DE19507456A1 - Continuous culture of aerobic microorganisms at high cell density - Google Patents

Abstract

Process for the continuous culture of aerobic organisms at high cell density. This is carried out in a vortex reactor with power input of 4-40 kW/m<3> and a vortex size:inlet dia. ratio of <10 and comprises:(a) injecting a liq. medium tangentially into the upper part of the reactor with a centrifugal force of <= 25 x g;(b) mixing an O2-contg. gas with the medium or introducing it via a jacket around the reactor;(c) withdrawing the liq. medium from the bottom of the reactor and readjusting it to the process conditions in an external recycling system, and(d) withdrawing accumulated gas from the top of the reactor. Also claimed is the appts. for carrying out the process above, where the reactor is integrated into a recycling system with elements for controlling the process conditions. The reactor has a gas outlet at the top, a liq. outlet at the bottom and a tangential inlet connected to a recycling pump whose power input provides the required centrifugal force. Aeration is provided by a device in the upstream of the tangential inlet and/or by perforated gas distributor surfaces in the reactor.

Description

The invention relates to a method for through management of biotechnological processes under cultivation aerobic organisms in high cell density in the continuous flow run in a cyclone reactor with a cyclone diameter ser: inlet diameter ratio <10, that at the top End tangentially liquid medium with an acceleration supply code (Centrifugal acceleration / gravitational acceleration) up to 25 is fed, mixed into the oxygen-containing gas and / or the oxygen-containing gas via the man Tel area of the reactor is supplied and that from the bottom Ren end of the reactor removed and an outer Recycling circulation brought to process conditions will accumulate while from the top of the reactor tes gas is removed, with a power input is worked in a cycle of 4-40 kW / m³, as well a suitable arrangement.

Biotechnological processes, especially fermentation processes involving the cultivation of microorganisms or cells expire, especially at work with high cell densities to an intensive oxygen drove.

There are therefore already different techniques for  Increasing the oxygen input capacity for fermen tion processes known individually or in combination tion can be applied:

• 1. An increase in the driving concentration cases by increasing the oxygen partial pressure in the Reactor with the greatest possible hydrostatic pressure ("Tower biology"), through reactions among higher ones Press ("pressure fermentation") and by gassing with oxygen-enriched air up to the use of pure oxygen.
• 2. An increase in the volume-specific substance exchange area a by dispersing the Gas phase in the entire reactor while avoiding Coalescence ("gap injectors", "radial flow nozzles" with Baffles).
• 3. A reduction in the mass transfer resistance on the liquid side 1 / k _L by increasing the energy input (eg "jet loop reactors").
• 4. Highest oxygen input rates are achieved in the centrifuge reactor: Here, a steam-sterilizable sieve centrifuge is used as the reactor (H. Voit, A. Mersmann, Chem. Ing. Techn. 61, 5 (1989) pp. 416-417). At 50 times the gravitational acceleration, oxygen entry rates of up to 100 g / (l _* h) can be achieved in the centrifugal bioreactor when gassed with air (Newtonian media, k _L a = 10 h ^-1 ). In addition to the intensification of the phase contact (k _{L increase} ) and the fine dispersion of the gas bubbles (a increase), this can be attributed to the local increase in the oxygen partial pressure (increase in the driving concentration gradient) in the centrifugal field.

A cyclone column reactor is also already known (P.S.S. Dawson, Biotechnol. & Bioeng. Symp. No. 4 (1974) 809-819), in which a cyclone column in one Circuit for the liquid medium is switched on, which is fed tangentially to the column head and itself screw-like as a "moving" film along the column wall moved downward with lively gas exchange with the am Gas supplied to the reactor bottom.

This cyclone column technology, which has been known for a long time was developed by J.D. Sheppard et al. a. (J. Chem. Tech. Biotech nol. 59 (1994) 83-89) picked up and in a scale up version of 75 l capacity of the reactor examined. In the scale-up unit, the column was shortened and for that with air injection in one died enlarged Circulation in at least two places while increasing the Rotation speed of the feed pump ensured. On in this way, one with the cyclone column reactor comparable fermentation performance achieved that as similar Lich referred to the conventional stirred tank reactor becomes.

A higher efficiency seems to be that of H.Voit et al. to have (see above) described centrifugal reactor, the in DE 39 05 609 A1 in connection with a cyclone defoamer is described in more detail.

A method that can be used for biotechnological processes alternatively with a cyclone operated in a cycle Actuator of the type according to the invention specified at the beginning has so far not been considered.

According to the invention is worked by intensive tangential entry into the cyclone reactor with a relatively low diameter ratio of cyclone to cyclone inlet with acceleration indicators up to 25 (in particular 2-25) using displacement pumps such as rotary lobe pumps, eccentric screw pumps, sine pumps or swash plate pumps or also peristaltic pumps and double mist pumps with the formation of oxygen transport coefficients K _La up to 3000 h ^-1 (with gas injection in circulation) or up to 25 000 h ^-1 (with gas supply via the cyclone jacket).

By arranging an impact body in the lower loading the formation of the secondary vertebra is richly promoted changes, so that the volume-specific Be Gassing amount can be reduced, which saves of energy and mitigation of foam and Leads to exhaust gas problems.

An impact body in the form of a is particularly expedient Tellers, if necessary at the same time - alone or in addition - can act as a gas distributor or fumigation device or in the form of a tip pointing downwards Cone, which may also - with a porous base - gas can serve.

However, the cyclone reactor according to the invention can also be used without Impact bodies are provided in the lower area, whereby then increased gas fractions get into the recycling circuit can, what sometimes for the sufficient oxygen ver care of sensitive cell systems also in recycling circle may be desired.  

Even with increased gas entry through the impact body or A perforated cylinder jacket of the cyclone can be used Part of the supplied gas is pressed into the circuit become. As a result, even at high cell densities Oxygen supply in the circuit ensured because the Storage capacity of the liquid phase for oxygen be very is bordered. An exact quantification is not possible since the gas / liquid material data is essential for the circulating gas content influence.

It is particularly useful to include a microphone filtration in the recycling circle to increase the Cell densities, e.g. B. worked with tube modules that can and the overflow velocity the tangential inlet into the cyclone reactor is adapted.

All probes for temperature, pH, pO₂, turbidity etc. as well as all inflows and outflows of the liquid phases (Substrates, correction agents, product) are preferred installed in circulation around the flow field in the Cyclone reactor not to interfere.

An arrangement for carrying out biotechnological processes according to the invention with the cultivation of aerobic organisms in high cell density is characterized by a culture liquid circuit with elements for regulating the process conditions for a reactor formed as a cyclone and integrated in the circuit with gas removal from the head and liquid removal from the bottom, whose upper tangential inlet is connected to a pump provided in the circuit for a power input for acceleration indicators (z _inlet ) up to 25 (centrifugal acceleration / gravitational acceleration) and in front of whose lower end an impact body for supporting the secondary vertebra can be arranged and by means of Fumigation in the circuit before the tangential inlet to the cyclone reactor and / or via perforated gas distributor surfaces in the reactor.

Areas of application for the technology according to the invention are:
bio-process engineering for the effective production of microbial low-molecular metabolites (e.g. organic acids, amino acids, vitamins) as well as exoenzymes (e.g. proteases, amylases) and for the effective production of recombinant proteins in high-cell density fermentations;
biotechnological research for the cultivation of high cell densities under defined, non-oxygen-limited conditions.

Further special features of the invention result from the patent claims and from the following Be description of exemplary embodiments with reference to the attached drawings, which are schematically four Variants of an arrangement according to the invention ben.

Referring to FIG. 1, a cyclone reactor 1 with bottom (hollow) baffle cone 2 in a culture fluid circulation is einbezo gene of the cyclone is passed branching off via a pump 4 from the lower end 3, with an outlet for cell-containing fluid 5 and a microfiltration 6, in particular in the form of tube modules for the delivery of filtrate over 7, while at 8 in particular supplementary culture medium can be fed. With 9 a gas injection is indicated for the gas supply over 10 . The liquid gas mixture then obtained enters the cyclone reactor tangentially at 11 , in which a liquid level is formed, as indicated, for example. The gas accumulated above the same exits the reactor at 12 via a dip tube 13 . This immersion tube can be provided with (not shown) foam destroyer elements below the gas outlet, for example in the form of triangles, which protrude into the gas space. Gas injection can also be provided between the pump and filter.

Fig. 2 and 3 show variants of the just explained embodiment in which takes place the fumigation, as indicated, on the porous formed cyclone wall and the porous base of the baffle cone.

According to FIG. 4, the fluid removal from the Zy is modified klonreaktor, namely liquid Kul is turmedium from the cyclone reactor via a dip tube 14 from the closed bottom of the cyclone reactor 1 taken constituted by the baffle cone 2 and the dip tube 13 is passed to gas sampling.

example Cyclone reactor for laboratory fermentations according to FIG. 4

This cyclone reactor has an inside diameter of 70 mm and a cylindrical length of 190 mm. The inlet diameter is 9 mm. The impact cone (angle in the cone tip 130 °) in front of the underflow has a diameter of 62 mm, so that the gas-free underflow can be sucked upwards into the axial tube via an annular gap of 4 mm ( Fig. 4), the diameter reduction from the cylindrical part of the cyclone reactor to the central tube (inner diameter 9 mm) takes place over a length of 10 mm. The immersion tube at the head of the cyclone reactor for withdrawing the gas phase is immersed 5 mm and has an inner diameter of 30 mm. The gas is withdrawn from the top of the immersion tube with a tube diameter of 9 mm. The suction pipe of the underflow is carried out centrally through the cover of the immersion pipe. The entire cyclone reactor is made of glass.

A SCHOTT GL screw connection is attached directly next to the immersion tube for receiving a continuous level probe (potentiometric measuring principle) is used to adjust the liquid phase volume in the cyclone reactor (Working volume 800 ml).

All other measuring probes (temperature pH, pO₂, turbidity), as well as all feed and Processes of the liquid phases (substrate, correction agent, cell-containing process) are in the Integrated circulation.

On the pressure side of the circulation pump (Filtron - Sinus - Pump) is a Microfiltration module (ceramic tube module with 19 channels, channel diameter 2.7 mm, exchange area 0.14 m²) installed. Then the air is introduced via a Sintered tube before the circulation flow tangentially in again via a heat exchanger the cyclone reactor is fed. This results in a total work volume of 1.5 l.

Determination of the oxygen transport coefficient (k _L a)

The k _L a value was determined using the stationary method with medium without cells, by removing the oxygen introduced into the cyclone reactor externally in a stripper using nitrogen (nitrogen desorption method).

The k _L a value was determined using different acceleration parameters z and different volumetric gas entry rates. The liquid volume flows were varied between 400 and 700 l / h, the air volume flows between 50 and 300 l / h. The results can be correlated with a potency approach:

k _L a = c _* (P / V) a _* (gas flow) ^b

The correlation coefficients were determined as follows (medium, see Tab. 1)

c = 1.3865, a = 0.5995, b = 0.4387
K _L a values up to 3000 l / h or oxygen input rates up to 21 g / (l _* h) were measured.

Cultivation of the aerobic bacterium Corynebacterium glutamicum in the cyclone reactor

For pre-cultivation, 1000 ml shake flasks with 4 baffles and one Working volume of 200 m used. The media components are at 30 min 121 ° C autoclaved (composition of the nutrient medium: Table 1). To Cooling to ambient temperature is carried out with 1 ml organism suspension Root position vaccinated. This is followed by a 24 hour incubation at 30 ° C.

The cyclone reactor system is chemically sterilized. This is dimethyl dicarbonate Diluted 1: 1000 with water and then pumped into the reactor system. At After 20 minutes the active ingredient has broken down into carbon dioxide and methanol at 20 ° C. After chemical sterilization of the reactor system, what remains is Water displaced by sterilized nutrient medium. The composition of the Culture medium for continuous cultivation is shown in Table 1. Salts and glucose must be autoclaved separately (30 min, 121 ° C). Vitamins and Amino acids are then added under sterile filtration.

To start the reaction, 200 m of incubated preculture are placed in the cyclone reactor pumped with a total volume of 1.5 l. To adapt the microorganisms the synthetic main culture medium follows a 12 hour batch operation of the cyclone Reactor. During this time, the cell mass concentration increases from 3 to 15 g Organic dry matter / l.

The volume flow of the circulation pump is constantly set to 500 l / h. The corresponds to a flow velocity v of the liquid at the tangential inlet of the cyclone reactor of 2 m / s.  

Table 1

Culture media for Corynebacterium glutamicum

The acceleration index z _inlet (centrifugal acceleration / gravitational acceleration) is calculated as follows:
z _inlet = 2 _* v² / (D _* g)
where v = empty pipe speed at the inlet (m / s)
D = inner diameter of the cyclone (m)
g = gravitational acceleration (9.81 m / s²).

This means that the acceleration index z _inlet = 11.7.

The supply air volume is set to 400 l / h. This results in a volume-specific energy input (power input of the circulation pump and kinetic energy of the gas input) of just under 10 kW / m³. Via the heat exchanger in circulation is a thermostat with external temperature control Temperature in the cyclone reactor system set to 30 ° C. The pH is determined by Addition of the correction agent (4N NaOH) regulated to 7.0.

With a dry biomass concentration of 15 g / l, continuous operation is possible be converted. The feed is adjusted to 300 ml / h. At a The total volume of the reactor system of 1.5 l corresponds to that of an average one Culture medium dwell time of 5 h in the reactor system. By circulating Integrated microfiltration unit can give the system a cell-free liquid flow be removed. The mean residence time of the microorganisms in the System are decoupled from the residence time of the nutrient medium.

A residence time factor F of 5 is set (the residence time factor F represents this Relationship between the residence time of the microorganisms and the residence time of the Nutrient medium). A residence time factor F of 5 means that the cell mass (the biocatalyst) is 5 times longer in the cyclone reactor system than that Nutrient medium. With an inlet volume flow of 300 ml / h, this is due to the Extraction of a cell-containing bleed flow of 60 ml / h and a cell-free one To achieve filtrate flow of 240 ml / h. The filtrate flow is fixed Specification of the bleed current via the level control.  

Results

After 3 cell mass dwell times (75 h) one becomes in steady state Cell mass concentration of 60 g BTM / l in the cyclone reactor reached. As pO₂ at this cell density has a value of 6% air saturation at the measuring point in circulation measured.

For biochemical control in the entire cyclone reactor system Adequate oxygen supply was applied to the fermentation medium Fermentation product lactate examined, which is only formed if not enough Oxygen is present. In the operating area examined, none Lactate formation can be determined.

Compared to the conventional stirred tank process, the use of Cyclone reactor the cell mass concentration in the reactor increased by a factor of 3 without causing undesirable oxygen limitations. At unchanged cell mass specific reaction rate of Amino acid formation can be used to determine volumetric productivity ("space-time Yield ") also compared to the continuous stirred tank process Increase factor 3.

Claims (14)

1. Procedure for performing biotechnological Processes under cultivation of aerobic organisms in high cell density in a continuous process in one Cyclone reactor with a cyclone diameter: on barrel diameter ratio <10, that at the top End tangentially liquid medium with a Be acceleration index (Centrifugal acceleration / gravitational acceleration) to to 25 is fed into the oxygen-containing Gas is mixed and / or the oxygen-containing Gas is supplied via the outer surface of the reactor is ent from the bottom of the reactor taken and via an external recycling loop brought to process conditions while from ent accumulated gas ent is removed, with a performance entry in Circuit of 4-40 kW / m³ is worked. 2. The method according to claim 1, marked by the use of a cyclone reactor with a Height: diameter ratio of 2.5-5 and a cyclone diameter: inlet diameter ver Ratio from 4-10. 3. The method according to claim 1 or 2, characterized, that liquid from the circulation through cross  current filtration is removed, its overflow speed with the inflow speed at Cyclone inlet is matched. 4. Arrangement for carrying out biotechnological processes under cultivation of aerobic organisms in high cell density, characterized by a culture liquid circuit with elements for regulating the process conditions for a cyclone designed, integrated in the circuit th reactor with gas extraction at the head and liquid extraction at the bottom, the upper tangent - Inlet with a pump provided in the circuit for a power input for acceleration indicators (z _inlet ) up to 25 (centrifugal acceleration / gravitational acceleration) and is connected by means of gassing in the circuit in front of the tangential inlet to the cyclone reactor and / or via perforated gas distributors in the reactor. 5. Arrangement according to claim 4, marked by one especially as a baffle plate or baffle cone-shaped impact body in front of the lower one End of the cyclone to support the secondary spine education. 6. Arrangement according to claim 4 or 5 marked by a ratio of cyclone diameter to inlet diameter of <10, especially between 4 and 10th   7. Arrangement according to one of claims 4 to 6, marked by a design of the pump for a performance load between 4 and 40 kw / m³. 8. Arrangement according to one of claims 4 to 7, marked by a ratio of length to diameter of the zy clone reactor between 2.5 and 5. 9. Arrangement according to one of claims 4 to 8, marked by a filtration module in the circuit for removal of a cell-free filtrate stream. 10. Arrangement according to one of claims 4 to 9, marked by an additional withdrawal point for cell-containing Liquid flow in the circuit. 11. Arrangement according to one of claims 4 to 10, marked by one provided at the top of the cyclone reactor tangential liquid inlet with upstream Fumigation equipment; lead one through the cyclone cover axial gas sampling nozzle, the one on the outer edge is provided with mechanical foam destroyer elements and one that supports secondary vertebrae formation Impact body in front of the lower open end of the cyclone, which leads to the pump provided in the circuit.   12. Arrangement in a modification of claim 11, marked by a cyclone reactor closed at the bottom, through the an immersion tube designed as a hollow cone reaches through for the removal of liquid, the coaxial reaching through the gas extraction nozzle to Liquid circulation leads. 13. Arrangement according to claim 11 or 12, marked by a porous surface of the cyclone for the gas supply, alternatively or in addition to fumigation establishment of the cycle. 14. Arrangement according to one of claims 5 to 12, marked by an upward porous impact body for the Gas supply, alternatively or in addition to the Bega circulation system.