DD234689A1 - PROCESS FOR OBTAINING BIOTENSIDES - Google Patents

Abstract

The invention relates to a method for obtaining biosurfactants. The aim is to increase the growth rate of P. aeruginosa, associated with increased excretion of surface-active substances. The object is seen to significantly promote the excretion of biosurfactants by P. aeruginosa strain by procedural measures. The object is achieved by the addition of (NH4) 2Fe (SO4) 2 to the preferably synthetic culture medium.

Description

Field of application of the invention

The invention relates to a method for obtaining extracellular lipids, in particular rhamnolipids, which can acquire significance as bioemulsifiers.

Characteristic of the known technical solutions

It is already known that strains of Pseudomonas aeruginosa secrete rhamnolipids into the medium upon growth on certain complex and synthetic media. The excretion begins in the late exponential growth phase. According to literature, the surface tension drops to a minimum value of 40mN / m. The growth dynamics of the microorganisms depends on the cultivation conditions. Through a - mostly empirically justified - growth optimization is shortened with complex supplements (yeast extract, peptone, meat extract u.a.) or with defined compounds (inorganic trace salts, vitamins), the logarithmic phase and increases the growth rate. The selection of a suitable C and N chain, the C: N ratio and the dynamics of growth have a significant influence on the biosynthesis and excretion of metaboiites. The following compounds have been predominantly used as inorganic trace salts and / or vitamins: corn steep liquor / yeast extract / iron / (II) sulfate, iron (III) chloride / zinc sulfate / manganese di sulfate / copper sulfate / cobaltil chloride / Sodium molybdate / potassium iodide / boric acid, peptone. The positive influence of these compounds or their combinations on the growth of P. aeruginosa is clear, however, the biosynthesis of rhamnolipid and / or the excretion into the culture medium - as the actually desired physiological performance - is partially or completely inhibited in some nutrient media, in none Case but increased. The precipitated surface-active compounds are usually separated by extraction with ether or ethyl acetate or by precipitation after acidification, but when using the latter method, a day-long standing of the solution in the cold must be accepted.

Despite many attempts to optimize the process, it has not hitherto been possible to stimulate the excretion of biosurfactants, in particular rhamnclipids, in portable yields with limited cultivation time.

Ziei the invention

The invention aims to increase the growth rate of P. aeruginosa and increase the excretion of surfactants.

Explanation of the essence of the invention

The invention has the object, by procedural measures, the excretion of biosurfactants by the used

P. aeruginosa strains essential to promote.

It has surprisingly been found that (NHa) ₂ Fe (SCUh has a substantial growth-promoting influence on P.

aeruginosa when added to a preferably synthetic culture media.

The stated object is therefore achieved in that strains of P. aeruginosa in a liquid synthetic medium using glucose, glycerol, alkanes as carbon source and ammonium sulfate u.a. be grown as a nitrogen source. According to the invention, ammonium iron (II) sulfate in a concentration of 0.1 to 100 mg / l, preferably 1 to 10 mg / l, is added to the culture medium as a growth-promoting additive.

The cultivation is carried out submerged in a batch process at 30 ⁰ C. The pH during cultivation is 6-7.5 ammonium iron (II) sulfate is added to the medium in a concentration of 0.01 mg / 100mi to 10mg / 100ml.

The invention will be explained in more detail below with reference to exemplary embodiments.

example 1

A synthetic medium adjusted to a pH of 7.2 is used, which consists of 7 g K ₂ HPO ₁ I, ₃ g KH ₂ PO ₄ , 0.1 g MgSO ₄ H ₂ O and 1 g (NH ₂ ) ₂ SO ₄ in 11 distilled water is produced. The only carbon source is glycerol (30 ml /!). This medium is mixed with ammonium iron sulfate in the following amounts. 100 ml of this medium are placed in 500 ml Erlenmeyer flasks and inoculated with an inoculum of P. aeruginosa 196 Aa, so that the optical density, measured at 60 nm, is between 0.03-0.05. The inoculum is recovered by flushing 18h old slant cultures with synthetic medium. The cultivation is carried out at 30 ⁰ C with shaking. The stalagmometrically measured surface tension of the cell-free culture filtrate is above the critical micelle concentration a measure of the biosurfactant concentration. The critical micelle formation concentration is 126 mg / l and, depending on the citation method, is achieved at a surface tension between 23-31 mN / m.

Table 1 breeding optical surface tension Ammonium iron (II) - duration density voltage If at H mN / m mg / l 24 0.45 66.7 - 40 0.85 32.4 - 24 0.8 29.2 0.2 24 1.15 35.4 2 24 1.1 34.9 10 24 1.1 33.8 50

Example 2

Example 1 is repeated, instead of P. aeruginosa 196 Aa is inoculated with P. aeruginosa M ₂ .

Table 2

Ammonium growth optical surface dilution

Iron (II) - permanent density voltage

sulfate

mg / l. , h mN / m 1:10

- 24 0.5 43.3

- 48 0.6 28.8 48.7 2 24 0.75

2 48 1.1 25.1 36.6

Excretion of biosurfactants also occurs by resting cells in buffered and aerated media with a carbon substrate.

Example 3

Example 1 is repeated without the addition of ammonium iron (II) sulfate. After 27 hours of culture, the bacterial suspension has reached an optical density of 0.72, the surface tension of the cell-free culture filtrate is 6OnIWm. The cells are harvested under sterile conditions and suspended in 100 ml of a sterile medium of the following composition 1/15 mol / l phosphate buffer pH = 5.0.2% glycerol. The batch is shaken at 30 ° C. After the times indicated, an aliquot is taken and, after centrifugation in the cell-free supernatant, the surface tension is measured.

Table 3

Time 19 43 67 91 115 139 163 288

Surface tension 26.3 27.7 27.7 26.3 28.5 -28.5 29.2 32.2 32.2

After 19 h, 10 times the critical peak concentration of rhamnolipid (1.3 g / l) is detectable. The strain Pseudomonas aeruginosa M ₂ used in Example 2 has the deposit number ZIMET 11,013th

Claims (7)

Invention claim: A process for obtaining biosurfactants, in particular rhamnolipids by culturing Pseudomonas aeruginosa in liquid synthetic media, characterized in that the culture medium (NH ^ FeiS. 2. The method according to item 1, characterized in that the strain P. aeruginosa 196 Aa is grown. 3. The method according to item 1, characterized in that the strain P. aeruginosa M _{2 is} grown. 4. The method according to item 1 to 3, characterized in that submerged is cultured discontinuously at a temperature near 30 ^c C. 5. The method according to item 1 to 4, characterized in that ammonium iron (II) ~ sulfate in amounts of 0.1 to 100mg / l is added. 6. The method according to item 5, characterized in that the Ammoniumseisenfllj-sulfate concentration is 1 to 10 mg / l. 7. The method according to item 1, characterized in that lines of P. aeruginosa are cultured in a medium with or without the addition of ammonium iron (II) sulfate until the detectable excretion of rhammolipid after separation from the culture medium as quiescent lines in a buffered and aerated Medium with a carbon substrate, e.g. As glycerol, incubated.