DK141338B - Process for aerobic culture of microorganisms and a fermentation apparatus for carrying out the process. - Google Patents

Description

(11) PRESENTATION 141338 (S) \ via / DENMARK <«> '"' ci · 3 c 12 «i '» * t (21) Note # 2707 / Υ ^ (22) Filed on 1 raaJ ^ 97 ^ ( 23) Life Day 16 · May 197 ^

(44) The note was submitted to the PC fpv iQftO

Submitted by the doctor

DIRECTORATE OF

PATENT AND TRADEMARKET (3 °) Priority requested from it,

May 16, 1972 # 23350/73 # OB

May 3, 1974, 25530/75 »GB

(71) IMPERIAL CHEMICAL INDUSTRIES LIMITED, Thames House North, Millbank,

London SW1P 4QG, GB.

(72) Inventor: Malcolm Ritchie Gibson, Norton Hall, The Green, Norton, Stockton-on-Tees, Teesside GB: Prank Peter Maslen # Norton Hall,

The Green, Norton, Stockton-on-Tees # Teesside, GB: Frank Cornelius Roes, clerk, Norton Hall, The Green, Norton, Stockton-on-Tees, Teesside, GB: Stu® art Raymond Leslie Smith, Norton Hall, The Green, Norton, Stockton * · on-Tees, Teesside, ΏΒ. - (74) Plenipotentiary during the proceedings:

The company Chas. Hude. ____ (54) Method for aerobic culture of microorganisms and a fermentation apparatus for carrying out the process.

The present invention relates to a method for aerobically culturing microorganisms and of the kind set forth in the preamble of claim 1, as well as a fermentation apparatus for carrying out the process and constitutes a further development of the invention disclosed in British Patent No. 1,353,008.

Reactions requiring intimate contact between a gaseous and a liquid, e.g. aerobic fermentations, have so far generally been carried out on an industrial scale in systems consisting essentially of a large tank into which or near the basis of which the gas is introduced. Intimate contact between the gaseous and the liquid is obtained by keeping the liquid in regular circulation by mechanical stirring means or by the gaseous introduced into the liquid or by a combination of these methods. In the case where the circulation is maintained by gaseous feeding, the circulation pattern in these prior art apparatus generally comprises a relatively rapid ascent and a slower descent phase. In fermentations conducted in this way, the oxygen supplied to the liquid culture is absorbed during the relatively short ascent phase, while the liquid culture during the more prolonged decay phase is subjected to anaerobic or almost anaerobic conditions.

The general industrial progress and especially the development in industrial fermentation, including the introduction of new processes for the production of proteins and other fermentation products, have led to an increase in the business and necessitated the introduction of factories with considerably increased capacity. The above-described apparatus cannot be adapted to meet modern requirements satisfactorily, since for various physical and biological reasons it is not commercially possible to increase the size of the tanks beyond certain limits.

In fermentations or ferments requiring a large supply of oxygen, it is difficult and expensive, especially when mechanical stirrers are used, to add the required amount of atmospheric air or other oxygen-carrying gases to the process of maintaining the various required properties of the liquid culture which is constantly circulated, at all points throughout the tank and everywhere to keep the liquid culture at an appropriate and constant temperature. This causes a tendency to limit the maximum volume of the tank and hence to maintain an economical design of the tank, the height is generally also limited (to about 15 m in most cases).

In recent years, great efforts have been made to develop methods for producing nutritional additives consisting of single cell protein by growing microorganisms on substrates containing cheap carbon sources. Frequently, such processes require a large supply of oxygen, especially when the microorganism is grown at high growth rate and when using a substrate, e.g. a hydrocarbon or partially oxidized hydrocarbon where the microorganism's oxygen demand is high. In order to do so through such a process, the product formed economically competitive with more traditional foods, it is necessary to carry out the fermentation in a large plant. For a favorable commercial operation, it is necessary to obtain a high rate of oxygen supply to the liquid culture during the process to ensure that the high oxygen demand for fast growing cultures with high cell mass values can be satisfied in a given volume fermenter. The rate of oxygen transfer which can be obtained in known fermenters of practically usable size is unsatisfactory for the operation of a commercially favorable process of this kind.

From the disclosure of British Patent No. 1,353,008, a method for aerobic fermentation of a substrate by means of microorganisms is known to be able to use the substrate for growth, and wherein the fermentation medium comprising the substrate and microorganisms is continuously circulated between two regions having different hydrostatic pressure. and where the atmospheric air and / or oxygen is dissolved in the fermentation medium in the area of greater hydrostatic pressure and where the carbon dioxide produced during the fermentation leaves the fermentation medium in the area of lower hydrostatic pressure.

From the specification of British Patent No. 1,353 * 008 there is also known a two compartment fermenter (a riser and a fall tube) and means for continuously circulating the fermentation medium between the compartments, there being means towards the lower end of one compartment (the stirrer). for introducing a gas into the fermentation apparatus and to produce a difference between the hydrostatic pressures at the lower ends of the two compartments sufficient to force the fermentation medium to circulate continuously between two regions of different hydrostatic pressure when the fermentation apparatus contains a fermentation medium , as well as gases for gaseous discharge from the upper part of the fermenter.

The article of British Patent No. 1,353 * 008 is suitably used in large scale fermentation processes as it allows a large degree of oxygen transfer into the liquid culture without a corresponding increase in power consumption over the known fermenters described above.

It has now been found that when the process of British Patent 4 1A1338 No. 1,353,008 is carried out in a fermenter having a height greater than a certain level and with certain relative dimensions of the riser or riser and the fall pipe and under certain conditions of the supply of gaseous species, in particular the supply of atmospheric air, obtains very satisfactory results (in terms of oxygen = transfer rate, power transfer ratio and microorganism growth) for large scale operation under conditions requiring an oxygen = high intensity or strength supply.

According to the present invention, the method referred to initially is characterized by that of the characterizing part of claim 1.

The fermentation apparatus of the kind specified in the preamble of claim 2 is characterized by the present invention as set forth in the characterizing part of claim 2.

It is convenient that during the start-up period of the process according to the invention, all or most of the amount of gases in the riser (s) is injected for a period of time to start circulating the liquid. This start-up procedure continues until the liquid circulates regularly at an appropriate rate, i.e. until the mean velocity of the liquid in the trap (s) is at least 100 cm / sec.

The present invention is suitable for aerobic fermentations requiring a very high intensity for the absorption of oxygen. Processes to which the present invention is very suitable include the preparation, of single cell protein, by growing microorganisms on hydrocarbon or partially oxidized hydrocarbon (e.g. methanol = containing) substrates, or the preparation of amino acids (e.g. ly = sin, glutamic acid and methionine) or the production of citric acid and enzymes (eg glucose isomerase) or the preparation of antibiotics (eg penicillin). In the specification, the term mean velocity of the liquid or liquid culture in the riser (s) or drop tube (s) is to be understood as the average velocity of the liquid or liquid culture in the riser (s) or drop tube (s) as measured over the full cross-section of the chamber. Since the internal cross-sectional area of these chambers, especially of the riser (s) need not be the same in its or their full length, the velocity of the liquid or liquid culture in both chambers may vary in their longitudinal direction.

141338 5

In the process of the present invention, the injection of gaseous into the liquid culture converts the entire circulating medium into a reduced density fluid consisting of gaseous bubbles, liquid and microorganisms, and the void or void difference between the riser (s) and the fall (s) caused by the injected gaseous announcer. this fluid has a velocity sufficiently large under normal operating conditions to render the dispersion of bubbles substantially homogeneous. In this form, the liquid culture circulates around the system essentially in a plug-like stream, i.e. a good flow in which a certain amount of mixing is effected by turbulence action.

During the process, all the liquid culture volume elements circulate quickly and uniformly between areas of high and low hydrostatic pressure in the lower and higher parts of the system, respectively. In the high-pressure region, the oxygen needed for the growth of microorganisms is absorbed into the culture, while the CO 2 gases produced during the process are released into the low-pressure region. In the system. the upper portion releases the spent gas from the circulating liquid culture in the low pressure range.

The oxygen transfer rate into the liquid culture obtainable in an aerobic fermentation process is an important factor as it determines the rate of production of the microorganisms obtainable in a given volume fermenter. In large-scale operation, it is preferred that the transfer rate is at least 2 kg 02 / m ^ h or more conveniently between 4 and 12 kg 02 / m ^ h.

As the transfer rate depends, among other things, on the total height of the system, it is important if an appropriate rate for microbiological growth and the productivity of the process is to be maintained, that the height must exceed a minimum size. In the fermenter according to the invention, a suitable transfer rate can be obtained when the total height is 20 m. However, heights of at least 30 m, especially heights of 40 to 60 m, are preferred.

The gas injected into the system, which may be oxygen, atmospheric air or another oxygen-containing gas species, serves several purposes: 1. It causes circulation of the culture within the system.

2. It supplies the oxygen required by the microorganisms.

3. By choosing the appropriate positions for the gas injection and the relative quantities of gas to be injected at the various points, a significant degree of control can be exercised for the process. In particular, the flow rate and thus the circulation time of the medium can be controlled. Also, the distribution of the dissolved oxygen concentration can be controlled.

4. The surface area of the Bohies formed by means of the injected gas is available for the desorption or emission of CO 2. The volume and composition of the injected gas can be used to control the distribution of the (?) Concentration in the liquid or liquid culture.

The total amount of gas injected, the positions at which it is injected, and the relative amounts injected at the various positions vary depending on the requirements of the microorganisms and the rates of circulation required. Gaseous can be injected into the fall pipe (s) at one or more levels, e.g. 2 to 5. Preferably, the gas is injected into the riser (s) at one level, although it can also be injected into the riser at a number of levels. Appropriate gas injection positions in each chamber are distributed along the drop tube (s), but concentrated at or near the base of the riser (s).

In some embodiments of the apparatus according to the invention, where the drop pipe (s) and the riser pipe (s) are completely separated and are connected by means of tubes at their upper and lower ends, the riser pipe type can be introduced into a lower connecting pipe which, for this purpose. purpose is considered to be part of the riser (s). When this is done, it is preferred that the lower connecting tube slopes upwardly towards the body of the riser (s). In order to more easily start the process according to the invention, a special gas supply is provided, in which gas is injected into the upper part of the stirrer (s). It is preferred that the amount of the gas injected or introduced into the trap (s) is between 20 and 40 [6] of the total air volume injected into the system.

7 141338

The exact geometric design of the fermentation apparatus can vary widely, as shown in the drawing. The riser (s) and the fall (s) are suitably disposed free of each other, either completely separated from each other, except connecting pipes at their upper and lower ends or placed within a single container internally divided by one or more partitions. with openings at the upper and lower ends. The riser (s) and the fall (s) may be cylinders arranged externally to one another and connected by tubes which may be horizontal, or in connection with the lower connection, U-shaped. Alternatively, the system may e.g. comprise two chambers which are coaxial, one chamber, preferably the riser, surrounding the other. It is preferred that the system comprise a plurality of risers located externally to and connected to a single fall tube, in particular 3 to 8 risers connected to a single fall tube.

In the latter case, each riser may have the same cross-sectional area as the drop pipe, so that the combined cross-sectional area of the riser becomes larger. Such a system is easily pre-made.

The velocities obtained by the liquid culture in the drop pipe (s) and in the riser pipe (s), respectively, depend on the cross-sectional area of these chambers, i.e. of their diameters, if they are cylindrical. The chambers do not necessarily have the same diameter in their full length, and in fact it is sometimes sometimes desirable that the riser (s) consist of two sections arranged vertically above each other, the lower section having a larger cross-sectional area. In this case, the cross-sectional area of the lower section (s) is preferably between three to eight times larger than the cross-sectional area of the upper section. It is preferred that the lower section constitute $ 30 to $ 60 of the total length of the riser. Alternatively, the cross-sectional area of the riser (one's) may decrease evenly with height.

In order to avoid the accumulation of large CO 2 volumes, the liquid culture is rapidly circulated between the high and low pressure areas of the fermentation apparatus. It is preferred that the time taken for the liquid culture to circulate completely around the system is between 0.5 and 5 minutes, preferably between 1 and 3 minutes. The velocity obtained by the liquid culture at any point in the riser (s) or drop (s) depends on the cross-sectional area of the chamber portion concerned. In order for the liquid culture to circulate substantially in a plug-like current, the minimum speed in the riser (s) must be greater than 10 cm / sec. Preferred mean velocities are 20 to 80 cm / sec, in the riser (s) (in the lower part if this chamber does not have a uniform cross-sectional area) and 200 to 500 cm / sec in the drop tube (s).

The fermenter can be operated either charge-displayed or continuously.

The liquid culture essentially circulates in a plug-like flow, enabling a high degree of homogeneity to be achieved. Almost the entire volume of the fermenter can be used to grow microorganisms, and spaces where anaerobic conditions prevail have been eliminated. This allows a high degree of control over the fermentation parameters, such as the temperature, pH and substrate concentration, to obtain an improved and more uniform product. For example, the method of the invention is used for the production of single cell protein, the microorganisms produced exhibit a high degree of morphological uniformity, and an improved conversion of substrate carbon to cell carbon is achieved. The invention also allows for efficient circulation and avoids the use of external circulation paths to dissipate the heat of reaction and fermentation.

The invention provides good results measured in achievable oxygen transfer rates and power consumption. The results obtainable as indicated by test results and calculations based on them are described below.

The oxygen volume transfer rates and the power transfer rates obtainable in different height fermenters were calculated theoretically for fermenters of the type shown in FIG. 1.

The oxygen volume transfer rate is the rate measured in kg 0o -x * - per hour and m of the liquid at which the oxygen dissolves in the liquid culture.

The power transfer rate is the ratio of oxygen transfer to the power consumption expressed in kg 0 ^ dissolved in the liquid culture per kilowatt working hour used to compress the air prior to injection into the liquid culture. The power transfer ratio decreases with increasing transfer rate and for the economical operation of a fermenter it is desirable that both parameters be as large as possible.

The calculations were performed by offsetting equations for the transfer of gases (especially oxygen and CC 2) between the gaseous bubbles and the liquid culture and also by calculating the pressure changes between the different parts of the system.

The force needed to compress the gas was calculated on the basis of the following assumptions: 1. The pressure in the upper part of the system (where the gas release occurs) is 1.5 bar absolute.

2. A pressure of 1 bar is lost between the compressor and the fermentation inlet opening.

3. It is assumed that atmospheric air with an initial state of 1 bar and 20 ° C is used as the source of gaseous oxygen supply. The compression is assumed to be adiabatic (isentropic) and sufficient to achieve a sufficient pressure rise to overcome the stated loss of 1 bar and inject the gas into the fermenter under this pressure (or pressure) existing at the point of injection ( or the points).

These assumptions result in an assessment of the compression work, which is realistic with regard to large axial compressor requirements for shaft power with two-stage cooling. It was assumed that up to 3 of the air was injected into the drop pipe.

The results obtained are given in the table.

10 1A1338

Transmission Power Transfer Fermentation Speed of the meter for oxygen ratio (m) to medium (kg / kWh) (kg / hm3) 2 2.7 20 6 3.0 30 8 2.7 40 10 2.5 50 12 2.3 60 14 2.1 70 16 1.9 80

The table is calculated on the basis of carefully employed assumptions regarding the mass transfer coefficient and mean bubble size.

It shows that the oxygen volume transfer rate increases with the height of the fermentation apparatus. The prior art best known to the inventors for prior art fermenters are transfer rates of approx. 3 kg per per hour No liquid culture, and these have been obtained only at lower power transmission ratios than those obtainable by the present invention. Thus, a fermenter according to the invention significantly improves the best known results as known by the inventors. It will be seen that the height of the fermentation apparatus is important in achieving this improvement.

It should be noted that the process of enriching the oxygen-delivering gas using pure oxygen or a mixture of pure oxygen with atmospheric air can lead to even higher transfer rates. This method is not a particular feature of the present invention, but can be simply combined with it. Similarly, the transfer rate can be increased by constructing the entire fermentation apparatus as a pressure vessel so that the pressure throughout the apparatus is raised above atmospheric pressure.

This method can also be combined with the present invention. However, both of these two known methods of increasing the transfer rate, namely the use of pure oxygen or a total pressure increase, also have deficiencies known to those skilled in the art. It is a particular advantage of the present invention that transfer rates of the size shown in the table can be obtained with atmospheric air as the gas supplying the oxygen and with a good power transfer ratio, and without raising the dissolved CO 2 concentration. in the liquid culture to a high level.

Preferred embodiments of the present invention are explained below with reference to the drawings. In this, FIG. 1 is a side view of a fermentation apparatus with a riser and a drop tube arranged side by side, and partly in cross section; FIG. 2 shows a fermentation means where the riser and the drop pipe are coaxial and the riser has an annular cross section, partially side view, partially in section; FIG. 3 is an alternative embodiment of the fermentation apparatus shown in FIG. 2, wherein the riser has a circular cross section; FIG. 4 is a side view of a fermenter, the riser having a constant cross-sectional area throughout the length of the fermenter, viewed from the side, partially in section; FIG. 5 shows a further embodiment of the fermentation apparatus shown in FIG. 4, wherein the riser has a circular cross-section, side view and partly in section; FIG. 6 is a section along the line x-x of FIG. 6A of an alternative embodiment of the fermentation apparatus shown in FIG. 1, wherein there are a number of riser pipes and the riser has a constant cross-sectional area throughout the length of the fermenter; FIG. 6A shows the fermentation apparatus of FIG. 6 is a top view of FIG. 7 is a partial section along the line x-x of FIG. 7A of a further embodiment of the embodiment shown in FIG. 1, in which there are a number of riser tubes, FIG. 7A a fermenter according to FIG. 7, the line Ύ-Υ in FIG. 7-

For ease of clarity, a suitable position for a heat exchanger for dissipating fermentation heat, or for removing or supplying necessary heat for chemical reaction to take place, is shown only in FIG. First

Fermentation means shown in FIG. 1, 2, 3 and 7 have one or more riser pipes 4f comprising cylindrical or annular upper and lower sections 1 and 2, interconnected by a reduction portion 3, the lower section 2 having a larger diameter than the upper section 1.

The 4, 5 and 6 have a riser or riser 4 comprising a cylinder or ring having a constant cross-sectional area.

The upper end of the riser is connected to the upper end of a drop tube 5 through an upper connecting portion 6 which may be horizontally cylindrical as shown in FIG. 1, 6 and 7, or comprise a vertical cylindrical section 7 and tapered connecting parts 8 and 9 forming an extension of the riser and the fall pipe, respectively, as shown in FIG. 2,3,4 and 5.

The lower end of the riser is connected to the lower end of the drop pipe by a lower connecting portion 10 which may be cylindrical as shown in FIG. 1, 6 and 7 or horizontally circular, forming a basis for the fermentation apparatus as shown in FIG. 2, 3, 4 and 5.

• A gaseous species, e.g. atmospheric air is fed into the lower section of the stirrer through feed tube 11 to produce continuous circulation of a liquid phase which fills the fermenter up to level A-A. The upper connecting portion 6 must not be filled completely with liquid to allow a free surface from which gaseous bubbles escape from the liquid and pass out through the outlet opening 12.

Extra gases, e.g. atmospheric air is fed into the trap through one or a plurality of feed tubes 13, two of which are shown in FIG. 1-7.

A food medium containing essential ingredients

Claims (2)

The fermentation is introduced into the fermenter through a tube 14 and the culture is removed from the fermenter through a tube 15. The riser section contains bubble decomposing means, as indicated by reference numeral 16. In FIG. 1, the heat exchanger 17 is shown in the drop pipe 5, although the heat exchanger can be placed at any point in the riser or in the drop pipe. Alternatively, the number of heat exchangers can be arranged in either the riser section or the fall pipe section or in both. The present patent does not cover the use of the process in the manufacture of human food. Claims. A method for aerobically culturing microorganisms in a nutrient medium preferably containing a hydrocarbon or a partially oxidized hydrocarbon as a carbon source, wherein the culture liquid is continuously circulated in a fermentation apparatus comprising at least one riser and at least one drop tube, wherein the total useful cross-sectional area of the drop tube, the drop pipes, respectively, are smaller than the total useful cross-sectional area of the riser, respectively the riser pipes, and where the upper and lower ends of the pipes, respectively, the pipes are connected to the respective upper and lower ends of the riser, respectively the riser pipes, and where the circulation of the liquid around the system is effected by injecting an oxygen-containing gas into it, especially at the bottom of the riser, but possibly also at points in the drop pipe, characterized in that the culture apparatus has a height of at least 20 m and that the liquid in the riser, respectively. the riser pipes have a mean velocity of at least 15 cm / sec. A fermentation apparatus for carrying out the method according to claim 1 comprising at least one riser (4) and at least one drop tube (5), wherein the total useful cross-sectional area of the drop tube (s) is less than the total useful cross-sectional area of the riser (s), and lower ends of the riser or riser are connected to the respective upper and lower ends of the riser or riser, characterized in that