DD210300A1 - METHOD FOR CONTROLLING PROCESSES OF BIOTECHNICAL FABRIC CONVERSION - Google Patents

Abstract

The invention is based on the achievement of low raw material and Energieverbraeuche, improving the quality of products and an increase in the stability of biotechnical processes. The invention has for its object to use appropriate information from the biological system of processes for biotechnical conversion for the control of these processes. According to the invention, the object is achieved in such a way that information is used which is obtained from distribution functions of one or more features of the microorganisms involved in the material conversion.

Description

title

Method for controlling biotechnical material conversion processes

Field of application of the invention

* The invention relates to a method for controlling processes of the biotechnical conversion material in which single-cell organisms (e.g. _e., Bacteria or yeasts), the well-can be genetically modified involved in pure or mixed culture ". These processes can be used for the extraction of biomass, the extraction of products, the disposal of waste or the extraction of energy sources. The invention is classified in the IPE C-12 Έ »

Characteristic of the known technical solutions .

The processes of biotechnical material transformation are characterized by an internal hierarchical structure. According to the known polynomials of cybernetics, such processes can not be clearly described if only their inputs and outputs are known (Uikolajev, PI, Kantere, VM, Surnal Vses. Chim. Obscestva in * Mendeleeva VJ_ (1972) 569) , Typical of these processes is a Hysteresisverhalten that is und.MultiStabilitäten due bi-, "overshoots" or oscillations (HAREISOH, DE3 ^1, lOPIWALA, HH, in. Adv Biochem Eng 2 (1974)...) ·

For this reason, the described "indirect measurement concept" (ΕΘΗΡΗΒΕΥ, AB, 'Process Biochem. 1977 (1977) 19) is only conditionally capable of providing information for checking and controlling

In this way it is not possible, according to this method, to unambiguously assign changes in the output variables to certain changes in the physiological state of the culture and to derive therefrom favorable measures for controlling the process.

Methods and devices which measure integrally at the metabolic level of such processes directly on the biological system and use them for control are also known (DD-WP 154449, DD-WP 154545). But they only provide averages over the populations and the information obtained is ambiguous with respect to the populations' states. Thus, for example, the distribution of the populations over the individual states of the cell cycle remains undetected and the information about the processes running in the cells can not be used for control because of their ambiguity.

Object of the invention

The aim of the invention is to achieve low consumption of raw materials and energy, to improve the product qualities and to improve the stability of the biotechnological. processes "

Explanation of the essence of the invention;

The object of the invention is to use suitable information from the biological system of processes for biotechnical, St off conversion for the control of these processes.

zen ». ·

According to the invention, the object is achieved in such a way that information is used for the control of biotechnical material conversion processes which has been obtained from distribution functions of one or more features of the microorganisms involved in the metabolic transformation. This information can be obtained from the and / or amount of maxima and / or minima of the obtained

Distribution functions or their form, which is described by statistical measures such as scattering, skew, Sscess or moments of higher order, are derived. The determination of the distributions- for the inventive

drive can be realized as follows:

1 · A representative part of the populations is examined according to morphological features and each cell or cell group examined is assigned to a particular discrete feature class or a continuous feature distribution is estimated. The examination can be carried out, for example, by counting on a microscope or by automatic image analysis.

2. In a representative part of the populations, physical properties, e.g. B * electrical or light-optical properties, measured and from this appropriate feature distributions "determined ·

3 · A representative part of the populations is stained for specific features and the individual cells are measured optically in absorbance or emission in the flow and from this the corresponding feature distributions are determined ·

From the parameters of the distributions determined according to 1 ×, 2 × and 3 ×, all the state properties of the process involved in achieving the object of the invention can be used

"microbial populations are determined.

The knowledge of the state allows a targeted influencing of the process by z. The dosage of the carbon and energy substrate or specific nutrients is also changed. A specific change of the throughput through the culture vessel, the temperature, the pH, the aeration rate and other parameters is possible depending on the type of process

Particularly, the method is suitable (ζ vgl-. B. DD-WP 140150), the process control parameters during dynamic Prozeßfü hr to determine Ungen

 The process is explained by the following examples.

example 1

In a Rührkesselfermentor yeasts of the species Saccharomyces cerevisiae are grown by the method of phase cultures (P, SS DAWSOLT, J. appl. Chem. Biotechnol, 22 (1972) 79) »As the carbon and energy source was glucose in a concentration of 30 g / The diet supplement was made as in V. MEYEEBURG elected (HK, τοη MEZESBtIRG, Arch, Mikrooiol 66 (1969) 289)

 Further fermentation parameters were:

Temperature 32 ⁰ G

Mass content of the fermentor 10 kg pH ·. 4.2

The state distribution was estimated by the protein content of the single cells. The protein content was determined cytometrically *

The ethanol producing subpopulation is characterized by a "peak" in the channel 90 of the histogram, the subpopulation using ethanol as a substrate by a "peak" in the channel 30.

The production of ethanol can thus be controlled by the ctometric measurement.

In the example, when a "peak" appeared in channel 30, it was ventilated with 50 l / kg »h" until a uniform population of peat lag again

Half of the medium. , ·

Example 2

In a Ruhrkesselfermentor the bacterial strain E. coli K12 was grown continuously at a Yerweilzeit of 1.5 h on glucose as the only carbon and energy source with the aim of obtaining protein-rich cell substance. The medium contained per liter:

3000 mg .H as ₄ 250 mg K as K ₂ GO

25 mg Mg as MgSO4

250 mg P as H ₃ PO ₄ (85

40 g of glucose

0.2 % yeast extract

and trace salts in sufficient quantity. Other fermentation parameters were:

Temperature 37 ^° C

Mass content of the fermenter 10 kg pH 7.0

The growth process was limited by the glucose supply. The media supply was controlled by means of a state distribution determined _by cytometric measurements of the protein content of the individual cells. When a "peak" appears in channel 25 of the histogram, which indicates an increase in the number of protein-poorer cells, the supply of medication was abruptly reduced. When the "peak" disappeared, the. Healing media supply increased again by leaps and bounds.

Through this targeted Uahrmedienzufuhr the crude protein content of the cell substance was increased from 70 % to SO% and the specific Glucoseyerbrauch from 2.0 g / g BTS to 1.8 g / g BTS be lowered.

' Example 3 '

The genetically manipulated bacterial strain Ξ. Coil C 600 was grown on glucose aerobically and semicontinuously with the aim of ascorbic acid recovery. The perforation parameters were the same as in Example 2.

After discontinuing the bacteria to a cell concentration of 20 g / l, half of the fermentation medium was drained from the fermentor and replaced with an equivalent amount of nutrient medium.

Lifting the usual fermentation parameters, the cell volume distribution was determined with a Coulter Counter ». When almost equal sxtrema of the volume distribution occurred, the temperature and / or the pH of the fermentation medium was changed once or several times over a short period of time.

When the ascorbic acid formation rate dropped below the maximum Yfert, the fermenter content was drained off and fed to the work-up.

Example 4 '

The genetically manipulated bacterial strain E, coli C 600 was grown on glucose aerobically and discontinuously with the aim of obtaining aseorbic acid. The perforation parameters were the same as in Example 2. By means of immunofluorescent staining of the cell membrane, the genetically manipulated strain was discriminated against the wild strain. The population was analyzed by means of flow cytometric analysis. The relative abundance of the engineered strain was controlled by population distribution. If a large proportion of the wild strain appeared, the process was stopped and re-inoculated.

Example 5 .

The Hefestamm- Candida utilis was on glucose as the sole carbon and energy source aerobically and continuously in a 10-liter fermentor at a Yerv / express time of 4 h, a temperature of 32 ⁰ C and a pH value of Permentationsmediums. from. 4 »2 bred. The aqueous ITährsBlzmedium contained per liter of water:

2000 mg IT as IiH ₄ Cl

300 mg P as H ₃ PO ₄ (85 %) 300 mg K as K ₂ CO ₃ 250 mg Mg as MgSO 4. .7H ₂ O; 40 g of glucose trace element compounds in sufficient quantity.

In order to be able to control the continuous cultivation according to the state of the population, the proportion of sprouting cells in the population was determined by means of an automatic scanner. When the majority of the cells were in the single-cell state, the nutrient supply was reduced. It was increased when the proportion of budding cells increased dramatically. By applying the method according to the invention, the specific glucose consumption during cultivation could be reduced.

Claims (6)

invention claim 1. A method for controlling processes of biotechnical material conversion, characterized in that the input variables of these processes are controlled according to the number, height and / or shape of the maxiina and / or minima of the distribution functions of one or more features of the cell populations involved in the process « 2. The method according to item 1, characterized in that the distribution functions on morphological features or physical properties of the population be true '- > the. 3 «Method according to item 1 and 2, characterized in that the control is effected by a change in the flow rate through the system, a change in the concentrations of individual substances in the inlet and / or a change in the gas flow through the system. 4. The method according to item 1, 2 and 3, characterized in that the occurrence of unwanted populations of accompanying organisms, which may also be undesirable mutations of genetically modified cells, by adjusting for these Accompanying organisms unfavorable growth conditions (z, 3. by Seffektorenmangel) suppressed can be" 5. The method according to item 1, 2 and 3, characterized in that in Permentaiionsphasen containing high levels of less effective growing cells, the carbon-containing substrates are dosed lower * 6 «method according to item 1, 2 and 3, characterized in that in the fermentation phases containing high levels of non or low producing cells, the growth conditions, z * B. by reducing the nitrogen and / or phosphate supply, be degraded.