CZ307082B6 - Strain of the microorganism Raoultella sp. KDF8 CCM 8678 degrading active pharmaceutical ingredients from the following group: diclofenac, codeine, ibuprofen and ketoprofen - Google Patents

Abstract

Raoultella is able to degrade morphine derivatives or metabolize the active ingredients non-steroidal anti-inflammatory drugs, drugs massively used in veterinary and clinical practice, which are pollutants especially surface waters: diclofenac, ibuprofen, ketoprofen and codeine. The present solution is further concerned using a particular strain of Raoultella sp. KDF8 for bioremediation of persistent pollutants pharmaceuticals active substances, a method for preparing the strain biomass of the microorganism Raoultella sp. KDF8 and its use for bioremediation of surface water and drinking water sources

Description

Microorganism strain Raoultella sp. KDF8 CCM 8678 degrading active pharmaceutical substances from the group: diclofenac, codeine, ibuprofen and ketoprofen

Technical field

Raoultella microorganism capable of degrading morphine derivatives or metabolizing the active substances of non-steroidal anti-inflammatory drugs, in particular codeine, diclofenac, ibuprofen and ketoprofen.

BACKGROUND OF THE INVENTION

Drugs widely used in veterinary and clinical practice pose a serious threat to the environment because their active substances can accumulate in the environment in the form of persistent pollutants. These include, for example, natural alkaloids such as codeine (Kod), morphine and thebaine, which are produced by poppy (Papaver somniferum), xenobiotics diclofenac (DF), which is an active pharmaceutical ingredient (API) in drugs such as non-steroidal anti-inflammatory drugs (Voltaren, Olfen, Dolmina, etc.). Ibuprofen (IBU) and ketoprofen (KTP) are other active substances found in the environment as pollutants, most of all in wastewater, and due to insufficient degradation of subsequently watercourses and soils. These substances are also among the APIs of non-steroidal anti-inflammatory drugs - ie anti-inflammatory drugs, and enter the environment through drugs. The need for effective and gentle disposal of such pollutants from the environment is based on the inclusion of these pollutants in the lists of undesirable pollutants that need to be monitored. Microbial transformation Kod has been described in both fungal and bacterial strains (Tsuda, K., 1964, Symposium on Microbiology No. 6, University of Tokyo, Tokyo, Japan). Morphine conversion has been described in Arthrobacter sp. (Liras, P. and Umbreit, W. W., 1975, Appl. Microbiol. 30, 262-266), Pseudomonas testosteroni (Liras et al., 1975, Appl. Microbiol. 30, 650-656), Bacillus sp. (Madhyasta and Ready, 1994, J, Chem. Soc. Perkin Trans. 1 (8), 911-912) and Rhizobium radiobacter (KYSLÍKOVÁ, Eva, et al. Biotransformation of 14-OH-codeine derivatives by Rhizobium radiobacter R89 Journal of Molecular Catalysis B: Enzymatic, 2013, 87: 1-5.), Which is capable of hydroxylating codeine to 14-OH-codeine and 14-OH-codeinone, but only on a complex LB medium whose nutrients it uses as source of energy and carbon. Thus, only the codeine is converted to other pharmaceutically active substances, not degraded and degraded.

SUMMARY OF THE INVENTION

It has been found that microorganisms of the genus Raoultella have the ability to degrade active pharmaceutically active substances. The presence of bacteria of this genus has been mapped in sewage treatment plants and, on closer inspection, has shown the ability of these bacteria to degrade active substances entering sewage treatment plants as persistent pollutants.

A new strain of Raoultella sp. KDF8, which, unlike Rhizobium radiobacter, is able to utilize codeine, diclofenac, ibuprofen and ketoprofen also in mineral medium as a carbon source and efficiently metabolize solutes. The original strain was initially selected by screening more than 300 soil isolates derived from compost soil containing dead parts of poppy plants. After targeted chemical mutagenesis and further selection, the strain with the best ability to degrade codeine (substance 1) and diclofenac (substance 2) was selected. This ability has not been described in Raoultella.

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Advantageous properties of Raoultella sp. KDF8 lies in its ability to efficiently metabolize the 5 wider spectrum of dissolved persistent pollutants (API) in the presence of low concentrations of cheap, energy-poor substrate or water in the form of API stock solution, utilizing these pollutants as a C source and energy or degrading them. The microorganism in this physiological state, i.e. metabolizing the dissolved API (in ethanol, methanol, DMSO or water), is utilized for the bioremediation of the API present in the aqueous environment.

Raoultella sp. KDF8 was classified according to morphological, growth and biochemical properties, which was later confirmed by phylogenetic analysis of 16S rDNA. This strain is currently deposited in the Czech Collection of Microorganisms at Masaryk University in Brno, under the designation CCM 8678. The strain properties are shown in Table 1.

Tab. 1 Morphological and biochemical properties of Raoultella sp. KDF8

Microscopy Morphology colonies Positive biochemical tests Negative biochemical tests Gram-negative ovoid cocci, tartlets, individually, in clusters Round, smooth, shiny, flat, creamy, border continuous Catalase, oxidative glucose acid, fermentative glucose acid, glucose gas, lysine decarboxylation, indole production, Simmons citrate, urease, acetoin, sodium malonate, ONPG and escaulin hydrolysis, mannitol, inositol, adonitol, cellobiose, rhamnose, sucrose , sorbitol threhalose, raffinose and melibiosis, growth at 37 ° C, methyl red test Oxidase, hydrogen sulfide production, decarboxylation omitins, arginine dihydrolase, deamination of phenylalanine, hydrolysis of Tween 80, gelatin and DNA, dulcitol acid, motility

Clarification of drawings

Giant. 1 Structural sample of substance 1 - codeine

Giant. 2 Structural formula of 2 - diclofenac

Giant. 3 Structural sample of 3 - 4-hydroxydiclofenac

Giant. 4 Structural sample of 4 - 2,6-dichloroaniline

Giant. 5 Structural formula of 5 - ibuprofen

Giant. 6 Structural sample of 6 - ketoprofen

Giant. 7 Diclofenac degradation chromatogram

Giant. 8 Codeine degradation chromatogram

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DETAILED DESCRIPTION OF THE INVENTION

Example 1

Isolation of codeine degrading strains.

Natural isolates were obtained from compost soil containing poppy waste. 5 g of soil was resuspended in 100 ml of LB medium (composition see List of used media) and shaken on a rotary shaker (200 rpm, 28 ° C) for 24 h. The grown culture on LB medium was diluted 1: 3 mineral medium BSB supplemented with TE (Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Mn ²⁺ , Fe ²⁺ , Co ²⁺ , Cu ²⁺ as described in the List of used media) and codeine (in concentration 1.0 g / 1) and cultured for 24 h on a rotary shaker (200 rpm, 28 ° C). Then, the suspension was re-diluted 1: 3 with fresh mineral BSBTE medium and the culture was repeated (24 h, 200 rpm, 28 ° C) under the same culture conditions. The resulting autochthon culture was diluted with saline and sown on LB or BSB solids containing Kod at 1.0 g / L. Individual morphologically different colonies, grown for 72 h at 28 ° C, were transferred to fresh Petri dishes with LB and BSB broth containing Kod (1.0 g / L). Each of the 300 colonies obtained was cultured in liquid BSBTE medium (composition see List of used media) supplemented with Kod (1.0 g / l). 20 ml medium in a 100 ml culture flask was inoculated with one colony each and the flasks were incubated on a rotary shaker (200 rpm, 28 ° C) for 48 to 120 h. Five isolates were obtained from 300 test colonies cultivated in BSBTE liquid broth. which were able to grow under these conditions, thus showing the ability to cometabolize Kod. The isolates were taxonomically ranked according to the iylogenetic analysis of the gene encoding 16S rDNA as Pseudomonas putida (4 isolates) and Raoultella sp.

Example 2

Preparation of Raoultella sp. KDF8 by targeted chemical mutagenesis

By Raoultella sp. KDF8 from Example 1 was inoculated with 20 ml LB medium in a 100 ml flask. The culture was incubated on a rotary shaker (200 rpm, 28 ° C) for 48 h. During the exponential growth phase (OD = 3), the biomass was separated from the culture medium by centrifuges (10,000 xg) under sterile conditions, 15 min), the cells were washed with saline, then centrifuged (10,000 xg, 15 min) and resuspended in 0.1 M phosphate buffer pH 7.5 in an equivalent volume of culture medium. This suspension was added to a solution of the N-methyl-N'-nitro-N-nitrosoguanidine mutagen in the same buffer so that the mixture contained 10 ⁸ cells per ml of the mixture and 150 µg of the mutagen per ml of the mixture. The mixture was stirred at 15 ° C for 60 minutes and then plated on LB agar. After incubation of the plates at 28 ° C for 24 and 48 h, monocolonium mutant isolates were obtained. 100 mutant isolates were obtained, which were transferred to selective solid broth BSB with DF or Kod (1.0 g / L). After 24 and 48 h, the single grown monocolonate isolates were inoculated into 20 ml of BSBTE medium supplemented with DF or Kod at concentrations of 1.0 g / L. The cultures were incubated at 28 ° C on a rotary shaker (200 rpm) for 48 h. The degradation of DF and Kod by HPLC was then verified (see Examples 3 and 4). A single monocolonate isolate having both Kod and DF degradation properties was obtained. This isolate was designated Raoultella sp. KDF8.

Example 3

Degradation of diclofenac by growing cells

Raoultella sp. KDF8 was cultured in two parallels in liquid BSBTE medium supplemented with DF sodium (1.0 g / L, 0.93 g / L pure). 20 ml of medium in a 100 ml culture flask was

Inoculated with monocolony from an agar plate and the flasks were incubated on a rotary shaker (200 rpm, 28 ° C) for 72 h. The biomass was separated by centrifugation and the supernatant was collected from the samples taken after 24 h, 48 h and 72 h. culture was treated as follows: TBME (tert-butyl methyl ether) was added to the supernatant in an equivalent amount and the mixture was extracted for 10 minutes. After centrifugation (14,000 xg, 10 min), the organic phase was used for the quantitative determination of diclofenac (compound 2), its metabolites 4-hydroxydiclofenac (compound 3) and 2,6-dichloroaniline (compound 4), ibuprofen (compound 5) and ketoprofen (compound 6) by HPLC (Fig. 7) under the following conditions:

Column: RPC18-Lichroprep Waters

Elution mixture: 70% MeOH: 30% water MilliQ + phosphoric acid (pH 3.0)

Flow. 0.8 ml / min

Application volume: 0.01 ml

Detection at 214 and 275 nm

Column temperature: 28 ° C

Analysis time: 20 min

Retention times:

2. diclofenac 11.2 min. 3. 4-hydroxydiclofenac 9.4 min. 4. 2,6-dichloroaniline 8.2 min. 5. Ibuprofen 9.8 min. 6. Ketoprofen 6.15 min.

substance 1

This strain showed 100% degradation of diclofenac dissolved in the medium in both samples without detection of the metabolites (Formula 3 and Formula 4) described in the literature for the degradation of diclofenac. The residual concentration of diclofenac in the individual samples as a function of culture time is documented in Table 2.

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Tab. 2 Determination of residual diclofenac concentration in Raoultella sp. KDF8 on HPLC

Time of cultivation 24 hours 48 hours 72 hours Residual DF concentration 50 mg / l 0 0

Example 4

Degradation of codeine by growing cells

Raoultella sp. KDF8 was cultured in two parallel in liquid BSBTE medium supplemented with codeine sulfate (pure - Kod 1.0 g / L). 20 ml of medium in a 100 ml culture flask was inoculated with monocolonium from an agar plate and the flasks were incubated on a rotary shaker (200 rpm, 28 ° C) for 72 h. 48 h and 72 h of culture were treated as follows: TBME and 0.1 ml NH ₄ OH were added to the supernatant in an equivalent amount and the mixture was extracted by shaking for 20 minutes at room temperature. After centrifugation (12,000 xg, 10 min), the organic phase was used for quantitative determination of Codeine (Compound 1) and its metabolites by HPLC (Figure 8) under the following conditions:

Column: RPC 18-Lichroprep Waters

Elution mixture: MilliQ water + 0.1% TFA: MeOH (4: 1 V / V), pH 3.0 (25% NH ₄ OH adjustment)

Flow rate: 1.0 mL / min

Application volume: 0.05 ml Detection at 220 and 214 nm Column temperature: 40 ° C Analysis time: 10 min

Retention times: Fabric 1. Codeine 4.7 min

This strain showed 22-30% degradation of codeine in the samples tested. The residual codeine concentration in each sample is documented in Table 3.

Tab. 3 Determination of residual codeine concentration in Raoultella sp. KDF8 on HPLC

Time of cultivation 24 hours 48 hours 72 hours Residual concentration Kod 780 mg 750 mg 700 mg

Example 5

Growth properties of Raoultella KDF8 strain on complex LB medium and defined BSB and M9 mediums supplemented by various carbon and energy sources

The selection of medium suitable for growth and degradation was carried out in shaking, single-shot cultures in a volume of 20 ml in 100 ml flasks. The composition of each media is shown in the Media Overview. Overgrown isolate of Raoultella sp. KDF8 on LB medium was used to prepare an inoculation culture that was cultured in 100 ml LB in 500 ml flasks for 24 h at temperature

-5 ° 307082 B6 ° C and 200 rpm. The inoculation culture thus prepared was seeded with 2% test media (20 ml) in 100 ml flasks. The cultures were incubated on a shaker (200 rpm, 28 ° C) for 48 h. Table 4 illustrates the effect of individual media on the growth of Raoultella sp. KDF8.

Tab. 4 Effect of media type supplemented by different carbon sources on the growth (OD ₆ oo) of Raoultella sp. KDF8 (28 ° C, 48h, 200rpm)

Media type Substrate Substrate concentration (g / l) ODgoo Final pH Growth rate (h ' ¹ ) M9 diclofenac 0.93 3.07 5.2 0.06 M9 codeine 1.0 0.238 6.8 0.003 BSBTE diclofenac 0.93 4.42 4.7 0.09 BSBTE codeine 1.0 0.333 6.6 0.005 BSBTE ’ glycerol 2.0 1.86 3.89 0.34 codeine 1.0 2.4 3.8 0.03 BSBTE glycerol 2.0 1.9 3.5 0.31 BSBTE yeast extract 10.0 5.5 7.8 0.41 BSBTE casein hydrolyzate 10.0 5.6 7.6 0.35 LB yeast extract 5.0 6.8 8.1 1.0 trypton 10.0

biphasic culture: codeine (1.0 g / l) was added to the culture after glycerol utilization (2.0 g / l).

Example 6

Effect of initial codeine concentration on its utilization by growing culture of Raoultella sp. KDF8

Based on the results shown in Tab. 4, the BSBTE mineral medium was selected for further optimization. Cultivations were carried out in shaken, single cultures - 20 ml medium in 10 ml flasks. Inoculation culture of Raoultella sp. KDF8, grown on LB agar, was prepared 20 in 100 ml LB medium at 28 ° C and 200 rpm for 24 h. The effect of initial codeine concentration on strain growth was tested in 20 ml BSBTE medium supplemented with different codeine concentrations, inoculated 2% culture grown in LB medium. Cultivation was performed with shaking at 200 rpm, 28 ° C for 120 hours. The results of the culture analyzes are shown in Tab. 5.

Tab. 5 Concentration effect of codeine on biomass growth in cultures of Raoultella sp. KDF8 (200 rpm, 28 ° C, 120h)

Carbon source Concentration (g / 1) θθόοο pH Codeine 0.5 0.150 6.9 1.0 0.369 6.6 1.5 0.289 6.8 2.0 0.125 7.0

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Example 7

Effect of initial concentration of diclofenac on its utilization by growing culture of Raoultella sp. KDF8

The concentration effect of diclofenac was monitored under the same conditions as in Example 6. Single culture - 20 ml medium in 100 ml flasks. Inoculation culture of Raoultella sp. KDF8, grown on LB agar, was prepared in 100 ml LB medium at 28 ° C and 200 rpm for 24 h. The effect of initial diclofenac concentration on strain growth was tested in 20 ml BSBTE medium supplemented with various concentrations of DF sodium salt , inoculated with a 2% culture grown in LB medium. The cultivation was carried out with shaking at 200 rpm, 28 ° C for 48 hours. 6.

Tab. 6 Concentration effect of diclofenac on biomass growth in cultures of Raoultella sp. KDF8 (200 rpm, 28 ° C, 48h)

Carbon source Concentration (g / 1) ODůoo PH Cdw * (mg / ml) DF 0.47 1.98 4.97 0.8 0.93 4.40 4.8 1.9 1.40 2.80 4.7 1.4 1.86 1.16 4.6 0.6

* dry biomass weight

Example 8

Effect of initial concentration of diclofenac on its biodegradation by whole-cell catalyst Raoultella sp. KDF8

The grown culture on LB medium of Example 5 was collected by centrifugation (12,000 x g, 10 min, 4 ° C), washed with saline, and resuspended in BSBTE medium to a final concentration (wet weight) of 10 wt. %. Biodegradation of DF was performed in 100 ml culture flasks with 2.836 g wet biomass and 25.524 ml BSBTE on a rotary machine (200 rpm and 28 ° C) for 72 h. DF sodium salt was added to the reaction mixture at t = 0 and its starting point. the concentration was 1.0 to 6.0 g / L. The degradation percentage calculated and averaged after the measurement of the supernatant and biomass samples by HPLC is shown in Table 7. The degradation percentage calculated after the supernatant samples were measured by HPLC is shown in Table 8. The tables show the difference in degradation between the supernatant samples and the supernatant + biomass pooled samples. In the supernatant, where diclofenac is dissolved, 100% degradation occurs.

Tab. 7 Time course of diclofenac degradation with 10% suspension of Raoultella sp. KDF8

initial concentration of DF (g / l) biodegradation (%) 24 h 48 h 72 h 0.93 91 94 95 1.86 87 93 98 2.79 67 71 75 3.72 47 65 75 5.58 55 63 70

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Tab. 8 Time course of diclofenac degradation in the supernatant of a 10% suspension of Raoultella sp. KDF8

initial concentration of DF (g / l) biodegradation (%) 24 h 48 h 72 h 1.0 99 100 ALIGN! 100 ALIGN! 2.0 99 100 ALIGN! 100 ALIGN! 3.0 99 100 ALIGN! 100 ALIGN! 4.0 97 99 100 ALIGN! 6.0 11 41 67

Example 9

Effect of initial codeine concentration on its biodegradation by whole-cell catalyst Raoultella sp. K.DF8

The grown biomass from LB medium of Example 5 was separated by centrifugation (12,000 x g, 10 min), washed with saline and resuspended in BSBTE medium to a final concentration (wet weight) of 10 wt. %. Biodegradation The Kod was run in 100 ml culture flasks with 1 g of wet biomass and 10 ml BSBTE medium on a rotary machine (200 rpm and 28 ° C) for 72 h. Codeine sulfate was added to the medium at t = 0 and the initial concentration was 0 , 2 and 1.0 g / l of pure substance. The percentage of degradation calculated and averaged after measurement of the supernatant and biomass samples taken by HPLC is shown in Table 9.

Tab. 9 Time course of codeine degradation by 10% suspension of Raoultella sp. KDF8

initial concentration Kod (g / 1) biodegradation (%) 24 h 48 h 72 h 0.2 30 45 49 1.0 22nd 27 Mar: 31

Example 10

Optimization of biomass concentration of whole cell catalyst for diclofenac degradation

Biomass from the grown culture on LB medium according to Example 5 was collected by centrifugation (12,000 x g, 10 min), washed with saline and resuspended in BSBTE medium to give a final concentration (wet weight) of 5 and 20 wt. %. Biodegradation of DF was performed in 100 ml culture flasks with 1.38 g wet biomass and 26.22 ml BSBTE (for 5 wt%) or 8.6 g wet biomass and 35.1 ml BSBTE (for 20 wt%). The cultures were shaken on a rotary machine (200 rpm and 28 ° C) for 72 h. DF sodium was added to the medium at t = 0 and its initial concentration was 1.0 to 3.0 g / l (pure 0.93 to 2.79 g / l for 5 wt%) or 2.0 to 6.0 g / l (pure 1.86 to 5.58 g / l for 20 wt%). The percent degradation calculated after HPLC sampling of the samples was documented in Table 10.

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Tab. 10 Time course of DF degradation with 5% and 20% Raoultella sp. KDF8

Suspension biomass initial concentration of DF (g / l) biodegradation (%) 24 h 48 h 72 h 5% 0.93 10 25 25 1.86 25 37 53 2.79 45 50 55 20% 1.86 49 57 65 3.72 28 55 62 5.58 25 43 70

Example 11

Degradation of ibuprofen and ketoprofen with whole cell catalyst Raoultella sp. KDF8

Biomass from the grown culture on LB medium according to Example 5 was collected by centrifugation (12,000 x g, 10 min), washed with saline and resuspended in BSBTE medium to a final concentration (wet weight) of 10 wt. %. Biodegradation of ibuprofen or ketoprofen was carried out in 100 ml culture flasks with 1 g of wet biomass and 10 ml of BSBTE medium on a rotary machine (200 rpm and 28 ° C) for 72 h. added at time t = 0 and the initial concentration was 1.0 g / l. Percentage of degradation calculated after

15 measurements of the samples taken by HPLC are shown in Table 11. ch ₃ O ch ₃ CH ₃ i / kz ^0H 1 1 O '0 substance 5 substance 6

Tab. 11 Time course of degradation of IBU and KTP with 10% suspension of Raoultella sp. KDF8

substance initial concentration (g / 1) degradation (%) 24 h 48 h 72 h IBU 1.0 34 40 52 KTP 1.0 31 48 50

Example 12

Effect of temperature on diclofenac degradation by whole-cell catalyst Raoultella sp. KDF8

The temperature effect on DF biodegradation was observed under the same culture conditions as 30 in Example 5 (single cultivation, DF addition, shaking 200 rpm) at ° C and 37 ° C for 140 h. Temperature effect on diclofenac degradation by strain Raoultella sp. KDF8 is shown in Tab. 12.

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Tab. 12 Time course of diclofenac degradation with 10% suspension of Raoultella sp. KDF8 at different temperatures

temperature initial concentration of diclofenac (g / l) biodegradation (%) 48 h 72 h 140 h Deň: 18 ° C 1.0 16 36 43 Deň: 32 ° C 1.0 35 41 70

Example 13

Growth properties of Raoultella bacteria on BSBTE mineral medium supplemented with various sources of carbon and energy

Bacteria of the genus Raoultella were cultured in two parallel in liquid BSBTE medium supplemented with sodium salt DF (1.0 g / l, 0.93 g / l pure), codeine sulphate (1.0 g / l pure), ibuprofen (1). , 0 g / l) and ketoprofen (1.0 g / l). An inoculation culture of Raoultella grown on LB agar was prepared in 100 ml LB medium at 28 ° C and 200 rpm for 24 h. 20 ml BSBTE medium in a 100 ml culture flask was inoculated with 2% culture of the genus Raoultella from liquid LB medium and cultures were incubated on a rotary shaker (200 rpm, 28 ° C) for 72 h. The biomass was separated by centrifugation and the supernatant and biomass were analyzed from samples taken after 24 h, 48 h and 72 h culture. . The percentage of degradation calculated after measurement of the samples by HPLC is shown in Table 13.

Tab. 13 Progress of degradation of Kod, DF, 1BU and KTP by growing culture of Raoultella

substance initial concen- tration; trace (g / 2) 24 h degradation (%) 48 h 72 h Code 1.0 20 May 24 28 DF 0.93 90 98 100 ALIGN! IBU 1.0 54 63 78 KTP 1.0 36 60 60

Overview of used media and solutions

Luria-Bertani Complex Medium (LB):

Trypton 10 g / l yeast extract 5 g / l

NaCl 10 g / l distilled water 1000 ml initial pH: 7.2 to 7.5

Mineral medium (BSB):

K ₂ HPO ₄ 1.0 g / L

KH ₂ PO ₄ 1.0 g / l (for examples 1-4)

NaCl 0.1 g / l

NH ₄ Cl 1.0 g / L

0.75 g / l (for Example 5) distilled water 1000 ml initial pH: 6.9 to 7.2

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Trace Elements (TE):

MgSO ₄ .7H ₂ O 200 mg / l

CaCl ₂ .2H ₂ O 50 mg / 1

FeSO ₄ .7H ₂ O 10 mg / l

ZnSO ₄ .7H ₂ O 67 mg / l

MnSO ₄ .H ₂ O 1.7 mg / l

CoCl ₂ .6H ₂ O 48 mg / 1

CuSO ₄ . 5H ₂ O 47 mg / 1

Na ₂ MoO ₄ · 2H ₂ O 45 mg / l

Mineral medium M9 Na ₂ HPO ₄ 14.6 g / l

KH ₂ PO ₄ 3.0 g / l

NaCl 0.5 g / l

NH ₄ Cl 1.0 g / L

MgSO ₄ .7H ₂ O 0.2 g / l distilled water 1000 ml initial pH: 6.9 to 7.2

Carbon and energy sources:

Glycerol 2.0 g / l

DF 1.0 g / L

Code 1.0 g / l yeast extract 10 g / l casein hydrolyzate 10 g / l

Active Substances: DF, Kod, IBU, KTP Stock DF: Stock Code:

IBU stock solution: KTP stock solution:

g of DF sodium salt in 10 ml of ethanol (EtOH) 0.23 g of codeine sulfate in 10 ml of dist. H ₂ O 0.23 g codeine sulfate in 10 ml DMSO 1 gIBU in 10 ml EtOH g KTP in 10 ml EtOH

Claims (7)

PATENT CLAIMS 1. A strain of the microorganism Raoultella sp. KDF8 deposited in the Czech Collection of Microorganisms under number 8678 degrading active pharmaceutical substances from the group: diclofenac, codeine, ibuprofen and ketoprofen. A process for degrading an active pharmaceutical substance, namely diclofenac, codeine, ibuprofen or ketoprofen, characterized in that the microorganism Raoultella sp. KDF8 CCM 8678 is cultured in a medium containing the active pharmaceutical ingredient or a mixture thereof at a temperature of 20 to 40 ° C, at a pH in the range of 4 to 7 and with stirring. A process for degrading an active pharmaceutical substance, diclofenac, codeine, ibuprofen or ketoprofen, according to claim 2, characterized in that the cultivation takes place for at least 20 hours. - 11 GB 307082 B6 A process for the degradation of an active pharmaceutical substance, diclofenac, codeine, ibuprofen or ketoprofen, according to claim 3, characterized in that the cultivation takes place for 20 to 72 hours. A process for the degradation of an active pharmaceutical ingredient, diclofenac, codeine, ibuprofen or 5 ketoprofen, according to claim 2, characterized in that the cultivation takes place at 28 ° C. A process for degrading an active pharmaceutical agent, namely diclofenac, codeine, ibuprofen or ketoprofen, according to claim 2, characterized in that the cultivation takes place at an initial concentration of active pharmaceutical agent of 0.5 to 6 g / l. A process for degrading an active pharmaceutical ingredient, diclofenac, codeine, ibuprofen or ketoprofen, according to claim 2, wherein the medium comprises an additional source C selected from the group: ethanol, dimethylsulfoxide, methanol. 15 8. Use of Raoultella sp. K.DF8 CCM 8678 for the degradation of an active pharmaceutical substance, namely diclofenac, codeine, ibuprofen or ketoprofen, from an aqueous medium.