0298 BATH ORIGINAL Blank page