EP0205546A1

EP0205546A1 - Process for increasing the solubility of gases in an aqueous medium and an emulsion for carrying out said process

Info

Publication number: EP0205546A1
Application number: EP19860900342
Authority: EP
Inventors: Estera Szwajcer; Andreas Institute of Biotechnology LEONHARDT; Klaus Institute of Biotechnology MOSBACH
Original assignee: Individual
Current assignee: Individual
Priority date: 1984-12-21
Filing date: 1985-12-20
Publication date: 1986-12-30
Also published as: WO1986003773A1; SE8406634D0

Abstract

On ajoute au milieu aqueux une emulsion de silicones comprenant une emulsion aqueuse d'un copolymère de silicone et d'un composé hydrophile, éventuellement un mélange avec une silicone. Est également décrite l'émulsion de silicones utilisée dans ce procédé.A silicone emulsion comprising an aqueous emulsion of a silicone copolymer and a hydrophilic compound, optionally a mixture with a silicone, is added to the aqueous medium. Also described is the silicone emulsion used in this process.

Description

PROCESS FOR INCREASING THE SOLUBILITY OF GASES IN AN AQUEOUS MEDIUM AND AN EMULSION FOR CARRYING OUT

SAID PROCESS

The present invention relates to a process for increasing the solubility of gases in an aqueous me¬ dium and an emulsion for increasing the solubility of gases in an aqueous medium. The low solubility of oxygen in water presents a problem in biotechnical processes which depend on oxygen concentration. This is especially the case when enzymes or cells are used for enzymatic conver¬ sions requiring oxygen as co-substrate. Also cell ^■ growth within immobilised systems is often slowed down due to diffusional in and out hindrance of gases, notably oxygen.

Attempts at solving this problem have been made. Thus, a photosyntheti.cally active alga has been co- immobilised with- a microorganism catalysing the oxida- tive dea ination of amino acids to α-keto acids with L-amino acid oxidase (P. Wikstrδm, E. Sz.wajcer, P. Brodelius, K. Nilsson, K. Mosbach (1982) "Forma¬ tion of h-keto acids from amino acids using immobiliz- ed bacteria and algae", Biotechnol . Lett. 4:153-153). Alternatively, a high catalase activity in a microor¬ ganism has been used to convert hydrogen peroxide in situ into oxygen (0. Hoist, S.O. Enfors, B. Mattiasson (1982) "Oxygenation of immobilized cells using hydrogen- peroxide; a model study of Gluconobacter oxydans con¬ verting glycerol to dihydroxyacetone", Eur. J. Appl. Microbiol. Biotechnol. 14:64-68). Furthermore, manga¬ nese dioxide (P. Brodelius, K. Nilsson, K. Mosbach (1981) "Immobilized cells of Trigonopsis variabilis containing D-amino acid oxidase", Appl. Biochem. Bio¬ technol. 6:293-308) as well as charcoal (E. Szwajcer, P. Brodelius, K. Mosbach (1982) "Immobilized whole cells of Providencia sp. PCM 1298 containing L-amino oxidase",Enzyme Microb. Technol. 4:409-413) have been co-immobilised with microorganisms both to eliminate hydrogen peroxide and, at the same time, to produce oxygen.

Literature also reports on the addition of oxygen carriers, such as perfluoro compounds, to the medium (P. Adlercreutz, B. Mattiasson (1982) "Oxygen supply by hemoglobin or emulsions of perfluorochemicals", Eur. J. Appl. Microbiol . Biotechnol. 16:165-170). However, none of these techniques has proved to be sufficiently efficient to improve to any essen¬ tial extent the gas transport into and out of cells.

The present invention provides a novel process and a means for increasing the solubility of gases in an aqueous medium, thereby to facilitate the supply of, for example, 0- to cells and the removal of CO- from cells. The process according to the invention is charac¬ terised in that there is added to the aqueous medium a silicone emulsion in the form of an aqueous emulsion of a copolymer of a silicon-, and a hydrophilic compound, optionally in mixture with a silicone. This silicone emulsion functions as a gas carrier and comprises silicone compounds which are nonpolar, chemically inert and have high solubility for gases like oxygen and carbon dioxide. Since these silicone compounds are insoluble in water, they must, for the purpose of the present invention, be copolymerised with strong¬ ly hydrophilic compounds. These copolymers readily form small micelles, in the nonpolar interior of which substances such as oxygen may be retained.

To further increase the effect of the copolymer emulsion, silicone may be added thereto.

The silicone emulsion according to the invention is characterized in that it comprises a copolymer of a silicone and a hydrophilic compound, optionally in mixture with a silicone. In the following Examples, use is made, as a model system, of immobilised cells of Providencia sp. PCM 1298 (E. Szwajcer, P. Brodelius, K. Mosbach (1982) "Immobilized whole cells of Providencia sp. PCM 1298 containing L-amino acid oxidase". Enzyme Microb. Technol. 4:409-413). These cells catalyse the following transformation:

R.CH.NH₂.COOH+0₂+H₂0 =R.CO.COOH+NH₃+H₂0₂

The copolymer employed was a copolymer between poly¬ dimethyl siloxane and polyethylene oxide:

_CH₃₀-(_CH_2CH₂0)_n-CH₂CH=CH₂ +

CH- CH, CH,

' . , '.. „ ' . _ H-PtCl_c/toluene

+ H-Si- OSi) -OS H 2 6 .

, , m , **

CH₃ CH₃ CH₃

CH₃ CH₃ CH₃ _CH₃₀-(_CH₂CH₂0)_n-CH₂CH₂CH₂-Si-(OSi)_ffl-OSi-

CH₃ CH₃ CH₃

-CH₂CH₂CH₂-0-(CH₂CH₂0)_n"CH₃ (I)

The silicone employed was:

CH.

CH₃-(SiO)_n-Si-(CH₃)₃ (ID CH-

MATERIALS AND METHODS

Chemicals: Alginate was obtained from Alginate Industry (Girvan, U.K.). L-Methionine was obtained from Merck (Darmstadt, Federal Republic of Germany). Polydimethyl siloxanes as well as perfluorodecalin were purchased from Fluka (Buchs, Switzerland) and "Pluronic F68" from Serva (Heidelberg, Federal Republic of Germany). All other chemicals were of analytical grade and ob¬ tained from diverse commercial sources. The copolymer was prepared by hydrosilylation with silicones contain¬ ing a Si-H end group and polyethylene oxide (PEO) functionalised with an allyl end group (see formula I). Details of the preparation of the copolymer have pre¬ viously been described by Kendrick et al (T.C. Kendrick, B.M. Kingston, N.C. Lloyd, M.J. Owen (1967) "The surface chemistry of polyurethane foam formation. 1. Eq^'uili- brium surface tensions of polysiloxane-polyether block copolymer solutions", J. Coll. Interface Sci. 24:135-140) Preparation of the emulsions: The aqueous phase (buffer or growth medium) , silicone and copolymer were mixed and autoclaved for 20 min. The amount of copolymer employed was always 5% by weight of the total volume.

The silicone contents were 5, 10, 15 and 25% by volume, respectively, of the total volume. The mixture was then sonicated (5 min, 350 V) using as probe a "A 350 G" sonicator from Ultrasonic Ltd. , U.K. The measurement of oxygen solubility was done according to Leonhardt (A. Leonhardt (1984) Thesis, University of Freiburg). The viscosity was determined with a Brookfield Synchro-Lectic Viscosimeter (Brook- field E. Laboratories, Straughlan, USA). Cultivation of Providencia sp. PCM 1298: Fermentation of the bacteria was performed essentially as previously described (E. Szwajcer, P. Brodelius, K. Mosbach (1982) "Immobilized whole cells of Providencia sp. PCM 1298 containing L-amino acid oxidase", Enzyme Microb. Technol. 4:409-413).

Enzym assay: The amino acid oxidase activity within whole cells of Providencia was assayed either by a colorimetric method using dinitrophenyl hydrazine or reversed phase HPLC. In all cases, L-methionine was used as substrate. The corresponding α-keto-γ- ethiol butyric acid formed by the amino acid oxidase present in the cells was established by these two methods ((P. Brodelius, K. Nilsson, K. Mosbach (1981) "Immobilized cells of Trigonopsis variabilis contain¬ ing D-amino acid oxidase", Appl. Biochem. Biotechnol. 6:293-308). Enzyme stability test: This test was performed with suspended free cells of Providencia. One sample was kept in 50 M Tris-HCl buffer, pH 7.5, alone, while the other was substituted with 10% by volume silicone and 5% by weight copolymer in the same buffer solu- tion. The two samples in the test tubes with screw caps were incubated on a rocking table for 2 days at

30°C.

Cultivation of immobilised cells: Beads (diameter 2 mm) containing cells of Providencia (2% by weight cells) were packed in a small reactor (total volume 5 ml ) , and growth medium containing 10% by volume silicone and 5% by weight copolymer were saturated continuous¬ ly with pure oxygen (20 ml/min.) and pumped continuous¬ ly through the reactor (8 ml/h). In a parallel experi- ment, growth medium containing 10% by volume perfluoro- decalin, formula

and 5% by weight "Pluronic", formula

I

CH.

was pumped through the reactor. Furthermore, growth medium alone wa^ pumped through the reactor as a re¬ ference. Cultivation was carried out at 28 C for 15 h. Test of cell viability and L-amino acid oxidase acti¬ vity: About 50 beads cultivated in the reactor were taken out and solubilised in 0.1 M sodium phosphate buffer (5 ml, pH 7.5). After shaking for 2 h at room temperature, the beads were completely solubilised. After centifugation (10 min., 10,000 g), the cells were examined. The viability of the cells was deter¬ mined with an oxygen electrode. As a measure of cell growth, the protein content of the sample was used, obtained by the method of Lowry et al (O.H. Lowry,

N.J. Rosebrough, A.L. Farr, R.J. Randall (1951) "Pro¬ tein measurements with the Folin-phenol reagent", J. Biol. Chem. 193:265-275). The amino acid oxidase activity was assayed according to the above procedure. The invention will be illustrated in more detail in the following Examples in conjunction with the drawings.

Fig. 1 shows the result of a L-amino acid oxidase stability test on free cells of Providencia sp. PCM 1298. Fig. 2 shows the effect on the L-amino acid oxidase activity due to different concentrations of oxygen- carrying additives and cells in alginate beads. Fig. 3 shows the effect of different silicone additives to 5% by weight copolymer on the relative enzyme activi- ty due to different cell concentrations in the alginate beads. Fig. 4 shows the effect of two different -oxygen carriers on the L-amino acid oxidase activity due to the cell concentration in the alginate beads. Fig. 6 shows the effect of different concentrations of oxygen carrier on the relative viscosity x , at 20°C. Fig. 6

^J rel ^ shows the oxygen solubility at 37°C in different sub¬ stances. EXAMPLE 1

Enzyme stability This test was performed to show any negative effect due to the presence of copolymer or silicone on the amino acid oxidase activity in the bacteria. Cell samples were taken at different intervals, washed with 50 mM Tris-HCl, pH 7.5, and then incubated with 5 mM L-methionine in the same standard buffer. The amount of α-keto acid formed was measured by a colorimetric method using 2,4-dinitrophenyl hydrazine. As will appear from Fig. 1, both samples showed the same relative activity after 3 h incubation of the cells. After 22 h, the activity in the reference sample had decreased to about 87%, whereas the acti- vity in the sample containing silicone and copolymer remained practically unchanged. After 40 h, a decrease of the activity was noted in both samples, but the sample with silicone and copolymer showed a higher remaining activity. The result of this test shows that*the L-amino acid oxidase activity did not de¬ crease in the presence of the polymers. EXAMPLE 2

Cell growth in alginate beads and enzyme activity after incubation in growth medium in the presence of different oxygen-carrying additives

The effect of a mixture of 10% by volume sili¬ cone and 5% by weight copolymer on the cell growth and the amino acid oxidase activity was determined. The initial concentration of the cells within the beads was 2% by weight. The total reactor volume was

5 ml . The growth medium was pumped through the reactor (8 ml/h) and saturated continuously with pure oxygen gas (20 ml/min. ) .

As will appear from Table 1, the cell growth in alginate beads increased somewhat after 15 h. In analogy therewith, also the amino acid oxidase acti¬ vity increased to some extent. With the medium con¬ taining perfluorodecalin and "Pluronic" these values showed a slight increase. On the other hand, the medium enriched with silicone and copolymer in accordance with the present invention showed a drastic increase in growth and enzyme activity. TABLE 1

Reference, growth medium free from oxygen carrier

PFD, growth medium containing 10% by volume perfluoro- decalin and 5% by weight "Pluronic".

_

'Silicone, growth medium containing 10% by volume sili¬ cone and 5% by weight copolymer.

EXAMPLE 3

The effect of different concentrations of oxygen-carrying additives and cells present in alginate beads, on the L-amino acid oxidase activity

Four different cell concentrations in alginate beads were used, ranging from 2 to 8% (Fig. 2). The beads (diameter 2 mm) were present in a small column (total volume 2 ml ) . 5 mmol L-methionine in 50 mM Tris-HCl buffer, pH 7.5, were used as substrate. In all tests, the solutions or emulsions were saturated with pure oxygen. Different oxygen carriers (see Fig. 2) were added to the substrate solution. Neither silicone nor perfluorodecalin alone could be used since they are not soluble, nor is it possible to obtain any emulsions thereof. Pure oxygen gas was used to saturate all solutions. The enzymatic activity of the beads containing 2% by weight cells is defined as the rela¬ tive activxty "1" for all activity tests using 5 mM L-methionine in 50 mM Tris-HCl buffer, pH 7.5.

Samples were taken after 90 min. of incubation and analysed with HPLC (P. Brodelius, K. Nilsson. K. Mosbach (1981) "Immobilized cells of Trigonopsis variabilis containing D-amino acid oxidase", Appl. Biochem. Biotechnol. 6:293-308).

In the case of the reference sample, an increase of the said concentrations led to an increase of mea¬ sured L-amino-acid oxidase activity by a factor of 1.8. Using "Pluronic" which is a common emulsifier for perfluoro chemicals, no influence on the enzyme activity was observed. The presence of copolymer alone increased the oxygen-carrying capacity. Most likely, the silicone part of the copolymer molecule is respons¬ ible for the observed increase (see also Fig. 6). The structure of "Pluronic" resembles that of poly¬ ethylene oxide which is a part of the copolymer (I). Thus, it is the silicone part which is responsible for the oxygen-carrying capacity here expressed as increased oxidase activity. The addition of silicone to the emulsion of the copolymer in water increases the amino acid oxidase activity (see Fig. 3), pre- sumably because it increases the oxygen solubility (see also Fig. 6). The effect of silicone together with copolymer is more pronounced when the cell den¬ sity increases in the beads (Fig. 4).

A clear difference in L-amino acid oxidase ac- tivity for higher concentrations of cells is shown between the sample containing copolymer together with silicone than for perfluorodecalin together with "Plu¬ ronic" (see Fig. 4). Since relatively high concentra¬ tions of these acid carriers are required to obtain significant effects, there is a risk of a viscosity increase. As will appear from Fig. 5, the silicone increases the viscosity only slightly, even at a con- centration of 25% by volume, in contrast to the effect which perfluorodecalin has on the viscosity. Conclusions

The use of silicone copolymers as herein described is of great potential interest to a number of biotech- nical processes, particularly those which depend on oxygen concentration. Thus, silicone emulsions accord¬ ing to the invention may be used to facilitate the gas transport process in bacterial cells, yeast fungus cells, plant cells and animal cells.

The advantage which silicone gives over perfluoro chemicals is, inter alia, its low effect on the viscosi¬ ty, allowing high concentrations as well as a low price. As indicated in the Examples, the cell growth in the present model system is far more efficient than with perfluoro compounds.

Added separately, silicone and "Pluronic" also lead to a substantial increase in oxygen solubility. However, only the copolymers described herein lead to stable micellar solutions maintaining the oxygen entrapped. Since the production of these compounds from inexpensive raw materials is relatively simple, the compounds are highly useful for both immobilised and free microorganisms, plant and animal cells.

Claims

1. A process for increasing the solubility of gases in an aqueous growth medium, c h a r a c t e r i s e d in that there is added to the medium a silicone emulsion in the form of an aqueous emulsion of a copolymer of a silicone and a hydrophilic compound, optionally in mix¬ ture with a silicone.

2. A process as claimed in claim 1, c h a r a c ¬ t e r i s e d in that the hydrophilic compound in the copolymer is polyethylene oxide.

3. A process as claimed in claim 1, c h a r a c ¬ t e r i s e d in that the silicone is polydimethyl siloxane.

4. A silicone emulsion for increasing the solu¬ bility of gases in an aqueous medium, c h a r a c - t e r i s e d in that it comprises a copolymer of a silicone and a hydrophilic compound, optionally in mixture with a silicone.

5. A silicone emuls-ion as claimed in claim 4, c h a r a c t e r i s e d in that the hydrophilic compound is polyethylene oxide.

6. A silicone emulsion as claimed in claim 4 or 5, c h a r a c t e r i s e d in that the silicone is polydimethyl siloxane.