DE19654942A1 - Bacterium of genus pseudomonas useful for biosynthesis of rhamnolipid-alginate polymer complexes - Google Patents

Abstract

A bacterium of the genus Pseudomonas obtained from soil samples from the oilfields of Borislav, Western Ukraine, by cultivation in inclined agar tubes or on agar plates in a nutrient broth having a composition (in g/l) of 20 g mannitol, 10 g NaNO3, 0.1 g CaCl2.2H2O, 0.4 g MgSO4.7H2O, 0.2 g cetyltrimethylammonium bromide, 0.005 g methylene blue, 0.44 g Na2HPO4.2H2O, 0.34 g KH2PO4, 15 g agar and trace salts (2 ml/l) 2.0 FeSO4.7H2O, 1.5 MnSO4.H2O, and 0.6 (NH4)6Mo7O24.4H2O with the addition of 1.l5 % Bacto-Agar (RTM) at a pH of 7.0, the bacterium being capable of the bioproduction of rhamnolipid-alginate polymer complexes.

Description

The present invention relates to rhamnolipid-alginate polymer complexes bacterial microorganisms of the genus Pseudomonas and a method for their Production, extraction and propagation. The invention also relates to a microbiological gische process for the preparation of rhamnolipid-alginate polymer complexes under Use of microorganisms of the genus Pseudomonas and new Rhanmolipid Alginate polymer complexes. The invention further relates to concentrates and solutions of Rhamnolipid-alginate polymer complexes with surface-active properties as well such concentrates or solutions stabilized emulsions. The invention also relates the use of concentrates containing rhamnolipid-alginate polymer complexes and solutions for stabilizing emulsions. This use can be found in the various technical areas are used.

Bacteria of the genus Pseudomonas are known to be aerobic, gram-negative, for Family of the Pseudomonadaceae belonging bacteria, which are mostly flagellated and polar can be optically recognized by their shape (straight or slightly curved rods). Like various other microorganism genera, bacteria are also of the genus Pseudomonas, especially some strains of Pseudomonas aeruginosa, are able to Assimilate hydrocarbons as a carbon source and convert them into valuable substances, for example in highly active biosurfactants. Growing bacteria from the Genus Pseudomonas, in particular of Pseudomonas aeruginosa, under production such biosurfactants, for example to produce rhamnolipids, is the subject numerous publications such as DE-A 21 50 375; "F. Wagner; in: Strategies for Biosurfactant Production; Fat Sci. Technol. 89 (1987) 586-591 ";" S. Lang et al .; in: Antimicrobial Effects of Biosurfactants; Fat Sci. Technol. 91: 363-366 (1989); R. K. Hommel; in: Formation and Physiological Role of Biosurfactants produced by Hydrocarbon utilizing microorganisms; Biodegradation 1 (1990), 107-119 ";" M. I. van Dyke et al .; in: Pseudomonas aeruginosa UG2 Rhamnolipid surfactants; Can. J. Microbiol. 39 (1993), 1071-1078 " and quotes to be found in it. From the mentioned publications and more, The prior art cited below is also known to be by culturing bacteria  Rhamnolipid biosurfactants obtained from the genus Pseudomonas for cleaning oil tanks or working up soil material that can be used efficiently by oil has been contaminated (see representative of other publications: I. M. Banat et al .; in: Biosurfactant Production and Use in Oil Tank Clean-up; World Journal of Microbiology and Biotechnology 7 (1991), 80-88 "; and" K. Disc arch; in: Enhanced Removal of Selected Hydrocarbons from Soil by Pseudomonas aeruginosa UG2 Biosurfactants and Some Chemical surfactants; J. Chem. Tech. Biotechnol. 59: 53-59 (1994)).

The rhamnolipids described in the cited documents have a structure that is built up from one (or more, possibly glycosidically linked) Rhamnose residues and regularly several, more than 8 carbon atoms Hydroxy fatty acid residues that have carbon atoms in the α-position to the carboxyl group the rhamnose residues are bound and ester-like with one another via the hydroxyl group can be linked.

From the prior art ("IW Sutherland; in: Biosynthesis of Microbial Exopolysac charides; Adv. Micr. Phys. 23 (1982), 80-142";"P.Gacesa; in: Alginates; Carbohydrate Polymers (1988), 161 -181 ";" LO Martins; in: Roles of Mn ²⁺ , Mg ²⁺ and Ca ²⁺ in Alginate Biosynthesis by Pseudomonas aeruginosa; Enzyme Microb. Technol. 12 (1990), 794-799 ") was also known that Pseudomonas aeruginosa can produce an extracellular alginate polysaccharide by assimilation of suitable carbon sources. This could be demonstrated in cell-free media from the cultivation of Pseudomonas aeruginosa under suitable conditions. This product also has advantageous properties, but was not previously known in connection with rhamnolipids, especially rhamnolipids produced by Pseudomonas aeruginosa.

The excellent surfactant properties of rhamnolipids can be applied, for example the values of the critical micell concentration (CMC) on the surface of example example, 0.01 to 1 g / l and the interface CMC values of, for example, 0.005 to 0.2 g / l (against hexadecane) and at the values for lowering the surface tension of Recognize water from, for example, about 70 to about 35 mN / m (van Dyke et al., Op. Cit.). These properties make the named connection class a preferred one  Target group of biotechnological syntheses, since the microbiological processes are easy in the be manufactured on an industrial scale using inexpensive starting substrates can and lead to products that are characterized by high efficiency even in low Concentrations, due to their biodegradability and their low toxicity award. The inexpensive receipt of such biosurfactants opens up new avenues Bio-surfactants opened that can be used in the technical fields of cleaning (including removal of hydrophobic hydrocarbon contamination with aqueous media and textile cleaning with aqueous media), building materials, metal processing, Manufacture of cosmetics, food and pharmaceuticals as well as pesticides Weed control, to name just a few.

An object of the invention was a new microorganism of the genus Pseudomonas to provide that in the context of its metabolism biosurfactants, especially rhamnolipid Alginate polymer complexes can produce. Another object of the invention was a process for the production or extraction of the new microorganism of the genus Pseudomonas specify, in particular, such a procedure that is easy and within the scope previously known technologies can be carried out using inexpensive substrates can.

Another object of the invention was to provide a method which would lead to new microbiologically producible rhamnolipid-alginate polymer complexes with good surfactant Properties leads, as well as such new rhamnolipid-alginate polymer complexes with good To provide surfactant properties.

Another object of the invention was concentrates and solutions or liquid systems such as providing emulsions that act as surfactants contain new rhamnolipid-alginate polymer complexes. It was also the job of Invention, uses of rhamnolipid-alginate polymer complexes containing Specify concentrates, solutions or other systems, for example for stabilization of emulsions or enabling technical processes that have not been done so far the desired efficiency or only at the risk of damaging the environment could be carried out.  

Surprisingly, it was found that a new bacterium of the genus Pseudomonas, which can be obtained inexpensively using conventional techniques Substrates can be grown on new rhamnolipid-alginate polymer complexes have a biotechnological path established. These new rhamnolipid alginate polymer Complexes are known in terms of their surface-active properties Rhamnolipids comparable or even better. In addition, can be partially as Waste materials in industrial cycles as substances arising as carbon sources use and lead to a meaningful use. The preserved new rhanmoli pid-alginate polymer complexes are particularly biodegradable and toxicological completely harmless what they do even in sensitive industrial areas such as cosmetics Industry or food technology usable.

The invention relates to those capable of producing rhamnolipid-alginate polymer complexes Bacteria of the genus Pseudomonas according to claim 1.

The invention further relates to a method for obtaining a for generating Rhamnolipid-alginate polymer complexes enabled bacteria of the genus Pseudomonas according to the following specification according to claim 5.

The invention further relates to a method for multiplying a for generating Rhamnolipid-alginate polymer complexes enabled bacteria of the genus Pseudomonas according to the following specification according to claim 6.

The invention also relates to a microbiological process for the production of Rhamnolipid-alginate polymer complexes under fermentation to produce Rhamnolipid-alginate polymer complexes enabled bacteria of the genus Pseudomonas ge according to the following specification according to claim 12.

The invention further relates to rhamnolipid-alginate polymer complexes of building blocks of Structure according to claim 20.  

The invention also relates to one or more rhamnolipid-alginate polymer complex (s) aqueous concentrates, emulsions or according to the following specification Solutions according to claims 23, 24 or 25.

Ultimately, the invention also relates to uses of the aforementioned, one or several rhamnolipid-alginate polymer complex (s) according to the specification below containing aqueous concentrates, emulsions or solutions according to the claims 26 and 29 to 34.

Preferred embodiments of the invention result from the subclaims.

The invention for the production of rhamnolipid-alginate polymer complexes competent bacterium belongs to the genus Pseudomonas. It was within the scope of the invention Surprisingly found in soil samples from the Borislav oil production area, western Ukraine. The bacterium was obtained using the following procedure: For the bacterium (Pseudomonas aeruginosa) was kept in a slant agar tube the standard nutrient broth from Merck (Darmstadt) with the following combination setting and with the addition of 1.5% Bacto Agar® (Difco, Detroit, U.S.A.) used. The medium was set up according to the supplied regulation and had to autoclaving a pH of 7.0. The same medium was used for the Manufacture of agar plates used. The medium had the following composition (Quantities in g / l): mannitol: 20.0; NaNO₃: 10.0; CaCl₂ · 2 H₂O: 0.1; MgSO₄7 H₂O: 0.4; Cetyltrimethylammonium bromide (CTAB): 0.2; Methylene blue: 0.005; Na₂HPO₄2 H₂O: 0.44; KH₂PO₄: 0.34; Agar: 15.0; Trace salts (2 ml / l): FeSO₄ · 7 H₂O: 2.0; MnSO₄ · H₂O: 1.5; (NH₄) ₆Mo₇O₂₄ · 4 H₂O: 0.6.

In order to assess the purity of the cultures used, there were intervals microscopic considerations performed. In addition, dilution series were made more sterile Saline solution, from which samples were spatulated out on agar plates. After 48- Hourly incubation at 30 ° C were only grown cultures in appearance counted as pure cultures.  

Rhamnolipids were present on the formation of ion associates with methylene blue of cationic surfactant (CTAB). This method was also applied to the Transfer agar plates: The agar plates were colored light blue, and when Rhamnolipids form dark blue courtyards around the colonies. The yard size was that Rhamnolipid concentration proportional to how different when applying Rhamnolipid concentrations were found. The agar plates with strains of Pseudomonas aeruginosa according to the invention were incubated at 30 ° C. After the colonies grew (48 h) the detection reaction could be observed.

Samples of the bacterium could be characterized based on the following properties:

• - Individuals: unicellular;
• - Shape: chopsticks (see Fig. 1);
• - Size: length 1.5 to 4.0 µm; Width: 0.8 to 0.9 µm;
• - Flagella: polar 1
• - movement: determined (+);
• - Grief behavior: Grief negative;
• - Lysis by 3% KOH: +;
• - Aminopeptidase (Cerny): +;
• - spores: none (-);
• - Oxidase: +;
• - Catalase: +;
• - ADH: +;
• - NO₂ from NO₃: +;
• - urease: +;
• - hydrolysis of gelatin: +;
• - aerobic;
• - substrate recycling:
  Adipate: +;
  Citrate: +;
  Malate: +;
  D-glucose: +;
  Mannitol: +;
  Acetamide: +;
  Geraniol: +.

The profile of cellular fatty acids is typical of the species Pseudomonas aeruginosa.

The partial sequencing of the 16SrDNA showed a high similarity to that of this Art.

According to the invention, identical bacteria of the genus Pseudomonas can also be obtained by that a bacterium of the genus Pseudomonas, for example Pseudomonas aeruginosa, with shaking (200 rpm) on a culture medium of the following composition (Amounts given in g / l): NaNO₃: 3.0; K₂HPO₄ · 3 H₂O: 2.0; KH₂PO₄: 1.5; MgSO₄7 H₂O: 0.5; Na citrate: 5.0; FeSO₄ · 7 H₂O: 0.0005; CaCl₂: 0.5; Carbon source: 20.0.

The above bacteria of the genus Pseudomonas with identical mor phological and physiological properties and equal ability to form Rhamnolipid-alginate polymer concentrates were given the designation ID 96-115 and on February 23, 1996 at the DSMZ-German Collection of Microorganisms and Zellkulturen GmbH under the deposit number DSM 10554.

According to the invention, such bacteria of the genus Pseudomonas were used to generate of rhamnolipid-alginate polymer complexes are able to extract from soil samples from the Petroleum prospect Borislav, western Ukraine. The extraction takes place according to the above described method.

The inventive for the production of rhamnolipid-alginate polymer complexes capable bacteria of the genus Pseudomonas can be Process for the cultivation of bacteria, for example in a moving submerged culture, breed or multiply by:

• - An aqueous, at least one assimilable carbon source, at least one Assimilable nitrogen source and known nutrient salts in suitable Prepares nutrient medium containing concentrations and sterilizes it;  
• - inoculates the culture medium thus obtained with a bacterium of the genus Pseudomonas, which has been obtained from a soil sample as described above or in For example, a mutation procedure from Pseudomonas aeruginosa was manufactured, and
• - the bacterium of the genus Pseudomonas at a pH in the range from 6.5 to 7.5
• - And at a temperature in the range of 20 to 35 ° C.
• - breeds aerobically in a moving submerged culture.

According to the invention, preference is given to those capable of forming rhanololipid Alginate polymer-equipped bacterium of the genus Pseudomonas used that with the designation ID 96-115 and on February 23, 1996 at the DSMZ - German collection of microorganisms and cell cultures GmbH under the back registration number DSM 10554 was deposited. This bacterium is particularly good for Formation of the desired rhamnolipid-alginate polymer complex capable and can assimilate a large number of economically interesting carbon sources.

In a further preferred embodiment of the invention, the assimilable Carbon source one or more compounds from the glucose, xylose, maltose, Sucrose, mannitol, glycerin, ethanol, n-paraffins, sodium citrate, sodium acetate, Group consisting of sodium succinate and fatty acid ester. Particularly preferred as Assimilable carbon sources are suitable for vegetable oils individually or in mixtures, rapeseed oil being particularly preferred as an inexpensive, regenerable product is and gives excellent results. Waste from the is also particularly preferred Margarine production. These not only deliver good results in fermentation, but also fall as waste products of industrial processes that would otherwise be elaborately disposed of would have to be inexpensive.

The carbon sources mentioned are suitable for fermentation in the nutrient solution Present quantities. These are preferably 10 to 50 g / l, even more preferably about 15 to 25 g / l, particularly preferably about 20 g / l, of the nutrient solution.  

The usual inorganic and / or organic nitrogen can be used as the nitrogen source sources are used, as they are generally used in fermentation processes. Examples of suitable nitrogen sources are nitrates, ammonium salts, urea and peptone. Amino acids are for the growth of the bacteria in the fermenta according to the invention ion process not essential.

The nitrogen sources mentioned are suitable for fermentation in the nutrient solution Present quantities. These are preferably 1 to 5 g / l, more preferably 2 to 4 g / l, particularly preferably about 3 g / l, of the nutrient solution.

In addition to the carbon sources and nitrogen sources mentioned above, the Nutrient medium or the fermentation broth also inorganic and / or organic salts added, as is known to those skilled in the art. Examples of suitable compounds (without to be limited) are phosphates such as dipotassium hydrogen phosphate trihydrate (e.g. in an amount of 1 to 5 g / l, especially of about 2 g / l), potassium hydrogen phosphate (e.g. in an amount of 1 to 5 g / l, especially 1.5 g / l); Sulfates such as magnesium sulfate heptahydrate (e.g. in an amount of 0.1 to 1 g / l, especially of about 0.5 g / l); Chlorides such as calcium chloride (e.g. in an amount of 0.01 to 0.1 g / l, especially of about 0.05 g / l); Iron salts such as iron sulfate heptahydrate (e.g. in an amount of 0.0001 to 0.001 g / l, especially of about 0.0005 g / l); Calcium salts such as the aforementioned calcium chloride; Salts of organic acids such as sodium citrate (e.g. in an amount of 1 to 10 g / l, especially about 5 g / l); etc. Also others known for fermentation Additives such as buffer substances can be known to those skilled in the art Amounts are used without needing to be mentioned here.

The pH of the nutrient solution moves during the process according to the invention Range 6.5 to 7.5, preferably in the range from 6.8 to 7.2, and in the stationary phase in Range from 8.0 to 9.0.  

The fermentation process according to the invention for increasing the to produce Rhamnolipid-alginate polymer complexes enabled bacteria of the genus Pseudomonas according to the invention is for a time under the conditions mentioned above between 0.5 and 5 days. The logarithmic growth phase achieves this System after about 20 hours.

As already mentioned above, the rhamnolipid-alginate polymer Complexes as metabolites of the new ones provided according to the invention View bacteria of the genus Pseudomonas. The microbiological invention Manufacturing process thus comprises the steps of:

• - An aqueous, at least one assimilable carbon source, at least one Assimilable nitrogen source and known nutrient salts in suitable Prepares nutrient medium containing concentrations and sterilizes it;
• - inoculates the culture medium thus obtained with a bacterium of the genus Pseudomonas, which has been obtained from a soil sample as described above or in For example, a mutation procedure from Pseudomonas aeruginosa was produced;
• - the bacterium of the genus Pseudomonas at a pH in the range from 6.5 to 7.5 and at a temperature in the range of 20 to 35 ° C aerobic in motion Submerged culture breeds;
• - the cells of the bacterium of the genus Pseudomonas in a manner known per se from separates from the aqueous medium; and if necessary
• - The rhamnolipid-alginate polymer complexes from the cell-free aqueous medium wins.

The first four steps of the process are the same as the breeding steps or multiplication of the microorganisms of the genus Pseudomonas according to the invention; the the above steps as well as the preferred embodiments described above these steps are therefore the same as the steps for producing the new ones Rhamnolipid-alginate polymer complexes according to the invention, and it can be repeated Description at this point.  

Upon completion of the manufacturing process, the cells of the bacterium become the genus Pseudomonas in a manner known per se from the aqueous medium of the nutrient solution severed. This can preferably be done by centrifuging off the cells from the aqueous phase or by membrane filtration. However, there are others Separation process applicable.

The rhamnolipid-alginate polymer complexes according to the invention can optionally be used be isolated from the cell-free aqueous medium thus obtained. Arrive here too known conventional methods of use. Examples are procedures below Freeze-drying or solution precipitation. If the products from the precipitated aqueous solution, this is in a particularly preferred Aus leadership form of the process, the aqueous solution z. B. acidified with 0.5 N HCl, for example to a pH of about 3.5, and cooled in the course of 2 hours, for example to a temperature of 15 ° C. The failed product is in itself filtered or centrifuged in a known manner.

In this way, the new rhamnolipid-alginate polymer complexes according to the Invention won. These are made up of building blocks of the following structures

• - Alginate polymer component (Alg): 1,4-linked copolymer of β-D-mannuronic acid and its C5 epimer α-L-guluronic acid:
• - Rhamnolipid component I (RL I): α-L-rhamnosyl-β-hydroxydecanoyl-β-hydr oxydecanoic acid
• - Rhamnolipid component II (RL II): α-L-rhamnosyl-α-L-rhamnosyl-β-hydr oxydecanoyl-β-hydroxydecanoic acid

Evidence can be found in the rhamnolipid-alginate polymer complexes according to the invention according to the invention two rhamnolipid components of the structures RL-I and RL-II and contain an alginate polymer component of the structure Alg. According to the invention particularly preferred the rhamnolipid-alginate polymer complexes as described above described methods as metabolites of the bacterium of the genus Pseudomonas are available, in particular the bacterium with the designation ID 96-115, as it was on 23. February 1996 at the DSMZ-German collection of microorganisms and cell cultures GmbH was deposited by the applicant under the deposit number DSM 10554.  

The analysis of the rhamnolipid-alginate polymer complexes according to the invention is carried out after methods known per se. The rhamnolipid content can be determined by Take off an aliquot (e.g. 10 to 50 ml) of the cell-free culture broth, several times Extract the withdrawn amount with an organic solvent (e.g. with CHCl₃ / MeOH in a volume ratio of 2: 1), combining the organic extracts and Stripping off the organic solvent. By adding anthrone a complex with the rhamnose component of the complex, whose concentration is photometric can be measured against a standard series of known concentrations.

The alginate content is determined, for example, gravimetrically after the complex has been precipitated Isopropanol determined in duplicate. To do this, an aliquot of the cell-free Culture broth, e.g. B. 10 to 50 ml, mixed with 2 parts of isopropanol. After completion of the Precipitation reaction, the alginate was centrifuged at 15,000 × g for 15 min. The supernatant was decanted off and the pellet was washed with 70% (v / v) isopropanol, at 48 h Dried and weighed 40 ° C in a vacuum drying cabinet. The invention Complexes are also characterized by elemental analysis, IR absorption spectra and other specific physico-chemical methods such as tensiometry, rheology, Thin layer chromatography, high pressure liquid chromatography (HPLC) and Mass spectroscopy. The following components of the complexes can be identified:

(a) Rhamnolipid I (RL I):
CMC: 50 g / l;
σ _s : 29.5 nN / m;
σ _i : 0.020 nN / m (n-heptane);

(b) Rhamnolipid II (RL II):
CMC: 20 g / l;
σ _s : 28.8 nN / m;
σ _i : 0.014 nN / m (n-heptane);

(c) Alginate polymer (Alg):
MG: approx. 400,000.

According to the invention, aqueous concentrates are provided for practical use, the one or more rhamnolipid-alginate polymer complex (s) according to the invention above structure included. The content in these aqueous concentrates is on one or more rhamnolipid-alginate polymer complex (s) 6.0 to 12.0 g / l.  

Alternatively, aqueous solutions can be provided according to the invention, which either aqueous systems after cell separation are as they result from the above described manufacturing process arise, or such aqueous solutions as they can be obtained, for example, by diluting the concentrates described above can. These aqueous solutions contain one or more of the invention Rhamnolipid-alginate polymer complex (s) of the structure given above. In these watery ones Solutions is the content of one or more rhamnolipid alginate polymer Complex (s) 0.1 to 15% by weight.

The invention also includes aqueous-based emulsions, the one or more Rhamnolipid-alginate polymer complex (s) according to the invention of the above-mentioned Structure included. The content of one or is in these aqueous emulsions several rhamnolipid-alginate polymer complex (s) 0.05 to 5 wt .-%. The above In addition to the components mentioned, emulsions can also, for example, oils, fats or contain other components that are not or only insufficiently miscible with water, unless the surface-active rhamnolipid alginate poly according to the invention mer complexes of the structure given above are present, but in the aqueous Phase a stable emulsion in the presence of the rhamnolipid alginate poly according to the invention form mer complexes.

The aforementioned concentrates, solutions or emulsions can be in a variety of Uses are used. An example is - without being limited to this - the Textile cleaning. In preferred applications, textiles from the various materials such as wool, fur, leather, synthetic leather, cotton, silk, Artificial silk or linen from traditional ways only poorly or not at all eliminating contaminants such as animal, vegetable or mineral fats, Oils, blood, lanolin, etc. When cleaning comes up essentially aqueous containing the rhamnolipid-alginate polymer complexes according to the invention Impact solutions.

Another advantageous use according to the invention lies in Cleaning of soil material such as earth, sand, debris, rock or similar from  Greases or oils, for example contaminating mineral oils. Such Use can play a role in leakage or leakage of oil-filled containers or in accidents where oil gets into the environment. With the help of the invention Concentrates or solutions can, for example, mineral oils in with high efficiency aqueous cleaning phases are bound in emulsion. The excellent surface-active properties of the rhaninolipid-alginate polymer according to the invention Complexes lead to a much better absorption of the oil contaminants in the aqueous Phase. The efficiency of conventional products can be significantly exceeded.

The same surprising effect can also be achieved when using the concentrates containing rhamnolipid-alginate polymer complexes according to the invention or Solutions for cleaning systems, machines, machine parts, tools and Instruments. In particular, sensitive instruments such as medical instruments can be found under Use of the rhamnolipid-alginate polymer complexes according to the invention with large Benefit treated and cleaned.

Surprisingly, it was also found that environmental disasters involving oil afflicted animals such as birds in more gentle and Carcasses not harmful with aqueous solutions that the invention Rhamnolipid-alginate polymer complexes contain, treated and so of oil residues can be exempted. The natural biosurfactants are particularly effective for aquatic animals not necessary counter-greasing of the feather dress or the skin, so that a far greater survival rate can be achieved than when using conventional ones Cleaning supplies.

Further preferred uses according to the invention also result from the highly efficient surface-active properties of the rhamnolipid according to the invention Alginate polymer complexes and the fact that they are non-toxic and biological Conditions are degradable. They can also be used to advantage in food technology and in cosmetics as efficient emulsifiers for the emulsification of Use fat phases and aqueous phases together.  

The invention is explained in more detail by the following examples, without being based on them to be limited.

Examples 1 to 4

The strain Pseudomonas aeruginosa DSM 10554 according to the invention was in a Culture medium of the following composition in shake culture (200 rpm) at 30 ° C for the in of the time given in Table 1 below (amounts in g / l):

NaNO₃: 3.0;
K₂HPO₄ · 3 H₂O: 2.0;
KH₂PO₄: 1.5;
MgSO₄ · 7 H₂O: 0.5;
Na citrate: 5.0;
FeSO₄ · 7 H₂O: 0.0005;
CaCl₂: 0.5;
Carbon source: 20.0.

The carbon source used in the individual examples is given in Table 1.

The cultivation was stopped after the time shown in Table 1, and the Culture broth was freed from cells of the microorganism.

The rhamnolipid-alginate-biopolymer complex (RABK) content is shown in Table 1. The values of surface-active tension σ _s and surface-active tension σ _{i of} the cell-free culture broth are also given in Table 1. The latter were determined using standard methods (ring measuring method) which are known to the person skilled in the art and therefore do not require any detailed explanation.

Table 1

Examples 1 to 4 show that based on easily accessible carbon sources, some of which are accessible as industrial waste, have a high biosurfactant concentration can be obtained in the culture broth which shows good surface-active properties.

Example 5

With the rhamnolipid-alginate-biopolymer complex obtained according to Examples 1 to 4 (RABK) the surface-active properties during washing were examined.

For this, a sheepskin sample with natural contamination was kept at 30 ° C for 30 minutes treated with constant agitation in an aqueous phase containing 1.5% RABK as it did was obtained according to Examples 1 to 4. After that, the sheepskin sample was taken rinsed three times with tap water and dried.

The dried sheepskin sample was tested three times to determine the cleaning result extracted with CHCl₃ / MeOH (2: 1; v / v). For the quantitative determination of the residual ver Soiling of the sheepskin sample, the extracts were combined and the extractant  was completely evaporated under vacuum. The gravimetric determination showed that a cleaning level of 97% had been reached (i.e. 97% of the total dirt were in the step of washing with the aqueous cleaning phase containing RABK removed, and 3% residual dirt was left on the sheepskin sample and only removed by extraction with organic solvent).

Examples 6 to 13

The procedure was as in Example 5. The material samples used, the additional applied contamination, the cleaning temperatures and the cleaning result (defined as in Example 5) are given in Table 2 below.

Table 2

As can be seen from the examples, the rhamnolipid-alginate-biopolymer complex shows excellent cleaning properties and is due to the excellent surfaces suitable for active properties, even problem dirt (artificial fat: 30% by weight lanolin, 30 wt .-% sunflower oil, 40 wt .-% crude oil) largely from fabrics, leather, synthetic leather or to remove from material surfaces. In addition, the invention shows Rhamnolipid-alginate-biopolymer complex has no irritant effect on the skin and is in the In view of its non-irritant properties conventional chemical Superior to surfactants.

Claims (12)

1. Bacterium des. Capable of producing rhamnolipid-alginate polymer complexes Genus Pseudomonas, obtainable from soil samples from the Borislav oil production area, western Ukraine, by cultivation in slant agar tubes or on agar plates in one Nutrient broth of the composition (quantities in g / l): mannitol: 20.0; NaNO₃: 10.0; CaCl₂ · 2 H₂O: 0.1; MgSO₄ · 7 H₂O: 0.4; Cetyltrimethylammonium bromide (CTAB): 0.2; Methylene blue: 0.005; Na₂HPO₄ · 2 H₂O: 0.44; KH₂PO₄: 0.34; Agar: 15.0; Trace salts (2nd ml / l): FeSO₄ · 7 H₂O: 2.0; MnSO₄ · H₂O: 1.5; (NH₄) ₆Mo₇O₂₄ · 4 H₂O: 0.6 with the addition of 1.5% Bacto-Agar® at a pH of 7.0. 2. Bacterium according to claim 1 with the following properties:

• - Individuals: unicellular;
• - Shape: chopsticks (see Fig. 1);
• - Size: length 1.5 to 4.0 µm; Width: 0.8 to 0.9 µm;
• - Flagella: polar 1
• - movement: determined (+);
• - Grief behavior: Grief negative;
• - Lysis by 3% KOH: +;
• - Aminopeptidase (Cerny): +;
• - spores: none (-);
• - Oxidase: +;
• - Catalase: +;
• - ADH: +;
• - NO₂ from NO₃: +;
• - urease: +;
• - hydrolysis of gelatin: +;
• - aerobic;
• - substrate recycling:
  Adipate: +;
  Citrate: +;
  Malate: +;
  D-glucose: +;
  Mannitol: +;
  Acetamide: +;
  Geraniol: +.
• - Profile of cellular fatty acids: typical of the species Pseudomonas aeruginosa;
• - Partial sequencing of the 16SrDNA shows a high similarity to that of this kind.

3. Bacterium according to claim 1, obtainable by a method in which a bacterium of the genus Pseudomonas, especially Pseudomonas aeruginosa, while shaking on a Culture medium of the following composition grows (details of the quantities in g / l): NaNO₃: 3.0; K₂HPO₄ · 3 H₂O: 2.0; KH₂PO₄: 1.5; MgSO₄ · 7 H₂O: 0.5; Na citrate: 5.0; FeSO₄7 H₂O: 0.0005; CaCl₂: 0.5; Carbon source: 20.0. 4. Bacterium according to one or more of claims 1 to 3, namely Pseudomonas aeruginosa ID 96-115, deposited with the DSMZ-German Microorganism Collection men und Zellkulturen GmbH on February 23, 1996 under the deposit number DSM 10554. 5. Process for obtaining a rhamnolipid-alginate polymer Complex capable bacterium of the genus Pseudomonas according to one of the claims 1, 2 or 4 from soil samples from the Borislav oil production area, western Ukraine, the method comprising the step of culturing the bacterium in slant agar tubes or on agar plates in a nutrient broth of the composition (quantities in g / l): Mannitol: 20.0; NaNO₃: 10.0; CaCl₂ · 2 H₂O: 0.1; MgSO₄ · 7 H₂O: 0.4; Cetyltrimethylam monium bromide (CTAB): 0.2; Methylene blue: 0.005; Na₂HPO₄ · 2 H₂O: 0.44; KH₂PO₄: 0.34;  Agar: 15.0; Trace salts (2 ml / l): FeSO₄ · 7 H₂O: 2.0; MnSO₄ · H₂O: 1.5; (NH₄) ₆Mo₇O₂₄ · 4 H₂O: 0.6 with the addition of 1.5% Bacto-Agar® at a pH of 7.0. 6. A method for multiplying a bacterium of the genus Pseudomonas capable of producing rhamnolipid-alginate polymer complexes according to one or more of claims 1 to 4, the method comprising the steps of

• - An aqueous, at least one assimilable carbon source, at least one assimilable nitrogen source and known nutrient salts in suitable concentrations containing nutrient medium is prepared and sterilized;
• - Inoculates the nutrient medium thus obtained with a bacterium according to one or more of claims 1, 2 or 4 and
• - the bacterium of the genus Pseudomonas at a pH in the range from 6.5 to 7.5
• - And at a temperature in the range of 20 to 35 ° C.
• - breeds aerobically in a moving submerged culture.

7. The method according to claim 6, wherein one or as the assimilable carbon source several compounds from the glucose, xylose, maltose, sucrose, mannitol, glyce rin, ethanol, n-paraffins, sodium citrate, sodium acetate, sodium succinate and fatty acid esters existing group can be used. 8. The method according to claim 7, wherein one or more as a carbon source vegetable oils are used, preferably rapeseed oil. 9. The method according to claim 7, wherein waste from the margarine as a carbon source Production can be used. 10. The method according to one or more of claims 6 to 9, wherein the pH is in the range from 6.8 to 7.2 and / or the temperature is in the range from 25 to 30 ° C. lies.   11. The method according to one or more of claims 6 to 10, wherein as a bacterium Pseudomonas aeruginosa ID 96-115, deposited with the DSMZ-German collection of Microorganisms and cell cultures GmbH on February 23, 1996 under the deposit number DSM 10554, is used. 12. Use of the bacterium according to one of claims 1 to 4 for the production of rhamnolipid-alginate polymer complexes.