DD149873A3 - METHOD FOR CONTROLLING THE CULTIVATION PROCESS OF MICROORGANISMS - Google Patents

Abstract

The invention relates to the field of the microbiological industry, especially to methods of controlling and regulating processes of culturing microorganisms. It is the object of the invention to simplify the control of cultivation processes and to reduce the time required for the determination of the factors which adversely affect the process. The goal is achieved by a method that enables the rapid recognition and accelerated correction of disorders in the stable process of microbial protein synthesis. It is based on detection of the increase in nitrite concentration beyond a set limit.

Description

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Title·:

Method for controlling the cultivation process of microorganisms

Field of application of the invention

The invention relates to the field of microbiological

Industry, especially methods for the control and regulation of processes of the cultivation of microorganisms »

Characteristic of the known technical solutions

There are methods for controlling cultivation processes O according to the biomass concentration (SU-US 302 363), according to, the amount of active cells (SU 365 373)> after the respiration intensity, according to the alcohol content of the exhaust gas (DT-AS 1 OSO 043), on the temperature of the fermentation medium (SU-US 506 611) and on, the amount of carbon dioxide formed during fermentation known (SU-US 344 352).

The disadvantage of the said method is that when changing the process parameter or Stoffwechseiprodukte used for the control, for. B · When reducing the amount of carbon dioxide produced, although a fault in the technological system is displayed, but to determine the causes all process parameters must be controlled, which is very time consuming *

Zie l of the invention

The purpose of the invention is to simplify the control of cultivation processes and the time required to determine the factors that negatively affect the process.

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influence, diminish «. ·

Explanation of the essence of the invention

The object of the invention is to use as a control variable an easily analyzed metabolite, which is excreted by the microorganisms "in case of deviations from the optimal process conditions and whose formation correlates with the change" of certain process parameters. In the cultivation of microorganisms, eg. As of methanassimilierenden bacteria, the excreted into the medium amount of nitrite is less than 0.3 mg N / l, when the ammonium nitrogen concentration 20 - 80 mg N / l, the flow rate is close to the critical flow rate and by setting appropriate partial pressures the oxygen concentration determines the growth rate.

It has been found that with inhibiting ammonium-nitrogen concentrations, limiting concentrations of the carbon source, and increasing degree of oxygen limitation, the nitrite concentration in the fermentation medium increases above the standard allowed for a given process. In this case, the increasing degree of oxygen limitation may be due either to a decreasing flow rate or decreasing oxygen partial pressure.

As the nitrite concentration increases, it is necessary to determine the concentrations of nitrogen, oxygen and the carbon source, the oxygen partial pressure and the flow rate of the aqueous nutrient solution and to correct deviations from the standard values.

The advantage of this method is that, in the event of deviations from the optimal process conditions, the nitrite concentration increases immediately and a malfunction of the process is signaled long before this disturbance becomes apparent from a change in other process parameters (eg * when the biomass yield is reduced) "The resulting nitrite has

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no negative influence on the growth of the microorganisms, as long as non-inhibitory concentrations greater than 20 mg nitrite nitrogen / l are achieved.

This control method can be used "in continuous and discontinuous fermentation processes, it is irrelevant whether the cultivation is carried out at atmospheric pressure or elevated system pressure." The determination of the nitrite concentration is carried out colorimetrically after diazotization with sulfanilic acid and coupling with OC-naphthylamine in one reaction step.

To prepare the detection reagent, 2 g of sulphanilic acid in 400 ml of 2NO acetic acid and 0.4 g of C-naphthylamine hydrochloride are dissolved in 80 ml of water with heating, mixed and added with 2N of acetic acid to the liter. Culture filtrate and reagent are mixed in the ratio 1: 1 (v / v), the red-violet color is measured after 10 minutes in the spectrophotometer at = 534 nm. The color intensity is compared with that of a Sichkurve. The test substance is anhydrous sodium nitrite. The detection range is 0.03 to 0.4 mg nitrite N / 1.

The nitrite determination can be carried out continuously according to the analytical principle using a Spekol of the VEB Carl Zeiss Jena with flow cell.

example 1

The methanutilizing bacterial strain Methylococcus capsulatus sp. was cultured in a continuous process under the following conditions: temperature: 40 ⁰ C.

 pH: 5.6 Fumigation intensity: 40 1 / lh. vO Gas to air ratio: 1: 3 flow rate; , 0,25 h "" ^

The composition of the nutrient solution was as follows: (for 1 g of biomass)

KCl 0.025 g

MgSO _4. 7H ₂ O 0.02 g

H ₃ PO ₄ (SCf.ig) 0.09 g

FeSO ₄ . 7H ₂ O 0.1 mg.

ZnSO ₄ . 7H ₂ O 0.3 mg

HnSO ₄ . 4H ₂ O 0.5 mg

CuSO ₄ . 5H ₂ O 0.5 mg. ·; ·.

HoBOo 0j3 mg

Ha ₂ MoO ₄ . 2H ₂ O 0.045 mg

The pH control was carried out with a 10% LTH ₁₁ OH solution, which at the same time served as the nitrogen supply of the organisms. Under these process conditions, an organic pale concentration of 10 g / l was achieved at steady state. Every half hour, the nitrite content of the culture liquid became Tdestimrat. In the 20th cultivation hour, the nitrite nitrogen concentration increased to values of 2.5 mg U / l. The ammonium hydroxide content of the culture fluid and the fumigation were checked. The analyzes showed that the ammonium nitrogen concentration was 400 mg N / l. To reduce this high nitrogen content of the fermentation medium, the pH adjustment was switched to a 10% NaOH solution. Every two hours, the ammonium nitrogen content was within optimal limits, and the 1-nitrite concentration decreased to <0.3 mg N / l. "

Example 2

The cultivation of the methanutilizing bacterial strain Llethylococcus capsulatus sp. was carried out as described in Example 1. At the 47th cultivation hour, the nitrite content of the culture fluid increased to 3 mg N / l. The ammonium-nitrogen content of the culture fluid and the fumigation were examined. It was found that.

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  the nitrogen concentration was within the optimal limits, but the ratio of natural gas: air was 1: 7 · The process was limited by methane.

After changing the ratio of natural gas: air to 1: 4, the Hitrit concentration of the culture liquid dropped to 0.15 mg N / 1.

Example 3

Methanutilizing bacteria of the strain Methylococcus capsulatus sp. were cultured in the batch process under otherwise identical conditions as in Example 1.

The addition of the nutrient salts took place gradually in the form of a concentrate.

The cell density increased exponentially to 9 S dry substance / 1, without an increase in the nitrite content was recorded. With a v / Eiteren increase in cell density and the nitrite content to values increased -y 1 mg N / l. The ammonium nitrogen content of the culture fluid was checked and found to be within the standard. By increasing the gas input, the nitrite content dropped to 4.0.3 mg N / l ^a "b. The time of commencement of gas limitation, which can only be determined with imprecision from a growth curve, could thus be determined with high accuracy.

Claims (3)

-b- 1 «Method for controlling the cultivation process of microorganisms at constant temperature and constant. pH, continuous aeration with an oxygen source and mixing with supply of a liquid or gaseous carbon source in an aqueous nutrient solution, after a metabolic product formed, characterized in that with the aim of simplifying the control and shortening the time to determine the factors controlling the process of culturing negatively, the control of the process after the concentration of the formed nitrites in the culture liquid is carried out so that as the concentration of nitrites increases over the standard admissible for a given process, the concentration of nitrogen in the culture liquid, the concentration of the carbon source and the oxygen in the culture liquid or in the exhaust gas and the flow rate of the aqueous nutrient solution in the case of continuous fermentation are determined and adjusted to the optimum for the given cultivating process t been discontinued 2. Control method according to item 1, characterized in that the optimum parameters of the cultivation process of different microorganisms are adjusted as a function of their consumption values for nitrogen, carbon, oxygen and their growth rate which are necessary for normal development 20 9 77 1 - * - Method for controlling according to item 2, characterized in that one cultivates methanutilizing bacteria for which the optimal conditions are: content in the culture liquid of ammonium nitrogen = 20 to 80 mg / l; <0.3 mg N / l, flow rate of the aqueous nutrient solution near the critical flow rate, limitation of the process by oxygen.