Liquid cation exchanger

Abstract

The present application relates to a process for removing an organic compound having one or more positive charges from an aqueous solution, comprising the steps a) provision of the aqueous solution comprising the organic compound and of a hydrophobic organic solution which comprises a liquid cation exchanger, where the liquid cation exchanger is hydrophobic, and where the liquid cation exchanger has one or more negative charges and an overall negative charge, b) contacting the aqueous solution and the organic solution, and c) separating off the organic solution from the aqueous solution.

Claims

1. A process for removing an organic compound comprising one or more positive charges from an aqueous solution, the process comprising: providing the aqueous solution comprising the organic compound and a hydrophobic organic solution comprising a liquid cation exchanger, wherein the liquid cation exchanger is a hydrophobic fatty acid but not a fatty acid ester, after the providing, contacting the aqueous solution and the hydrophobic organic solution, and separating off the hydrophobic organic solution from the aqueous solution, wherein the organic compound comprises at least two amino groups and no negatively charged functional group, wherein the organic compound is a compound of Formula H.sub.2N(CH.sub.2).sub.xNH.sub.2, wherein x is 1 to 20; or wherein the organic compound is a cyclic sugar comprising at least two amino groups, and wherein the process does not employ a membrane.

2. The process according to claim 1, wherein a temperature of the contacting is from 28 to 70 C.

3. The process according to claim 1, wherein a pH of the contacting is from 6 to 8.

4. The process according to claim 1, wherein a quantitative ratio of the liquid cation exchanger to the organic compound is at least 1.

5. The process according to claim 1, wherein a volume ratio of the hydrophobic organic solution to the aqueous solution is from 1:10 to 10:1.

6. The process according to claim 1, wherein the liquid cation exchanger is a fatty acid comprising more than 12 carbon atoms.

7. The process according to claim 1, wherein the liquid cation exchanger is an unsaturated fatty acid.

8. The process according to claim 1, wherein the aqueous solution further comprises a biological agent having a catalytic activity.

9. The process according to claim 8, wherein the biological agent is a cell.

10. The process according to claim 1, wherein the hydrophobic organic solution further comprises an organic solvent.

Description

(1) The present invention is furthermore illustrated by the following figures and limiting examples, which reveal further features, embodiments, aspects and advantages of the present invention.

(2) FIG. 1 shows a control experiment for confirming that LSME does not have a toxic effect, investigated with a E. coli W3110 strain and compared to potassium phosphate buffer (Kpi) as negative control.

(3) FIG. 2 shows the viability of the strain E. coli W3110 in the form of the number of CFUs which the strain can form in the absence of a liquid cation exchanger and in the presence of different liquid cation exchangers after 0 h, 4 h and 24 h.

(4) FIG. 3a shows the results of removing diaminoisoiditol (DAI) and diaminoisosorbitol (DAS) from an aqueous phase using D2EHPA as liquid cation exchanger.

(5) FIG. 3b shows the results of removing DAI and DAS from an aqueous phase using oleic acid as liquid cation exchanger.

(6) FIG. 3c shows the results of removing DAI and DAS from an aqueous phase without using a liquid cation exchanger.

(7) FIG. 4 shows the effect of using a liquid cation exchanger on the toxicity by reference to the change in living cell count of an E. coliW3110 strain in the presence of ALSME 0.2%, with ammonia adjusted to DEHPA (D2EHPNH3 2%) or a DEHPA/LSME mixture (2%/98%) (D/L) in the presence of ALSME 0.2%.

(8) FIG. 5 shows the effect of different liquid cation exchangers on the OTR of methyl aminolaurate producing E. coli strain. The experiment was carried out as described in Example 5.

(9) FIG. 6 shows the influence of different liquid cation exchangers on the yield of methyl aminolaurate which an E. coli strain with suitable genetic modification produces. The experiment was carried out as described in Example 5.

EXAMPLE 1

Investigation of the Toxicity of the Solvent LSME which is Used in Compositions with Liquid Cation Exchanger

(10) This experiment was used to show the relatively low toxicity of LSME with regard to biotechnologically relevant microorganisms, which makes LSME a suitable organic solvent for the process according to the invention.

(11) Before the determination of the CFU was able to be carried out, a plate LB (10 g/L peptone from casein, 5 g/L yeast extract, 10 g/L NaCl) was streaked with E. coli BW3110 and incubated for 24 h. On the evening of the following day, a preculture was inoculated from this previously streaked plate. This preculture had a volume of 50 mL LB medium and was incubated overnight for ca. 16 h. On the following day, the preculture with an OD.sub.600 of 0.2 in 200 mL of M9 medium (Na2HPO4 6.79 g/L; KH2PO4 3.0 g/L; NaCl 0.5 g/L; NH4Cl 1 g/L; 1 mL/L trace element solution, pH 7.4. Trace element solution: HCl 37% (=455.8 g/L) 36.50 g/L; MnCl2*7H2O 1.91 g/L; ZnSO4*7H2O 1.87 g/L; Na EDTA*2H2O (Titriplex III) 0.84 g/L; H3BO3 0.30 g/L; Na2MoO4*2H2O 0.25 g/L; CaC12*2H2O 4.70 g/L; FeSO4*7H2O 17.80 g/L; CuC12*2H2O 0.15 g/L) was transinoculated with 3% glucose (w/v) and incubated for ca. 20 h. After the incubation of the main culture, the cells were harvested, centrifuged at 5258 g and 4 C. for 10 min and resuspended with an OD.sub.600 of 30 in 10 mL 50 mM Kp, buffer at pH 7.4 (or 25 mM HEPES buffer pH 7.4 if CFU determinations were carried out with ALSME). Both buffer solutions used contained 5% glucose (w/v). The bacteria suspension was then transferred to the shake flasks and treated with the respective substance solutions. After thorough mixing has taken place by swirling the flask, 100 L of the suspension was pipetted out and placed into 900 L, of previously charged sterile saline. This corresponded to sampling at time point t.sub.0. Then followed incubation of the preparations at 250 rpm and 30 C. The CFUs were determined over a period of 22 h. The samples were taken firstly at time points t.sub.0, t.sub.3, t.sub.6 and t.sub.22. For some preparations, a further sampling time point t.sub.15 was added and, in addition to this, a further additional dilution series was plated out in order to minimize deviations.

(12) The OD.sub.600 was 60. The cells were resuspended in 10 mL of Kp, buffer and then mixed in the flask with 5 mL of LSME 98% (w/w). One dilution stage per preparation was plated out. The number of CFU/mL remained constant over a period of 6 h. After 22 h, a percentage decrease in living cell count of just 30.3% was recorded.

EXAMPLE 2

Comparative Experiments Relating to the Toxicity of Various Liquid Cation Exchanger Towards Biotechnologically Relevant Microorganisms

(13) This example shows the lower toxicity of unbranched fatty acids compared with other liquid cation exchangers such as DEHPA as well as branched and unbranched saturated fatty acids.

(14) Firstly, a preculture comprising 20 ml LB medium was inoculated in a 100 ml baffled flask with a cryoculture of the corresponding strain. The culture was cultured overnight at 37 C. and with shaking at 200 rpm used on the next day in order to inoculate an identical main culture to an OD of 0.2. The main cultures (each 30 mL of LB medium) were then further incubated under identical conditions. At an OD of 0.4 to 0.5, the main culture was covered with in each case identical volumes (30 ml) of solvent and then further incubated.

(15) To determine the number of CFU (colony-forming units) 0.1 ml samples were taken in the following experiments and diluted in sterile 0.9% NaCl solution. Suitable dilution stages were plated out on LB agar plates. After incubation at 34 C. overnight, the colonies formed were counted and the CFUs determined.

Experiment 1: Comparison of the Toxicity Between DE2HPA and a Saturated Fatty Acid as Liquid Cation Exchanger

(16) 50% DEHPA and lauric acid (15%), each dissolved in LSME and laden equimolar or 25 mol % with ALSME, were contacted as liquid cation exchanger with a E. coli BL21 (DE3) strain, and the influence of these two compounds on the ability of the strain to form colonies, expressed in CFUs, was investigated. Preliminary experiments showed that methyl lauratewhich could not function as a liquid cation exchanger on account of a lack of chargeis well tolerated by the strains used.

(17) TABLE-US-00001 TABLE 1 Experi- Number of CFUs ment Liquid cation after 22 or 24 h No. E. coli strain used exchanger used relative to t = 0 h 1a E. coli BL21(DE3) None 244% 1b E. coli BL21(DE3) DEHPA .sup.0% 1c E. coli BL21(DE3) Lauric acid 1.2%

(18) It is found that both liquid cation exchangers significantly reduce the number of CFUs, but when using lauric acid in contrast to DEHPA still a few viable cells are present and the saturated fatty acid is therefore to be preferred as liquid cation exchanger.

Experiment 2: Comparison of the Toxicity Between Branched Saturated Fatty Acids and Various Amounts of Oleic Acid as Liquid Cation Exchanger

(19) For this, two different concentrations of oleic acid were used and the volume was adapted by adding the corresponding amount of LSME (methyl laurate).

(20) TABLE-US-00002 TABLE 2 Experi- Number of CFUs ment Liquid cation after 22 or 24 h No. E. coli strain used exchanger used relative to t = 0 h 2a E. coli BL21(DE3) Isononanoic acid 0 2b E. coli BL21(DE3) 2-Ethylhexanoic acid 0 2c E. coli BL21(DE3) LSME/25% oleic acid 11% 2d E. coli BL21(DE3) LSME/75% oleic acid 18% 2e E. coli W3110 Isononanoic acid 0 2f E. coli W3110 2-Ethylhexanoic acid 0 2g E. coli W3110 LSME/25% oleic acid 29% 2h E. coli W3110 LSME/75% oleic acid 17%

(21) It is found that the number of viable cells when using the unsaturated fatty acid oleic acid together with LSME is consistently significantly higher than when using branched saturated fatty acids.

Experiment 3: Comparison of the Toxicity Between Unbranched Saturated Fatty Acids and Unsaturated Fatty Acids as Liquid Cation Exchanger

(22) Here, different amounts of an unsaturated fatty acid were compared with an unsaturated fatty acid as regards their toxicity when being used as liquid cation exchanger compared. On account of the lower solubility of the unsaturated fatty acid lauric acid, this was used in a smaller amount. The volumes of the different cation exchangers were made the same with the LSME. The number of CFUs was determined at the start, after 4.5 h and after 24 h.

(23) As FIG. 2 reveals, the addition of the saturated fatty acid as liquid cation exchanger, even at a lower concentration than that of the unsaturated fatty acid, brings about a decrease in the CFUs, whereas in the case of the unsaturated fatty acid an increase in the CFUs is established.

(24) Overall, a decrease in the toxicity for the various investigated liquid cation exchangers in the following order is found: DEHPA>saturated fatty acids>unsaturated fatty acids.

EXAMPLE 3

Removal of Diaminoisoiditol (DAD and Diaminoisosorbitol (DAS) Using Various Liquid Cation Exchangers from an Aqueous Phase

(25) This example shows that a liquid ion exchanger, here D2EHPA or oleic acid, as constituents of an organic hydrophobic phase increases the absorption capacity of the hydrophobic phase for a positively charged compound.

(26) The organic solvents used here are kerosene (K), benzyl benzoate (B), methyl laurate (ML), cis-9-ocitadecen-1-ol (OD). As liquid cation exchangers, oleic acid and D2EHPA were compared in terms of their effect with the respective pure solvent.

(27) The experimental procedure proceeded identically in all cases: the aqueous phase comprised 2% by weight of diamine and the organic phase consisted of the corresponding solvent and 20% by weight of the respective liquid cation exchanger. The volume ratio of the two phases was 1:1 (in each case 4.5 ml). At a constant temperature of T=30 C., the samples were shaken for 2 hours in a water bath. Then, after centrifugation and phase separation, the phases were analyzed. The pH was adjusted in all cases with aqueous ammonia or sulphuric acid.

(28) FIG. 3a shows the results of the reactive extraction with D2EHPA as liquid cation exchanger. Without the addition of acid or alkali, the degree of extraction in the case of a pH range from 5.4 to 6.2 is ca. 100%. The addition of acid leads to a considerable reduction in the degree of extraction. In the event of adding a small amount of alkali, no influence on the degree of extraction is evident for the experiments with benzyl benzoate. If a large amount of alkali was added, starting pH from 11 to 12, the experiments could not be evaluated. When using DAS with cis-9-octadecen-1-ol as solvent, a reduction in the degree of extraction with increasing pH is observed, although the samples were milky/cloudy, in the event of the extraction of DAI, upon the addition of a small amount of alkali no significant influence on the degree of extraction is evident, in the case of an initial pH of 11 to 12 the degree of extraction drops significantly, the two phases remaining milky/cloudy despite repeated centrifugation, a ca. 1.5 cm thick white layer was evident at the phase boundary. When methyl laurate was used as solvent, the addition of a small amount of alkali, initial pH 10 to 11, led to a slight drop in the pH, and the phase was milky/cloudy. The addition of a large amount of alkali led to the formation of a single clear phase.

(29) The results of using oleic acid as liquid cation exchanger are shown in FIG. 3b. In the course of the experiments, a degree of extraction of 100% was not achieved. The experiments with methyl laurate as solvent could only be evaluated if sulphuric acid was used during the preparation to lower the pH. The maximum degree of extraction achieved was 4% in the case of the extraction of DAS and 48% in the case of that of DAI. The experiments as regards the extraction of DAS with cis-9-octadecen-1-ol as solvent could likewise only be evaluated if the starting pH was reduced with H2SO4. A maximum degree of extraction of 5.5% at pH 6 was achieved. In the case of the extraction of DAI, a maximum degree of extraction of 85% was achieved at pH 7.9 if neither alkali nor acid was added. The addition of alkali led to a significant decrease in the degree of extraction. When using kerosene as solvent for the extraction of DAS, a maximum degree of extraction of 85% at PH 8.5 and, in the case of the extraction of DAI, a maximum degree of extraction of 77.5% at pH 8.4 are achieved. The addition of alkali leads to a very slight reduction in the degree of extraction. The experiments with benzyl benzoate could be evaluated only in cases where the pH of the aqueous phase had been lowered with sulphuric acid before the start of the experiment. The maximum degree of extraction in the case of the extraction of DAS is 61.5% at pH 7.3, that of DAI 15.3 at pH 1.1. Above pH 6, a further lowering leads to an increase in the degree of extraction. The aqueous phase remained cloudy even after centrifugation for 45 minutes.

(30) The degrees of extraction obtained in the corresponding comparative experiments with the same solvents but without liquid cation exchanger are shown in FIG. 3c, plotted against the pH. No clear dependency of the degree of extraction on the pH is evident. In every case, the degrees of extraction are significantly below 20%.

EXAMPLE 4

Reduction in the Toxicity of an Organic Compound with Positive Charge by Contacting with a Liquid Cation Exchanger

(31) This experiment shows that through the presence of a liquid cation exchanger the toxic effect of a positively charged organic compound in an aqueous phase which is a fermentation liquor can be reduced by extracting this compound into the organic phase.

(32) The fundamental experimental procedure corresponded to that in Example 1.

(33) Since ALSME 0.2% (w/v), dissolved in aqueous systems, had a bactericidal effect, this experiment was carried out again in combination with D2EHPNH3/LSME 2/98% (w/w) in the shake flask, D2EHPNH3 here meaning D2EHPA laden quantitatively with ammonium. The use of the liquid ion exchanger improves the transfer of ALSME into the organic phase, as a result of which its concentration in the aqueous phase, in which the cells are also located, is reduced. In order to reduce a toxic effect caused by D2 EHPA, low concentrations of 2% (w/w) D2EHPNH3 were used.

(34) The bacteria were firstly resuspended in 5 mL (corresponds to half of the buffer volume). A further 5 mL of buffer were optionally admixed with 0.4% (w/v) ALSME and then optionally vortexed with 5 mL of D2EHPNH3/LSME 2/98% (w/w) for 1 min at 3000 rpm. This solution was added to the initially charged bacteria suspension in the shake flask and mixed. The first sample was then taken.

(35) The solution had a foamy consistency at the start of the experiments, although this had disappeared in both experiments at the time of taking the second sample. The abbreviation D/L was used for D2EHPNH3 (D2EHPA laden with ammonia)/LSME 2/98% (w/w). Between the sampling t.sub.0 and t.sub.1.5 h the number of CFU/mL increased by 34.3%. From the sampling (t.sub.1.5) to the last sampling (t.sub.22) the number of CFU/mL reduced by 54.9%. Compared to the preparation with D2EHPNH3/LSME 2/98% (w/w) without the addition of ALSME 0.2% (w/v), the number of reproducible cells after 22 h was 4.5 times higher and, at 3.4%, not significantly lower than the average value of the control preparations in HEPES buffer (see FIG. 4). Compared to the preparation with ALSME 0.2% (w/v) in the shake flask, without the addition of an organic phase, the number of CFU/mL was 2800 times higher.

(36) It is found that the presence of the liquid cation exchanger reduces the toxicity of the positively charged compound, determined here via the number of remaining CFUs.

EXAMPLE 5

Comparative Experiments Relating to the Toxicity of Various Liquid Cation Exchangers Towards a Microorganism Producing -Aminolauric Acid (ALS) and the Methyl Ester (ALSME)

(37) The biotransformation of methyl laurate to methyl aminolaurate was tested in an 8-fold parallel fermentation system from DasGip with different ion exchangers.

(38) 1 L reactors were used for the fermentation. The pH probes were calibrated using a two-point calibration with measurement solutions of pH 4.0 and pH 7.0. The reactors were filled with 300 mL of water and autoclaved for 20 min at 121 C. in order to ensure sterility. The pO2 probes were then polarized overnight (at least 6 h). On the next morning, the water was removed under the clean bench and replaced by high cell density medium with 50 mg/L kanamycin and 34 mg/L chloramphenicol. Subsequently, the pO2 probes were calibrated with a one-point calibration (stirrer: 600 rpm/gassing: 10 sL/h air) and the feed, correctant and induction means stretches were cleaned by means of clean-in-place. For this, the tubes were flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralised water, and finally filled with the respective media.

(39) The ALS and ALSME producing E. coli strain BL21 (DE3) Tlr pBT10 pACYC:Duet[TAcv] was firstly grown from cryoculture in LB medium (25 mL in a 100 mL baffled flask) with 50 mg/L kanamycin and 34 mg/L chloramphenicol overnight at 37 C. and 200 rpm for ca. 18 h. Then, 2 mL in each case were transinoculated in high cell density medium (glucose 15 g/L (30 mL/L of a separately autoclaved 500 g/L stock solution with 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), (NH.sub.4).sub.2SO4 1.76 g/L, K.sub.2HPO.sub.4 19.08 g/L, KH.sub.2PO.sub.4 12.5 g/L, yeast extract 6.66 g/L, trisodiumcitrate dihydrate 11.2 g, ammonium iron citrate solution 17 mL/L of a separately autoclaved 1% strength stock solution, trace element solution 5 mL/L of separately autoclaved stock solution (HCl (37%) 36.50 g/L, MnCl.sub.2*4H.sub.2O 1.91 g/L, ZnSO.sub.4*7H.sub.2O 1.87 g/L, ethylenediaminetetraacetic acid dihydrate 0.84 g/L, H.sub.3BO.sub.3 0.30 g/L, Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/L, CaCl.sub.2*2H.sub.2O 4.70 g/L, FeSO.sub.4*7H.sub.2O 17.80 g/L, CuCl.sub.2*2H.sub.2O 0.15 g/L)) (325 mL in a 100 mL baffled flask) with 50 mg/L kanamycin and 34 mg/L chloramphenicol and incubated for a further 6 h at 37 C./200 rpm.

(40) The 3 cultures were combined in a shake flask and the optical density was determined at 7.2. In order to inoculate the reactors with an optical density of 0.1, in each case 4.2 mL were drawn up in a 5 mL syringe and the reactors were inoculated by means of canulae via a septum.

(41) The following standard program was used:

(42) TABLE-US-00003 DO regulator pH regulator Preset 0% Preset 0 ml/h P 0.1 P 5 Ti 300 s Ti 200 s Min 0% Min 0 mL/h Max 100% Max 40 mL/h N XO2 (gas F (Rotation) From To mixture) From To (gas flow) From To Growth and 0% 30% Growth and 0% 100% Growth and 15% 80% biotrans- 400 rpm 1500 rpm biotrans- 21% 21% biotrans- 6 sL/h 72 sL/h formation formation formation Script Trigger sharp 31% DO (1/60 h) Induction IPTG 2 h after feed start Feed trigger 50% DO Feed rate 3 [mL/h]

(43) The experiment carried out can be divided into two phases, the cultivation during which the cells should reach a certain optical density, and the subsequent biotransformation during which the expression of the genes required for the biotechnological process for producing ALSME was induced. The pH values were regulated to pH 6.8 on the one hand with ammonia (12.5%). During cultivation and biotransformation, the dissolved oxygen (DO) in the culture was regulated at 30% via stirrer speed and gassing rate. The fermentation was carried out as a feed batch, where the feed start, 5 g/Lh glucose feed (500 g/L glucose with 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), was triggered via a DO peak. With feed start, the temperature was also reduced from 37 C. to 30 C. The expression of the transaminase was induced 2 h after feed start via the automatic addition of IPTG (1 mM). The induction of the alk-genes was carried out by the manual addition of DCPK (0.025% v/v) 10 h after feed start. Before the start of the biotransformation, the optical density of the culture broths was determined.

(44) The start of the biotransformation phase was carried out 14 h after feed start. For this, 150 mL of a mixture of methyl laurate and the respective ion exchanger (10% w/w) were added as a batch to the fermentation broth. The ion exchangers used were di(2-ethylhexyl)phosphoric acid (DEHPA), lauric acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid and a mixture of free fatty acids from the saponification of globe thistle oil. In order to provide an amino group donor for the transaminase, at the same time as the addition of the organic phase, 10.7 mL of an alanine solution (125 g/L) were added to the fermentation broth. For the sampling, 2 mL of fermentation broth were removed from the reactor and part of it was diluted 1/20 in an acetone/HCl mixture (c(HCl)=0.1 mol/L) and extracted. Samples were taken from all 8 reactors at 1.25 h, 3 h, 5 h, 20 h, 22 h and 25 h after the start of the biotransformation. The transfer rates for oxygen (OTR=oxygen transfer rate) and carbon (CTR=carbon transfer rate) were determined during the fermentation via offgas analysis on the DasGip systems. The fermentation was ended 22 h after the start of the biotransformation.

(45) Quantification of ALS, ALSME, DDS, DDSME, LS, LSME, HLS, HLSME, OLS and OLSME in fermentation samples was carried out by means of LC-ESI/MS.sup.2 by reference to an external calibration for all analytes and using the internal standard aminoundecanoic acid (AUD).

(46) For this purpose, the following instruments were used: HPLC instrument 1260 (Agilent; Boblingen) with autosampler (G1367E), binary pump (G1312B) and column oven (G1316A) Mass spectrometer TripelQuad 6410 (Agilent; Boblingen) with ESI source HPLC column: Kinetex C18, 1002.1 mm, particle size: 2.6 m, pore size 100 (Phenomenex; Aschaffenburg) Precolumn: KrudKatcher Ultra HPLC In-Line Filter; 0.5 m filter depth and 0.004 mm internal diameter (Phenomenex; Aschaffenburg)

(47) The samples were prepared by pipetting 1900 L of solvent (acetone/0.1 N HCl mixture=1:1) and 100 L of sample into a 2 mL reaction vessel. The mixture was vortexed for ca. 10 seconds and then centrifuged at ca. 13 000 rpm for 5 min. The clear supernatant was removed using a pipette and analyzed following appropriate dilution with diluent (80% (v/v) ACN, 20% double distilled H.sub.2O (v/v), +0.1% formic acid). For each 900 L sample, 100 L of ISTD were pipetted in (10 L for a sample volume of 90 L).

(48) The HPLC separation was carried out using the aforementioned column or precolumn. The injection volume was 0.7 L, the column temperature 50 C., the flow rate 0.6 mL/min. The mobile phase consisted of eluent A (0.1% strength (v/v) aqueous formic acid) and eluent B (acetonitrile with 0.1% (v/v) formic acid). The following gradient profile was used

(49) TABLE-US-00004 Time [min] Eluent A [%] Eluent B [%] 0 77 23 0.3 77 23 0.4 40 60 2.5 40 60 2.6 2 98 5.5 2 98 5.6 77 23 9 77 23

(50) The ESI-MS.sup.2 analysis was carried out in the positive mode with the following parameters of the ESI source: Gas temperature 280 C. Gas flow 11 L/min Nebulizer pressure 50 psi Capillary tension 4000 V

(51) Detection and quantification of the individual compounds was carried out with the following parameters, with in each case one product ion being used as qualifier and one being used as quantifier:

(52) TABLE-US-00005 Collision Precursor ion Product ion Residence time energy Analyte [m/z] [m/z] [ms] [eV] DDSME 245.2 167.1 25 6 DDSME 245.2 149.1 50 8 HLSME 231.3 181.2 15 2 HLSME 231.3 163.2 25 5 DDS 231.2 213.2 50 0 DDS 231.2 149.1 25 9 ALSME 230.3 198.1 25 10 ALSME 230.3 163.2 15 10 OLSME 229.2 197.2 50 0 OLSME 229.2 161.1 25 5 HLS 217.2 181.2 35 0 HLS 217.2 163.1 20 4 OLS 215.2 161.2 25 0 OLS 215.2 95.2 60 13

(53) Results:

(54) If DEHPA as described in the prior art is used as cation exchanger, then directly after adding the compound to the culture, this results in a drop in the OTR. The curve drops within a short time to 0, which indicates that metabolically active cells are no longer present in the culture. DEHPA thus has a high grade toxic effect on cells.

(55) If lauric acid is used as liquid cation exchanger instead of DEHPA, then although this likewise leads to a drop in the OTR, this is not so severe, and in the course of the next 22 h the cells recover and exhibit increasing metabolic activity. Accordingly, lauric acid is notably less toxic than DEHPA.

(56) Even better results can be observed when using saturated fatty acids with longer carbon chains. If palmitic acid and stearic acid are used, then the OTR curve falls significantly more shallowly than in the case of using lauric acid or even DEHPA. It can be concluded from this that these fatty acids have a considerably lower toxic effect.

(57) The use of unsaturated fatty acids such as palmitoleic acid, saponified globe thistle oil (contains predominantly linoleic acid) and oleic acid surprisingly leads to even better results. These fatty acids surprisingly exhibit an even lower toxicity than the saturated fatty acids.

LITERATURE SOURCES

(58) J. Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, Vol. 2 of Wiley Series in Solution Chemistry, John Wiley & Sons, Chichester, 1997 Asano, Y., Fukuta, y., Yoshida, Y., and Komeda. H. (2008): The Screening, Characterisation, and Use of w-Laurolactam Hydrolase: A New Enzymatic Synthesis of 12-Aminolauric Acid, Biosc. Biotechn. Biochem., 72 (8), 2141-2150 DE10200710060705 (2007): Recombinant cells producing w-aminocarboxylic acids or their lactams