DD210173A3 - METHOD FOR CONSERVING MICROORGANISMS - Google Patents

Abstract

The invention relates to a method for culturing microorganisms by alternately exposing the microorganism-containing culture fluid to an oxygen source and maintaining the oxygen as a limiting factor in the growth rate, while simultaneously introducing the trace elements catalyzing the oxygen-transferring cell regulation mechanisms according to the amount of oxygen transferred or specifically required become. It has been found that the most favorable cell substance yields and favorable specific oxygen requirements are achieved under alternately oxygenated conditions at growth rates of my / my max> 0.6, when the amount of trace elements to be introduced is calculated after a factor F.

Description

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Title of the invention Procedure train? Breeding of microorganisms Field of application of the invention

The invention relates to a method for oxygen-limited aerobic culture of microorganisms. It can be applied in the microbiological industry in the extraction of cell substance, where with a minimum of oxygen, energy source and technical effort, a high cell substance yield should be achieved.

Characteristic of the known technical solutions

In almost all processes for microbial cell substance synthesis, the principle of chemostatic environment design is an imperative requirement for achieving optimal results with respect to predetermined target functions, e.g. In known processes, for example, the trace elements catalyzing the oxygen-transferring enzyme systems are supplied in correlation to the biomass formed or, according to the chemostatic principle, are kept within defined eoncentration limits.

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Disorders of the chemostatic environment system, z. B _e the effect of temporary concentration in nutrient components, the power source or the Säuerst off supply, causing cell physiological responses (Katinger, Physiological response of Candida tropicalis grown on η-paraffin to mixing in a tubular closed loop fermenter in European J. appl "Microbiol» 3, 103-114, 1976).

These feedback reactions of the cell become visible in the form of time-decaying oscillations of the material-transforming and energy-producing reaction processes and thus ultimately result in increased entropy production. The entropy production leads in each case to an increased heat formation and thus to losses in the cell substance synthesis.

In industrial production plants, the unrestricted chemostatic principle can only be approximated for many reasons; B ·.

- There are no absolutely ideal mixed systems

The thermostabilization of the exothermic reaction processes requires the removal of large amounts of heat, which in

""; technical systems with reasonable effort only on external circuits can be reached, in which it must necessarily come to temperature gradients.

Another essential moment of aerobic culture is the need to convert oxygen from the air or from oxygen-containing gases into the aqueous culture medium. Numerous methods and technical systems are known, such as surface aeration, self-priming circulating systems, submersible jet systems, turbocharged agitator systems, etc. "All of these systems are inherently energy intensive, which are transmitted amounts of sediment

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systementsprechend limited, so that almost all procedures must be operated under oxygen limitation. Oxygen for these reasons becomes the limiting factor of growth speed ·

In addition, it is known that oxygen excess and energy source limitation can lead to higher rates of inhalation and thus lower cell substance yields. It is known that the greater the internal surface area of the dispersed oxygen-containing gas, the more effective the coalescence of the dispersed gas bubbles is, and the better the oxygen transfer is, the more effective the oxygen transfer. convective mass transfer within the liquid decor with dark ^v - 'tet can be.

But these requirements can only with highly effective mixing and dispersing, the. are aimed at a favorable energy transfer from the functional technical elements to the! liquid.

High absolute amounts in the energy input and a favorable momentum transfer are only achieved with gas-free liquid flows. In the gassed state cause the. decreased density, compressibility and reduced viscosity significant loss of effectiveness. Thus, the energy transfer factor in turbine blade

For example, stirrers in the 3? agitated liquids agitate to 20 % compared to unfilled liquids. In order to avoid this disadvantage, the liquid is degassed before impulse imparting, however, the disadvantages of an alternating S'auerstoffversorgung occur in the form of regular disturbances of the chemostatic principle with respect to the oxygen supply.

Thus, large-scale processes of aerobic Mikroorganismenzüchtung either have the disadvantage that the dimensioning of the functional elements for the energy transfer in the desired maintenance of the chemostatic principle for the oxygen supply disproportionately large

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be measured or that 'in the opposite case, the results from the periodic disturbances of the chemostatic principle resul animal losses in the cell substance synthesis are effective.

Object of the invention

The object of the invention Desteht in the realization of a method with the technical advantages of a primary pulse transmission to the degassed liquid with resulting alternating supply of oxygen with simultaneous y tigern effect certain benefits of the chemo-static principle of the oxygen supply.

The aim is thus an economical method of cell culture under pulsating oxygenation with simultaneous oxygen limitation.

Explanation of the essence of the invention

It was found that these objects can be achieved if the process is carried out by passing the culture fluid in the largely degassed state to a path forced by physical means, then gassing it with oxygen and, in this state, oxygen is held as a limiting factor of the growth rate, wherein at the same time the trace elements catalyzing the oxygen-transferring cell regulation mechanisms do not function according to the correlation principle to the biomass formed or constant in concentration. but after-transferred or, specifically required amount of oxygen to be supplied _o

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In order to determine the required trace element levels, it was assumed that the most favorable cell substance yields and the most favorable specific acid substance consumption were under ideally mixed, constantly acid-supplied. Conditions can be reached at growth rates of , a / η max of ^ 0.6 *

Under these conditions, the minimum possible specific oxygen demand in grams of oxygen per gram of desired cell substance is to be determined in oxygen tests in trial runs.

Under analogous conditions, under the technical conditions of the alternating supply of oxygen, a demand for oxygen increases.

The increased oxygen demand correlates with an increased demand for energy source, usually a carbonaceous nutrient substrate.

It has been found that the increased oxygen demand under these conditions can be reduced if the trace element quantity to be introduced is calculated according to an I'actor ir, which is determined from the quotient a of the ideally required specific oxygen amount and the real specific oxygen demand multiplied by the factor b as

ν quotient of the specific oxygen transfer in grams of oxygen per kilogram of culture fluid

· '", And hour and the specific oxygen demand determined in ideal conditions in grams of oxygen per gram of biomass formed. This numerical value is multiplied by the element limit factor c, which assumes the following numerical values for the individual elements:

copper i 0.04- zinc JS 0.28 iron ^ 0.025 manganese ^ 0,2

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example

In a nearly ideally mixed, constantly aerated 250 l stirred tank fermentor, iodderomyces elongisporus was grown under known optimum conditions on n-paraffin-containing petroleum distillate

The residence time was 4 hours, the growth rate / U / ai max was accordingly * 0.7 above 0.6. Under approximately ideal chemostatic conditions, a specific oxygen demand of 2.3 g OVg HTS (yeast dry matter = HTS) was determined.

In another 250 1 - Permentor, in which also iodderomyces elongisporus was bred, an alternate oxygen supply was realized as follows: From the lower part of the fermenter was continuously withdrawn a volume of 3 000 l / h, degassed in an intermediate vessel and an air-sucking Injector pumped back into the permentor. With this system, an oxygen transfer performance of 12.4 g Oo / kg culture fluid was achieved.

The specific oxygen demand was initially 2.7 g Op / g HTS '·. '-

After determining this. Values were changed to the nutrient solution in which the trace elements, in quantitative ratio of the aforementioned Grenzkonzentrationszahlwerte were included, as follows:

M = grams of element per kilogram of culture fluid and hour

M = P · c

P = 0.85. 5.39

P 's 4,85 M * = 4,85 · c

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The specific oxygen demand of the biomass then decreased from 2.7 to 2.45.

The yield coefficient j increased from 0.8 kg dry biomass per kg n-alkane to 0.9ο

Claims (1)

233171 O invention claim Process for the growth of microorganisms, in which the microorganism-containing culture fluid on a by Γ) technical devices caused web is guided, characterized in that it is exposed alternately to a source of oxygen and the oxygen is the limiting Paktor-growth rate, wherein the supply of the trace elements copper, zinc, iron and manganese in g element per kilograms of a mm culture liquid and hour after a factor F and a - trace element limit factor c is controlled, the factor F is calculated from a quotient a of the under continuous anemostatic conditions of oxygen supply at growth rate. of u. / yes max of ^ 0.6 specific oxygen demand and the specific oxygen demand obtained in real conditions in grams of oxygen per J grams of absolute dry biomass multiplied by a factor b. which represents a quotient of the real oxygen transfer amount obtained in the alternating oxygen supply in grams of oxygen per kilogram of culture fluid and hour and the specific oxygen demand obtained under ideal conditions, the elementary boundary coefficient c for the individual elements having the following values: