DD155329A1 - METHOD FOR REGULATING THE CULTIVATION OF MICRO-ORGANISMS - Google Patents

Abstract

The invention relates to a method for controlling the cultivation of microorganisms under aerobic conditions in an aqueous culture medium containing, besides the carbon source, nitrogen, phosphorus, potassium, magnesium, iron, copper, manganese and zinc. The aim of the invention is, by using the concentration of the phosphate ions contained in the culture medium as a control, to ensure compliance with a certain phosphorus concentration setpoint, whereby the cultivation process stabilized, the ash content of the biomass lowered and the amount of nutrient salts to be used are reduced. In this case, the target value of the phosphorus concentration (preferably from the range 20 to 60 mg / l) and the proportion of the essential elements in the culture medium in relation to the phosphor according to their specific stoichiometric consumption values should be such that the phosphorus is the first Naehrelement limited. The invention can be applied to fermentation processes with or without liquid recirculation, independently of the microorganism used, the carbon source used and the fermenter used.

Description

Title of the invention

Method for regulating the cultivation of microorganisms

Field of application of the invention

The invention relates to the field of the microbiological industry, especially to methods of controlling the cultivation of microorganisms under aerobic conditions in an aqueous culture medium independent of the carbon source and the type of per-menter. It belongs in the IPK C1;

Characteristic of the known technical solutions

Methods for cultivating microorganisms under aerobic conditions are known in large numbers. The various carbon sources, microorganisms and cultivation methods are described (DE-OS 2,633,451, DD-PS 129 804, SU-US 452 236).

Also known are concrete methods for controlling or regulating cultivation processes according to the exhaust gas composition (DE-PS 1 080 048), according to the temperature of the fermentation medium (SU-US 506 611) and according to several parameters, such. B. Gas throughput, energy input and substrate supply (Jefferis, R, P., Proc. Biochem W (1975) 3, 15).

Common to all methods is the need for stable supply of the microorganisms with the elements necessary for growth. This is achieved by supplying a nutrient solution containing these elements.

In SU-US 452 236, this problem is only a function of the bioma concentration in the culture medium

required concentration range for the whole nutrient solution to be used, d. H. the nutrient solution quantity is supplied relatively independently of the actual nutrient requirement.

In the method claimed in US Pat. No. 5,066,111, although the substrate and nutrient solution are metered in a common input stream as a function of the fermentation temperature (ie simultaneous productivity-dependent supply and temperature control), since the nutrient solution concentration and composition are not considered in the feed, the do not solve this task. In addition, in principle, the specified method is applicable only to small laboratory equipment with liquid substrates and for a limited outdoor temperature range.

Although in DE-OS 2,323,416 methods and arrangements for the regulation and analysis of biochemical processes are claimed in a very general manner, but as a result of the generality of the formulations, instructions for technical action are given in no case, but only global principles of procedure are listed.

The disadvantage of the mentioned methods thus lies in the inaccurate determination of the dosage amount for the nutrient solution, since overdosage with nutrient solution can cause an extracellular concentration of certain elements (eg copper), ranging from merely uneconomic operation via inhibition of growth to poisoning which can cause microorganisms. Although certain ranges are sometimes given within certain ranges within which the concentration of these elements in the culture fluid must move

3iO, however, the dosage amount for the nutrient solution is only reduced or increased according to empirical values when over or under very egg tion of the predetermined concentration ranges, which is the more difficult to realize the farther the cultivation process is away from the steady state. This is associated with a labile process control, a high ash content of the biomass and too high a raw material use in the nutrient solution.

In the method claimed in DE-PS 1 080 048, although the feed of the nutrient solution is likewise regulated, the invention specified therein can in principle only be applied to the cultivation of baker's yeast, where the regulation can be derived from the alcohol content of the waste gas.

Aim of the invention;

The object of the invention is to ensure such a nutrient supply, which increases the stability of the cultivation process, lowers the use of raw materials and reduces the ash content of the biomass.

Explanation of the essence of the invention

The object of the invention is to use a suitable ingredient of the nutrient solution as a controlled variable to arrive at a process that guarantees a nutrient solution dosage adapted to the respective process state.

This object is achieved by realizing a control in which the concentration of the phosphor Cj-p (t) contained in the culture medium represents the controlled variable and the desired value of the nutrient solution throughput Vtt constitute the manipulated variable. The concentration Стр (t) is thereby determined by means of known methods. The other elements of the nutrient solution necessary for the cultivation process are dimensioned according to the specific stoichiometric consumption values in relation to the phosphor, so as to ensure that the first occurring nutrient salt limitation is a phosphor limit.

The phosphorus balance of the process is established to derive the regulatory law and thus to determine the necessary nutrient solution throughput. For the controlled variable Cj-n (t), a setpoint Отъа is specified. Assuming that the values of the phosphorus concentration C-rp (t) and the productivity χ of the microorganisms are determined at intervals Δt and the actual values are present at the time t = t, the difference between the instantaneous value can be determined Cvp (t) and

the target value Cj ^ ß using the phosphorus balance equation of the target value of the broth volume / time unit V ^ t such that at a certain time t = t ^ (tj> t _Q), the phosphorus concentration Cj ₁ P (t) the setpoint С-у ^ reached. If this time t - identical to the next measurement time t β t + Δ t, then a strong overshoot of the controlled variable and thus a restless process regime would result. For this reason, for the purpose of smoothing, a time buffer к · Δ t is given, where k / is a fixed, freely selectable number (useful

XO is moderate к я 2 or к = 3 to choose), whereby the time t ^ is set to t- β t + к · Δι «The concrete calculation of the manipulated variable V ^ is a function of the time behavior of the unbegastened fermentation volume Vp, the residence time tr and productivity &.

Under the conditions

- the mean residence time T is considered constant in / * t, t +

At7 accepted,

the productivity is approximately constant in / * t _Q , t _Q + & tj ,

- The volume of perforation Vp is constant in / * t _o > t + At / SO results z. B. the setpoint of nutrient solution amount / time unit Vj ₁ T from the phosphor balance

- ^C LP ⁽ v

t + к

The symbols not used previously have the following meaning: PlJL-specific phosphorus content of the nutrient solution (mg / l)

OtE stoichiometric consumption value (kg P / kg biomass) This dosage is maintained only for the time interval & t and recalculated when the new measured values are present (at time tj = t _Q + At), in which case the concentration ^C LP ^^ with respect to the interval / * t ^, t ^ + к. Δt / regulated and the newly determined setpoint for the time interval / "t», tj + AiJ is maintained.

Using other prerequisites (eg temporal change of the fermentation volume) results in other values for ν ,, - γ, a change in the principle does not follow.

The method may also be applied to fermentation processes with partial recycling of the liquid phase, taking into account the initial input concentration of the biomass and the altered phosphorus input, but here again there are no fundamental changes.

With the dosage of the nutrient solution according to the invention, the concentration of phosphorus dissolved in the culture medium is maintained in the vicinity of the desired value. This makes stable process control possible by prescribing a low solubility value C pg (suitably from 20 to 60 mg / l) The ash content of the biomass is reduced and the nutrient solution and thus the raw materials used as needed without excess

 The inventive adjustment of the setpoint for the Nährlösungsdurchchsatz can both by means of process computers as well as by hand by humans using tables or nomogr arms, which are created using the provision of the invention, take place. The invention is illustrated by the following examples:

example 1

In a continuous stirred tank fermentor, the bacterial strain GB 21 - IMET B 385 was grown on methane. The fermentation parameters were:

dwell time: 6 h Temperature: 38 ⁰ C PH value: 5.5 Ungasasted filling volume: 0.1 m ³ stirrer speed: 1000 rpm Print: 1 ... 4 at Productivity: 1.3 ... 5.8 kg / nAi

The concentrate of the mineral nutrient solution contained per liter

90 ml H ₃ PO ₄ (85%) 40 ml KOH (50 %) 66.7 g MgSO ₄ . 7H ₂ O 4.48 g PeSO ₄ . 7H ₂ O 3.65 g MnSO ₄ . 4H ₂ O 2.58 g H ₃ BO ₃

and the trace elements molybdenum, chromium, nickel, aluminum, calcium, cobalt, copper and zinc in sufficient quantity.

Purely the experiment, 5 l of concentrate were made up with water to 100 l, so that a specific phosphorus content of% L ^{= 2} »° 4 ² S / l resulted.

The other parameters were:

At = 30 min, к = 3

Setpoint CjjLs = 30 mg / 1 (default).

In long-term experiments, the dosage of the nutrient solution according to the invention enabled the concentration of dissolved phosphorus to be kept in the range of 15 × 50 mg / l, with 85.8 %

BO of all yrs ranged from 20 to 40 mg / l.

Without using the method according to the invention, the value of the phosphorus concentration in this period varied between 0 and 180 mg / l.

Example 2

In a continuous stirred tank fermentor, the bacterial strain MB 58 - IMET B 346 was grown on methanol.

The permentation parameters were:

Residence time: 7h

Temperature: 32 ^° C

pH value: 4.0

Ungasasted volume: 0.1 m ^

Stirrer speed: 1000 rpm

Pressure: 2.5 at

Productivity: 3,5 - 4,5 kg / іЛ

The concentrate of the mineral nutrient solution contained per liter

75 ml H ₃ PO ₄ (85 %)

53.2 ml of KOH (50%)

60.82 g of MgSO ₄ . 7H ₂ O

2.1 g of PeSO ₄ . 7H ₂ O 1.14 g MnSO ₄ . 5H ₂ O

1.02 g H ₃ BO ₃

and the trace elements molybdenum, cobalt, copper and zinc in sufficient quantity.

For the experiment, 10 1 concentrate with V / ater was brought up to 100, so that a specific phosphorus content of ^P HL ^{= 3 '4} ^ ^{1 er} S ^from · The other parameters were At »1 h, к" 3 setpoint CjjLg = 40 mg / 1 (specified).

In a long-term experiment was at the beginning of the dosage according to the invention a value of C ^ s HO mg / 1 before.

Within 3 hours, the environment of the target value (40 mg / l) was reached and from then on held in the test period 95.8 % of all values in the range 20 ... 60 mg / 1 and 72.9 % of all values in Range 30 ... 50 mg / 1.

Without using the method according to the invention, the concentration of dissolved phosphorus in the culture medium fluctuated in the range 0 ... 145 mg / l ·

Claims (3)

claim 1. A method for controlling the cultivation of гкгоOrganismen under aerobic conditions in an aqueous culture medium containing, besides the carbon source necessary for growth elements, characterized in that the concentration of phosphorus contained in the culture medium is used as a controlled variable necessary for growth Elements are added according to their specific stoichiometric consumption of the culture medium and are measured in relation to the phosphor so that the first Nährsalzlimitation occurring is a Phosphorlimitatioii and the nutrient solution to be dosed from the Phosphorbilanzgleichung under specification of the process parameters and the setpoint of the controlled variable is determined so that Expiration of a smoothing serving buffer time, the concentration of phosphorus contained in Hährmedium reaches the predetermined setpoint, wherein the buffering time a predetermined multiple of the sampling interval width of Pho Sphorkonzentrationsmessung is. 2. The method according to item 1, characterized in that the desired value of the concentration of phosphorus contained in the culture medium in the range of 20 to 60 mg / 1 is set. 3 · Method according to points 1 and 2, characterized in that after separation of the biomass from the fermentation medium, a partial recycling of the aqueous phase takes place in the permentor.