DE2300478B2 - METHOD FOR AEROBIC BREEDING OF YEAST IN SYNTHETIC MEDIA - Google Patents

Description

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The invention relates to a method according to the preamble of claim 1.

So far, such cultivations have been carried out in synthetic media that contain all the necessary nutrients. of which potassium and phosphorus oxide in the form of potassium dihydrogen phosphate and nitrogen in the form of an ammonium salt of a strong mineral acid are present, carried out so that the neutralization the mineral acid released from the ammonium salt during yeast protein synthesis and for maintenance the required base of the specified optimum pH value together with the carbon source is added. The simultaneous metered addition of base and carbon source is done by means of a the optimal pH value is controlled by the set standard pH meter. This pH meter gives an impulse for the Dosage delivered when the pH of the culture medium falls below the setpoint value.

The fact that the consumption of ammonium ions from the salt of the strong mineral acid, the amount of acid released corresponds to the amount of yeast formed with a certain content of nitrogenous substances is proportional. This amount is also the equivalent Amount of base that is used to regulate the pH of the culture medium to the optimum value, nrnnortional. If now the average nitrogen content of the yeast mass formed and the yield coefficient are known to the culture, the ratio of the added amount of base to the optimal amount of the carbon source to be added can be calculated. As a result, you can by metering in parallel of base and carbon source in the course of the cultivation the optimal pH value and the optimal Maintain amount of carbonaceous nutrients.

However, optimal control of the supply of the carbon source can only be achieved if that required proportion of base and carbon source is correctly determined, which is only possible with gui researched and mastered cultivation processes in which a stable nitrogen content in the produced Biomass and constant values for the yield coefficient can be achieved. With less known cultivations for which it is not possible. in advance a corresponding ratio of base and Determining the carbon source accurately can result in exhaustion or an overdose of the carbon source koi .ien. If no carbon source up front that is, the cultivation process now ends. while an excess of carbon source leads to the formation of undesirable metabolites and lowering of the yield.

When culturing is carried out using the above-mentioned method in such cases If the carbon source is exhausted, the pH meter does not react and the metering stops. As a result, of course, the cultivation process also stops because of the absence of carbonaceous Nutrients up. The previous method with a one-sided standard pH meter can therefore be used for breeding which the pH value does not change in such a way in the absence of a carbon source that the Normal pH meters would not be applied.

Namely, depending on the composition of the culture media, the lack of carbonaceous Nutrients in some cases noticeable in that the drop in pH stops and in other cases by the fact that the pH of the culture medium rises violently.

The method according to the invention eliminates the stated disadvantages.

The invention is defined in claim 1.

Claims 2 and 3 represent advantageous embodiments of the invention.

So it happens B. in the cultivation of the yeast Candida utilis in a medium with synthetic ethanol in the moment in which the microorganisms consume or use up the ethanol, in a flash Increase in pH. Within a few minutes the pH increases by up to 0.6. With this type of culture the pH meter for two-sided control is set with one for metering the carbon source narrow control range of 0.1 to 0.6 pH, preferably 0.3 pH, used.

When the pH falls below the lower value of the set range, a solution of the Base, preferably ammonia, or a solution of the base and the carbon source are metered in, the The ratio of the amount of the carbon source to the amount of the base is lower than that of the assumed one Nitrogen content in the biomass and the yield coefficient corresponds to the ratio calculated (cf. to this the patent claim 1). If you are absent or

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Consumption of the carbon source is now increasing. the pH value radch, and when exceeding the upper At the limit of the set pH range, the carbon source is added. By adding the carbon source the biosynthesis of proteins is restored, the pH value drops rapidly to the lower value of the and the process repeats.

As an example of another type of culture in which, in the event of a lack of carbon source, there is no rapid rise in the pH value, but only in the absence of a fall in this value the cultivation of the yeast Candida lipolytica in a medium with isolated n-alkanes are used. For this The second type of culture is a single-sided pH meter for automatic metering of the carbon source Regulation with a connected timer controlled by an external pulse from the pH meter applied.

If the carbon source is present in the nutrient medium, the pH value has _{a decreasing tendency due to the biosynthesis of the proteins and the gradual consumption of the NH 4} ⁺ ions from the ammonium salt of the strong mineral acid. When the pH falls below the set value, the solution of the base, preferably of the ammonia or the solution of the base and the carbon source, is added, the ratio of the amount of carbon source to the amount of base in turn being lower than that from the assumed nitrogen content in the biomass and the yield coefficient corresponds to the calculated ratio (cf. claim 1).

After the carbon source has been used up, the synthesis of proteins and the decrease in the pH value, and the pH value stabilizes at a value that is only slightly higher than that value set on the pH meter. The timer shows the time of the last addition of the base measured; if within 1 to 10 minutes, preferably within 1.5 to 2 minutes, not to one further metering in of the solution of the base (optionally metering in of the base and the carbon source) comes, the timer gives an impulse to meter in the carbon source.

By adding the carbon source, the synthesis of proteins is quickly restored (according to 25 to 50 seconds), the pH value drops below the set value Value, and the metered addition of the solution of the base, possibly also the carbon source, begins again.

An advantage of the method according to the invention is that it is generally applicable to the majority of Cultivation types can be used without disturbing the metering of the carbon or carbon dioxide. nitrogenous nutrients and the cultivation process comes to a standstill.

The new method for growing yeasts with automatic metering of the carbon source is explained by the following examples.

example 1

A one-off Inflow cultivation of the yeast Candida lipolytica carried out in a medium with isolated paraffins. Of the The fermentation vat was fitted with mechanical stirrers at a rate of oxygen dissolution 280 mol / 1 · h. The isolated paraffins contained 99.5% by weight of n-alkanes, with the hydrocarbons with Cm to Cn with a specific gravity of 0.77 g / ml predominated.

That for the assumed increase in yeast dry matter of 10 g / l applied basic medium contained per liter:

4 g (NH ₄ I ₂ SO ₄ ,
0.8 g KH ₂ PO ₄ ,
0.25 g MgSO ₄ · 7 H ₂ O,
O ₁ OIgZnSO ₄ -7H ₂ O.

To the 151 of the basic medium in the fermentation vessel with a pH of 4.5 became an inoculum. that of an initial concentration of yeast dry matter of 2 g / l and 10 g of n-alkanes added, which are necessary for the beginning of the biosynthetic process. Then the real thing began Cultivation at a temperature of 34 to 35 ° C and a pH of 4.5.

A pH meter was used to regulate the pH value and to automatically meter the n-alkanes unilateral control (by adding the base) used with a set pH of 4.5. When sinking the pH value of the medium below the value of 4.5, a pulse emitted by the pH meter set the coupled dosing device for ammonia solution and isolated n-alkanes in operation.

The required amount of ammonia and isolated n-alkanes was required for an increase in the yeast dry matter of 10 g / l in the course of the entire cultivation process, for an assumed nitrogen content in the dry matter of the biomass of 9.5% and calculated for a yield coefficient of 1. For the direct metering by means of the pH meter were 80% of the calculated amount of n-alkanes and the entire calculated amount of ammonia is used. The ammonia was added to the volume with water the n-alkanes were diluted to the same volume and the coupled dispensers were adjusted so that they when the pH falls below 4.5, the same volume of ammonia solution and the isolated one metered in n-alkanes.

There was an electronic timer on the pH meter, activated by an output pulse from the pH meter connected to measure the time that has elapsed since the last metering. If now within no further metering took place after 90 seconds, the time element gave an impulse to open the Released valve, which was used to regulate the supplementary inflow of the isolated n-alkanes. For due Supplementary dosing was used approximately 20% of the calculated required amount of n-alkanes. The timer was only activated after a few ammonia additions, which marked the beginning of yeast growth signaled, put into action.

The cultivation was ended after 6.5 hours, in which time the assumed increase in yeast dry matter was achieved. The mature medium contained 188 g yeast dry matter, what corresponds to a yield coefficient of 1.05. The biomass produced contained in the dry matter 9.0% N, 6.2% crude lipids and 0.25% residual hydrocarbons.

Example 2

The cultivation of the yeast Candida utilis on synthetic ethanol as a carbon source was carried out in the same way

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Fermentation vessel and carried out using the same basic medium as in Example 1. The crude synthetic ethanol used contained 90% by volume, i.e. 85.7% by weight of ethanol.

To 15 liters of basic medium was added ·; the inoculum is added in an amount corresponding to the initial concentration of 2 g of dry yeast substance in one liter and 15 ml of synthetic ethanol. The actual cultivation was then started at 30 ^° C., at which the pH was maintained in the range from 4.5 to 4.8. To regulate the pH value and to automatically control the synthetic ethanol inflow, the pH meter for two-sided regulation with a set control range of 4.5 to 4.8 was used. When the pH value fell below the value of 4.5, over the pH meter (or under the control of the pH meter) a mixed solution of ethanol and ammonia is added.

The required amount of ammonia and synthetic ethanol was required for an increase in the yeast dry matter of 10g / l, i.e. 150g, from the assumed nitrogen content in the dry matter of the biomass

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of 10% and the yield coefficient of 0, b5 calculated for the preparation of the ethanol-ammonia solution became 80% of the calculated amount of synthetic ethanol and the whole calculated amount of ammonia used

The lack of a carbon source in the medium manifested itself in a rapid rise in pH a value of 4.8 within 2 to 4 minutes after the last addition, whereupon the pH meter then turns on Impulse to open the "acid" valve, via which supplementary synthetic ethanol was added to the medium, was delivered. After the addition of the synthetic ethanol, the pH of the medium fell within b0 to 90 seconds again to the value of 4.5, and the metered addition of the ethanol-ammonia mixture was resumed. In the course of the cultivation, about 20% of the metered in calculated amount of synthetic ethanol

The cultivation was terminated after 6 hours. It was a yield of 64% dry matter of the Biomass obtained from 100 g of absolute ethanol; the yeast dry matter contained 102% N.

Claims (3)

22 00 claims: 1. Process for aerobic cultivation of yeast in synthetic media containing all the necessary nutrients contain, of which potassium and phosphorus oxide in the form of potassium dihydrogenphosphhal and the nitrogen in the form of the ammonium salt of a strong mineral acid are present with automatic Maintaining the optimal pH value in the culture medium through continuous metering the inorganic base, preferably of alkali hydroxide or ammonia, and the optimal amount of the carbon-containing nutrients through continuous metering of the carbon source, thereby characterized in that the base as well as the carbon source, the latter in a 20% below that amount which corresponds to that from the average nitrogen content in the generated yeast biomass and the yield coefficient of the cultivation process Corresponds to the ratio of base and carbon source, is added when the pH value of the Culture medium below the specified value or below the lower limit of the specified regulated Area decreases, the width of which is 0.1 to 0.6 pH, and that the remaining carbon source is metered in is when the decrease in pH after a period of 1 to 10 min after the the last addition of the base does not take place or stops above the specified value or if the pH of the culture medium exceeds the upper limit of the regulated range '. 2. The method according to claim 1, characterized in that the period of time is 90 to 120 seconds. 3. The method according to claim 1 or 2, characterized in that the width is 0.3 pH.