WO1998007879A1

WO1998007879A1 - Process to obtain polyhydroxy alkanoates and the use thereof

Info

Publication number: WO1998007879A1
Application number: PCT/DE1997/001772
Authority: WO
Inventors: Klaus Metzner; Marion Sela; Dietmar Runkel; Torsten Sembritzki
Original assignee: Buna Sow Leuna Olefinverbund Gmbh
Priority date: 1996-08-20
Filing date: 1997-08-19
Publication date: 1998-02-26
Also published as: DE19633475A1; DE19633475C2

Abstract

The process enables polyhydroxy alkanoates to be obtained in an amorphous state from the cell structures of an alkanoate accumulating microorganism. According to said process the cellular mass in aqueous phase is first subjected to mechanical forces, subsequently undergoes alkaline treatment at temperatures above 70 °C and lysis, and is then treated with hydrogen peroxide at temperatures of 70-95 °C, non-polymer cellular components being separated between the processing stages.The hydroxyalkanoate particles so obtained can be used as a polymer dispersion for surface coating, as a polymer-processing auxiliary agent or as supporting material in medicine.

Description

Process for the production of polyhydroxyalkanoates and their use

The process is used to obtain polyhydroxyalkanoates (PHA) in the form of polyhydroxybutyric acid, polyhydroxyvaleric acid or copolymers of these polyhydroxyalkanoic acids, which are contained in the cell mass of microorganisms or plant biomass which accumulate these polyhydroxyalkanoic acids as internal cellular reserve.

To obtain PHA from the cell mass of a microorganism, solution and precipitation processes are known in which the PHA is removed from the cell structure in dissolved form and separated from the residual biomass. According to US 3044942, 1,2-dichloroethane in a mixture with ethanol, according to EP 024810 dichloromethane and according to US 32756 0 chloroform are used as solvents for the PHA. Using suitable precipitants, the PHA is obtained in a solid form from the PHA-containing solution separated from the cell mass. According to DD 229428, polyhydroxybutyric acid is extracted from the biomass with acetic anhydride at elevated temperatures, here at 100 to 140 ° C. The polyhydroxybutyric acid precipitates out of the polymer solution in a powdery state by lowering the temperature. The handling of large amounts of solvents and the associated recycling and disposal costs have a disadvantageous effect. This applies in particular to chlorinated hydrocarbons as solvents. In addition, these processes are often associated with severe molecular weight loss and the formation of a crystalline PHA fraction.

Patent application WO 95/08620 describes a recovery process for a solid product contained in a cell mass, in which the cell mass is converted into a dissolved form and is separated off together with the aqueous fermentation broth in such a way that the insoluble product, for example indigo contained in the cell mass , is in solid form. The cell substances are dissolved by adding an alkali metal hydroxide solution at a treatment temperature between 40 and 100 ° C. The extraction process for cell constituents can also be carried out by lysing the cell substances by means of an enzymatic treatment, in particular by using lysozyme, proteases, DNAsen, RNAsen or a combination of these.

To obtain a plastic from a microorganism cell that accumulates such a substance, patent application WO 94/24302 describes a method which uses an oxidizing medium in the presence of a complexing agent to dissolve the cell shells of the microorganisms. For example, a hydrogen peroxide at a treatment temperature between 60 and 180 ° C is used as an oxidizing agent for the production of polyhydroxybutyric acid. As a preliminary stage to the oxidative treatment, a physical preparation of the cell material is possible, in particular through a temperature treatment in the range between 100 and 200 ° C.

Another effect of peroxides, for example hydrogen peroxide, is used in patent application WO 94/10289 for a process for the recovery of products from an aqueous composition for the decomposition of nucleic acids, if the amount of nucleic acid is sufficient to increase the viscosity such that the following Process steps are affected. The concentration of the nucleic acids dissolved in water is given as> 0.1 g / l, for example 0.5-20 g / l.

With regard to the stated task, there are different disadvantages with these known methods. The handling of large amounts of solvent is problematic in the extraction process. Usual amounts, based on the biomass, are in the range from 8: 1 to 20: 1. These solvents have to be disposed of properly or laboriously worked up, which causes considerable costs and additional equipment. Residual solvents in the product, especially when using poorly environmentally compatible substances, must be removed in additional cleaning steps. The requirements for product purity are very high, especially in applications in the food sector and in medical applications. Another disadvantage of extractive processes is the formation of a crystalline fraction in the polyhydroxyalkanoate structure. The polymers present in the microorganism in completely amorphous form experience a change in some physical properties. These can have a negative influence on the film formation and the crosslinkability of the molecules, for example in dispersion applications. The other processes have a very specific effect, ie they cause denaturation, lysis or separation of a few cell components and are generally not sufficiently suitable for the production of a pure product in one treatment step, so that a meaningful combination of different processing steps is necessary.

The object of the invention is to obtain a polyhydroxyalkanoate, in particular polyhydroxybutyric acid, polyhydroxyvaleric acid or a copolymer of these with a particle size of less than 1.5 μm from an aqueous cell suspension of a microorganism that accumulates these polymers and to use them. It is necessary that the granules of the polymer embedded in the cells of the microorganisms in the amorphous state are retained as unchanged as possible in this state and that a proportion of the crystalline structure of the polymer is excluded or kept negligibly low.

The object is achieved by the invention presented in the claims. The present method is characterized in that, without the use of PHA solvents, a dispersion with a high proportion of polyhydroxyalkanoates is formed by combining mechanical, chemical and enzymatic treatments of the bacterial biomass suspension with intermediate separation of the extracted cell components. The PHA content achieved depends on the PHA content of the biomass and the number of processing steps used. In the multi-stage process, the cell walls are first destroyed by the action of mechanical forces on the microorganism cells in the aqueous phase. Such mechanical forces can be generated in known devices by the generation of shock, friction, shear or pressure forces. are caused. It is also possible to let such forces act on the cell material in their pure form, as well as in superimposed or alternating form.

In the first step, the bacterial biomass is subjected to mechanical cell disruption, for example in an agitator ball mill or a high-pressure homogenizer. The disruption serves to destroy the solid cell envelope and release water-soluble or water-rinsable cell components. Furthermore, an enlarged surface for the attack of biological or chemical substances is created. The separation between the treatment steps is carried out by separation, centrifugation or by decanter. This removes dissolved substances, fine cell fragments and impurities in the clear run, which in each case leads to a concentration of the polyhydroxyalkanoate granules. The following step is followed by an alkaline treatment, for example with sodium hydroxide solution, in a pH range of 8-12. This treatment is accompanied by heating the suspension to a temperature above 70 ° C and leads to the hydrolysis of certain cell components.

After neutralization, an enzymatic purification, preferably of a proteolytic character, can be used for the lysis of released cell components. In contrast to enzymatic digestion processes, a lower residual biomater content and thus a purer product, high storage stability and the possibility of producing easily resuspendable dispersion powders, which represent the most stable storage form, are achieved. Mixtures of cell wall-lysing enzymes in combination with pure proteases have proven successful. After the lysed cell components have been separated off, hydrogen peroxide bleaching is carried out at a temperature between 70 ° C. to 95 ° C., followed by washing. Mixing ratios of one part of polyhydroxyalkanoate granules with residual cell components to 5 to 100 parts of H ₂ O ₂ in concentrations of 1 to 33% are used. This treatment results in the denaturation of residual proteins, the achievement of an optically pure white product and a preservation which improves the storage stability and the susceptibility to putrefaction. Depending on the microorganism and the desired product purity, treatment stages can be omitted, this applies mainly to the complex and expensive enzyme treatment, which is only necessary in exceptional cases. For the processing of polyhydroxybutyric acid, the sequence of treatment steps has proven to be the optimal treatment method. Prewashing with alkalis in the sense of an alkaline disruption or mechanical cell destruction in an alkaline medium did not improve the work-up result. If the processing steps are used in a different order, depending on the microorganism used, the result may deteriorate considerably. A reduction in the separation steps between the treatments is rarely useful to obtain a very pure product.

Such polymer particles obtained from cells of microorganisms by the method according to the invention are particularly suitable for the production of polymer dispersions. This can be done directly or by resuspending the PHA particles present in dried form according to the recovery process described in a liquid medium, preferably in water. It is possible to use dispersing aids, film formers, anti-foaming agents, etc. The proportion of solid phase to liquid phase depends on the intended application of the corresponding dispersion.

These dispersions are suitable for the production of coatings or coatings on surfaces. In particular, these can be surfaces of a cellulose material, such as cardboard or paper, since in addition to the barrier properties, this ensures that these coated materials are completely biodegradable. In addition to being applied to surfaces, this effect can also be achieved by introducing the dispersion into the paper pulp.

Likewise, a dispersion produced by the process according to the invention can be used as an auxiliary for the processing of polymers, in particular when processing PVC. The nitrogen content in the polyhydroxyalkanoate has a stabilizing effect on the polymer to be processed. Especially because of the extremely good human or veterinary compatibility of the polyhydroxyalkanoic acids, dispersions made from them are suitable as a carrier material for the introduction of medical contrast agents and active ingredients.

The process according to the invention is explained in more detail using the following examples:

A fermentation process in a batch process cultivates microorganisms through a corresponding growth phase with subsequent initiation of accumulation for the storage of polyhydroxybutyric acid in the cell. The polymer concentration that can be achieved depends very much on the type of microorganism and the fermentation conditions. For technically relevant processes, a polymer content of more than 50% of the dry cell mass is assumed.

1st example

By cultivating the microorganism Methylobacterium rhodesianum, a PHB content of 52% was achieved after the fermentation process had ended. The starting biomass concentration based on the dry matter was 50 g / l.

1st step: cell disruption in a high-pressure homogenizer (850 bar, 2 passages) and separation of cell fragments via a plate separator (5000 g)

2nd step: Sodium hydroxide solution treatment at 85 ° C and pH 11 for 2 hours

3rd step: hydrogen peroxide bleaching with H ₂ O (33%) in a ratio of 1:10 for 2 h

85 ° C

Step 4: washing and thickening using a plate separator

Dispersion: average Particle diameter: 1.34 μm solids content: 38 g / l

PHB content of the solid: 78% nitrogen content: 0.7% The target grain range is clearly achieved. The indication of the nitrogen content in the product reflects the residual biomass content and serves as a measure of the product purity.

2. Example according to example 1, but:

1st step: mechanical cell disruption in an agitator ball mill (2 passages) and separation of cell fragments using a plate separator (5000 g)

2nd step: sodium hydroxide solution treatment at 85 ° C. and pH 9 for 3 h, washing over

Dish separator

3rd step: hydrogen peroxide bleaching with H ₂ O ₂ (33%) in a ratio of 1:10 for 4 h

75 ° C

Step 4: washing and thickening using a plate separator

Dispersion: average Particle diameter 1.22 μm solids content 39 g / l PHB content of the solid 82% nitrogen content 0.4%

3. Example according to example 1, however

2nd step: Sodium hydroxide solution treatment at 85 ° C. and pH 8 for 4 h, washing over

Dish separator

3rd step: enzyme treatment by Sfreptomyces species in connection with

Proteases

4th step: 3-fold washing and thickening using a plate separator

Dispersion: average Particle diameter: 1.12 μm solids content: 38 g / l PHB content of the solid: 92%

Nitrogen content: 0.3%

4th example

In the fourth example, the possibility of producing a dispersion powder with subsequent resuspension was investigated. The crude dispersion prepared in Example 2 was used for this.

according to example 2, but:

Step 5: spray drying the dispersion to produce a dipersion powder

(average particle diameter reached 5.2 μm)

Step 6: Resuspending in water using Ultraturrax

Dispersion: average Particle diameter: 1.43 μm solids content: 35 g / l

PHB content of the solid: 82% nitrogen content: 0.7%

5th example

In the last example, the experiment was carried out on the basis of a biomass with a significantly higher starting PHB content. By cultivating the microorganism Alcaligenes eutrophus, a PHB content of 76% was achieved after the fermentation process had ended. The starting biomass concentration based on the dry matter was 52 g / l. In contrast to the previous examples, the fermenter broth was already concentrated here using a plate separator and brought to a dry biomass content of 137 g / l.

1st step: cell disruption in a high-pressure homogenizer (1000 bar, 1 passage) and separation of cell fragments via a plate separator (5000 g)

2nd step: Sodium hydroxide solution treatment at 75 ° C. and pH 10 for 2 h, washing over

Dish separator

3rd step: hydrogen peroxide bleaching with H ₂ O ₂ (33%) in a ratio of 1:10 for 2 h 80 ° C

4th step: washing and thickening dispersion: medium. Particle diameter 0.95 μm solids content 42 g / l PHB content of the solid 95% nitrogen content 0.02%

Claims

claims

1 process for the production of polyhydroxyalkanoates, which are accumulated as granules in the course of a fermentation process in the cells of a microorganism, from a cell mass contained in an aqueous phase by one or more digestion of the cells and separation of cell components, characterized in that

a) in the aqueous phase, the cell walls of the microorganisms are first exposed to friction, impact, shear and / or pressure forces, b) the parts of the cell mass and polyhydroxyalkanoate granules remaining in the suspension are subjected to an alkaline treatment with a pH value from 8-12 at a temperature above 70 ° C for a period of 1 to 4 h and then the aqueous phase is neutralized, c) the remaining cell mass is subjected to lysis of the crushed and released cell components, d) the polyhydroxyalkanoate granules with residual components of the Cell mass in suspension in a ratio of 1 100 to 1 5 with hydrogen peroxide concentration 1% to 33% at a temperature of 70 ° C to 95 ° C, treated and e) the polyhydroxyalkanoate dispersion and f) optionally in a conventional manner be dried,

wherein part or all of the cell fragments obtained and the soluble substances of the cell mass converted into the liquid phase are separated from the fermenter broth between the process stages

2 The method according to claim 1, characterized in that the polyhydroxy alkanoate is a polyhydroxybutyric acid, a polyhydroxyvaleric acid or a copolymer thereof

3. The method according to claim 1 and 2, characterized in that the mechanical cell destruction takes place in an agitator ball mill or a high-pressure homogenizer.

4. The method according to claim 1 to 3, characterized in that the alkaline

Treatment of the cell mass contained in the aqueous phase with sodium hydroxide solution at a temperature greater than 70 ° C.

5. The method according to claim 1 to 4, characterized in that the lysis of the comminuted and released cell components is carried out by treatment with enzymes of Sfreptomyces species.

6. The method according to claim 1, characterized in that the dried polyhydroxyalkanoate particles are optionally redispersed with the addition of dispersing agents.

7. Use of a dispersion prepared according to claims 1 to 6 of polyhydroxyalkanoates for the production of coatings or coatings on surfaces.

8. Use of a dispersion of polyhydroxyalkanoates prepared according to claims 1 to 6 as a biodegradable filler.

9. Use of a dispersion prepared according to claims 1 to 6 of polyhydroxyalkanoates for use as processing aids for polymers.

10. Use of a dispersion of polyhydroxyalkanoates prepared according to claims 1 to 6 as a carrier material for introducing contrast media or pharmaceutical active ingredients in the medical field.