DE1642689C3 - Process for the recovery of microorganisms - Google Patents

Description

• It is known that too little is currently available for the Ver for smoke required by humans and animals Protein, especially cheap animal E. white available is. With the intention of remedying this E.we.ß-Knapphe.t, various bio-synthetic methods have recently been launched Procedure developed by the Sbende. Protein by growing yeast mushrooms », Nd / or bacteria on different carbonaceous Substrates are made available to ensure adequate growth of the Need microorganisms.

This biosynthetic technology iecnn Finer wide application in the hydrocarbons as nutrient sources, ^ _{however, is of} serious

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Another recently developed and more promising Method for the biosynthetic production of protein which is useful for nutrition by growing microorganisms on petroleum substrates. The latter pro-one synthesis is usually carried out on an aqueous culture medium, the hydrocarbon soil, an inoculation with the microorganisms to be grown, Contains oxygen and essential cellular nutrients. With this type of prolein synthesis can

to use the hydrocarbon culture media are less consuming than carbohydrates and the im generally no complex growth factors such as vitamins, amino acids and the like

^nk ^ _a uchlichen biosynthesis is a microorianism on an aqueous culture medium, 55 ^ '^^^ "' ^^^ organism is inoculated and one that with ^d « «™ JJ ^{|| C} ™ ⁰ ^ *" _and oxygen as well

S _n S2? Stop. bred. When the i has reached the _extent>

_{Evidence of} culture medium removed,

^ J ^^^ f ^ .p ^^ separated, ab w ^ «J» · * ^^ ^ '^ permanent liquid, kills and' 8 «το« η «. _Fermentation tank, in

w.rd m ~ al S ⁿ ^ ¹ ", _uruckge, surmounts _e. erfindem further ge ^{we zucttet} _wt ^ t n _nne of micro-" 5 d "" ^^^ ^^^ _u ^Z _{b e} T ^U _wherein , ° f ™ ^{men 1} ^ ¹ ^ S ^ medium contained microdass one ^ine ^ ^em * ^aU "^ Γ methylene chloride, tetra-organisms with Chloro.orm, Metnyien

chlorocarbon, methylene bromide, trichlorofluoromethane, Dichlorofluoromethane, l-bromo-2-chloroethane, 1,1-dibromoethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1-dichloroethane, 1,2-dichloroethane, pentachloroethane, 1'1,1,2-tetrabromoethane, 1,1,1,2-tetrachloroethane,

1 [ 1 '2,2-tetrachloroethane, 1,1,2,2-tetrachloro-1,2-dif luorä'th'an, ethyl bromide, 1,1-dichloropropane, 1,2-dichloropropane, 1,3-dichloropropane , 2,2-dichloropropane, bromotrichloromethane, 1,1,2-tribromoethane, 1,1,1-dichloro-ithane, 1,1,2-trichloroethane, 1,1,2-trichloro-1,2,2-trifluoroethane , l-bromo-2-chloropropane, 2-bromo-l-chloropropane, 1,2-dibromopropane, 1,3-dibromopropane, 2,2-dibromopropane, 1,2,3-trichloropropane, 1-ch'.orbutane, 2-bromobutane or 2-bromo-2-methylpfopan extracted and the microorganism diene isolated from the organic extract.

The subclaims contain advantageous developments of the method according to the invention.

As a result of this extraction, the cells of the microorganisms agglomerate, and it has the anao appear to be forming a kind of stabilized precipitate in the form of an emulsion with the organic liquid. However, it is not yet certain how the agglomeration will take place. Since the organic liquid is immiscible with the aqueous phase, the extract can be easily separated, because the total mass settles into an aqueous and an organic phase. The two phases can be separated from one another in a simple manner, for example by centrifuging or decanting and the microorganism-cell product can be isolated from the organic phase, for example by evaporating the organic solvent and drying the cells.

In the process according to the invention, one of the above-mentioned organic liquids is used, which wets the microorganism cells.

The extraction of the aqueous phase containing the microorganisms with the said immiscible organic liquid can take place at a temperature which is at or above the growth temperature of the microorganism cells, as is usually set in a suitable fermentation vessel. Temperatures above the cell growth temperature can be used, but they should be below a temperature at which noticeable denaturation of the proteins occurs. For example, the organic liquid is at a temperature between about 20 and about 55 ^C C, admixed most preferably about 35 to about 45 C, and in particular at about 40 C. The extraction can be carried out in one or more mixing containers and then the mixture can be allowed to settle in one or more further containers; customary procedures can also be used, for example stirring and the like. The aqueous liquid can also be extracted with the organic liquid using countercurrent columns or using columns having rotating contact trays.

The denaturation of protein occurs when the molecules of the cell protein and the essential amino acids break into parts with a lower molecular weight and / or by-products with a lower nutrient content, z. B. by acid hydrolysis. Such by-products do not contribute to the nutritional value of the Fiweißstoffe and / or amino acids of the no longer living microorganism cells or other for the nutritional valuable products that are contained in such cells.

The residence time, i.e. H. the length of time that the water-immiscible liquid is in contact with the aqueous liquid is generally about 1 minute to 1 hour or more, most preferably between about 10 and about 30 minutes. No significantly improved results are obtained if the residence time is exceeded extended for a period of more than 30 minutes, however, such extended periods can be used if desired readily use as such residence times are not critical.

The amount of water immiscible liquid used to extract the aqueous liquid is used, can vary over a wide range, in general the proportion is from organic liquid to aqueous liquid between about 1:20 and about 100: 1. Most favorable a ratio of organic liquid to aqueous liquid between about 1:10 is used and about 10: 1, especially between about 1: 3 and about 3: 1. The amount of organic used Fluid is not critical and varies with theirs physical properties.

As stated above, the microorganism cells agglomerate in the organic liquid and form a stabilized precipitate or emulsion when the aqueous liquid with the organic Liquid is brought together. Depending on the organic liquid used, they float Agglomerates or they sink to the bottom. In most cases, 60 to 70% of the amount remains in the original aqueous liquid present water in the aqueous phase, and the remaining 30 to 40% form a suspension with the microorganism cells and the organic liquid. When the dwell time is longer than 15 minutes, then breaks the suspension and, in connection therewith, further water passes over into the aqueous phase. However it was found that virtually all of the cells present in the initial aqueous fluid are, immediately after mixing the organic liquid with the aqueous phase and after after settling have passed into the organic phase in two phases.

Since the organic liquid is immiscible with the aqueous liquid, the Extraction an aqueous phase and an organic phase. These phases quickly separate from one another after the mixing has taken place, and you can therefore easily use common working methods, be separated from one another by decanting, for example. The organic liquid that which contains microorganism cells, is then subjected to a further separation (filtration, centrifugation) and / or subjected to evaporation, customary procedures being used, for example in a freezer, a drum dryer, a spray dryer, a hot dryer, etc. worked and the cells of the microorganisms are recovered. The evaporated organic Solvent can, if desired, be recovered and washed in the usual way or if necessary, refurbished and then reused for new purposes.

After the microorganism cells have been separated from the organic liquid, can

the cells are placed in a conventional drying device, for example a spray dryer, and therein dry the cells and stop the cells from living. The dried Zeil product can be used as Food additive can be used. However, it is not absolutely necessary to dry the cells, because one can also use the separated cells as such directly for purposes other than food use, for example, for the production of intercellular chemicals such as esters. Furthermore came. one can stop the life in the cells by means other than drying and then the product use for food purposes.

The inventive method for recovering microorganism cells can be in Following any fermentation process used to grow microorganisms on carbon containing substrates, for example carbohydrate and petroleum substrates, uses. Especially The recovery according to the invention is suitable method for such fermentation processes in which hydrocarbon culture media are used. the The present description therefore contains many references to such a fermentation.

The inventive improved method for as Recovery of microorganisms can be applied to all microbiological cultivations. So can any microorganisms that are used for microbiological cultivation by means of the invention Process can be recovered. This includes bacteria, fungi and yeast.

Although the method of the invention is capable of working for a wide range of different microorganisms is, there are nine microorganisms that are very specific for hydrocarbon assimilation suitable. These microorganisms are listed below along with their appropriate ATCC registration number listed.

Name of the microorganism ATCC number

Micrococcus cerificans 14,987

Pseudomonas ligustri 15 522

Pseudomonas pseudomallei 15 523

Pseudomonas orvilla 15 524

Alcaligenes sp 15 525

Cellumonas galba 15 526

Brevibacterium insectiphilium 15 528

Corynebacterium sp 15 529

Corynebacterium pourometabolum ..15 530

50 Example 1

In an aqueous biosynthesis bath that is 1.0 percent by weight Containing n-hexadecane as the hydrocarbon feed medium became Micrococcus continuously cerificans bred.

In a 7.5 liter biosynthesis container 4 liters of the aqueous nutrient medium which inoculates bacteria with 1.0 percent by weight Micrococcus cerificans had been filled. Sufficient air was allowed to bubble through the inoculated slurry so that that the oxygen demand of the bacteria was met before that of hydrocarbons and nutrient feed consisting of inorganic salts (containing phosphoric acid and ammonium hydroxide) was admitted. A typical composition for the biosynthetic bath in a given typical The stage of continuous biosynthesis is as follows:

Components g / liter

n-hexadecane 10

H ₃ PO ₄ 5

KCl 1

CaCl ₂ 0.5

MgSO ₄ . 7 H ₂ O 0.2

FeSO ₄ -7 H..O 0.2

NaCl 0.2

NH ₄ OH, added in sufficient amount to maintain a pH of 7.0.

The temperature in the biosynthesis bath was kept at 35 ± 2 C during the growth and the pH became essentially neutral; H. at about 7.0 ± 0.1 during the entire biosynthesis time regulated. The conversion of the hydrocarbon feed to cells was kept at 90% and higher. After a residence time of about 2 hours, a flow of the aqueous, sludge became continuous Fermentation product withdrawn from the biosynthesis bath. This stream contained the aqueous fermentation product about 1 percent by weight of bacterial cells in addition to unreacted hydrocarbons, inorganic salts, Nutrients etc.

Example 2
Approach 1

^{30 cm 3 of} the aqueous, muddy fermentation product from the biosynthesis container of Example 1 were poured into a narrow glass column, which had an inlet for charging in the middle and outlets at the top and bottom. Then 10 cm ^{3 of} methylene chloride (dichloromethane) were added to the glass column and the mixture was mixed with the aqueous, sludgy fermentation product. The temperature during the admixture was about 21.1 ° C. Spontaneous agglomeration of the microorganism cells in the methylene chloride occurred when methylene chloride was introduced. The mixture was then allowed to settle for about 30 minutes. Most of the aqueous phase remained as a supernatant layer. The resulting organic phase was separated from the aqueous phase and the microorganism cells were isolated by stripping to remove the methylene chloride and trapped water. Practically the entire amount of the microorganism cells contained in the aqueous, muddy fermentation product used was recovered. The agglomerated product contained about 40% of the original aqueous sludge.

Approach 2

The procedure of batch 1 was repeated, but with the difference that 30 cm ^{3 of} methylene chloride were used. Virtually all of the microorganism cells were recovered from the aqueous sludge, and about 42% of the original aqueous sludge was contained in the agglomerated product.

Approach 3

The procedure of batch no. 1 was repeated, with the difference that 90 cm ^{3 of} methylene chloride were used. All cells could be removed from the aqueous sludge, and about

50% of the original aqueous sludge was present in the agglomerated product.

Example 3

The procedure was as described in Example 2, batch 1, except that 10.30 or 90 cm ^{3 of} methylene chloride were added to the aqueous sludge at a temperature of about 38.degree. Virtually all of the cells were removed from the aqueous sludge in each of the three approaches.

Example 4

The procedure described in Example 2 was followed, but with the difference that as organic liquids Chloroform and carbon tetrachloride were used in different approaches. At all

Solvents used in this example, the results were about the same as in the examples 2 and 3 had been achieved.

Example 5

Again, as in Example 2, batch 1 was described worked, but with the difference that acetone, ethanol, hexane and an aliphatic hydrocarbon solvent were used. None

ίο these organic liquids led to agglomeration of the microbacterial cell product and therefore gave up when the cells were separated from the aqueous Sludge no effect. Acetone and ethanol are liquids that can be mixed with the aqueous sludge.

»5 th, whereas hexane and the aliphatic hydrocarbon solvent do not wet the microorganisms.

609 622 /:

Claims (3)

Patent claims: 1. Procedure for the recovery of micro-organisms, characterized in that it is contained in an aqueous medium Microorganisms with chloroform, methylene chloride, carbon tetrachloride, methylene bromide, inchlorofluoromethane, Dichlorofluoromethane, l-bromo-2-chloroethane, 1,1-dibromoethane, 1,1-D.chlorathane, 1,2-dichloroethane, 1,1-dichloroethane, 1,2-chloroethane, Pentachloroethane, 1,1,1,2-tetrabromoethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, 1,1,2,2-tetrachloro-1,2-difluoroethane, ethyl bromide, 1,1-dichloropropane, 1,2-dichloropropane, 1,3-dichloropropane, 2,2-dichloropropane, bromotor chloromethane, 1,1,2-tribromoethane, 1,1,1-chloroethane, 1,1,2-trichloroethane, l, l, 2-trichloro-l, 2 2-tr.fluoroethane, l-bromo-2-chloropropane, 2-bromo-l-chloropropane, 1,2-dibromopropane, 1,3-dibromopropane 2 2-dibromopropane, 1 ^, S-trichloropropane, 1-chlorobutane, 2-bromobutane or 2-bromo-2-methylpropane extracted and from the organic extract the Microorganisms isolated. 2. The method according to claim 1, characterized in that the organic liquid in a volume ratio to the aqueous medium on the order of between about 1:20 and about 100: 1 are used. ,, ■, 3. Process according to Claims 1 and 2, characterized in that the organic Liquid used in a volume ratio to that of the aqueous medium of about 1: 3.