DE1442051B2 - Process for the cultivation and purification of microorganisms on hydrocarbon fractions - Google Patents

Description

or partly from straight-chain hydrocarbons existing starting material and an aqueous nutrient medium and treatment of the cultivation product to reduce the proportions of residual Hydrocarbons that are bound to the microorganisms, which is characterized is that the separated from the culture medium, the microorganism, aqueous nutrient medium and fraction containing hydrocarbons treated with a surfactant, centrifuged and then, optionally after drying, subjected to a solvent extraction.

Particularly suitable solvents are ethyl alcohol, isopropanol, including light hydrocarbons Benzene and lighter platform fractions, ethyl ether, acetone, chlorinated solvents and Petroleum liquid gases such as butane and propane.

The term “microorganism” used here also includes mixtures of microorganisms. As the microorganisms grown according to the invention, there are yeast, fungi or bacteria in question.

ι Using yeast is this preferably to the family Cryptococcacceae, in particular to the subfamily Cryptococciodeae. If necessary, however, for example asocsporegenic yeasts of the subfamily Saccharomycoideae can be used. The preferred genera of the subfamily Cryptococcoideae are Torulopsis (also known as Torula), Candida, and Mycoderma. The preferred yeast strains are listed below. Particularly . the strains whose deposit number is given in the table below are preferred. The strains marked with CBS can be obtained from the central office in front of Schimmelculture, Baarn, Holland, and the strains marked with INRA from the Institut National de la Recherche Agronomique, Paris.

Candida lipolytica CBS 599

Candida pulcherrima CBS 610

Candida utilis CBS 890

Candida utilis, Variati major CBS 841

; Candida tropicalis CBS 2317

Candida arborea

Torulopsis colliculosa CBS 133

Hansenula anomala CBS 110

Oidium lactis

Neurospora sitophila

Mycoderma cancoillote INRA;

STV 11

Of the above-mentioned strains, Candida lipolytica is particularly preferred.

If necessary, fungi can be used as microorganisms. A suitable strain is Penicillium expansum.

Bacteria can also be used as microorganisms. The bacteria are expediently the Orders Pseudomonales, Eubacteriales, and Actinomycetales used.

Preferably used bacteria are the groups Bacillacea and Pseudomonadacea. Preferred species are Bacillus megaterium, Bacillus subtilis and Pseudomonas aeruginosa. Other suitable strains are:

Bacillus amylobacter,
Pseudomonas natriegens,
Arthrobacter sp.,
Micrococcus sp.,

Corynebacterium michiganense, Pseudomonas syringae,
Xanthomonas begoniae,
Flavobacterium devorans,
Acetobacter sp.,
Actinomyces sp.,
Agrobacterium sp.,
Aplanobacter sp.

In addition to the feedstock, an aqueous nutrient medium is required for the growth of the microorganisms and a source of oxygen, preferably in the form of air, is required. A suitable nutrient medium for yeast (and mushrooms) has the following composition:

Diammonium phosphate 2 g

Potassium chloride 1.15 g

Magnesium sulfate · 7H ₂ O 0.65 g

Zinc sulfate 0.17 g

Manganese sulfate · 1H ₂ O 0.045 g

Ferrous sulfate · 7H ₂ O 0.068 g

Tap water 200 g

Yeast extract 0.025 g

Distilled water to 1000 cm ³

A typical nutrient medium for growing Nocardia has the following composition:

Ammonium sulfate Ig

Magnesium sulfate 0.20 g

Ferrous sulfate · 7H ₂ O 0.005 g

Manganese sulfate · 1 ~ H ₂ O 0.002 g

Monopotassium phosphate 2 g

Disodium phosphate 3 g

Calcium chloride 0.1 g

Sodium carbonate 0.1 g

Yeast extract 0.008 g

Distilled water to 1000 cm ³

For other bacteria, for example, a nutrient medium with the following composition is suitable;

Monopotassium phosphate 7 g

Magnesium sulfate · 7H ₂ O 0.2 g

Sodium chloride 0.1 g

Ammonium chloride 2.5 g

Tap water (trace elements) .... 100 cm ³

Yeast extract 0.025 g

Distilled water to 1000 cm ³

Another suitable nutrient medium for growing bacteria has the following composition:

NH ₄ Cl 0.5 g

NaCl 4 g

MgSO ₄ 0.5 g

Na ₂ HPO ₄ 0.5 g

KH ₂ PO ₄ 0.5 g

Water ad 1000 cm ³

Microorganisms, especially yeast, sometimes grow only with difficulty when they first grow under Using hydrocarbon fractions as starting material can be grown. Sometimes it is required to use a microorganism for inoculation that was previously grown on the to using hydrocarbon fraction has been adapted. It is also possible that the microorganisms despite the cultivation in the presence of an aqueous mineral medium, which is the appropriate

5 6

Containing nutrients only grow with difficulty, It has been found that when using yeast because the hydrocarbon fraction is not the wax of the species Candida lipolytica in a process according to Contains factors that are used in starting materials of the invention, in which the ventilation by "vortex the base of carbohydrates are present. ventilation «takes place, a high growth rate unless these growth factors will be reached in time, whereby the generation time is occupied, range from 2 to 5 hours.

In the case of batch operation, the microorganisms will grow. The microorganism will preferably regrow initially with a slow increase in cell density, possibility of centrifuging the main mass (This growth period is hereinafter separated as the »carrying phase of the liquid phase. It is usually called phase «). After that, the growth- io water-based cream or paste obtained, which still increases speed to a higher value. The period is contaminated by hydrocarbons. with a higher growth rate than this water-containing and adhering carbon "Exponential phase" called. Subsequently, the hydrogen is still contaminated microorganism cell density constant again (the "stationary phase"). paste is made with an aqueous solution of an above-one Washed a certain amount of the microorganism for the surface-active agent and then centrifuge the next batch is preferably fuged before. The micro termination obtained by centrifugation the exponential phase decreased. The organism paste is then the solvent incubation is usually subjected to extraction before the stationary phase. You can be as continuous canceled. In this phase the microorganism is extracted or with successive washing Usually from the bulk of the aqueous solvent and subsequent phase separation Nutrient medium and from the main mass of which are not carried out. Extraction is useful used fractions separated. in a stationary one equipped with a paddle stirrer The growth of the microorganisms used container at a stirring speed of preferred favored when going to the nutrient medium a wise less than 10 rpm or in one around one very small amount of a yeast extract (carried out a container rotating through 25 horizontal axis. Hydrolysis of yeast obtained in vitamins of the With continuous solvent extraction the Group B rich industrial product) or whole extract continuously withdrawn and continuously generally on vitamins of group B and / or biotin or in batches at normal pressure or reduced gives. This amount is preferably in the size pressure distilled. The solvent becomes continuous of 25 parts per million based on the 30 borrowed returned to the extraction apparatus. Under aqueous culture medium. They can be higher or lower these conditions the yeast can continuously be imported and withdrawn depending on the loading or batch type selected for the incubation, conditions. In solvent extraction, the solution is The growth of the microorganism takes place on medium, preferably with periodically changing Ge costs the feed fraction introduced into the extraction apparatus with intermediate formation, of bodies - mainly fatty acids - with this has the consequence that this fluid flow Acid function so that the pH of the aqueous pulsates. Those going through the solid material mineral medium decreases progressively. Without correct liquid surges, an oscillation and door growth comes to a standstill pretty quickly - limited movement of every grain of solid in mind, and the concentration of the microorganism 40 in relation to its neighbor, and this is equivalent in the medium or the cell density no longer increases, with a mechanical stirring of the whole. the end so that a so-called stationary phase is reached. Because of this, the products become a lot overall

The aqueous medium is therefore preferably extracted more quickly and more completely,

by gradually or continuously adding an expedient in the liquid feed stream is a

aqueous medium with a high pH-value placed at the device, which generates liquid surges,

maintained the desired pH. Usually the amplitude and frequency of which is used experimentally for

The pH value of the nutrient medium is adjusted to the most favorable value in each special case when using

Mushrooms or yeasts, especially when using is made. These bumps come with any

Candida lipolytica, in the range from 3 to 6, preferably produced with known device, preferably with

kept wise from 4 to 5, (bacteria require a 50 of a piston pump whose valves have been removed

higher pH of usually 6.5 to 8). As are. The number of impacts is preferably between

Alkaline substances for adding to the seasoning are 1 and 60 per minute.

Sodium Hydroxide, Potassium Hydroxide, Disodium Hydro- Suitable solvents for the procedure were

gene phosphate and ammonia in free form or in already mentioned. If necessary, you can use a

aqueous solution. 55 first extraction stage a polar solvent,

The optimal temperature of the growth medium is e.g. B. an alcohol such as ethanol or isopropanol,

differently, depending on the type of yeast used, and then use the partially purified micro-

and is usually in the range from 25 to 35 ° C. In the organism in a second extraction stage with one

Using Candida lipolytica, a Tempe hydrocarbon, e.g. B. normal hexane, or one

Temperature range from 28 to 32 ° C preferred. 60 light platform fraction or benzene, as a solution

The uptake of oxygen is treat medium for growth. Preferably in the second

the yeast is essential. The oxygen is usually used as a solvent in a mixture stage

supplied as air, to the growth rate to a greater extent from hydrocarbons and to the

To keep it high, the air supplying the oxygen has to be a minor fraction of a polar solvent

be divided into fine bubbles by stirring. The air 65 consists. The azeotropic is expediently used

can be introduced through a frit, but a mixture of hexane with isopropanol or ethanol,

the system known as "vortex aeration" can also be used.

ventilation must be applied. must be carried out naturally.

It is believed that using one of hydrocarbon and polar solvent existing mixture the function or one of the functions of the polar solvent the attenuation the binding of the material to be extracted (also the binding of hydrocarbons in the polar Solvents are not soluble).

When using alcohol in the first stage of a two-stage extraction to the above described manner, the water content of the cream or paste of the microorganism is significantly reduced. As a result, the material introduced into the second stage, which contains the microorganism, a water content so low that it is ensured that non-aqueous impurities are still present are miscible with the solvent used in the second stage. Each extraction stage can comprise one or more sub-stages resulting from a wash with the solvent used in the stage and subsequent separation exist.

If only a single wash is used in the first stage, the amount of ethanol or isopropanol will be 1.5 to 3 times the volume of water in the cream or paste of the microorganism is available. However, two washes with ethanol or isopropanol can optionally be used be made, in the first wash a volume of solvent equal to the volume of water in the cream or paste, and in the second wash a lower amount of ethanol or isopropanol, for example half the amount used in the first wash is used.

In between washes of each stage or sub-stage, the liquid is left out of the cream or paste run off, for example by filtration. Some of the remaining solvent is then preferred removed by vacuum filtration.

In the second stage, the amount of solvent used is in the (or each) laundry usually 2 to 20 times the volume of the dry microorganism obtained.

The solvent is preferably removed in the last stage by evaporation, expediently under reduced pressure and advantageously in a stream of inert gas, e.g. B. nitrogen or superheated Steam.

By using a mixture of hydrocarbon and polar solvent as the solvent in the extraction stage, the composition of the solvent of the second stage, which takes up in each polar solvent from the first extraction stage, can be stabilized. An enrichment in polar solvent can be avoided by a distillation stage in which the solvent of the second stage is worked up, whereby a) polar solvent for recycling into the first extraction stage and b) a mixture of hydrocarbon and polar solvent for recycling into the second stage is separated subtracted from. The extract obtained in the second extraction stage is expediently distilled in a second distillation stage, with a) as top product a mixture of hydrocarbon, polar solvent and water for recycling into the second extraction stage and b) as bottom product a fraction containing polar solvent, water and the contains extracted substances. This fraction is preferably mixed with the extract obtained in the first stage before this is used in the distillation, whereby all the impurities obtained by the solvent extraction are removed as the bottom fraction in this distillation stage. Ethanol or isopropanol is expediently used as the polar solvent. An azeotropic mixture is preferably used as the solvent for the second stage.
The optimal contact time usually changes

ίο reversed with the extraction temperature. It is usually impractical to use temperatures above 70 ° C as higher temperatures lead to a cause some degradation of the product.

When the cream or paste of the microorganism is partially dried before solvent extraction one usually comes in the first extraction stage with a single wash with one alcoholic solvents and with a smaller amount of solvent than would be necessary,

ao if no drying had been carried out. If the degree of drying is significant, there is one first extraction stage using a polar solvent, e.g. B. an alcoholic solvent, not mandatory. In this case, one-step extraction using a solvent can be used be carried out, which consists exclusively of hydrocarbons or a mixture of Hydrocarbon and polar solvent, e.g. B. an alcohol or ketone or chlorinated hydrocarbon, consists.

In general, it is convenient to avoid drying under harsh conditions as this will partial decomposition of the microorganism, e.g. B. by destruction of vitamins and oxidation of unsaturated compounds. Furthermore, the decomposition products are in the extractive Solvents used in distillation are soluble, so that they are lost from the product or further Recovery stages are required.

When the microorganism is dried before the solvent extraction, a drying ^{temperature of 70 °} C. is preferably not exceeded. The invention is illustrated by the following examples. Experiments 1 and 2 were not carried out according to the invention and are described for comparison purposes only.

example 1

A yeast of the species Candida lipolytica is prepared using a paraffinic gas oil on the in the German patent application P 14 70 490.6 described method grown. (According to this procedure the microorganism is first - preferably by centrifugation - from the larger part of the closed separated aqueous phase. The resulting water-containing yeast paste, on the cells of which oil adheres is mixed with an aqueous surfactant solution, followed by centrifugation and washed with water.) The yeast obtained in this way contains 44% proteins and 18.5% lipids. she has a pungent and rancid taste, common to yeast made from hydrocarbon fractions is characteristic. The lipids are from this yeast in a Soxhlet extraction apparatus with a

Extracted mixture of 80% hexane and 20% ethyl alcohol. After evaporation of the solvent a yeast is obtained which contains 52% proteins and 3% lipids.

309 544/5

From 100 g of yeast, 16 g of extract are obtained by solvent extraction, mainly consists of oxidized and non-oxidized fatty acids, esters and sterols. The way of that Most of its lipid-freed yeast has a perfectly neutral taste, which makes its use as animal feed.

Example 2

A yeast cream was produced according to the method of German patent application P 14 70 490.6, which is described on the basis of the associated flow diagram. It should be noted that in the present Case material used is the yeast cream that after washing with surfactant Means and with water before the drying stage, which is described on the basis of the above diagram, was obtained.

In a series of experiments, a solvent was continuously poured into an extraction apparatus which contained a batch of the yeast cream. A filter drum served as the extraction apparatus, which rotated around its horizontal axis.

Yeast cream containing 2 parts by weight of dry yeast and 8 parts by weight of water was added to the extraction apparatus with 16 parts of ethanol. The mixture was ^{kept at 60 °} C. for 30 minutes while the drum was rotating.

The solvent was evaporated using a vacuum at the end. Was obtained in this way, a mixture of 2 parts of yeast and 2 parts of solvent consisting ^of water and to Vs% of ethanol and contained some remaining impurities.

This mixture was treated with 10 parts by weight of a mixed solvent that was 80 percent by weight Normal hexane and 20 percent by weight

ίο

Ethanol. The mixture was ^{kept at 60 °} C. for 15 minutes, whereupon the solvent - finally under vacuum - was stripped off. The washing with this solvent was repeated several times (if a yeast product of high purity is desired, the washing can be repeated up to 10 times, for example). Finally, the yeast product is dried in superheated steam.

The composition of the yeast cream before

ίο solvent extraction and the product obtained is given in the following table:

Nitrogen, weight percent
the dry yeast 7%

Total weight of lipids based on the weight of the
Dry yeast 10%

Example 3

Yeast cream

Yeast product

7.8% 0.5 «/ ο

The yeast cream described in Example 2 was extracted continuously in a drum that was around its vertical axis rotated and was fitted with a paddle stirrer rotating at 10 rpm. In separate experiments served as the solvent

1. Ethanol.

2. Ethanol followed by normal hexane.

3. Isopropanol.

The extract was continuously distilled to separate the lipids and hydrocarbons and that Solvent returned to the extraction apparatus. The conditions and results obtained are listed in the following table:

4th After
treatment Attempt no.
7th After
treatment 8th After
treatment Solvent and treatment
duration Ethanol
(11 hours)
600 cm ³ / hour
60 ° C
1000 140
9.0
5.0
72 Ethanol (20 hours)
Hexane (9 hours)
600 cm ³ / hour
6O ⁰ C
1000 200
7.45
4.2
200 Isopropanol
(40 hours)
600 cm ³ / hour
6O ⁰ C
1000 200
8.50
6.9
100 Amount of solvent
temperature Before the
treatment Before the
treatment Before the
treatment Weight of the yeast paste (grams) .. 210
6.05
41.2 210
5.56
42.9 210
. 5.65
42.9 composition Dry yeast
Weight (gram)
% Nitrogen Total weight of lipids in the
Yeast (weight percent)
extract
Weight (gram)

For comparison, two tests are described below which are not carried out according to the invention became.

Attempt 1

The experiment described in Example 1 was repeated with the exception that the yeast Saccharomyces cerevisia, which was grown in a known way on sugar beet molasses as a substrate, was used. It did not become noticeable

Improvement of the characteristic taste of this yeast achieved.

Attempt 2

A similar experiment was carried out starting from a Torula yeast which was found in known to have been grown on bisulfite waste liquor. Again, there was no noticeable improvement the characteristic taste of this ίο yeast noted.

Claims (1)

In French patent 1 009 198 is Claim: mentions that microorganisms that live on an in Water-insoluble nutrient medium, for example carbon Process for the production of microorganisms lenwasserstoffen that can be cultured mechanically by cultivating such organisms in counter-5 manner, by decanting, washing, extraction or even partially separating them from the substrate by straight-chain filtration.
In the French patent 1 297 033 is used for rials and an aqueous nutrient medium and obtaining a pure yeast as follows of the cultivation product for the method: the original emulsion, d. H. the one with reducing the proportion of residual carbon to yeast enriched culture broth is an emulsifier, Hydrogen, which is attached to the microorganisms. H. a surfactant added, after which are bundles, which are centrifuged by notation. By centrifuging once that one is separated from the culture medium in the presence of the surfactant the microorganism, aqueous nutrient medium and not yet received a pure product, rather Hydrocarbons-containing fraction with a 15 must be followed by further purification steps in the form treated with surface-active agents, centrifuged by washing with water or again with surface-active and then, optionally after drying, connect a surface-active agent, in each case the Subject to solvent extraction. Washes followed by separations (centrifugation) are. By washing several times either with pure water or with a medium that has one Contains emulsifier, a pure product is obtained. Yeasts grown with hydrocarbons as the sole source of carbon have one The invention relates to a method for obtaining significantly higher lipid content than yeasts, which on the of microorganisms through cultivation in counter-traditional substrates (molasses, waste liquor, bisulfite ware one wholly or partially from straight-chain liquor) can be bred. Lipids are essentially made up of hydrocarbons existing starting material borrowed from fatty acids, esters (with higher or and an aqueous nutrient medium and treatment of a lower degree of oxidation including the fats of the cultivation product to prevent the anabolic and sterols). parts of residual, to the microorganisms 30 It has now been found that microorganisms, bound hydrocarbons. especially yeast, on hydrocarbons as one When cultivating yeast on hydrocarbons, you can grow carbon sources and thereby a substances, in particular on petroleum hydrocarbons, the product received for which the breeding is based on as a carbonaceous substrate, hydrocarbons are generally characteristic as a substrate a yeast obtained, which after washing and 35 taste attenuates or eliminates and continues The microorganism has a peculiar pungent and rancid taste from the clinging hydrocarbon taste that exempts the use of yeast substances by removing them from the culture Can prevent feed. The medium separated on molasses, the microorganism next to Waste liquors or bisulphite liquors remove traditional aqueous nutrient medium and hydrocarbons grown also have a peculiar taste, 40 holding fraction with a surfactant that of that of the agents grown on hydrocarbons is treated, centrifuged and finally, yeasted is different. Several methods have been used, optionally after drying, a solvent Attenuation of this taste, especially subject to extraction. the sharpness, suggested, but none of these properties. The fact that the one containing the microorganism driving was really successful. 45 fraction after separation from the culture broth, From "Die Brauerei / Wissenschaftliche Beilage", September 1961, before being subjected to solvent extraction, a method is known in which washing is carried out with surface-active agents, a first step from the culture medium being carried out in a simple manner - without being a large number of filtration a fraction is obtained, the micro- of wax stages must follow - a process that can also be carried out in organisms, a little aqueous medium and the remaining 50 large-scale industrial scale contains hydrocarbons, whereupon in a second for complete separation and purification of the stage the so separated Microorganism provided to out-bred microorganisms,
holding fraction of a solvent extraction to the opposite of a solvent extraction is the Removal of lipids and hydrocarbons progress of the claimed process is also subjected. In detail, a method 55 is described in its feasibility on an industrial scale, in which a culture medium, which consists of a stab. If the entire, non-concentrated culture mixture of yeast cells, extracellular oil return medium is extracted with a solvent, then there would be an aqueous medium a large amount of solvent. If, on the other hand, the culture is separated into two layers 60 after being left to stand, before solvent extraction, in which the upper layer is concentrated about 20 to 25 volume broth by filtration, then it turns out that the original medium is a percentage of the original medium. This this filtration as difficult because the culture broth upper layer containing the produced yeast, residual kogasin an oil-in-water emulsion, which is contained by small and aqueous medium, is separated and yeast particles are stabilized. Such an emulsion is filtered. Then this layer is plugged with acetone 65 the well-known industrial filters,
mix and centrifuge. This step can again - the subject matter of the invention is therefore a method The product is then dried for the production of microorganisms by cultivation. four such organisms in the presence of a whole