DE2236596A1 - NEW PROCESS FOR FERMENTATION IN INVERSE EMULSION - Google Patents

Description

DAT7PI 68 MANNHEIM 1, -den 13.7.72

DR. GERHARD RAIZ-CL 2236596 SeckenheimerStr.36α-Tel. (0621) 406315

PATENT Attorney PoiMeli eefck ο η toi Frankfurt / M.Nr. 8 »3

Bank. Deutsche Bank Mannheim No. 72/000 " 7115 Telegr.-Codet Qorpat

INSTITUT FRANCAIS DU PETROIE DES CARBURANTS ET LUBRIFIANTS

1 & 4 »Avenue de Bois-Preau 92502 - RÜEIL-MALMÄISON

and

ENTREPRISE DE RECHERCHES ET D'ACTIVITES PETROLIERES

7, Rue Nelaton 75 - PARIS (15 & ne)

The present invention relates to an economical process which is the growth of every single-celled living being a hydrocarbon substrate serving as a carbon source and an aqueous nutrient medium,

The cultivation of microorganisms on hydrocarbons is already known; However, it has been found, that die.Mikroorganismen for growing on a chemically reduced subabrab need much more oxygen like a hydrocarbon than e.g. for growing on molasses or wood hydrolyzate,

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The fermentation process with hydrocarbons as substrates are carried out in emulsions in which the water is continuous Phase and the hydrocarbon is the dispersed phase.

A maximum of 30% hydrocarbons are used in these fermentation processes, since a higher percentage increases the viscosity of the emulsion, slowing down the transfers required for growth.

It is also known to produce water-in-oil emulsions in which the water is dispersed in the hydrocarbon phase 1st; the degree of liquidity of these emulsions, referred to below as inverse emulsions, depends on the extent to which the hydrocarbon phase is present dominates.

However, these water-oil systems have not yet gone into fermentation has been used because it is problematic in terms of regulation in the individual droplets of the inverse emulsion are (each drop of the dispersed phase behaves in isolation, since it has no overall contact with the aqueous phase); the regulation of the pH value in these systems is particularly important practically impossible.

There is, however, great interest in the use of these inverse emulsions since it is known that the solubility of oxygen In paraffins it is about ten times greater than in water with a temperature of 30 ° C. The oxygen transfer has a limiting effect in the process.

In the method described in the present invention, one makes these conditions of optimal ventilation in the Use of hydrocarbon medium.

For one has been surprised and found that it is possible to find a hold between the aqueous phase and the coal water , which one easily asked of one

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System in inverse emulsion to a system in direct emulsion can pass "

It has also been found that the microorganisms are in the aqueous phase are located in the water / oil boundary layer and thus both for the hydrocarbon and for the oxygen transfers are particularly favorable, so that the energy required for these transmissions is minimal »

These considerations reside in the procedure described below basic *

According to the natural method, the yeast grows in a fermentation vessel in inverse emulsion. At least part of the contents of this permentation vessel becomes continuous passed into a decanter in which the emulsion breaks itself. In this way one obtains the hydrocarbon above, practically no water and no yeast, which is returned to the main fermentation vessel, while a direct one below Oil-in-water emulsion. Which contains the microorganisms contains.

The direct emulsion is then regenerated, i.e. it is treated so that they are put back into the> permentation vessel can; this is preferably done after decanting into a container in which the 'emulsion is stirred' and in which, above all, one on the one hand regulate the pH by acidic or basic additives and on the other hand those necessary for the growth of the microorganisms Can add elements (especially mineral salts).

In all cases, the direct emulsion thus obtained is in the Main fermentation device recycled.

The advantages of the method compared to the system in direct Emulsion are the following!

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1) Better oxygen transfer, the greater productivity per liter of the aqueous phase used.

2) Saving water due to the increased cell concentrations.

3) Saving energy needed for oxygen and hydrocarbon transfer is required.

4) Better control of foam formation due to the very high Hydrocarbon content.

The present process is characterized in that a microorganism is cultivated in an aerated and stirred culture medium which consists of a water-in-oil emulsion and contains at least 50% by weight of a liquid hydrocarbon phase and a maximum of 50% by weight of an aqueous nutrient phase, that at least part of the emulsion is decanted in a stationary zone, that the decanted oily phase is recycled into the fermentation zone, that the decanted aqueous phase is subjected to a regenerating treatment and that the regenerated aqueous phase is recycled to the fermentation zone.

It is preferable to work as follows:

It is preferable to use a strain of microorganisms previously grown in a nutrient medium which

a) a source of nitrogen, a source of phosphorus and a source of assimilable potassium,

b) a source of trace elements,

c) growth factors,

d) contains essential mineral elements.

The aforementioned aqueous medium, which contains the microorganism, is then added to a hydrocarbon charge which contains at least 10% linear paraffins. The proportion of hydrocarbons is in any case greater than $ 50 of the total and is preferably between $ 65 and $ 85. The temperature is between

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20 and 45 ° G and preferably between 20 and 40 ° C and in particular between 28 and 37 ° 0. Forms under these conditions the hydrocarbon is the continuous phase and the aqueous phase is the dispersed phase. The pH will be between 2 and 5.5 and preferably held between 3 and 5.

This inverse emulsion is continuously withdrawn and passed into a decanter, from which a direct emulsion is produced at the bottom with at most $ 50 and preferably $ 10 to $ 30 hydrocarbon withdraws while the predominantly hydrocarbon upper phase is recycled to the main fermenter will.

The direct emulsion is fed into a container in which the pH is brought below 7 and, in particular, kept between 3 and 5. Since the medium generally tends to become acidic in the course of the growth process of the microorganisms, it is particularly advisable to keep the pH at the optimal value, e.g. by regularly adding alkaline solutions (e.g. ammonia), which happens when that aqueous medium contains consumable ammonium ions, as set out below. The medium required for growth is also added at this stage. The aqueous nutrient medium essentially contains a nitrogen source and a source of assimilable phosphorus, for example in the form of NH. ⁺ - and PO. """- Ions, trace elements, eg iron and copper compounds, growth factors of the type of B vitamins, which are obtained eg in the form of yeast extracts, and ions which are essential for growth and reproduction and the type of which depends on the strain of the microorganisms being cultivated.

One draws part of the direct emulsion, also called yeast cream, which is 10 to 50 $ and preferably 20 to 30 $ hydrocarbon contains, from ,. This yeast cream is treated with a process suitable for the batch used to remove the yeast to separate from the hydrocarbons.

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Most of the direct emulsion is recycled into the Main fermentation vessel in which the direct emulsion is broken and dispersed in the continuous hydrocarbon phase.

It should be explained at this point that, among the microorganisms used according to the invention, the yeast, the bacteria and the molds or mixtures thereof are to be understood.

As non-limiting examples of these three categories of microorganisms the following are mentioned:

The Endomycetaceae family, and particularly the subfamily of the Saccharomycetoideae, to which the genera £ ichia, Sä2l 2 £ SHlä »2S5ä £ i2Si2S2 belong to the subfamily of the Lipomycetoideae and especially the genus Lipomyces.

The family of the Cryptococcoideae and of them the genera Torulopsis and Candida, the subfamily of the Rhodotoruloideae and thereof the genus Rhodotorula.

The order of the Pseudomonales, especially the Pseudomonadaceae family and thereof the genus Pseudomonas and thereof the following Tribes:

- Pseudomonas fluorescens

- Pseudomonas ovalis

- Pseudomonas cruciviae.

The order of the Eubacterialles, including the family Aohromobaoteraceae and especially the genus Achromobaoter, the genus Flavobaoterium and the following strains ι

- Flavobncterium aquatile

- Rl avobnc torium luLnnorma

- Flavobaotorl. To mnrinuin,

dia family of the Miorocoocaceae, especially the strains Microooccua luteus and Micrococous flavus, the family of Brevlbacteriaoeae

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and thereof the genus Brevibacterium.

The order of the Actinomycetales and thereof the families Mycobacteriaceae and Actinomycetaceae,

- The family of Mucoracees and of them the genus Rhizopus.

- The FernΙ.Π Io dor Anporgillalen and of it the genus Aspor-

and Penlcillium.

The present invention is explained with reference to FIGS. 1 and 2. FIG. 1 shows a mode of implementation in which a main fermentation vessel 1 for the inverse emulsion, a decanting vessel 2 and a regulating vessel 3 are used for the treatment of the direct emulsion. D _e m fermentation vessel is fed through line 4 and air through conduit 5 hydrocarbon. The air flows out through line 6. The reaction mixture passes through line 7 into decanter 2: the hydrocarbon (upper phase) is recycled through line 8, and the direct emulsion (lower phase) is passed through line 9 into regulating vessel 3. After adjusting the pH and adding the medium necessary for the growth through line 10, part of the emulsion is recycled through line 11 and part is withdrawn through line 12 for the purpose of recovering the yeasts in a known manner. In Figure 2, the decanter and the regulating container are combined in a single container 13; the feed takes place through line 7. The hydrocarbon is recycled through line 8 in a mixture with a part of the direct emulsion formed in container 13 at the top away from the liquid phase. In contrast, the direct emulsion phase is predominant at the bottom of the container 13. Part of it is withdrawn through line 12 to obtain the yeast.

According to a variant of the process, a single heterogeneous apparatus is used, in the lower part of which there is a more direct zone Emulsion can be obtained, since the proportion of hydrocarbons there is low, while the remaining contents of the fermentation vessel

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exists as an inverse emulsion. The regulation of the pH and the addition of media takes place in this part of the reaction _vessel in which there is continuous recirculation with the remaining contents of the fermentation vessel.

Another variant of the present invention is a Part of the direct emulsion coming from the fermentation vessel into a classic fermentation device in direct emulsion which is called the fermentation device on the 2nd floor. It has been found that in this fermentation vessel in which the yeast likewise the hydrocarbons are consumed, the transition into an inverse emulsion the hydrocarbon transfer in this Production fermentation tanks favored. Skipping from the direct to the inverse emulsion allows good contact between the hydrocarbon droplets and the yeast, and therefore, the transmission takes place in a satisfactory manner.

The following experiment was carried out:

Into the main fermentation vessel one injects 400 cm aqueous Nährlünung with a content of 7, 5 ß / iiter l'Candida lipolytioa in the continuous hydrocarbon phase (about 2 liters). This fermentation vessel is gently stirred and aerated to the extent necessary. The hydrocarbon is an η-paraffin with 12 to 18 carbon atoms. The temperature is 30 ° C.

The aqueous nutrient medium has the following composition!

Acid ammonium phosphate, 2.9 g

Magnesium sulfate., 0.9 g

Potassium Chloride 1.32g

Yeast extract · .300 g

(Growth factor)

Tap water. ..... 300 g

(with trace elements)

Distilled water 700 g

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The pH of the medium is adjusted to 3.5 by adding ammonia.

The liquid coming out of this fermentation vessel will pumped into the decanter in which the emulsion is broken so that a direct emulsion of the following composition is obtained below subtracts: 75 $ aqueous phase, 25 $ hydrocarbon, the aqueous phase containing the yeasts. The hydrocarbon withdrawn above is returned to the fermentation vessel. This direct emulsion is homogenized in a connected container, in which their pH by adding ammonia in normal solution is set to 3.5; it is then returned to the growth fermentation vessel.

The growth of the microorganisms over time was measured and it was found that the cell concentration in the aqueous phase after 9 hours of culture was 30 g per liter. The degree of growth of the microorganisms (if X and X are the amounts of yeast present at the beginning and after the lapse of 9 hours) is expressed by jut- = 1 / t log X / Xo. 0.15 was calculated for μ.

In the present system there is therefore an appropriate development the yeast instead.

If one also measures the instantaneous production of the yeast, at dx = JU.-X. dt, one obtains dx / dt = 4 _f 5 g per liter and per hour, based on the aqueous phase.

It is known that the productivity of classic fermentation vessels, based on one liter of aqueous phase, is in the order of 1 to 2 g per liter and per hour.

In this experiment, the process according to the invention made it possible to consume two to three times less water than in the classic process, with a degree of growth of 0.15.

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The amount of agitation required for these transfers became m reduced by four to five times, ^ appoint on that Stir effort, which in the hydrocarbon fermentation in direct Emulsion with mechanical stirring is required.

Beis |) iel_2

The apparatus described is for use in continuous Culture has been modified. A pump system has been used, with which 50 to 100 cm of fresh water per hour Nutrient medium, namely the one described in Example 1, in the regulating container can be directed. Otherwise, the same amount of aqueous phase has been drawn off, while the hydrocarbon phase is recycled into the main fermentation vessel. The process took 24 hours, with five complete renewals of the aqueous medium and one yeast production of 20 g. The productivity of this system was 3 g per liter and per hour, based on the aqueous phase, and the degree of growth the yeast was between 0.1 and 0.15.

It should be mentioned in connection with Example 2 that the continuously withdrawn aqueous phase can also be passed into a fermentation vessel in direct emulsion, in which the fermentation is continued under conventional conditions. It was found that there was excellent transfer of the hydrocarbons in this fermentation vessel due to the previous going into the inverse system. It has been found, for example, that the stirring speed, which must be very high in a classical fermentation, became superfluous if the fermentation in direct emulsion was preceded by fermentation in inverse emulsion. The required for the transfer of hydrocarbons in the production fermentation vessel Energieaufw _a nd could be reduced by half; this is a considerable advantage when one considers the large amount of energy that is generally required for these transmissions. Thus, the present invention also relates to a method for culturing microorganisms, wherein a fermentation apparatus in an inverse emulsion with a

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Fermentation device is connected in direct emulsion, wherein the inverse emulsion fermentation device does the job has to stimulate the growth of microorganisms and the transfer of hydrocarbons in the fermentation device in direct emulsion to facilitate.

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Claims (5)

■ 1 λ) New process for the fermentation of microorganisms, thereby characterized in that a microorganism is cultivated in an aerated and stirred culture medium that consists of a Wasaerin-oil emulsion and at least 50% by weight of a liquid hydrocarbon phase and a maximum of 50% by weight of an aqueous phase Nutrient phase contains that at least a part of the emulsion is subjected to decanting in a stationary zone that one the decanted oily phase is recycled into the fermentation zone, so that the decanted aqueous phase is subjected to a regenerative treatment and that the regenerated aqueous phase is recycled to the fermentation zone. 2) Method according to claim 1, characterized in that the hydrocarbon $ 65 to $ 85 and the aqueous nutrient phase $ 15 to 35 $ by weight of the emulsion. 3) Method according to claim 1 or 2, in which the hydrocarbon in the form of η-paraffin or gas oil with at least IO56 η-paraffins is used. 4) Process according to claim 1, characterized in that the aqueous phase withdrawn from the decantation zone is passed at least partially into a fermentation zone in direct emulsion will. 5) Process for the fermentation of microorganisms, characterized in that that one stimulates the growth of microorganisms in a fermentation zone in inverse emulsion and that one that Growth of the microorganisms in a fermentation zone in direct Emulsion continues 309807/0676