DD229854A3 - METHOD FOR THE FERMENTATIVE PRODUCTION OF SECONDARY METABOLITES - Google Patents

Abstract

The invention relates to a process for the fermentative production of secondary metabolites. It is used for the non-procedural execution of fermentations for the production of secondary metabolites. The process is carried out by using the continuously detectable and controllable global process parameters p H value, liquor dosing rate, pO 2 value, reaction heat and exhaust gas composition. The current liquor dosage rate as a measure of the physiologically effective amount of phosphate is used to control the dosage of substrates. Specifically, ammonium hydroxide is used as the liquor.

Description

Field of application of the invention

The invention relates to a method for the fermentative production of secondary metabolites, preferably antibiotics, whose formation characteristics can be influenced by controlling the metabolism of the production organisms by direct or / and indirect feeding of various, for example, phosphate-containing substances during the fermentation. Thus, the invention is of importance to the microbial industry.

Characteristic of the known technical solutions

It has already been proposed, in addition to the setting, in particular reduction, the concentration of available phosphate in the starting medium of fermentations to obtain secondary metabolites phosphate-containing substances to fermentation beginning and / or admit dosed during the course of the fermentation while avoiding further limitations to regulate the interactions of the biological system with the aim of increasing the rate of product formation.

With regard to the application of the above-mentioned invention in so-called real-time fermentations, there is the difficulty of applying to the continuously measurable global process parameters established in fermentation technology, such as pH, pO ₂ value, metering rates, exhaust gas composition (CO ₂ formation), heat generation, etc., instead of the first to have to dodge substrate concentrations that have to be subsequently determined by time-consuming chemical analysis as primary regulatory process parameters. This necessity results from the given state of the automated measuring technology in the biotechnology (Sukatsch, DA, Nesemann, G .: Automatic parameter capture in industrial fermentations, Chemie-Technik 6 [1977], 261).

Object of the invention

The invention includes the object of non-procedural fermentation for the production of secondary metabolites.

Explanation of the essence of the invention

The invention has for its object to describe a method for the production of secondary metabolites, which is a technically advantageous solution for the real-time control of industrial fermentations with direct and / or indirect substrate dosage, especially phosphate, carbon sources, nitrogen sources, trace elements and substances with effector effect includes. Here, the control of the substrate doses of continuously detectable and controllable global process parameters such as pO ₂ value, pH, heat and CGyBildung and derived specifically from the Dpsierraten zurpH control and the formation rates for heat and CO ₂ and the realization of the allow effective product formation rate over a long period of fermentation.

Surprisingly, it has been found that the time course of the liquor metering rate in the pH setpoint control maps the time course of the concentration of physiologically active phosphate for the conversion of carbon sources, nitrogen sources, trace elements and substances with effector effect, as long as these substrates and the dissolved oxygen content Limitation freedom exists. By this is meant that the liquor necessary to maintain a selected lower p-control threshold, said caustic dosing rate, is proportional to the physiologically effective phosphate concentration. The image scale thus depends on the determination of the lower control limit of the pH value; the onset of the lye dosage is determined by the buffering of the starting nutrient medium.

In the further development of the invention is used as lye metering ammonium hydroxide for controlling the dosage o.g. Substrates used.

The procedure is carried out as follows:

At the control device for the pH, the lower pH control threshold is set, for example pH 6.2. This results in the fermentation process, the equivalent ammonium hydroxide metering rate as a function of time. Because of the described functional relationship between this dosage rate and the concentration of the physiologically active phosphate, the measure of the metabolic activity of the fermentation is shown. The consumption of C sources, N sources, trace elements and substances with effector effect corresponding to this metabolic activity is compensated by addition. The process can also be carried out so that the most effective for product formation time course of the phosphate concentration is realized by direct phosphate dosing by adjusting the most favorable for the course of the process ammonium hydroxide dosing. With regard to the direct addition of phosphate in the form of the addition of phosphate-containing substances from an external reservoir and the indirect phosphate dosing as providing phosphate compounds by enzymatic or hydrolytic digestion complex nutrient components such as starch, soybean meal, corn steep liquor u.a. It has been found that the amount of ammonium hydroxide dosing rate indicates transitions between direct and indirect phosphate dosing by significant increase or decrease, respectively. The size of the change is determined by the phosphate influx. The direct phosphate dosing is also minimally necessary by achieving the physiological-regulatory reasons

^ 3ek5stsstoffstoffgehalt determined or codetermined.

In addition, the fact that the time course of the heat of reaction in the fermenter due to the activity of the microorganisms and the CGy formation with the time courses of the ammonium hydroxide

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Dosage rate and the concentration of the physiologically active phosphate corresponds. For this reason, the current heat of reaction and the current CO ₂ formation and thus also the formation rates associated with these variables are also control variables for the dosage of phosphate and the other substrates.

embodiment

The invention will be explained by an embodiment with reference to diagrams.

A lyophilically dried on glucose gelatin mycelium (gross weight of the contents about 300mg) of the selectant BF 32 of Nourseothricin-forming strain IMETJA 3890b is suspended in 3 ml of a 0.9% sodium chloride solution. 0.5ml of this suspension is used as inoculum of 400ml pre-breeding of the first stage. The pre-breeding medium contains per liter of tap water the following starting materials: 40g glucose, 15g deoiled soybean meal, 0.3g KH ₂ PO ₄ , 5g NaCl and 3g CaCO ₃ . The acidity is adjusted to pH 6.5 before sterilization. This medium is filled in 2500ml, sealed with cotton stopper breast bottles in quantities of 400ml each. Sterilization is carried out by heating for 35 minutes at 121 ^° C. in an autoclave.

The cooled to room temperature and inoculated mixtures are incubated for 48 hours as shaking cultures (180min ~ ¹ ) at 29 ° C. With 800 ml of the culture solution of the first Vorzucht thus obtained as a second stage of Vorzucht a 180 liter heat sterilized medium (60 minutes at 121 ⁰ C) of the same composition as in the first pre-culture stage filled, equipped with devices for sterile ventilation and a stirrer Edelstahlfermenter ( 250 liters gross volume). The cultivation is carried out over the period of 42 hours (pH 5.2 reached) at 27 ⁰ C to 29 ° C, a stirrer speed of 240min ~ ¹ and a ventilation rate of 1 liter of air per liter of culture solution per minute. The previous information relates to the necessary preparatory work.

The main culture is a stainless steel fermenter (4200 liters gross volume) equipped with sterile aeration devices and a central agitator, filled with 2500 liters of nutrient medium of 1 liter of tap water: 32g potato starch, 60g glucose, 11g de-oiled soya flour, 11g ( NH ₄ J ₂ SO ₄ , 2g MgSO ₄ -7H ₂ 0.1g NaCl, 6g CaCO ₃ and 0.5g ZnSO ₄ 0.5g of silicone-based defoaming agent is added per liter of culture solution and the acidity is adjusted to pH before sterilization The sterilization of the nutrient medium (without glucose) is carried out in situ by preheating with mangle steam to 75 ° C and subsequent heating by direct steam to 120 ⁰ C for 16 minutes After cooling to 29 ⁰ C is in the form of a 50% aqueous solution is added separately by sterilizing glucose sterilized under aseptic conditions for 30 minutes at 120 ° C. Before the inoculation, the St acidity with sulfuric acid so that after inoculation the starting pH value is 7.4. The main crop fermentation batch seeded with 4% inoculum from the second preemergence stage is 120 hours at 29 ° C, an aeration rate of 1250 liters per minute (0th to 12th hour) and 2500 liters per minute (12th to 120th hour), respectively. at 0.03 MPa overpressure with stirring at 180 min ~ ¹ cultured.

The recorded time courses show:

Diagram 1 Time course of the antibiotic nourseothricin produced and the dry weight of the biomass Diagram 2 Time curves of the pH value, the glucose and ammonium nitrogen concentration Diagram 3 Time behavior of the ammonium hydroxide dosing rate, the phosphate concentration and the dissolved oxygen content Diagram 4 Time curves of the exhaust gas composition from oxygen and carbon dioxide and the Reaction heat Diagram 5 Time courses of the specific product formation rate and the specific growth rate. The fermentation is conducted in terms of the control interventions by direct phosphate dosing and glucose dosing according to the behavior of the ammonium hydroxide dosing rate. The pH set point interval is set at 6.2 + 0.05 and the lower limit, pH 6.15, is reached after 38 hours of fermentation time.

For direct phosphate dosing a sterile aqueous solution of potassium dihydrogen phosphate is used. From the 2nd to the 32nd hour, this solution is metered in so that a total of 42 milligrams of phosphorus in the form of phosphate is used to stimulate metabolic activity per liter of culture solution.

Determined by the selected pH setpoint interval, the buffering state of the nutrient medium and the lye concentration starts at the 38th hour, the ammonium hydroxide (25%) dosage. The observed decrease in the increase in ammonium hydroxide dosing rate triggers further direct phosphate doses of 8.5 milligrams of phosphate-phosphorus per liter of culture solution between the 47th and 48th hour and the 53rd and 54th hour, respectively.

As a result, the dosing rate reaches its maximum value at the 62nd hour and then falls accordingly to the transition from direct and indirect phosphate dosing. In this way, the fermentation is about 20 hours near the favorable specific dosing rate of 230 milliliters of 25% ammonium hydroxide solution per cubic meter of culture solution and hour. The ammonium hydroxide solution to be added is sufficient to keep the nitrogen concentration in the physiologically favorable range. Therefore, no further nitrogen sources are added. With respect to the carbon source, the increase in the ammonium hydroxide dosing rate, increased after the second phosphate dosing, gives rise to the addition of 48 grams of glucose per liter of culture solution at the 56th hour. Indication of the transition to indirect phosphate dosing by the slight increase of the ammonium hydroxide dosing rate around the 95th hour is to trigger the second glucose dose of 18 grams of glucose per liter of culture solution.

In the case of direct phosphate dosing, there is no restriction with regard to the dissolved oxygen content. Reaction heat and CO ₂ formation have the course of the fermentation phases analogous to the ammonium hydroxide metering rate and serve to avoid substrate limitations.

The final nourseothricin level of 10.5 grams per liter of culture is achieved by setting the effective specific product formation rate between 7-10 " ³ per hour and 8-10 ~ ³ per hour over the 45 hour period, and the glucose used becomes full ausfermentiert and not exceeded the reprocessing set biomass limit.

2nd embodiment

The spore canned (mulled millet) of the select DG 1 strain of the strain Penicillium chrysogenum serves as inoculum for the inoculum of two major bencylpenicillin culture fermentations by transferring a spore suspension with the total porcine number of about 4-10 ⁹ spores into the 42.5 hour seed culture , The Impffermentor filled with 2.5 liters of nutrient medium (stainless steel glass stirred fermentor with 4.51 gross volume) is sterilized for 60 minutes at 121 ⁰ C in an autoclave. The nutrient medium of the seed culture contains per liter the feedstocks:

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0.5 g, MgSO ₄ - 7H ₂ O 0.1 g, ZnSO ₄ -7H ₂ O 0.02 g, MUSO. ₄ - 5H ₂ O 0.007 g, sunflower oil 5g, polypropylene 0.2 mL front Sterilization is carried out adjusting the pH With sodium hydroxide to 6.5, after sterilization, the pH of 6.25 in at 25 ° (process temperature of Impfkultur- and Hauptkulturfermentationen) recooled medium before. During inoculation culture, the continuously measured and registered pO ₂ value is constantly kept greater than 60% of the saturation concentration by adjusting the speed. The pH is also continuously measured and recorded. First, the pH drops and goes through a relative pH minimum. Then it shows a local increase and then the pH reaches the phase of its minimum value, followed by a rapid increase. At pH 7.2, the maturation of the seed culture is achieved and 500 ml each are used to inoculate the main cultures in stirred fermentors of 4.51 gross volume. After the addition of the seed culture, the working volume is 3 liters each. The medium batches of 2.5 liters contain the components cottonseed flour 120 g, glucose 45 g (NH ₄ J ₂ SO ₄ 7.5 g, CaCO ₃ 12 g, sunflower oil 15 g, Na ₂ S ₂ O ₃ 5 H ₂ O 24 g, polypropylene glycol 0.6 g and are sterilized in the autoclave for 60 minutes after being filled in the fermenter without prior adjustment of the pH at 121 ° C. After sterilization, the pH is 7.2.

At the pH controller, the lower control threshold is set to 6.6. For pH control, 12.5% ammonium hydroxide is used as lye (original 200 ml, sterile-filtered). As further dosing substrates each 10% phenylacetic acid ^ adjusted to pH 7.0 with potassium hydroxide solution, 350 ml) and 70% glucose solution (1 000 ml) stored. The sterility of these solutions is produced simultaneously with the sterilization of the fermentors, since the storage vessels are connected before autoclaving. A fermentor is equipped with another dosing tank containing 400 ml phosphate solution with a total of 2.63 g KH ₂ PO ₄ .

To combat foaming, 20 ml of polypropylene glycol are stored sterile each time. Of these, in the start phase of the main cultures, 5 ml each are added to the fermentors to suppress foaming tendency. There is a rapid drop in the pH value, the control threshold is reached after half an hour. This starts the NH ₄ OH dosage; the accumulated Laugedosierrate is detected every hour. Diagram 6 shows the time courses for the NH ₄ 0H rate and, derived therefrom, dosages for PO ₄ -P and glucose for one of the two main culture fermentors. The leachate rate drops rapidly (corresponding to the decrease in soluble PO ₄ -P by the growing culture).

This drop tendency is reduced by means of external phosphate dosing from the 2nd hour. The P0 ₄ -P dosage is increased only to the extent that the NH ₄ OH rate remains monotonically decreasing. Their zero from the 6th hour indicates under these conditions that the PO ₄ -P replenishment does not fully meet the demand after the PO ₄ -P start depot (87 mg / l free-sterilized) is consumed. This partial PO ₄ -P limitation is used for targeted formation of the extracellular phosphatase activity, ie the indirect PO ₄ -P dosage by enzymatic exploitation of the complex substrates is adjusted to the steady state.

From the NH ₄ OH zero point, the dosage of the phenylacetic acid solution starts at 0.65 ml / lh. From the 11th hour, the lye dosage resumes and the external PO ₄ -P dosage is reduced, since the internal PO ₄ -P supply is induced. After 14.5 hours, the external PO ₄ -P dosage is completely stopped. After 15 hours, the NH ₄ OH rate indicates the depletion of starting glucose over another zero point. This zero point is thus the starting point for the glucose dosage. It is initially adjusted at 0.55 g / lh and increased to 1.25 g / lh from the 15th hour to stabilize the NH ₄ OH rate at 0.1 ml / lh. These settings cause zero carbon and nitrogen supply restriction during the duration of the fermentation, which is the basic cycle of a cyclic benzylpenicillin fermentation. The reference fermentor is operated without using the NH ₄ -OH dosing rate for the real-time control of dosages according to the standard procedure. The Phenylessigsäuredosierung is carried out from the 11th hour with 0.65 ml / lh, the glucose dosage runs from the 23rd hour with 1.25 g / lh. No external phosphate dosage is included. During the fermentations, the pO ₂ value is always kept greater than 50% by means of speed change. The dosing rates are adjusted according to the volume increase

 During the basic cycle, the following time profiles result for the product yield:

Bencylpenicillin (IU / ml)

Time (h) Fermentor with Fermentor without

Real-time control Real-time control

22 1750 900

35 4000 3450

47 6700 4800

59 8600 6100

71 13800 8 200

There is thus an increase in product yield of about 60% at the end of the base cycle. The yield of 8000 to 9000 IU / ml after about 70 hours is prior art.

Claims (3)

-1- 559 44 Invention claim: 1. A method for the real-time control derfermentativen production of secondary metabolites with substrate dosage and addition of the amount of liquor necessary for compliance with the selected lower pH control threshold, characterized in that the per unit time added amount of liquor (liquor dosing) to control the dosage of Carbon sources, nitrogen sources, trace elements and substances used with effector effect and Limitationsfreiheit is set. 2. The method according to item 1, characterized in that the nitrogen is metered by the use of ammonium hydroxide over the liquor itself. 3. The method according to item 1 and 2, characterized in that the lye metering rate is adjusted by feeding phosphate-containing substances. For this 6 pages drawings