CA1312568C - Method for the aeration, without exit gases, of fermentation media - Google Patents
Method for the aeration, without exit gases, of fermentation mediaInfo
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- CA1312568C CA1312568C CA000583807A CA583807A CA1312568C CA 1312568 C CA1312568 C CA 1312568C CA 000583807 A CA000583807 A CA 000583807A CA 583807 A CA583807 A CA 583807A CA 1312568 C CA1312568 C CA 1312568C
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- medium
- fluid carrier
- oxygen
- fermentation
- carrier medium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
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Abstract
Abstract of the disclosure:
A method for the aeration, without exit gases, of fermen-tation media Aeration, without exit gases, of fermentation media is pos-sible by oxygen being transported, with the aid of fluid carrier media, into the fermentation medium and entering the fermentation medium via the liquid/liquid phase bound-ary, with, subsequently, the fluid carrier medium which has been depleted in oxygen and enriched in carbon dioxide being again enriched with oxygen, without emission of exit gas streams, and with the carbon dioxide which is displaced in this way being desorbed and removed.
A method for the aeration, without exit gases, of fermen-tation media Aeration, without exit gases, of fermentation media is pos-sible by oxygen being transported, with the aid of fluid carrier media, into the fermentation medium and entering the fermentation medium via the liquid/liquid phase bound-ary, with, subsequently, the fluid carrier medium which has been depleted in oxygen and enriched in carbon dioxide being again enriched with oxygen, without emission of exit gas streams, and with the carbon dioxide which is displaced in this way being desorbed and removed.
Description
` ~3~2~8 H~ECHST AKTIENGESELLSCHAFT Dr.SW/gm HOE 87/F 346 Description A ~ethod for the aeration, ~ithou~ exit gases, o~ ~er-~entation ~edia The invention relates to a me~hod for the aeration, ~ith-out exit gases, of fermentation media ~i~h oxygen or oxygen-containing gases, the oxygen being transported by means of fluid carrier media into the fermentation medium, and there entering the fermentation med;um via the liquidl liquid phase boundary, and subsequently the fluid carrier med;um which has been depleted in oxygen and enriched in carbon dioxide being again enriched with oxygen, without emission of exit gas streams, with the rarbon dioxide ~hich is displaced thereby being desorbed and removed.
The cultivation of live cells, especially in large amounts, is nowadays an essential constituent of the production of, for example, antiviral substances such as interferon, or of biologically active substances such as hormones. What are called hybridoma cells - a fusion product of antibody-producing cells with myeloma cells - are cultivated in large amounts especially for the preparation of monoclonal antibodies which have the ability ~o recogni~e very parti-cular epitopes of a protein (of a macromolecule) and to bind thereto.
In conventional laboratory methods for the cultivation of cells, the latter are suspended in a medium in a Petri dish. Oxygen and carbon dioxide diffuse through the sur-face of the med;um into the cells. The depth of the cul-ture medium influences the ra~e of diffusion of oxygen and carbon dioxide to the cells. As the depth of the medium increases, the ratio of the surface to the volume of the medium decreases. At a particular time the concentrations of oxygen and carbon dioxide in the air over the dish, and/
or the rate of dissolution in the medium, are insufficient ~ 3 ~
to supply the total volume of the medium with sufficient gas and to satis~y the requirements of the growing cells, i.e. ~he available oxygen becomes the factor limiting growth. Accordingly, the state of the art teaches that the depth of the medium ought to be in the region of mm for static cultures. In general, the upper limit of cell density for cultures of this type is about 105 cells/ml of culture medium when the air ouer the culture medium is used to supply oxygen.
When methods for the cultivation of cells are carried out on the industrial scale it is difficult to make available sufficient amounts of oxygen to satisfy the needs of the cells~ Gases are frequently bubbled directly through the medium in order to supply the cells with the required gas concentrations. ~owever, direct bubbling through is un-suitable especially for the cultivation of sensitive cells.
These cells are physically damaged when a gas is bubbled through at a sufficient rate to mainta;n appropriate gas concentrations in the medium, or by mechanical stirrers because of the high shear stress. In addition, protein constituents of the medium, uhich are normally necessary in all cell cultures, form a foam, ~hich may trap the cells, on the surface of the medium. The cells entrapped in the foam rapidly die It is possible to add antifoam agents to the medium but these are occasionally toxic for the cells in the culture and, in addition, can be separated from the product during working-up only with difficulty.
An elegant method of cultivating even sensitive cells on the industrial scale and, at the same time, ensuring the necessary supply of oxygen is the introduction of oxygen into the culture medium by means of a flùid carrier medium.
It is known that, for example, fluor;nated hydrocarbons are able to dissolve oxygen physically about 20 times better than can water. Thus~ if such fluorinated hydro-carbons are saturated with oxygen (or air) they can idealLybe used as a fluid carrier medium for transporting oxygen into a culture medium. The oxygen then migrates across -- ~3~
the liquid/liquid phase boundary from the fluorinated hydrocarbon ;nto the aqueous culture medium. The oxygen-depleted fluor;nated hyclrocarbon can then, because it is immiscible with water, easily be removed from the culture medium, enriched with oxygen again and returned to the culture medium. Methods of this type are described~ for example, in the American Patent US 3,850,753. Th;s des-cribes aerobic fermentation with simul~aneous aeration, stirring and/or shaking in a system composed of water and inert solvents immiscible with water, such as perfluoro-C1-Czo-alkanes. S. ~ang, ~iotechnol. Lett. Vol. 7 (1985) pages 81-86 is concerned with the aerob;c fermen-tation of E. coLi ~PP01 in the presence of perfluoromethyl-decalin. The organic solvent is introduced through a nozzle, and continuously removed from the system, in a downward direction. No foam format;on occurs~ The use of a polylysine-stabilized perfluor;nated hydrocarbon emul-sion (FC-70, a tr;(C1s-perfluoroalkyl)am;ne, FC-77, a mix-ture of C8F18 and cyclic C8F16O), which is utilized as a microcarrier for the cultivation of adherent cells or is employed as a blood substitute, is described in Science, Vol. 219, pages 1448-1449 (1983). European Patent Appli-cation EP 0,16~,813 describes a method for the cultivation of animal or plant cells whose oxygen requirement during fermentation is covered by an oxygen-saturated fluorinated hydrocarbon solution. One of the two phases - ferment-ation medium or fluid oxygen-carrier medium - can be oper-ated continuously.
Despite all ~he advantages associated with the industrial fermentation of live cells, there is one aspect which should not be forgotten and which appears especially on fermentation of cells manipulated by genetic engineering:
the exit air which is produced by such fermenters and which may also contain small amounts of cellular mater;al. Ex-treme care must be taken, especially on fermentation ofmicroorganisms wh;ch have been modif;ed by genetic engin-; eering, to ensure that the exit air no longer contains ~ cellular material. The industrial elaboration reguired ~i2~
for this is at present great tsafety fermentation).
The object of the invention was to provide a fermenta~ic,nmethod in which both the supply of oxygen to the fermen-tation medium is ensured in an optimum manner and, at the same time, the requirements of safety fermentation (absence of exit gas) are met in an economic manner.
This object is achieved by a method for introducing oxygen into fermentation media with the aid of fluid carrier media by transport of the oxygen into the fermentation Medium across the liquid/Liquid phase boundary, where the oxygen - which is removed by the fermentation medium in the oxygen-depleted carrier medium is replenished in an aeration device ~ithout emission of exit gas streams, and the carbon dioxide which is formed cluring the fermentation and which is likewise dissolved in the carr;er medium is partially or completely desorbed and removed.
Fermentation media are defined as the generally customary, usually aqueous, culture media which, besides the suspen-ded cells, contain the nutrient med;um with suitable sour-ces of carbon and nitrogen. E~ample~ of cells ~hich canbe cultivated are animal, plant or microbial cells, which ; can also be immobilized or encapsulated. Flu;d carrier media are defined as those liquids which, at the least, a) are only slightly ~iscible or, preferably, are com-pletely immiscible with water b) are able to bind oxygen reversibly better than can ~ater c) are inert to the cells used, and, furthermore, preferably also d) have a higher specific gravity than the culture medium (water) 2~
e) at the same time are also able to bind carbon dioxide reversib~y better than can waterO
Examples of suitable fluid carrier media are perfluorinated straight-chain or branched C1-C2û-alkanes, perfluorin-S atecl Cs-C14-cycloalkanes, perfluorinated tetrahydro~
furan, perfluorinated tetrahydropyran, perfluorinated adamantane and the perfluors-Cs-C14-cycloalkanes, per fluorotetrahydrofurans, perfluorotetrahydropyrans and perfluoroadamantanes substituted by C1-Cs-perfluoroalkyl, as well as perfLuoro-C1-C20-alkoxy compounds and per-fluorinated polyethers such as C10F222 ( ~ HOSTINERT
130), C13F2gO3 (HOSTINERT 175), C16F3404 (HOSTINERT
216) and Cz2F4606 (HOSTINERT 272, manufactured by Hoechst AG).
Examples which may be mentioned are:
perfluorinated derivatives of heptane, octane, nonane, tri-butylamine, N-methylmorpholine, 1- methyldecalin, deca-lin, naphthalene, methyldecalin, methylnaphthalene, 2-butyl-, 2-pentyl-, Z-hexyl- and 2-heptylfuran, and 2-butyl-, Z-pentyl-, 2-hexyl or Z-heptyltetrahydrofuran.
However, also suitable are silicones such as dimethyl- or phenylmethylsilicones, polyvinylpyrrolidones or those sub-stances which are not toxic to the cells and can be used as blood substitutes (see DMW No. 35, volume 105, Stuttgart, Aug. 29, 1980~ pages 1197-1198 and J. Fluorine Chemistry~
9 t1977), 137-146).
Exchange of gases between the fer0entation media and the oxygen-carrier medium takes place across the liquid/
liquid phase boundary. It has been demonstrated in this connection that the oxygen-transfer rate is considerably higher than in the case ~f a gas/liquid phase transfer such as takes place, for example, when oxygen tair) is bubbled through the fermentation medium. The release of oxygen to the fermentation medium takes place simultaneouslY
~L3~2~
,, , with uptake of carbon dioxide by the carrier medium be-rause, usually, carbon dioxide is also more ~physically) soluble in the oxygen-carr;er medium than in the aqueous culture broth.
The best procedure for the method according to the inven-tion is such that, for example, the fluid carrier medium is treated with oxygen or air or an oxygen/air mixture in a bubble column. The fluid carrier medium ~hich has been saturated ~ith gas in this way is now contac~ed with the culture medium, when the exchange of gases described above takes place. This can be carried out in such a way, for example, that the medium which has a higher specific gra-vity is allowed to drip in a down~ard direction through a column of liquid medium with a lower specific gravity, or else the medium which has a lower specific gravity is allowed to rise in an upward direction through the medium which has a higher specific gravity. In both cases the phases separate again so that the oxygen-depleted and carbon dioxide-enriched fluid carrier medium can be drawn off and passed to the bubble reactor, where the physically dissolved carbon dioxide is driven off by the excess oxy-gen (air) bubbling through, and the fluid carrier medium is again enriched with oxygen. It is essential to the invention that the carbon dioxide which is driven off is dra~n o~f together with excess oxygen (air3 from the gas chamber of the bubble column, and the carbon dioxide is completely or partially chemically or physically desorbed~
This can be carried out, for example, by scrubbing with an alkali metal hydroxide solution, ~hen solid alkali metal carbonate is formed. The residual gas (~ainLy composed of oxygen) is, according to the invention, returned to the bubble column. This completes the gas circulation, and no exit gas is able to escape. An advantage of this method is that it is necessary tv replenish only the oxygen actu-ally consumed by the culture medium. Another advantage ofthe method is that it is possible, by means of a bypass around the carbon dioxide scrubber, to control the carbon dioxide concentration in the gas and thus in the liquid ;
~3~25~
., .
carrier medium and thus, in turn, in the culture medium, which makes it possible to control the pH accurately~
A further advantage of the method according to the inven-tion is that both the fermentation solution and the fluid carrier medium can be operated continuously, i.e. it is possible during the fermentation process both for the aqueous culture medium to be continuously rene~ed and -where appropriate with a stationary ~ermentation medium -for the fluid carrier medium to be continuously regener-1û ated. The relevant phase - the fermentation medium and/or the fluid carrier medium - is accordingly designated the "continuous phase". In the case where the fluid carrier medium is dripped through a column of the liquid fermen-tation mediu~ (irrespective of whether the fluid carrier medium now has a lower or higher specific gravity than the fermentation medium), it suffices merely to introduce the oxygen-carrier medium in order to bring about adequate mix-ing of the fermentation medium. However, it is also pos-sible, where appropriate, to mix the fermentation medium by the ~se of suitable conducting devic~s in the fermenter or by an external cycling (c;rculation).
A preferred embodiment of the method can be carried out with the device depicted in F;gure 1. A fluid oxygen-carrier medium, which has a higher specific gravity ~han waterO for example a fluorinated hydrocarbon, ;s treated in a bubble column ~1), via a supply line (2) with a gas mixture containing mainly oxygen. The oxysen-enriched fluorinated hydrocarbon is taken off via a discharge line t3) and passed via an opening (4) into a fermenter t5) whose temperature can be controlled. The oxygen-enriched fluorinated hydrocarbon is dripped through a perforated plate (6) (diameter of the perforations bet~een 0.5 and ~ mm) into the aqueous culture medium (7) which can, where appropriate, be operated continuously via the supply (8) and discharge (9) lines. ~hile the fluorinated hydro-carbon, which has a higher speci~ic gravity, is dripping do~n ;t releases oxygen to the aqueous culture medium and, ~2~6~
. .
at the same time, is enriched ~ith carbon dioxide from the culture medium. The fluorinated hydrocarbon which has been depleted/enriched in this way is taken off via an opening (10) and returned via a leveling loop t11) (which controls the level of liquid fluorinated hydrocarbon in the fermenter) and a pump (12) through the opening (13) to the bubble column. A circula~ing pump (14) provides~ in conjunction with the suppLy and discharge lines (15) and (16), for thorough mixing of the fluorinated hydrocarbon in the bubble column. The heat exchanger (17) included in the circulation adjusts the temperature of the fluid car-rier medium to correspond to that of the fermentation.
The excess pressure of oxygen, which continuously prevails in the bubble column, drives out the carbon dioxide which is present physically dissolved in the recycled, oxygen-depleted fluorinated hydrocarbon. At the same time, the fluorinated hydrocarbon is again enriched with oxygen.
The exit gas, which mainly contains carbon diox;de and excess oxygen (air), is taken off v;a a line (18) and passed by means of a bLower (19) ts a carbon dioxide scrubber (20) which is filled, for example, with sodium hydroxide soLution.
The carbon dioxide is precipitated here as a carbonate, and the gas which now mainly contains only oxygen (air) is returned v;a the supply line (2) to the bubble column. A
def;ned carbon dioxide concentrat;on in the system can be provided for by a bypass (21) around the carbon dioxide scrubber, by ~hich means it is possible to control the pH
in the fermenter.
The sterile system is charged with the fluid carrier med-ium via the connector (a) with simultaneous opening of the valves (v). The valves tv) are closed during the fermen-tation. The temperature of the fermenter (5) can be con-trolled via the supply and discharge lines (22) and ~Z3).
((24) = sterile filter, (25) - flo~ meter, (26), ~27) =
supply and discharge lines, respectively, for charging/
emptying the carbon d;oxide scrubber).
-` ~L3~2~
_ 9 Ex~ples The apparatus depicted in Figure 1 was used for aerobic fermentations with per~luorinated hydrocarbons as oxygen carriers~
5 Fermenter tS) : diameter 220 mm height 220 mm Perforated disk (6) : material: stainless steel diameter of perforations 1 mm Gas flow through the bubble colu~n: 100 - 500 l/h Oxygen carrier:
Hostinert 216 Molecular mass: 902 g/mol; boiling point: 216C; surface tension (25C)~ 15.4 mN/m; dynamic viscosity: 8.78 mPa s (20C); mlO2/100 ml: 51.4 (25C); density: 1.839 kg/l (20C).
Hostinert 175 Molecular mass: 736 g/mol; boiling point: 175C; surface tension ~25C) 14~6 mN/m; dynamic viscosity: 4~39 mPa-s;
(2ûC~ mlO2/100 ml: 57 (25C); density: 1.816 kg/l (20C) Hostinert circulation flou I: (16)-(14)-(15) ~see Figure I]
180 l/h Hostinert circulation flow II: (4)-(6)-t1û) [see Figure I]
20-150 l/h.
A) GeneraL infor~ation on the ~ethod The sterilized system and the reactor (see Figure 1) are charged with sterile fluid carrier medium in accordance with the setting on the leveling looP, the temperature is controlled, and aeration is carried out in the bubble column with nitrogen/oxygen mixtures (0 to 100% 2, 100 to 0X Nz) and with addition of carbon dioxide. The reac-tor is subsequently charged with 10 l of nutrient solution, the temperature is controlled, and homogeneous mixing and ~` ~3~ 2~
oxygen enrichment are carried ou~ via the flow of Hostinert (~t-6-10). The fermenter is inoculated with the preculture and, during the fermentation, parameters such as pH, and concentrations of substrate and product are monitored and controlled. The dissolved oxygen content is controlled via the flow of Hostinert (4-6-10). The carbon dioxide formed during the fermentation can be entirely or partially removed in the scrubber ~20), and the consumed oxygen can be replenished.
Ex~mple 1 Fermentation of Escherichia coli K 12 Nutrient medium: 0.8 % meat peptones (trypsin digest) û.8 % casein peptone (trypsin digest) 0.3 % yeast extract 0.5 % sod;um chloride 1 ~ glucose Temperature: 37C; pH: 7.0 The fermentation was carried out in the manner described in section A. The fluid carrier medium used was Hostinert 216, which is saturated with air in the bubble column.
The fermentation medium is inoculated with 100 ml of pre-culture, and the oxygen supply is ensured by introducing Hostinert 216 (60 l/h). The Hostinert flow is increased up to 150 l per hour as appropriate for the growth of the cells in the exponential phase of growth, with the satur-ation of the disperse phase being carried out with a nitro-gen/oxygen mixture.
A generation time of 30 minutes is achieved in the expo-nential phase of growth, with a dry mass content of 0.32 g/l at the end of the exponential phase.
~3~L2~68 Exa~ple 2 Fermen~ation of bakers' yeast (Saccharomyces cerevisiae) Synthetic medium containing 1 % glucose and 2.48 g/l (NH4)2HP04 0.4 g/l MgS04 . 7 H20 0.2 g/l KCl 0.12 g/l CaCl2 . 2 H20 20 mg/l NaCl 20 mg/l m-inositol 1.2 mg/l Ca pantothenate 0.8 mg/l H3~03 û.4 mg~l ZnS04 . 7 H20 0.4 mg/l MnS04 . 7 H20 O.Z mg/l FeCl3 . 6 ~l2 0.2 mg/l Na2Moo4 . 2 ~l2 0.12 mg/l NaI
0.04 mg/l CuS04 . 5 H20 0.02 mg/l biot;n.
The temperature of the fermentation solution and the ~ Host;nert 216 is controlled at 30C, and the PH ;s maintained constant at S.O during the fermentation by addi-tion of 1 normal sodium hydroxide solution~ ~fter inocu-lation with 100 ml of preculture (24 hours old, synthetic medium containing 1 X glucose), the Hostinert flow through the reactor is adjusted from ~ l/h to 120 l/h, and the fermentation is operated continuously u;th a dilution rate of D = 1 h 1 (inflow rate f = 1 l/h, connectors t8) and (9)). Once equilibrium has been reached the biomass amounts to 4.5 g/l.
Exa~p~e 3 Hybridoma cells are microencapsulated as in Example 11 of German Patent Application P 37 060 10.4, to which express reference is made at this pointJ
~3~ 2~
The reactor is charged with Dulbecco's medium, the tempera-ture is controlled at 37C, and the fermentation solu-tion is enriched with oxygen using ~ Hostinert 175 which is saturated with a nitrogen/oxygen t1:1) mixture contain-ing 5 ~ carbon dioxide. The fermentat;on medium is inocu-Lated with 10 ml of capsules (capsule diameter 500 ~m;
104 capsules) per liter, and the medium is changed dis-continuously during the fermentation.
Day Cell count per mlug of antibody per ml reactor volumereactor volume _ __ _ 3 2 x 105 8 4* 4 x 105 20 9.6 x 105 30 8* 7 x 105 S0 1510 1.7 x 106 110 13 1.û4 x 106 90 * The medium was changed on these days.
The antibody content (mouse IgG) in X by weight was deter-mined using an enzyme immunoassay (Behring ~erke AG, Marburg).
The cultivation of live cells, especially in large amounts, is nowadays an essential constituent of the production of, for example, antiviral substances such as interferon, or of biologically active substances such as hormones. What are called hybridoma cells - a fusion product of antibody-producing cells with myeloma cells - are cultivated in large amounts especially for the preparation of monoclonal antibodies which have the ability ~o recogni~e very parti-cular epitopes of a protein (of a macromolecule) and to bind thereto.
In conventional laboratory methods for the cultivation of cells, the latter are suspended in a medium in a Petri dish. Oxygen and carbon dioxide diffuse through the sur-face of the med;um into the cells. The depth of the cul-ture medium influences the ra~e of diffusion of oxygen and carbon dioxide to the cells. As the depth of the medium increases, the ratio of the surface to the volume of the medium decreases. At a particular time the concentrations of oxygen and carbon dioxide in the air over the dish, and/
or the rate of dissolution in the medium, are insufficient ~ 3 ~
to supply the total volume of the medium with sufficient gas and to satis~y the requirements of the growing cells, i.e. ~he available oxygen becomes the factor limiting growth. Accordingly, the state of the art teaches that the depth of the medium ought to be in the region of mm for static cultures. In general, the upper limit of cell density for cultures of this type is about 105 cells/ml of culture medium when the air ouer the culture medium is used to supply oxygen.
When methods for the cultivation of cells are carried out on the industrial scale it is difficult to make available sufficient amounts of oxygen to satisfy the needs of the cells~ Gases are frequently bubbled directly through the medium in order to supply the cells with the required gas concentrations. ~owever, direct bubbling through is un-suitable especially for the cultivation of sensitive cells.
These cells are physically damaged when a gas is bubbled through at a sufficient rate to mainta;n appropriate gas concentrations in the medium, or by mechanical stirrers because of the high shear stress. In addition, protein constituents of the medium, uhich are normally necessary in all cell cultures, form a foam, ~hich may trap the cells, on the surface of the medium. The cells entrapped in the foam rapidly die It is possible to add antifoam agents to the medium but these are occasionally toxic for the cells in the culture and, in addition, can be separated from the product during working-up only with difficulty.
An elegant method of cultivating even sensitive cells on the industrial scale and, at the same time, ensuring the necessary supply of oxygen is the introduction of oxygen into the culture medium by means of a flùid carrier medium.
It is known that, for example, fluor;nated hydrocarbons are able to dissolve oxygen physically about 20 times better than can water. Thus~ if such fluorinated hydro-carbons are saturated with oxygen (or air) they can idealLybe used as a fluid carrier medium for transporting oxygen into a culture medium. The oxygen then migrates across -- ~3~
the liquid/liquid phase boundary from the fluorinated hydrocarbon ;nto the aqueous culture medium. The oxygen-depleted fluor;nated hyclrocarbon can then, because it is immiscible with water, easily be removed from the culture medium, enriched with oxygen again and returned to the culture medium. Methods of this type are described~ for example, in the American Patent US 3,850,753. Th;s des-cribes aerobic fermentation with simul~aneous aeration, stirring and/or shaking in a system composed of water and inert solvents immiscible with water, such as perfluoro-C1-Czo-alkanes. S. ~ang, ~iotechnol. Lett. Vol. 7 (1985) pages 81-86 is concerned with the aerob;c fermen-tation of E. coLi ~PP01 in the presence of perfluoromethyl-decalin. The organic solvent is introduced through a nozzle, and continuously removed from the system, in a downward direction. No foam format;on occurs~ The use of a polylysine-stabilized perfluor;nated hydrocarbon emul-sion (FC-70, a tr;(C1s-perfluoroalkyl)am;ne, FC-77, a mix-ture of C8F18 and cyclic C8F16O), which is utilized as a microcarrier for the cultivation of adherent cells or is employed as a blood substitute, is described in Science, Vol. 219, pages 1448-1449 (1983). European Patent Appli-cation EP 0,16~,813 describes a method for the cultivation of animal or plant cells whose oxygen requirement during fermentation is covered by an oxygen-saturated fluorinated hydrocarbon solution. One of the two phases - ferment-ation medium or fluid oxygen-carrier medium - can be oper-ated continuously.
Despite all ~he advantages associated with the industrial fermentation of live cells, there is one aspect which should not be forgotten and which appears especially on fermentation of cells manipulated by genetic engineering:
the exit air which is produced by such fermenters and which may also contain small amounts of cellular mater;al. Ex-treme care must be taken, especially on fermentation ofmicroorganisms wh;ch have been modif;ed by genetic engin-; eering, to ensure that the exit air no longer contains ~ cellular material. The industrial elaboration reguired ~i2~
for this is at present great tsafety fermentation).
The object of the invention was to provide a fermenta~ic,nmethod in which both the supply of oxygen to the fermen-tation medium is ensured in an optimum manner and, at the same time, the requirements of safety fermentation (absence of exit gas) are met in an economic manner.
This object is achieved by a method for introducing oxygen into fermentation media with the aid of fluid carrier media by transport of the oxygen into the fermentation Medium across the liquid/Liquid phase boundary, where the oxygen - which is removed by the fermentation medium in the oxygen-depleted carrier medium is replenished in an aeration device ~ithout emission of exit gas streams, and the carbon dioxide which is formed cluring the fermentation and which is likewise dissolved in the carr;er medium is partially or completely desorbed and removed.
Fermentation media are defined as the generally customary, usually aqueous, culture media which, besides the suspen-ded cells, contain the nutrient med;um with suitable sour-ces of carbon and nitrogen. E~ample~ of cells ~hich canbe cultivated are animal, plant or microbial cells, which ; can also be immobilized or encapsulated. Flu;d carrier media are defined as those liquids which, at the least, a) are only slightly ~iscible or, preferably, are com-pletely immiscible with water b) are able to bind oxygen reversibly better than can ~ater c) are inert to the cells used, and, furthermore, preferably also d) have a higher specific gravity than the culture medium (water) 2~
e) at the same time are also able to bind carbon dioxide reversib~y better than can waterO
Examples of suitable fluid carrier media are perfluorinated straight-chain or branched C1-C2û-alkanes, perfluorin-S atecl Cs-C14-cycloalkanes, perfluorinated tetrahydro~
furan, perfluorinated tetrahydropyran, perfluorinated adamantane and the perfluors-Cs-C14-cycloalkanes, per fluorotetrahydrofurans, perfluorotetrahydropyrans and perfluoroadamantanes substituted by C1-Cs-perfluoroalkyl, as well as perfLuoro-C1-C20-alkoxy compounds and per-fluorinated polyethers such as C10F222 ( ~ HOSTINERT
130), C13F2gO3 (HOSTINERT 175), C16F3404 (HOSTINERT
216) and Cz2F4606 (HOSTINERT 272, manufactured by Hoechst AG).
Examples which may be mentioned are:
perfluorinated derivatives of heptane, octane, nonane, tri-butylamine, N-methylmorpholine, 1- methyldecalin, deca-lin, naphthalene, methyldecalin, methylnaphthalene, 2-butyl-, 2-pentyl-, Z-hexyl- and 2-heptylfuran, and 2-butyl-, Z-pentyl-, 2-hexyl or Z-heptyltetrahydrofuran.
However, also suitable are silicones such as dimethyl- or phenylmethylsilicones, polyvinylpyrrolidones or those sub-stances which are not toxic to the cells and can be used as blood substitutes (see DMW No. 35, volume 105, Stuttgart, Aug. 29, 1980~ pages 1197-1198 and J. Fluorine Chemistry~
9 t1977), 137-146).
Exchange of gases between the fer0entation media and the oxygen-carrier medium takes place across the liquid/
liquid phase boundary. It has been demonstrated in this connection that the oxygen-transfer rate is considerably higher than in the case ~f a gas/liquid phase transfer such as takes place, for example, when oxygen tair) is bubbled through the fermentation medium. The release of oxygen to the fermentation medium takes place simultaneouslY
~L3~2~
,, , with uptake of carbon dioxide by the carrier medium be-rause, usually, carbon dioxide is also more ~physically) soluble in the oxygen-carr;er medium than in the aqueous culture broth.
The best procedure for the method according to the inven-tion is such that, for example, the fluid carrier medium is treated with oxygen or air or an oxygen/air mixture in a bubble column. The fluid carrier medium ~hich has been saturated ~ith gas in this way is now contac~ed with the culture medium, when the exchange of gases described above takes place. This can be carried out in such a way, for example, that the medium which has a higher specific gra-vity is allowed to drip in a down~ard direction through a column of liquid medium with a lower specific gravity, or else the medium which has a lower specific gravity is allowed to rise in an upward direction through the medium which has a higher specific gravity. In both cases the phases separate again so that the oxygen-depleted and carbon dioxide-enriched fluid carrier medium can be drawn off and passed to the bubble reactor, where the physically dissolved carbon dioxide is driven off by the excess oxy-gen (air) bubbling through, and the fluid carrier medium is again enriched with oxygen. It is essential to the invention that the carbon dioxide which is driven off is dra~n o~f together with excess oxygen (air3 from the gas chamber of the bubble column, and the carbon dioxide is completely or partially chemically or physically desorbed~
This can be carried out, for example, by scrubbing with an alkali metal hydroxide solution, ~hen solid alkali metal carbonate is formed. The residual gas (~ainLy composed of oxygen) is, according to the invention, returned to the bubble column. This completes the gas circulation, and no exit gas is able to escape. An advantage of this method is that it is necessary tv replenish only the oxygen actu-ally consumed by the culture medium. Another advantage ofthe method is that it is possible, by means of a bypass around the carbon dioxide scrubber, to control the carbon dioxide concentration in the gas and thus in the liquid ;
~3~25~
., .
carrier medium and thus, in turn, in the culture medium, which makes it possible to control the pH accurately~
A further advantage of the method according to the inven-tion is that both the fermentation solution and the fluid carrier medium can be operated continuously, i.e. it is possible during the fermentation process both for the aqueous culture medium to be continuously rene~ed and -where appropriate with a stationary ~ermentation medium -for the fluid carrier medium to be continuously regener-1û ated. The relevant phase - the fermentation medium and/or the fluid carrier medium - is accordingly designated the "continuous phase". In the case where the fluid carrier medium is dripped through a column of the liquid fermen-tation mediu~ (irrespective of whether the fluid carrier medium now has a lower or higher specific gravity than the fermentation medium), it suffices merely to introduce the oxygen-carrier medium in order to bring about adequate mix-ing of the fermentation medium. However, it is also pos-sible, where appropriate, to mix the fermentation medium by the ~se of suitable conducting devic~s in the fermenter or by an external cycling (c;rculation).
A preferred embodiment of the method can be carried out with the device depicted in F;gure 1. A fluid oxygen-carrier medium, which has a higher specific gravity ~han waterO for example a fluorinated hydrocarbon, ;s treated in a bubble column ~1), via a supply line (2) with a gas mixture containing mainly oxygen. The oxysen-enriched fluorinated hydrocarbon is taken off via a discharge line t3) and passed via an opening (4) into a fermenter t5) whose temperature can be controlled. The oxygen-enriched fluorinated hydrocarbon is dripped through a perforated plate (6) (diameter of the perforations bet~een 0.5 and ~ mm) into the aqueous culture medium (7) which can, where appropriate, be operated continuously via the supply (8) and discharge (9) lines. ~hile the fluorinated hydro-carbon, which has a higher speci~ic gravity, is dripping do~n ;t releases oxygen to the aqueous culture medium and, ~2~6~
. .
at the same time, is enriched ~ith carbon dioxide from the culture medium. The fluorinated hydrocarbon which has been depleted/enriched in this way is taken off via an opening (10) and returned via a leveling loop t11) (which controls the level of liquid fluorinated hydrocarbon in the fermenter) and a pump (12) through the opening (13) to the bubble column. A circula~ing pump (14) provides~ in conjunction with the suppLy and discharge lines (15) and (16), for thorough mixing of the fluorinated hydrocarbon in the bubble column. The heat exchanger (17) included in the circulation adjusts the temperature of the fluid car-rier medium to correspond to that of the fermentation.
The excess pressure of oxygen, which continuously prevails in the bubble column, drives out the carbon dioxide which is present physically dissolved in the recycled, oxygen-depleted fluorinated hydrocarbon. At the same time, the fluorinated hydrocarbon is again enriched with oxygen.
The exit gas, which mainly contains carbon diox;de and excess oxygen (air), is taken off v;a a line (18) and passed by means of a bLower (19) ts a carbon dioxide scrubber (20) which is filled, for example, with sodium hydroxide soLution.
The carbon dioxide is precipitated here as a carbonate, and the gas which now mainly contains only oxygen (air) is returned v;a the supply line (2) to the bubble column. A
def;ned carbon dioxide concentrat;on in the system can be provided for by a bypass (21) around the carbon dioxide scrubber, by ~hich means it is possible to control the pH
in the fermenter.
The sterile system is charged with the fluid carrier med-ium via the connector (a) with simultaneous opening of the valves (v). The valves tv) are closed during the fermen-tation. The temperature of the fermenter (5) can be con-trolled via the supply and discharge lines (22) and ~Z3).
((24) = sterile filter, (25) - flo~ meter, (26), ~27) =
supply and discharge lines, respectively, for charging/
emptying the carbon d;oxide scrubber).
-` ~L3~2~
_ 9 Ex~ples The apparatus depicted in Figure 1 was used for aerobic fermentations with per~luorinated hydrocarbons as oxygen carriers~
5 Fermenter tS) : diameter 220 mm height 220 mm Perforated disk (6) : material: stainless steel diameter of perforations 1 mm Gas flow through the bubble colu~n: 100 - 500 l/h Oxygen carrier:
Hostinert 216 Molecular mass: 902 g/mol; boiling point: 216C; surface tension (25C)~ 15.4 mN/m; dynamic viscosity: 8.78 mPa s (20C); mlO2/100 ml: 51.4 (25C); density: 1.839 kg/l (20C).
Hostinert 175 Molecular mass: 736 g/mol; boiling point: 175C; surface tension ~25C) 14~6 mN/m; dynamic viscosity: 4~39 mPa-s;
(2ûC~ mlO2/100 ml: 57 (25C); density: 1.816 kg/l (20C) Hostinert circulation flou I: (16)-(14)-(15) ~see Figure I]
180 l/h Hostinert circulation flow II: (4)-(6)-t1û) [see Figure I]
20-150 l/h.
A) GeneraL infor~ation on the ~ethod The sterilized system and the reactor (see Figure 1) are charged with sterile fluid carrier medium in accordance with the setting on the leveling looP, the temperature is controlled, and aeration is carried out in the bubble column with nitrogen/oxygen mixtures (0 to 100% 2, 100 to 0X Nz) and with addition of carbon dioxide. The reac-tor is subsequently charged with 10 l of nutrient solution, the temperature is controlled, and homogeneous mixing and ~` ~3~ 2~
oxygen enrichment are carried ou~ via the flow of Hostinert (~t-6-10). The fermenter is inoculated with the preculture and, during the fermentation, parameters such as pH, and concentrations of substrate and product are monitored and controlled. The dissolved oxygen content is controlled via the flow of Hostinert (4-6-10). The carbon dioxide formed during the fermentation can be entirely or partially removed in the scrubber ~20), and the consumed oxygen can be replenished.
Ex~mple 1 Fermentation of Escherichia coli K 12 Nutrient medium: 0.8 % meat peptones (trypsin digest) û.8 % casein peptone (trypsin digest) 0.3 % yeast extract 0.5 % sod;um chloride 1 ~ glucose Temperature: 37C; pH: 7.0 The fermentation was carried out in the manner described in section A. The fluid carrier medium used was Hostinert 216, which is saturated with air in the bubble column.
The fermentation medium is inoculated with 100 ml of pre-culture, and the oxygen supply is ensured by introducing Hostinert 216 (60 l/h). The Hostinert flow is increased up to 150 l per hour as appropriate for the growth of the cells in the exponential phase of growth, with the satur-ation of the disperse phase being carried out with a nitro-gen/oxygen mixture.
A generation time of 30 minutes is achieved in the expo-nential phase of growth, with a dry mass content of 0.32 g/l at the end of the exponential phase.
~3~L2~68 Exa~ple 2 Fermen~ation of bakers' yeast (Saccharomyces cerevisiae) Synthetic medium containing 1 % glucose and 2.48 g/l (NH4)2HP04 0.4 g/l MgS04 . 7 H20 0.2 g/l KCl 0.12 g/l CaCl2 . 2 H20 20 mg/l NaCl 20 mg/l m-inositol 1.2 mg/l Ca pantothenate 0.8 mg/l H3~03 û.4 mg~l ZnS04 . 7 H20 0.4 mg/l MnS04 . 7 H20 O.Z mg/l FeCl3 . 6 ~l2 0.2 mg/l Na2Moo4 . 2 ~l2 0.12 mg/l NaI
0.04 mg/l CuS04 . 5 H20 0.02 mg/l biot;n.
The temperature of the fermentation solution and the ~ Host;nert 216 is controlled at 30C, and the PH ;s maintained constant at S.O during the fermentation by addi-tion of 1 normal sodium hydroxide solution~ ~fter inocu-lation with 100 ml of preculture (24 hours old, synthetic medium containing 1 X glucose), the Hostinert flow through the reactor is adjusted from ~ l/h to 120 l/h, and the fermentation is operated continuously u;th a dilution rate of D = 1 h 1 (inflow rate f = 1 l/h, connectors t8) and (9)). Once equilibrium has been reached the biomass amounts to 4.5 g/l.
Exa~p~e 3 Hybridoma cells are microencapsulated as in Example 11 of German Patent Application P 37 060 10.4, to which express reference is made at this pointJ
~3~ 2~
The reactor is charged with Dulbecco's medium, the tempera-ture is controlled at 37C, and the fermentation solu-tion is enriched with oxygen using ~ Hostinert 175 which is saturated with a nitrogen/oxygen t1:1) mixture contain-ing 5 ~ carbon dioxide. The fermentat;on medium is inocu-Lated with 10 ml of capsules (capsule diameter 500 ~m;
104 capsules) per liter, and the medium is changed dis-continuously during the fermentation.
Day Cell count per mlug of antibody per ml reactor volumereactor volume _ __ _ 3 2 x 105 8 4* 4 x 105 20 9.6 x 105 30 8* 7 x 105 S0 1510 1.7 x 106 110 13 1.û4 x 106 90 * The medium was changed on these days.
The antibody content (mouse IgG) in X by weight was deter-mined using an enzyme immunoassay (Behring ~erke AG, Marburg).
Claims (11)
1. A method for introducing oxygen into a fermentation medium in a closed system with the aid of a fluid carrier medium by transport of oxygen into said fermentation medium which comprises:
a) release of oxygen from the fluid carrier medium across a liquid/liquid phase boundary to said fermentation medium with the simultaneous uptake of carbon dioxide, formed during fermentation, by the fluid carrier medium;
b) transporting the oxygen-depleted, carbon dioxide-enriched fluid carrier medium to an aeration device wherein the fluid carrier medium is re-oxygenated and carbon dioxide is driven off from said fluid carrier medium and combined with excess oxygen from the aeration device to form an exit gas stream;
c) recycling the re-oxygenated fluid carrier medium to said fermentation medium;
d) transporting said exit gas stream collected from said aeration device to a desorption device wherein the carbon dioxide is completely or partially desorbed leaving a residual exit gas stream; and e) recycling said residual exit gas stream to said aeration device, without the emission of any gas to the environment.
a) release of oxygen from the fluid carrier medium across a liquid/liquid phase boundary to said fermentation medium with the simultaneous uptake of carbon dioxide, formed during fermentation, by the fluid carrier medium;
b) transporting the oxygen-depleted, carbon dioxide-enriched fluid carrier medium to an aeration device wherein the fluid carrier medium is re-oxygenated and carbon dioxide is driven off from said fluid carrier medium and combined with excess oxygen from the aeration device to form an exit gas stream;
c) recycling the re-oxygenated fluid carrier medium to said fermentation medium;
d) transporting said exit gas stream collected from said aeration device to a desorption device wherein the carbon dioxide is completely or partially desorbed leaving a residual exit gas stream; and e) recycling said residual exit gas stream to said aeration device, without the emission of any gas to the environment.
2. The method as claimed in claim 1, wherein the exit gas flow is recycled with the carbon dioxide being entirely or partially removed from the exit gas flow.
3. The method as claimed in claim 1 or 2, wherein the fluid carrier medium has a higher density than the fermentation medium.
4. The method as claimed in claim 1 or 2, wherein the fluid carrier medium has a lower density than the fermentation medium.
5. The method as claimed in claim 1, wherein either the fluid carrier medium passes in the form of small drops through the fermentation medium, or the fermentation medium passes in the form of small drops through the fluid carrier medium.
6. The method as claimed in claim 5, wherein the dispersion into drops of the fluid carrier medium or of the fermentation medium is carried out by one of: capillaries; a perforated disk; and capillaries and a sieve plate.
7. The method as claimed in claim 6, wherein the dispersion is carried out by one of capillaries; and capillaries and a sieve plate, and wherein the capillaries and the perforations of the sieve plate, when present, each have a diameter of 0.5 - 4 mm.
8. The method as claimed in claim 1, wherein perfluorinated hydrocarbons, perfluorinated polyethers, perfluoroalkoxy compounds, silicones, polyvinylpyrrolidone or blood substitutes are used as fluid carrier medium.
9. The method as claimed in claim 1, wherein the fermentation medium is mixed in a controlled fashion by use of line devices or by an external circulation.
10. The method as claimed in claim 1, wherein the fermentation is operated continuously.
11. The method as claimed in claim 1, wherein microorganisms, mammalian cells, plant cells, species which have been modified by genetic engineering, or immobilized or encapsulated cells of the type described above, are cultivated.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3739621.8 | 1987-11-23 | ||
| DE19873739621 DE3739621A1 (en) | 1987-11-23 | 1987-11-23 | METHOD FOR THE EXHAUST-FREE BEGINNING OF FERMENTATION MEDIA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1312568C true CA1312568C (en) | 1993-01-12 |
Family
ID=6341042
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000583807A Expired - Fee Related CA1312568C (en) | 1987-11-23 | 1988-11-22 | Method for the aeration, without exit gases, of fermentation media |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0317854B1 (en) |
| JP (1) | JPH01168274A (en) |
| AT (1) | ATE96166T1 (en) |
| CA (1) | CA1312568C (en) |
| DE (2) | DE3739621A1 (en) |
| DK (1) | DK651688A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04122412A (en) * | 1990-09-12 | 1992-04-22 | Hitachi Ltd | Method and equipment for dissolving oxygen |
| US7718405B2 (en) | 2005-09-19 | 2010-05-18 | American Air Liquide, Inc. | Use of pure oxygen in viscous fermentation processes |
| WO2015059798A1 (en) * | 2013-10-24 | 2015-04-30 | 学校法人東京電機大学 | Cell or tissue transport device |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US1732921A (en) * | 1926-03-06 | 1929-10-22 | Anheuser Busch | Process for aerobic fermentation |
| US3846245A (en) * | 1969-09-22 | 1974-11-05 | Mobil Oil Corp | Process for growing microorganisms with low pressure air |
| JPS572678A (en) * | 1980-06-06 | 1982-01-08 | Hitachi Ltd | Device for aerated cultivation |
| DE3584317D1 (en) * | 1984-06-14 | 1991-11-14 | Teijin Ltd | METHOD FOR BREEDING ANIMAL OR VEGETABLE CELLS. |
| FR2588271B1 (en) * | 1985-10-08 | 1988-08-12 | Commissariat Energie Atomique | CONTINUOUS AEROBIC OR ANAEROBIC FERMENTATION PLANT AND METHOD |
-
1987
- 1987-11-23 DE DE19873739621 patent/DE3739621A1/en not_active Withdrawn
-
1988
- 1988-11-12 AT AT88118888T patent/ATE96166T1/en not_active IP Right Cessation
- 1988-11-12 EP EP88118888A patent/EP0317854B1/en not_active Expired - Lifetime
- 1988-11-12 DE DE88118888T patent/DE3885064D1/en not_active Expired - Fee Related
- 1988-11-22 DK DK651688A patent/DK651688A/en not_active Application Discontinuation
- 1988-11-22 CA CA000583807A patent/CA1312568C/en not_active Expired - Fee Related
- 1988-11-22 JP JP63293762A patent/JPH01168274A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DK651688A (en) | 1989-05-24 |
| EP0317854A3 (en) | 1990-05-23 |
| EP0317854B1 (en) | 1993-10-20 |
| EP0317854A2 (en) | 1989-05-31 |
| DE3739621A1 (en) | 1989-06-01 |
| DK651688D0 (en) | 1988-11-22 |
| DE3885064D1 (en) | 1993-11-25 |
| ATE96166T1 (en) | 1993-11-15 |
| JPH01168274A (en) | 1989-07-03 |
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