EP0932660A1

EP0932660A1 - Method of cultivating biomass

Info

Publication number: EP0932660A1
Application number: EP97910254A
Authority: EP
Inventors: Olaf Lehmann; Christiane Berndt; Klaus-Dieter Menzel; Dominik Driesch; Christian Wulfes; Toralf Gliem; Arnulf Christner; Matthias Hilliger; Karl Miersch
Original assignee: Buna Sow Leuna Olefinverbund GmbH
Current assignee: Dow Olefinverbund GmbH
Priority date: 1996-10-12
Filing date: 1997-10-04
Publication date: 1999-08-04
Also published as: JP2000514313A; WO1998016624A1; DE19642105A1

Abstract

Methylotrophic micro-organisms are cultivated in known manner on the carbon substrate, methanol, using continuous measuring and secondary feeding processes in order to restrict the methanol concentration to very low values. According to the invention, a cultivation method comprising a plurality of cultivation stages is carried out in agitated and aerated bioreactors, the average methanol concentration decreasing from one stage to the next owing to suitable feeding methods. To that end, the methanol fed in during the cultivation stage (k-2) is proportional to the alkaline solution added in metered amounts during the regulation of the pH, in the cultivation stage (k-1), as the specific dosage, and in the final cultivation stage (k) first as the specific and then as the pO2-regulated dosage. This method prevents an accumulation of toxic intermediates and avoids the need to measure and regulate the methanol concentration during the entire cultivation period, such that costs are considerably reduced, in particular when the method is applied on an industrial scale.

Description

Process for growing biomass

The invention is used in the cultivation of methylotrophic microorganisms on the carbon substrate methanol and can be used both in research and in industry.

Methanol as a carbon source is an inexpensive substrate, which is also a waste product. However, methanol has a growth-inhibiting effect if it acts on the microorganisms in higher concentrations and / or over a long period. The reason for this presumably lies in the formation of toxic intermediates, e.g. B. formaldehyde, and their accumulation with excess methanol. Concentrations greater than 0.5 g / l must already be considered as excess methanol. Conventional fed-batch processes, in which a larger amount of the C substrate is placed in the culture medium and added or supplemented after it has been consumed, can therefore hardly be used. For example, it is not possible to cultivate Methylobacterium at a permanent methanol concentration of 0.5 - 10 g / l for a longer period than 20 - 30 h without stagnating growth. Methods are therefore known in which the concentration of the methanol is determined by constant measurement and is kept at values below 0.5 g / l by cyclic or continuous additions. These processes require a high level of human and technical effort.

Methods are also known in which the methanol concentration is regulated at low values by using an in-situ methanol probe. Suzuki et. al. [Appl. Microbiol. Biotechnol. (1986) 24, S 370 - 374] regulate the methanol concentration at 0.5 ± 0.2 g / l. To do this, they use a methanol probe consisting of a porous Teflon membrane and a flame ionization detector. The PID controller for the methanol dosage is implemented on a PC. The control parameters for controlling the methanol dosage are estimated via the (^ concentration in the exhaust gas, which is determined via an exhaust gas analysis. Pomerleau et. Al. (Preprints of the 6th International Conference on Computer Applications in Biotechnology, Garmisch-Partenkirchen, Germany , May 14-17, 1995) use in In principle, the same arrangement, but do not indicate which methanol probe you use.

Disadvantages of this procedure are the high costs of the probe, its insufficiently secured long-term stability and the relatively expensive

Control strategies.

For the industrial production of biomass on a larger scale, processes are required in which the mass of the microbiological starting material (inoculated preserves, shake cultures) is increased by a factor of 100,000 or more. These mass increases require multi-stage cultivation processes with the simplest possible procedure.

The invention has for its object to develop a method for growing methylotrophic microorganisms on the substrate methanol with a large increase in mass of the starting material (inoculated preserves, shake cultures). The cultivation is to be carried out in stirred and ventilated fermenters and the biomass is to be used to form cellular products or storage materials.

The object is achieved by the method presented in claims 1-9. For this purpose, the microorganisms are grown in stirred and aerated bioreactors in a k-stage fed-batch process, the average methanol concentration decreasing from stage to stage by the cultivation stage (k-2) with excess methanol, the cultivation stage (k-1) in first section with excess methanol and then with methanol rate limitation and the cultivation step (k) from the beginning under methanol rate limitation. Rate limitation is a state in which the rate of the inflowing methanol is equal to the rate of the methanol consumed by the culture and there is no accumulation of methanol in the culture medium. The cultivation method according to the invention can be carried out in such a way that, before the cultivation stage (k-2), further cultivation stages are carried out, which are carried out according to the same cultivation principle of the cultivation stage (k-2). Consequently In the case of multi-stage cultivation, the cultivation process always takes place in the last three cultivation stages - in those with the highest mass increase - according to the process steps according to the invention.

It has been found that the growth-inhibiting effect of methanol is avoided using this procedure.

The average methanol concentration in the cultivation stage (k-2) is 2 to 6, preferably 2.5 g / l, in the cultivation stage (k-1) 0.5 to 2, preferably 1 g / l and in the cultivation stage (k ) 0 to 0.5 g / l. The cultivation is carried out in all stages while avoiding growth oxygen limitation; a decrease in the dissolved oxygen concentration below 10% of the saturation concentration is avoided by known measures.

In the cultivation stage (k-2), methanol is added to the culture medium before the inoculation in an amount of 2 to 10, preferably 3 to 6 g / l. Then the inoculation is carried out with a microorganism culture from shake cultures, canned vaccines or with culture solution, which was taken from one of the subsequent stages. The cultivation is carried out at a constantly regulated pH with stirring and aeration until a biomass concentration sufficient for the inoculation of the cultivation stage (k-1) is reached. To replenish the methanol consumed during growth, proportioning to the alkali metered in via the pH control is metered in in a ratio of 7 to 10, preferably 8.5, mol of methanol to 1 mol of alkali. An alkali can be, for example, NaOH, KOH or NH ₄ OH, although divalent alkalis can also be used. This ratio dosage can also be achieved by carrying out the titration with a mixture which contains 7 to 10 moles of methanol and at least 0.3 liters of water per mole of lye. It has been found that the concentration of methanol in the culture medium is almost constant or slightly decreasing over a longer period of time. The cultivation is stopped before the methanol reaches a very low, growth-limiting concentration. Thus, the cultivation in this stage is carried out continuously with methanol shot at a maximum specific growth rate corresponding to the other cultivation conditions (temperature, pH, etc.).

In the cultivation stage (k-1), methanol is added to the culture medium before the inoculation in an amount of 2 to 10, preferably 3 to 6 g / l. Then the inoculation is carried out with the complete culture solution from the cultivation stage (k-2), or with part of it. The cultivation is carried out at a constantly regulated pH with stirring and aeration until a biomass concentration sufficient for the inoculation of the following cultivation stage (k) is reached. At a certain point in time within the 1st to 10th, preferably 3rd to 7th cultivation hours, an initially constant methanol dosage of 0.5 to 1.5, preferably 0.8 grams of methanol per liter of culture solution and hour is started. The flow rate of this dosage can be increased by a factor of 1.5 to 10, preferably 3 to 4, until the end of the second cultivation stage. It was found in this procedure that there is an excess of methanol until about 8 to 15 hours after the inoculation and then that the methanol is consumed by reducing its concentration to a growth-limiting value close to 0. Furthermore, the growth of the culture is limited in methanol, that is to say that the amount of methanol flowing in is used up immediately and there is no accumulation of methanol in the culture medium. The amount of the methanol inflow rate determines the growth rate and the achievable biomass concentration.

In the last cultivation stage (k), no methanol is added to the culture medium before inoculation or at the beginning of the cultivation. The inoculation is carried out with the complete culture solution from the cultivation stage (k-1) or with a part thereof. The cultivation is carried out at a constantly controlled pH with stirring and aeration until a sufficient biomass concentration is reached.

The methanol supply of the last cultivation stage (k) is carried out from the beginning as a continuous dosage, the inflow rate of the methanol being so is chosen that a flow equilibrium called rate limitation is established.

This is achieved by a specific methanol addition of 0.05 to 1.05, preferably 0.2 to 0.4 g of methanol per g of biomass (dry matter) and hour. Such a dosage can be implemented, for example, by increasing the rate of the methanol feed in the same time units by the same factor in the range of approximately 1.1 to 1.7 per hour (exponential profile). The methanol dosage is only increased to such an extent that, given the maximum values for aeration, stirrer speed and fermenter pressure, the dissolved oxygen value does not fall below 10% of the saturation concentration.

Embodiment

Figures 1 - 3 show the course of the biomass growth, the alkali / methanol mixture or the methanol dosage, the methanol concentration and the pθ2 value in the 3 cultivation stages described.

Figure 1: 1st cultivation level (k-2) Figure 2: 2nd cultivation level (k-1) Figure 3: 3rd cultivation level (k)

For the production of polyhydroxybutyric acid with Metylobacterium spec. A biomass of approx. 42 kg dry matter is required on the C-substrate methanol in order to initiate the product formation process at a biomass concentration of approx. 20 g / l (dry matter) in a 4000 liter fermenter with a 2100 liter filling volume. To obtain this amount of biomass, a three-stage successive cultivation process (k = 3) in a 30 l fermenter with a 20 l filling volume, in a 300 l fermenter with a 200 l filling volume and in a 4000 l fermenter with an initial filling volume of 2000 l is carried out from shake cultures carried out. A mineral salt medium is used as the culture medium, which in the 1st stage contains all required mineral salts and trace elements for biomass growth up to 5 g / l (dry matter), in the 2nd stage all required mineral salts and trace elements except nitrogen for biomass growth up to 10 g / l l (dry matter) and in level 3 contains all required mineral salts and trace elements except nitrogen for biomass growth up to 30 g / l (dry matter). For defoaming, 0.1 ml / l polypropylene glycol (PPG) is added to the medium before sterilization, further additions of this defoamer are carried out during the fermentation if necessary.

The cultivation takes place in all stages at a temperature of 31 - 32 ° C, an aeration rate of 0.4 to 0.6 vvm and an overpressure of 200 - 600 mbar as well as a constantly regulated pH value of 6.7 to 6.8 .

The first cultivation stage in the 30 l fermenter begins with the inoculation of the medium from a shaking culture carried out for 25 hours at 32 ° C., consisting of 6 shaking bottles with a filling volume of 400 ml each. A biomass starting concentration of 0.1 g / l (dry mass) is thus achieved. Before the inoculation, an amount of 60 g of methanol corresponding to 3 g / l is added to the culture medium. A mixture consisting of 75 ml of 45% sodium hydroxide solution, 427.5 ml of methanol and 475 ml of fully demineralized water is used to regulate the pH. Increasing the stirrer speed prevents the pO value from dropping below 20% of the saturation concentration. After 17 hours, the cultivation is ended at a biomass concentration of approx. 4 g / l (dry matter). The methanol concentration at the end of the cultivation is 2 g / l. When the entire content of the 30 l fermenter is inoculated into the 300 l fermenter, the second cultivation stage begins with a biomass starting concentration of approx. 0.4 g / l (dry matter). Before the inoculation, an amount of 600 g of methanol corresponding to 3 g / l is added to the culture medium. 25% ammonium hydroxide solution is used to regulate the pH. Increasing the stirrer speed prevents the pO ₂ value from dropping below 20% of the saturation concentration. From the 4th hour, a constant methanol dosage of 160 g / h corresponding to 0.8 g / l and h started. From the 10th to the 22nd cultivation hour, this dosage is increased linearly up to 600 g / h, corresponding to 3 g / l and h. The methanol concentration drops steadily until the 13th hour, at which point the methanol is used up and continues until the end of cultivation. In this state, known as rate limitation, the amount of methanol flowing in is equal to the amount of methanol consumed by the culture, and the methanol concentration can no longer be measured. At the 22nd hour of cultivation, the fermentation is ended at a biomass concentration of 10.4 g / l (dry matter).

With the inoculation of the entire content of the 300 I fermenter into the 4000 I fermenter, the third cultivation stage begins with a biomass starting concentration of approx. 1 g / l (dry matter). 25% ammonia solution is used to regulate the pH. The culture is now supplied with methanol exclusively via continuous metering. The methanol dosage starts with an inflow rate of 600 g / h corresponding to 0.3 g / l and h immediately after the inoculation. From the second hour of cultivation, this inflow rate is increased continuously by a factor of 1.136 per hour. The increase is controlled by a computer every minute, by 0.2134% each. The pO ₂ value drops steadily and reaches a value of 30% around the 9th hour of cultivation. From this point on, the pO ₂ value is kept in a range from 30 to 50% by gradually increasing the stirrer speed from 100 to 200 rpm. After reaching the upper stirrer speed of 200 rpm, the pO ₂ value drops to 20% by the 23rd cultivation hour. From this point in time, when the inflow rate of the methanol has reached an amount of about 8100 g / h, the pO ₂ value is regulated in a range of 10-20% of the saturation concentration via the methanol dosage. The dosage is reduced when the pO ₂ value drops below 15% and increased when the pO ₂ value rises above 15%.

During the entire cultivation in the third stage, the culture is in the state of methanol rate limitation, as in the second half of stage 2. Only in this way is it possible to react immediately to an increase in the pO ₂ value or to reduce the methanol flow rate and thus to avoid oxygen limitation of growth.

At the 37th cultivation hour, a biomass concentration of 20.7 g / l (dry matter) was reached. The cultivation of the biomass is thus ended, the further process management serves exclusively the formation and accumulation of polyhydroxybutyric acid by suitable measures.

Claims

claims

1. Process for growing biomass from methylotrophic microorganisms which are used for product formation with methanol as carbon source in stirred and aerated bioreactors in a multi-stage process, characterized in that the biomass is grown in a k-stage fed-batch process , the average concentration of the methanol decreasing from stage to stage by the cultivation stage (k-2) under excess methanol, the cultivation stage (k-1) in a first section under methanol excess and in a second section under methanol rate limitation, the cultivation step ( k) is carried out from the beginning under methanol rate limitation and the cultivation stage (k-2) is preceded by cultivation stages which are carried out according to the same principle of the cultivation stage (k-2).

2. The method according to claim 1, characterized in that the average methanol concentration of the cultivation stage (k-2) 2 to 6, preferably 2.5 g / l, in the cultivation stage (k-1) 0.5 to 2, preferably 1 g / l, and in the cultivation stage (k) is 0 to 0.5 g / l.

3. The method according to claim 1 and 2, characterized in that the dissolved oxygen concentration is not below 10% of the saturation concentration.

4. The method according to claim 1 to 3, characterized in that the culture medium at the beginning of the cultivation stages (k-2) and (k-1) each an amount of 2 to 10, preferably 3 to 6 g of methanol per liter is added.

5. The method according to claim 1 to 4, characterized in that in the cultivation stage (k-2), methanol is added in proportion to the alkali metered in via the pH control in a ratio of 7 to 10 mol.

6. The method according to claim 5, characterized in that a mixture of water, methanol and NaOH, KOH or NH ₄ OH is used for pH control, which contains at least 0.3 I water per mole of NaOH, KOH or NH ₄ OH.

7. The method according to claim 1 to 4, characterized in that in the cultivation stage (k-1) there is a feed of methanol by a continuous methanol dosage from the 1st to 10th, preferably 3rd to 7th, in particular 4th cultivation hour a flow rate of 0.5 to 1.5, preferably 0.8 g / lh of methanol is started, which can be increased by a factor of 1.5 to 10, preferably 3 to 4, until the end of the cultivation stage.

8. The method according to claim 1 to 3, characterized in that the methanol supply in the cultivation stage (k) is carried out from the start by a continuous metering, the inflow rate of the methanol being chosen so that a flow equilibrium called rate limitation is established.

9. The method according to claim 1 to 8, characterized in that the continuous methanol dosage in the cultivation stage (k) by a specific substrate supply from 0.05 to 1.05, preferably 0.2 to 0.4 g of methanol per g of biomass and hour is realized.