EP4638769A1

EP4638769A1 - Method for preparing poly-3-hydroxybutyrate, method for producing low molecular weight poly-3-hydroxybutyrate biopolymer, m9 medium, gms medium, use of m9 medium with edta and gms medium for microorganism cultivation

Info

Publication number: EP4638769A1
Application number: EP23907930.4A
Authority: EP
Inventors: Wies aw SWIETNICKI; Otton Roubinek; Dariusz CHUDY; Renata KUREK; Katarzyna KOZAK; Joanna Chojniak-Gronek; Piotr KIERYL; Marzena KRZEK; Diana MIKIEWICZ; Agnieszka ROMANIK-CHRUSCIELEWSKA; Natalia Lukasiewicz; Iwona Sokolowska; Katarzyna FLORYS-JANKOWSKA; Micha SYPNIEWSKI; Marek Kubiszewski; Marcin Cybulski; Piotr Krzeczynski; Olga Michalak; Jolanta Janiszewska
Original assignee: Siec Badawcza Lukasiewicz - Instytut Chemii Przemyslowej imienia Profesora Ignacego Moscickiego
Priority date: 2022-12-22
Filing date: 2023-12-07
Publication date: 2025-10-29
Also published as: PL443234A1; WO2024136685A1

Abstract

The subject of the invention is a method for preparing poly-3-hydroxybutyrate, a method for producing low molecular weight poly-3-hydroxybutyrate biopolymer, a culture method, M9 medium, GMS medium, the use of M9 medium with EDTA and GMS medium for microorganism cultivation. More specifically, the invention provides a solution to the problem of polymer brittleness. Bacterial cultivation in special conditions and thermal degradation of the extracted polymer in unique conditions allow for a drastic reduction in molecular weight from above 200 kDa to below 10 kDa.

Description

Method for preparing poly-3-hydroxybutyrate, method for producing low molecular weight poly-3-hydroxybutyrate biopolymer, M9 medium , GMS medium, use of M9 medium with EDTA and GMS medium for microorganism cultivation

The subject of the invention is a method for preparing poly-3-hydroxybutyrate, a method for producing low molecular weight poly-3-hydroxybutyrate biopolymer, M9 medium, GMS medium, the use of M9 medium with EDTA and GMS medium for microorganism cultivation. More specifically, the invention provides a solution to the problem of polymer brittleness. Bacterial culture in specific conditions and thermal degradation of the extracted polymer in unique conditions allow for a drastic reduction in molecular weight from above 200 kDa to below 10 kDa.

Synthetic plastics based on styrene, propylene and ethylene polymers are cheap to produce, but they have a significant drawback: degradation in the environment by natural decomposition lasts for decades. The long environmental decomposition time for synthetic polymers and the lack of effective waste recycling have resulted in the plastic accumulation in the environment, often causing a serious problem for human and animal populations. For this reason, attempts have been made to produce plastics that are quickly degraded in the environment.

The most popular bioplastic is the poly-3-hydroxybutyrate (P3HB) polymer, the biosynthetic pathway of which has been described for the bacterium Ralstonia eutropha (Brigham et al., 2012). The production pathway was transferred to commercial E. coli to produce P3HB using standard genetic engineering methods and biotechnological solutions (Insomhun et aL, 2016; Li et al., 2016). The polymer is quickly biodegradable in the natural environment (Al-Khattaf et aL, 2022; Vodicka et aL, 2022), potentially solving the problem of environmental pollution. Unfortunately, the obtained P3HB biopolymer is brittle and has a melting point close to the decomposition temperature (Zarzyka et aL, 2021), which limits its applications in industry.

Poly[(R)-3-hydroxybutyrate] (P3HB) is a biopolymer widely distributed in nature, and its physicochemical and processing properties are closely related to, among others, the value of the number-average (Mn) and weight-average (Mw) molecular weight. From the point of view of polymer processing, it is beneficial to develop a method for controlled production of low molecular weight/oligomeric P3HB through controlled degradation of high molecular weight P3HB obtained from the biotechnological process. The thermal degradation of bacterial poly[(R)-3-hydroxybutyrate] was of interest to many research groups [Grassie et al. Polym Degrad Stab 1984;6:47, 6:95, 6:127; Kunioka et al. Macromolecules 1990;23:1933; Lehrle et al. Macromolecules 1994:27:3782; Kopinke et al. Polym Degrad Stab 1996;52:25; Lee et al. Macromol Chem Phys 2001 ;202: 1257; Aoyagi et al. Polym Degrad Stab 2002;76:53; Nguyen et al. Biomacromolecules]. It was observed that the early phase of thermal degradation occurs at temperature above 160°C only by random chain scission (cis-elimination). Therefore, when processing polymer obtained from bacterial strains, at temperatures just above the melting point, an unfavorable, rapid loss of biopolymer molecular weight is observed.

One way to achieve controlled thermal degradation of polymer is to carry out the process in the presence of metal cations. Kawalec et al. showed that thermal degradation of P3HB can be induced by the formation of sodium, potassium, tetrabutylammonium salts at the carboxyl ends of polymer chains after adding appropriate acetates to P3HB having free carboxyl groups. In the experiments performed, sodium or potassium acetate or tetrabutylammonium acetate and the biopolymer were mixed together in the presence of methylene chloride, and after evaporating the solvent the obtained samples were then degraded in an oven at 150°C. Kang Ju Kim et al. (Polymer Degradation and Stability 93, 776-785, 2008) investigated the effect of metallic additives on the degradation of P3HB. Isothermal (at 180°C) and non-isothermal (up to 250°C) degradation reactions were performed by adding 400 ppm of metal chloride: NaCI (dehydrated), CaCh (dehydrated), ZnCL (dehydrated), SnCL (dehydrated) and AlCh (dehydrated) to polymer samples. Sodium and calcium cations accelerated a random chain scission of P3HB molecules, also decreasing the thermal degradation temperature. The effect of Zn, Sn and Al compounds on the thermal degradation of P3HB was small. Kang Ju Kim et al. (Polym Degrade Stab 2006;91 :769) also showed that the presence of calcium or magnesium enhances the depolymerization reaction of P3HB molecules, while zinc (Zn) compound practically does not catalyze depolymerization.

Many attempts have been made to change the polymer properties, and the best known are those involving the use of a second copolymer in the biosynthesis process in organisms (Volova et al., 2021 ; Zhila et al., 2022; Shen et al., 2018; Das et al., 2019; Hokamura et al., 2015; Sohn et al., 2020). The obtained bioplastics had reduced brittleness, allowing for better forming in technological processes. Another solution was to combine P3HB secretion with biosynthetic blocking of the polymerization process of the secreted polymer (Miyahara et al., 2019; Sakurai et al., 2022).

The biosynthetic polymer P3HB has a molecular weight of over 100 kDa. There were also attempts to reduce the brittleness of the P3HB biopolymer in the finished product through controlled thermal decomposition using the {3-elimination mechanism in the polymer (Wang et al., 2016; Liu et al., 2009). The described process is long-lasting and requires controlled reaction conditions.

Patent Application US2008299627A1 (2008-05-16) discloses a method for producing polyhydroxyalkanoates (PHAs) using Bacillus sp. with succinate as a carbon source. The PHAs comprise more than 95% of poly (3-hydroxyvalerate-co-4-hydroxyvalerate) (P3HV-co-P4HV). High purity polyhydroxyvalerate (PHV) is also disclosed, which includes PHV produced by microorganisms using succinate as a carbon source.

The European patent EP2780461 B1 (filed on November 17, 2011) describes a method for producing microbial copolyesters, particularly microbial hydroxyalkanoate copolymers, from two different sucrose-containing raw materials. The inventors faced the problem of producing biodegradable polyesters characterized by a combination of material strength and ductility so as to make them suitable for a broad range of applications. A method for producing hydroxyalkanoate copolymers, which comprises: (i) pre-treating a sucrose-containing raw material in an acidic solution; (ii) feeding the pre-treated raw material into a bioreactor containing polyhydroxyalkanoate producing microbial cells; (iii) cultivating the polyhydroxyalkanoate producing microbial cells to form a cell mass containing hydroxyalkanoate copolymers; (iv) recovering of hydroxyalkanoate copolymers from the cell mass.

BRPI0501139A (filled 2005-04-04) describes the process of producing polyhydroxyalkanoates using bacteria selected from the genera Burkholderia, Waltersia and Alcaligenes, grown in culture media in which the main carbon source is glycerol or residues containing significant amounts of it, derived from the biodiesel production of vegetable oils such as soybean oil, corn oil, cottonseed oil, peanut oil; palm oil, castor oil; or from a mixture of residues from different sources rich in glycerol. The process consists of four steps, and in steps I and II bacteria selected from the genera Burkholderia, Waltersia and Alcaligenes are grown in a culture medium containing said carbon sources and supplemented with other nutrients to provide nitrogen, phosphorus, sulfur, magnesium, potassium and oxygen, and trace elements such as manganese, cobalt, zinc, molybdenum, nickel, copper, boron, so that balanced biomass growth occurs in which there is practically no accumulation of intracellular polyhydroxyalkanoate.

Then, step III of the process begins, which is the phase of PHA synthesis itself, by limiting or depleting of one or more of the nutrients in the culture medium together or separately. During step III, the produced PHA consists mainly of 3-hydroxybutyrate monomers. The synthesis process proceeds until the intracellular PHA content reaches 20% to 80% of the dry mass of the produced biomass, and then the biomass is concentrated, and the polymer material is extracted using conventional methods described in the literature.

The patent description CA2314151A1 (filed 1998-12-22) discloses biocompatible polyhydroxyalkanoate compositions, in particular based on biotechnologically produced homo- and copolymers of 4-hydroxybutyric acid, with controlled degradation rates. In one embodiment, the polyhydroxyalkanoates contain additives, such as for example, pore forming agents to alter the degradation rates. In another embodiment, the polyhydroxyalkanoates are formed from mixtures of monomers or contain pendant groups or modifications in their backbones to alter their degradation rates. In yet another embodiment polyhydroxyalkanoates are chemically modified. Methods of manufacturing devices which increase porosity or exposed surface area can be used to alter degradability. As shown in the examples, these polyhydroxyalkanoate compositions have extremely favorable mechanical properties as well as are biocompatible and degrade within desirable time frames under physiological conditions. These polyhydroxyalkanoate materials provide a wider range of polyhydroxyalkanoate degradation rates than currently available.

Methods of processing these materials, particularly for therapeutic, prophylactic or diagnostic applications, or into devices that can be implanted or injected, are also described.

The patent description KR102067342B1 ( filed 2018-08-17) describes E. coli for the polyhydroxybutyrate production, a method for its manufacturing and a method for producing polyhydroxybutyrate. E. coli for the polyhydroxybutyrate production produces a high concentration of polyhydroxybutyrate, and thus it can be effectively used for the polyhydroxybutyrate production.

The patent descriptions KR102408174B1 , KR102408178B1 and KR102408177B1 (filed 2020-06-08) describe a recombinant microorganism for the production of poly-3- hydroxybutyrate (PHB). Regarding the production method, since the recombinant microorganism is characterized by an excellent cell growth rate and polyhydroxybutyrate production efficiency in the presence of wood hydrolyzate, it is possible to increase the efficiency of bioplastics production.

The patent application WO2022091685A1 (filed 2021-09-30) discloses a PHB copolymer with which the aggregation of PHB copolymer particles during the production process is inhibited and which has high bulk density. It is also an object to provide a production method for said copolymer. The PHB copolymer production method comprises: (a) a step of performing an enzymatic treatment using a specific enzyme on a microorganism containing a PHB copolymer having a composition ratio that is a 3HB unit/a hydroxyalkanoate unit other than a 3HB unit and is within the recommended range; (b) a step of adding a surfactant after adjusting the pH to a specific range by adding an alkaline aqueous solution; (c) a step of preparing an aqueous suspension with pH of at most 7.0 and a shear viscosity within a specified range; and (d) a step of spray drying.

The patent description EP2346922 (filed 2008-10-15) discloses a process for controlled degradation of polyhydroxyalkanolates and products prepared from them. The process for producing foamed polyhydroxyalkanolate (PHA) oligomers and/or polymers with reduced molecular weight comprises reacting at least one PHA with at least one carbonate salt at a temperature of 50°C to 300°C. Furthermore, oligomers prepared according to the above process with a mass of 1.2 kDa to 25 kDa in the form of a foamed substance are disclosed. The degradation process was carried out without the use of a solvent in the extruder, to obtain the polymer after degradation in the form of a brittle foam.

Due to the homogeneity of the solution and the extended degradation time compared to the difficult to control minute degradation times described in EP2346922, degradation in a solvent allows the process to be controlled to obtain a range of oligomers and/or polymers with various values of average number molecular weight (Mn) and average weight molecular weight (Mw), depending on the needs arising from the further use of these compounds. Moreover, the process from EP2346922 carried out directly in the extruder on the high molecular weight polymer itself leads to anionic moieties in the resulting oligomer and/or polymer, which are less stable, as shown in patent EP19991898 (filed on 2006-03-20). Only their transformation into polymers containing non-degradable terminal groups obtained through the chemical protonation reaction protects the polymers against further thermal degradation during processing. Despite the prior art solutions described above, there is a constant need to obtain an effective solution to the problem of brittleness of polyhydroxybutyrate polymers, which depends on its molecular weight and, above all, on the crystal structure and secondary crystallization processes. The higher the degree of crystallinity and the higher molecular weight, the more brittle and more difficult to process is the polymer due to its rheological properties. These parameters are critical for further processing of the P3HB polymer. The solution to the problem involving a change in the crystal structure combined with a drastic molecular weight reduction of the polymer has not yet been provided.

The aim of the present invention is to solve the problem of polymer brittleness by using a process that allows obtaining a polymer with a molecular weight of less than 10 kDa. Bacterial culture in special conditions and thermal degradation of the extracted polymer in unique conditions allow for a drastic molecular weight reduction from more than 200 kDa to less than 10 kDa.

Surprisingly, the solution according to the invention is an excellent response to such market demand. Shorter time of the thermal degradation process in the presence of chemical compounds and the high efficiency of the process made it possible to obtain the P3HB biopolymer with the molecular weight of less than 10 kDa and a polydispersity index (PDI) of approximately 3.0. The obtained polymer can be used for further technological processes.

The implementation of a such specified goal and the solution of the problems described in the prior art related to the brittleness of polyhydroxy butyrate polymers were achieved in the present invention by modifying P3HB.

The subject of the invention is a method for preparing poly-3-hydroxybutyrate, characterized by the following steps: a) culturing the bacterial inoculum in GMS medium; b) adding the bacterial inoculum to the bioreactor containing M9 medium with EDTA; c) disintegration of the bacterial mass; d) extraction of P3HB biopolymer from bacterial mass.

Preferably, the culture of the inoculum comprises culture in GMS medium in an amount of up to 10% of the volume in the bioreactor, at a temperature in the range of 20 to 42°C, preferably 37°C, for 10 to 16 hours, preferably between 12 and 14 hours, and the optical density ODeoo of the obtained inoculum is in the range of 0.5 to 1 .5 AU, and it is carried out on shakers in the range from 50 to 300 rpm, preferably 180 rpm. Preferably, the culture in the reactor is carried out in a semi-batch, batch, flow or sequential manner, preferably semi-batch manner.

Preferably, the culture in the reactor is carried out in a semi-batch manner, and the glucose concentration during feeding of the medium in the reactor is in the range of 100 to 300 mg/dL.

Preferably, mechanical, hydraulic or pneumatic mixing is used in the culture in reactor. Preferably, the bacterial inoculum is added to the bioreactor containing M9 medium with EDTA, the pH value of the culture is in the range of 6.5 to 8.5, preferably 7.2, and it is adjusted with an ammonia solution at a concentration of 10 - 30%, preferably 16%, the initial concentration of glucose in the reactor is in the range of 50 to 2000 mg/dL, preferably 500 mg/dL, the culture is aerated by continuously supplying air to the system, wherein its amount per 1 dm³ of medium is in the range of 0.01 to 3.0 (Ndm³/min) of air/1 dm³ of medium, and mechanical mixing is also used in the range of 50 to 1000 rpm using turbine, propeller and blade type mixers, preferably of Rushton type.

Preferably, the culture is stopped when the value of the optical density ODeoo of the obtained culture in the reactor remains constant after 3 consecutive measurements.

Preferably, the step of the bacterial mass disintegration comprises:

- suspension of the bacterial mass in 1 to 2 volumes (w/v) of demineralized water;

- cooling the suspension to a temperature of 0-30°C;

- sonication of the mixture for at least 10 minutes with cooling.

Preferably, in the step of the extraction of the P3HB biopolymer from the bacterial mass:

- the broken bacterial mass is extracted with boiling acetone for at least 30 minutes;

- the solid is retained after extraction with acetone;

- the solid is dried;

- the solid is extracted with boiling chloroform for at least 60 minutes; and then it is filtered on a Celite-type silica bed and chloroform is removed;

- solvent residues are removed in a dryer under reduced pressure and at a temperature not exceeding 90°C for at least 2 hours.

Preferably, the poly-3-hydroxybutyrate is a high molecular weight poly-3- hydroxybutyrate biopolymer.

Another subject of the invention is a method for producing a low molecular weight poly- 3-hydroxybutyrate biopolymer from a high molecular weight poly-3-hydroxybutyrate biopolymer, characterized in that, in addition to the steps defined above, the preparation of the low molecular weight P3HB biopolymer includes the step of the degradation of the high molecular weight P3HB polymer to a low molecular weight polymer.

Preferably, the controlled P3HB degradation reaction is carried out in the presence of sodium and potassium cations, in the organic solvent dimethyl sulfoxide (DMSO), to obtain after degradation the reaction mixture in the form of a homogeneous solution from which the degradation product is precipitated with an antisolvent, so that the pH of the mixture thus obtained is under 6.

Preferably, the controlled P3HB degradation reaction is carried out in the presence of sodium and potassium cations in the form of acetates.

Preferably, the controlled P3HB degradation reaction is carried out in the organic solvent dimethyl sulfoxide (DMSO) at a temperature of 120 to 150°C.

Preferably, the degradation product is an oligomer and/or polymer.

Preferably, the antisolvent is water with the addition of inorganic and/or organic acid. Preferably, the obtained product does not contain halogenated solvents.

Preferably, the degradation is carried out in a solvent at a temperature 30-60°C lower than the temperature of high molecular weight P3HB degradation.

Another subject of the invention is M9 medium, characterized in that the M9 medium comprises: a) M9 salt solution (10x) in an amount of 50 to 250 mL/L of medium; b) 50% glucose in an amount of 5 to 100 mL/L of medium; c) 1 M MgSO₄ in an amount of 0.5 to 5.0 mL/L of medium; d) 1 M CaCL in an amount of 0.1 to 2.0 mL/L of medium; e) 1 mg/mL of biotin in an amount of 0.5 to 5.0 mL/L of medium; f) 1 mg/mL of thiamine in an amount of 0.5 to 5.0 mL/L of medium; g) a trace element solution (100x) in an amount of 2 to 30 mL/L of medium; h) 100 mg/mL of proline in an amount of 1 to 20 mL/L of medium; i) 5 mg/mL of tetracycline in an amount of 0.5 to 20 mL/L of medium.

Preferably, M9 salt solution (10x) comprises: a) NazHPCU in an amount of 60 to 120 g/L of solution; b) K₂HPO₄ in an amount of 20 to 60 g/L of solution; c) NaCI in an amount of 2 to 20 g/L of solution; d) NH4CI in an amount of 2 to 20 g/L of solution. Preferably, the M9 medium comprises: a) M9 salt solution (10x) in an amount of 100 mL/L of medium; b) 50% glucose in an amount of 10 mL/L of medium; c) 1 M MgSO₄ in an amount of 1 mL/L of medium; d) 1 M CaCl₂ in an amount of 0.3 mL/L of medium; e) 1 mg/mL of biotin in an amount of 1 mL/L of medium; f) 1 mg/mL of thiamine in an amount of 1 mL/L of medium; g) trace element solution (100x) in an amount of 10 mL/L of medium; h) 100 mg/mL of proline in an amount of 8 mL/L of medium; i) 5 mg/mL of tetracycline in an amount of 2.5 mL/L of medium.

Preferably, M9 salt solution (10x) comprises: a) Na₂HPO₄ in an amount of 75.2 g/L of solution; b) KH₂HPO₄in an amount of 30 g/L of solution; c) NaCI in an amount of 5 g/L of solution; d) NH4CI in an amount of 5 g/L of solution.

Preferably, the pH value of the salt solution for M9 (x10) is in the range of 6.5 - 9.0, preferably 7.2.

Preferably, the trace salt solution for M9 (100x) comprises: a) EDTA in an amount of 2 to 20 g/L of solution; b) FeCl₃-6H₂O in an amount of 0.2 to 1.5 g/L of solution; c) ZnCh in an amount of 0.02 to 0.2 g/L of solution; d) CUCI₂-2H₂O in an amount of 0.005 to 0.05 g/L of solution; e) COCI₂-2H₂O in an amount of 0.002 to 0.02 g/L of solution; f) H₃BO₃ in an amount of 0.002 to 0.02 g/L of solution; g) MnCl₂-4H₂O in an amount of 0.0005 to 0.004 g/L of solution.

Preferably, the trace salt solution for M9 (100x) comprises: a) EDTA in an amount of 5 g/L of solution; b) FeCl₃-6H₂O in an amount of 0.83 g/L of solution; c) ZnCfe in an amount of 0.084 g/L of solution; d) CUCI₂-2H₂O in an amount of 0.013 g/L of solution; e) C0CI₂-2H₂O in an amount of 0.010 g/L of solution; f) H₃BO₃ in an amount of 0.010 g/L of solution; g) MnCl₂-4H₂O in an amount of 0.0016 g/L of solution. Preferably, the pH value of the trace salt solution for M9 (x100) is in the range of 6.5 - 9.0, preferably 8.2.

Preferably, the M9 medium is intended for the cultivation of microorganisms producing poly-3-hydroxybutyrate.

Another subject of the invention is GMS medium for inoculum cultivation, characterized in that the GMS medium comprises: a) K₂HPO₄ in an amount of 2 to 20 g/L of solution; b) K₂HPO₄ in an amount of 0.1 to 5 g/L of solution; c) NH4CI in an amount of 0.1 to 10 g/L of solution; d) FeNH4 citrate in an amount of 0.05 to 1 g/L of solution; e) Na citrate in an amount of 0.1 to 10 g/L of solution; f) K₂SO₄ in an amount of 0.1 to 5 g/L of solution; g) trace salts for GMS in an amount of 1 to 15 mL/L of solution;

Preferably, GMS medium comprises: n amount of 8 g/L of solution; n amount of 2 g/L of solution; amount of 3 g/L of solution; d) FeNH4 citrate in an amount of 0.15 g/L of solution; e) Na citrate in an amount of 3 g/L of solution; f) K₂SO₄ in an amount of 0.9 g/L of solution; g) trace salts for GMS in an amount of 3 mL/L of solution.

Preferably, the following components are sterile added to the GMS medium: a) 5 mg/mL of tetracycline in an amount of 50 to 500 μL/50 mL of medium; b) 1 mg/mL of thiamine in an amount of 0.5 to 20 μL/50 mL of medium; c) 50% glucose in an amount of 0.1 to 10 mL/50 mL of medium; d) 50% MgSO₄ in an amount of 50 to 500 μL/50 mL of medium; e) 1.68% CaCl₂ in an amount of 50 to 500 μL/50 mL of medium; f) 100 mg/mL of proline in an amount of 50 to 1000 μL/50 mL of medium.

Preferably, the following components are sterile added to the GMS medium: a) 5 mg/mL of tetracycline in an amount of 125 μL/50 mL of medium; b) 1 mg/mL of thiamine in an amount of 5 μL/50 mL of medium; c) 50% glucose in the amount of 500 mL/50 mL of medium; d) 50% MgSO₄ in the amount of 190 μL/50 mL of medium; e) 1.68% CaCl₂ in the amount of 190 μL/50 mL of medium; f) 100 mg/mL of proline in an amount of 300 μL/50 mL of medium.

Preferably, the trace salt solution for GMS (100x) comprises: a) MnSCUxHzO in an amount of 0.1 to 5 g/L of medium; b) ZnSO₄x7H₂O in an amount of 0.1 to 10 g/L of medium; c) COCI₂X6H₂O in an amount of 0.1 to 10 g/L of medium; d) Na₂NMoO4x2H₂O in an amount of 0.1 to 10 g/L of medium; e) CaCl₂x6H₂O in an amount of 0.5 to 15 g/L of medium; f) CUSO₄X5H₂O in an amount of 0.1 to 10 g/L of medium; g) H₃BO₃ in an amount of 0.1 to 5 g/L of medium; h) 32% HCI in an amount of 50 to 300 mL/L of medium.

Preferably, the trace salt solution for GMS (100x) comprises: a) MnSCuxH₂O in an amount of 1 g/L of medium; b) ZnSO₄x7H₂O in an amount of 2.78 g/L of medium; c) COCI2X6H₂O in an amount of 1.86 g/L of medium; d) Na₂NMoO₄x2H₂O in an amount of 2 g/L of medium; e) CaCl₂x6H₂O in an amount of 4.5 g/L of medium; f) CUSO₄X5H₂O in an amount of 1.85 g/L of medium; g) H₃BO₃ in an amount of 0.5 g/L of medium; h) 32% HCI in an amount of 100 mL/L of medium.

Preferably, the GMS medium is intended for the cultivation of microorganisms producing poly-3-hydroxybutyrate.

Another subject of the invention is the use of M9 medium with EDTAfor the cultivation of microorganisms producing poly-3-hydroxybutyrate with a controlled content of heavy metals in polymer.

Another subject of the invention is the use of GMS medium for the cultivation of microorganisms producing poly-3-hydroxybutyrate with a controlled content of heavy metals in the polymer.

Preferably, the high molecular weight poly-3-hydroxybutyrate is degraded to a low molecular weight polymer under controlled conditions.

The invention is illustrated in the drawing, wherein: figure 1 shows a map of the pIBA/phaCABchro plasmid; figure 2 shows the ¹H NMR spectrum of the substance: P3HB; figure 3 shows the spectrum of H055 sample (before degradation); Figure 4 shows a comparison of the ¹H NMR spectrum of H055 sample (before degradation, lower) and H055/6/124/1 sample (after degradation, upper);

Figure 5 shows an exemplary molecular weight distribution for H055 sample before (H055) and after thermal decomposition (H055/6/124/1) in gel permeation chromatography tests.

In order to better understand the invention, the solution is illustrated in the embodiments presented below. These examples are not intended to limit the invention, but only to provide a more detailed understanding of its possible embodiments.

Examples

Construction of a plasmid for expression of the P3HB polymer in E. coli

The genetic construct used to synthesize the poly-3-hydroxybutyrate (P3HB) polymer in E. coli cells has the gene system shown in Fig. 1. The synthesis of the P3HB polymer occurs with the participation of enzymes encoded by the phaC, phaA and phaB genes constituting the phaCAB operon, which has been described in Ralstonia eutropha (also known as Cupriavidus necatof). The plasmid contains the phaCAB operon (without optimized codons), hybrid promoter and chromosomal DNA fragments.

Example 1

Bacterial cultures

M9 medium in a volume of 6 liters was prepared in the bioreactor, with the following composition: dissolving 75.2 g of Na₂HPO<, 30 g of K₂HPO₄, 5 g of NaCI, 5 g of NH4CI in 800 mL of demineralized water, adjusting the pH to 7.2 with NaOH, filling with demineralized water to the volume of 1 L and autoclaving (15 min, 121 °C).

**The solutions were filtered through a 0.22 pm filter.

***A solution of trace elements for M9 medium (100x concentrated) in a volume of 1 L was prepared by dissolving 5 g of EDTA in 800 mL of demineralized water and adjusting the pH to 8.2 with NaOH. Then, 0.83 g of FeCl₃-6H₂O, 84 mg of ZnCI₂, 13 mg of CUCI₂-2H₂O, 10 mg of COCI₂-2H₂O, 10 mg of H₃BO₃, 1.6 mg of MnCl₂-4H₂O were added to the solution, dissolved by stirring and filled with demineralized water to the volume of 1 L and autoclaved (15 min, 121°C).

Bacterial cultures for bioreactor inoculation were carried out in sterile GMS medium composed of:

*Trace salt solution for GMS in a volume of 1 L was prepared by dissolving 1.0 g of MnSO₄xH₂O, 2.78 g of ZnSO₄x7H₂O, 1.86 g of CoCI₂X6H₂O, 2.0 g of Na₂NMo04x2H₂0, 4.5 g of CaCl₂x6H₂O, 1.85 g CuSO₄x5H₂O, 0.5 g H₃BO₃ in 800 mL of demineralized water, 100 mL of 32% HCI was added, filled up with demineralized water to the volume of 1 L and autoclaved (15 min, 121°C).

Components filtered through a 0.22 pm filter were sterile added to the GMS medium:

Bacterial inoculum culture in a total volume of 500 mL (divided into 50 mL flasks) was incubated at 37°C under shaking (180 rpm ) for 14 hours. The optical density (ODeoo) was then measured, and it was 0.7-0.8 AU. The bacterial inoculum was sterile added to the bioreactor.

The culture was carried out in a semi-batch manner - for the first period time of 5 hours, the culture was carried out in a batch manner, and then medium with glucose was added to the culture in order to maintain the appropriate level of glucose in the bioreactor. The initial volume of the medium was 6 L, and after adding the remaining ingredients, i.e. magnesium and calcium salts and inoculum, the volume of the medium was approx. 7 L. Approximately 1.5 L of medium with glucose was added to the reactor during cultivation.

Measurements (readings) were made every hour - the values of (i) optical density of the culture ODeoo, (ii) glucose concentration mg/dL, (iii) pH, (iv) oxygen concentration DO%, (v) stirrer speed (mixing) RPM and (vi) airflow AirFlow were recorded. The pH value = 7.2 of the culture was adjusted with a 16% ammonia solution, previously filtered. During monitoring of culture conditions, when the glucose level dropped below 100 mg/dL, external dosing of fresh M9 medium with 40% glucose and other ingredients was started. When the biomass increased, the pump flow was controlled to maintain the glucose concentration at the level of about 100-200 mg/dL. The glucose concentration should be maintained at a level that provide optimal growth of microorganisms. The minimum concentration providing good growth of microorganisms is about 100 mg/dL. The cultures were stopped when the spectrophotometrically measured ODsoo value remained at a constant level after three consecutive measurements, which indicated the termination of biomass growth.

Example 2

Disintegration of the bacterial mass

1. The total culture volume was centrifuged and the supernatant was discarded;

2. The precipitate was frozen at -20°C for a minimum of 12 h.

3. The precipitate was suspended in demineralized water in a volume as close as possible to the volume of bacterial biomass (proportion close to 1 :1);

4. The suspension was autoclaved at 121°C for 20 minutes, then cooled;

5. The suspension was sonicated using an ultrasonic sonicator U400st (Hielscher), sonotrode H14, for 15 minutes in portions no larger than 150 mL, the vessel was covered with ice, and the temperature was maintained below +10°C;

6. The suspension was frozen at -20°C for a minimum of 12 h.

Example 3

Extraction of P3HB biopolymer from bacterial mass on a small scale

After disintegration of the bacterial mass described above, the P3HB polymer was extracted by a procedure utilizing acetone and chloroform. For small scale, the protocol described below was followed each time:

1. Transferring the precipitate (biomass) to a round-bottom flask equipped with a reflux condenser;

2. Adding acetone in an amount 3 times greater than the biomass (i.e. 3 g of biomass - 12 mL of acetone);

3. Heating to boiling (the boiling point of acetone is 56.05°C) and stirring intensively at boiling for 30 min;

4. Filtering off the precipitate using a Buchner funnel (on paper);

5. Drying (20 min - may be shorter, 90°C);

6. Transferring the precipitate to a round-bottom flask equipped with a reflux condenser;

7. Adding chloroform in an amount 12 times greater than the mass of the precipitate (i.e. 3 g of precipitate -36 mL of acetone);

8. Heating to boiling (boiling point of chloroform is 61.2°C) and stirring intensively at boiling for 60 min; 9. Filtering off the precipitate using a funnel with paper and celite (approx. 2-3 cm);

10. Evaporation of the filtrate on a rotary evaporator under vacuum;

11. Drying the flask content (evaporation of residual solvents) for at least 2 hours at 90°C (pressure 30 mBa);

12. Submitting for ¹H-NMR/GPC analysis.

Example 4

NMR analysis of P3HB polymer

The tested samples are slightly soluble in chloroform. To dissolve the substance, the sample (maximum 5 mg of the tested substance) was poured with deuterated chloroform (approx. 0.7 mL) and dissolved in a tightly closed glass bottle on a water bath at a temperature of about 30°C using ultrasound for about 30 minutes.

The P3HB polymer is well characterized by nuclear magnetic resonance (NMR). Characteristic signals in the ¹H NMR spectrum for the substance: P3HB (in CDCI₃) (fig.

2)

Signal (5 ppm in CDCI3): Identification

1.28 doublet, 3H; CH3 group

2.47 multiplet, 1 H; CH2 group

2.61 multiplet, 1 H; CH2 group

5.26 multiplet, 1 H; CH group

In the solution according to the invention, it was found that the controlled P3HB degradation reaction can be carried out in the presence of sodium and potassium cations, preferably in the form of acetates, in the organic solvent dimethyl sulfoxide (DMSO), preferably at a temperature of 120-150°C, to obtain after degradation the reaction mixture in the form of a homogeneous solution, from which the degradation product, i.e. oligomer and/or polymer, is precipitated with an anti-solvent, preferably water with the addition of inorganic and/or organic acid, so that the pH of the mixture obtained in this manner is below 6. The product obtained in this manner, after filtration, washing with water and drying, has the form of free-flowing, non-dusty powders, and does not contain halogenated solvents that are difficult to remove, but are commonly used in extraction methods for the separation of high molecular weight Poly[(R)-3- hydroxybutyrate P3HB from bacterial biomass in biotechnological processes. An important aspect of the invention is to conduct the degradation in the solvent at a temperature lower than the temperature of high molecular weight P3HB decomposition (by 30-60°C), which is only about 10°C higher than its temperature, and this has a beneficial effect on the reaction control. Moreover, the oligomers and/or polymers obtained in the degradation process in DMSO can be used for special purposes, e.g. in pharmacy and biomedicine, due to the elimination of residual halogenated solvents during the workup of the post-reaction mixture after degradation, and on the other hand, high acceptable limits for the residual solvent DMSO in special products.

Example 5

Degradation of high molecular weight P3HB polymer to low molecular weight polymer

0.82 g of P3HB polymer (Mn=471.1 kDa; Mw =941.1 kDa; PDI=2) in dimethyl sulfoxide (DMSO, 15 mL) was placed in the flask and heated to 120°C. Potassium acetate (0.0025 g) was added to the mixture. It was heated under reflux with stirring using magnetic stirrer for 4 hours. Heating was stopped and a solution of 150 pL of 1 M HCI in 60 mL of water and was added in portions to the stirring hot solution. After cooling the polymer suspension to room temperature, P3HB was filtered off. The polymer was washed with water (5 x 30 mL) and dried under vacuum (20 mBar, 70°C). 0.65 g of low molecular weight P3HB was obtained (Mn = 2.9 KDa; Mw = 7.9 Kda; PDI = 2.72). Degradation yield 80%.

Example 6

Physico-chemical analysis of the P3HB polymer after degradation - analysis using the NMR technique

The identity of the polymer after degradation was analyzed using the NMR technique. The identity of the polymer (H055) was confirmed before degradation. The obtained ¹H NMR spectrum contains all the bands characteristic for the P3HB polymer (fig. 3). The sample (H055/6/124/1) was then tested after degradation. The spectrum (fig. 4) confirms the presence of P3HB polymer in the sample with a purity above 95%.

Example 7

Physico-chemical analysis of the P3HB polymer after degradation - analysis using the gel permeation chromatography (GPC) method The P3HB polymer was analyzed by gel permeation chromatography (GPC). The study was performed at the Faculty of Materials Science and Engineering of the Warsaw University of Technology. The technique is based on the analysis of the interactions of the polymer with the column bed, which was derivatized with carbon chains. The P3HB polymer dissolved in an organic solvent is hydrophobic and the degree of its interaction with the hydrophobic bed depends on the length of the polymer: the longer the polymer, the stronger the interactions with the bed and the greater the retention on the column. Column calibration with the use of polymers of known molecular weight allows the determination of the molecular weight range for the P3HB polymer. A description of the test procedures and an exemplary sample analysis are provided below (fig. 5).

The samples were dissolved in HPLC-grade chloroform (Chempur) for ~36 h: for the first 24 h at 37°C, followed by another 12 h at 50°C due to the fact that not all samples dissolved. The solutions had a concentration of 2 mg/mL.

The number average (Mn) molecular weight and weight average (Mw) molecular weight were determined by GPC. Measurements were performed on an Agilent 1200 series HPLC modular system with a refractive index detector (RID). The system was equipped with two PLgel 5 pm MIXED-C columns (300 x 7.5 mm) connected in series. Calibration was performed on 12 polystyrene standards with weights (Mp) in the range of 474 g/mol-1,800,000 g/mol.

Measurements were made at 35°C. HPLC-grade chloroform was used as the mobile phase with a flow rate of 0.7 mL/min. Before analysis, the previously prepared solutions were filtered through a PTFE membrane with a pore size of 0.2 pm.

Data were recorded using “ChemStation for LC” and analyzed using “ChemStation GPC Data Analysis Software".

GPC analysis (fig. 5) of the P3HB sample after thermal decomposition (H055/6/124/1 ) showed a number average (Mn) molecular weight of 2.9 kDa, weight average (Mw) molecular weight of 7.9 kDa and a polydispersity index (PDI) of 2.72. However, the P3HB sample before thermal decomposition (H055) had a number average (Mn) molecular weight of 471.1 kDa, a weight average (Mw) molecular weight of 941.1 kDa and a polydispersity index (PDI) of 2.41. The results of the GPC analysis confirm the reduction of the molecular weight of the P3HB polymer from high molecular weight to low molecular weight. NMR analysis confirms the identity of the substance before and after thermal degradation. These data confirm that the thermal degradation procedure of the P3HB polymer provides a polymer with a significantly reduced molecular weight and a purity of 95% (proton NMR) with respect to the original substance.

Example 8

0.82 g of P3HB polymer (Mn = 471.1 kDa; Mw = 941.1 kDa; PDI = 2) in dimethyl sulfoxide (DMSO, 15 mL) was placed in the flask and heated to 120°C. Potassium acetate (0.025 g) was added to the mixture. It was heated under reflux with stirring using magnetic stirrer for 4 hours. Heating was stopped and a solution of 150 pi L of 1 M HCI aq in 60 mL of water was added in portions to the stirred hot solution. After cooling the polymer suspension to room temperature, P3HB was filtered off. The polymer was washed with water (3 x 30 mL) and dried under vacuum (20 mBar, 70°C). 0.53 g of low molecular weight P3HB was obtained (Mn = 1.1 ; Mw = 2.0; PDI = 1.82). Degradation yield 62%.

Example 9

0.82 g of P3HB polymer (Mn = 471.1 kDa; Mw = 941.1 kDa; PDI = 2) in dimethyl sulfoxide (DMSO 15 mL) was placed in the flask and heated to 120°C. Potassium acetate (0.00085 g) was added to the mixture. It was heated under reflux with stirring using magnetic stirrer for 4 hours. Heating was stopped and a solution of 150 pL of 1M HCI aq in 60 mL of water was added in portions to the stirred hot solution. After cooling the polymer suspension to room temperature, P3HB was filtered off. The polymer was washed with water (3 x 30 mL) and dried under vacuum (20 mBar, 70°C). 0.7 g of low molecular weight P3HB was obtained (Mn = 6.3 kDa; Mw = 715.2 kDa; PDI = 2.41). Degradation yield 85%.

Example 10

9.95 g of P3HB polymer (Mn = 152 kDa; Mw = 401 kDa; PDI = 2.64) in dimethyl sulfoxide (DMSO, 300 mL) was placed in the flask and heated to 120°C. Potassium acetate (0.030 g) was added to the mixture. It was heated under reflux with stirring using magnetic stirrer for 4 hours. Heating was stopped and a solution of 1 mL of 1 M HCI aq in 1200 mL of water was added in portions to the stirred hot solution. After cooling the polymer suspension to room temperature, P3HB was filtered off. The polymer was washed with water (3 x 400 mL) and dried under vacuum (20 mBar, 70°C). 7.96 g of low molecular weight P3HB was obtained (Mn = 2.7 kDa; Mw = 7.0 kDa; PDI = 2.59). Degradation yield 80%.

Example 11

16.6 g of P3HB polymer (Mn = 471.1 kDa; Mw = 941.1 kDa; PDI = 2) in dimethyl sulfoxide (DMSO, 300 mL) was placed in a flask and heated to 120°C. Potassium acetate (0.051 g) was added to the mixture. It was heated under reflux with stirring using magnetic stirrer for 4 hours. Heating was stopped and a solution of 1.2 mL of 1 M HCI aq in 1200 mL of water was added in portions to the stirred hot solution. After cooling the polymer suspension to room temperature, P3HB was filtered off. The polymer was washed with water (3 x 500 mL) and dried under vacuum (20 mBar, 70°C). 14.66 g of low molecular weight P3HB was obtained (Mn = 60 kDa; Mw = 499.4 kDa; PDI = 5.76). Degradation yield 88%.

SUBSTITUTE SHEET (RULE 26)

Claims

1. A method for preparing poly-3 hydroxybutyrate, characterized in that it comprises the steps: a) culturing the bacterial inoculum in GMS medium; b) adding the bacterial inoculum to the bioreactor containing M9 medium with EDTA; c) disintegration of the bacterial mass; d) extraction of P3HB biopolymer from bacterial mass.

2. The method for preparing poly-3-hydroxybutyrate according to claim 1 , characterized in that the culture of the inoculum comprises culture in GMS medium in an amount of up to 10% of the bioreactor volume, at a temperature in the range of 20 to 42°C, preferably 37°C, for 10 to 16 hours, preferably between 12 and 14 hours, and that the optical density ODeoo of the obtained inoculum is in the range of 0.5 to 1.5 AU, and it is carried out on shakers in the range of 50 to 300 rpm, preferably 180 rpm.

3. The method for preparing poly-3-hydroxybutyrate according to claim 1 , characterized in that the culture in the reactor is carried out in a semi-batch, batch, flow or sequential mode, preferably semi-batch mode.

4. The method for preparing poly-3-hydroxybutyrate according to claim 3, characterized in that the culture in the reactor is carried out in a semi-batch mode, and the glucose concentration during feeding of the medium in the reactor is in the range of 100 to 300 mg/dL.

5. The method for preparing poly-3-hydroxybutyrate according to claim 3, characterized in that mechanical, hydraulic or pneumatic mixing is used in the culture in reactor.

6. The method for preparing poly-3-hydroxybutyrate according to claim 3, characterized in that the bacterial inoculum is added to a bioreactor containing M9 medium with EDTA, wherein the pH value of the culture is in the range of 6.5 to 8.5, preferably 7.2, and it is adjusted with an ammonia solution at a concentration of 10 - 30%, preferably 16%, the initial concentration of glucose in the reactor is in the range of 50 to 2000 mg/dL, preferably 500 mg/dL, the culture is aerated by continuously supplying air to the system, wherein its amount per 1 dm³ of medium is in the range of 0.01 to 3.0 (Ndm³/min) of air/1 dm³ of medium, and wherein the

SUBSTITUTE SHEET (RULE 26) mechanical mixing is also used in the range of 50 to 1000 rpm using turbine, propeller and blade type mixers, preferably Rushton type mixer.

7. The method for preparing poly-3-hydroxybutyrate according to claim 3, characterized in that the culture is stopped when the value of the optical density ODeoo of the obtained culture in the reactor remains constant after 3 consecutive measurements.

8. The method for preparing poly-3-hydroxybutyrate according to claim 1 , characterized in that the step of the bacterial mass disintegration comprises:

- suspending the bacterial mass in 1 to 2 volumes (w/v) of demineralized water;

- cooling the suspension to a temperature of 0-30°C;

- sonication the mixture for at least 10 minutes with cooling.

9. The method for preparing poly-3-hydroxybutyrate according to claim 1 , characterized in that in the step of extracting the P3HB biopolymer from the bacterial mass:

- the solid is retained after extraction with acetone;

- the solid is dried;

10. The method for preparing poly-3-hydroxybutyrate according to claim 1 , characterized in that the poly-3-hydroxybutyrate is a high molecular weight poly-3- hydroxy butyrate biopolymer.

11. A method for producing a low molecular weight poly-3-hydroxybutyrate biopolymer from a high molecular weight poly-3-hydroxybutyrate biopolymer, characterized in that, in addition to the steps defined in claims 1 to 10, the production of the low molecular weight P3HB biopolymer comprises the step of the degradation of the high molecular weight P3HB polymer to the low molecular weight polymer.

12. The method for producing the low molecular weight P3HB biopolymer according to claim 11 , characterized in that the controlled degradation reaction of P3HB is conducted in the presence of sodium and potassium cations, in an organic solvent dimethyl sulfoxide (DMSO), to obtain after degradation the reaction mixture in the form of a homogeneous solution from which the degradation product is precipitated

SUBSTITUTE SHEET (RULE 26) with an antisolvent, so that the pH of the mixture obtained in this manner is below 6.

13. The method for producing low molecular weight P3HB biopolymer according to claim 12, characterized in that the controlled P3HB degradation reaction is carried out in the presence of sodium and potassium cations in the form of acetates.

14. The method for producing low molecular weight P3HB biopolymer according to claim 13, characterized in that the controlled P3HB degradation reaction is carried out in the organic solvent dimethyl sulfoxide (DMSO) at a temperature of 120 to 150°C.

15. The method for producing low molecular weight P3HB biopolymer according to any of claims 11 to 14, characterized in that the degradation product is an oligomer and/or polymer.

16. The method for producing low molecular weight P3HB biopolymer according to any of claims 11 to 15, characterized in that the antisolvent is water with the addition of inorganic and/or organic acid.

17. The method for producing low molecular weight P3HB biopolymer according to any of claims 11 to 16, characterized in that the obtained product does not contain halogenated solvents.

18. The method for producing low molecular weight P3HB biopolymer according to any of claims 11 to 17, characterized in that the degradation is carried out in a solvent at a temperature 30-60°C lower than the temperature of high molecular weight P3HB decomposition.

19. M9 medium, characterized in that the M9 medium contains: a) M9 salt solution (10x) in an amount of 50 to 250 mL/L of medium; b) 50% glucose in an amount of 5 to 100 mL/L of medium; c) 1 M MgSCU in an amount of 0.5 to 5.0 mL/L of medium; d) 1 M CaCL in an amount of 0.1 to 2.0 mL/L of medium; e) 1 mg/mL of biotin in an amount of 0.5 to 5.0 mL/L of medium; f) 1 mg/mL of thiamine in an amount of 0.5 to 5.0 mL/L of medium; g) solution of trace elements (100x) in an amount of 2 to 30 mL/L of medium; h) 100 mg/mL of proline in an amount of 1 to 20 mL/L of medium; i) 5 mg/mL of tetracycline in an amount of 0.5 to 20 mL/L of medium.

20. M9 medium according to claim 19, characterized in that M9 salt solution (10x) contains:

SUBSTITUTE SHEET (RULE 26) a) Na₂HPCU in an amount of 60 to 120 g/L of solution; b) K₂HPO₄ in an amount of 20 to 60 g/L of solution; c) NaCI in an amount of 2 to 20 g/L of solution; d) NH4CI in an amount of 2 to 20 g/L of solution.

21. M9 medium according to claim 19, characterized in that it preferably contains: a) M9 salt solution (10x) in an amount of 100 mL/L of medium; b) 50% glucose in an amount of 10 mL/L of medium; c) 1 M MgSC>4 in an amount of 1 mL/L of medium; d) 1 M CaCL in an amount of 0.3 mL/L of medium; e) 1 mg/mL of biotin in an amount of 1 mL/L of medium; f) 1 mg/mL of thiamine in an amount of 1 mL/L of medium; g) trace element solution (10Ox) in an amount of 10 mL/L of medium; h) 100 mg/mL of proline in an amount of 8 mL/L of medium; i) 5 mg/mL of tetracycline in an amount of 2.5 mL/L of medium.

22. M9 medium according to claim 20, characterized in that the M9 salt solution (10x) preferably contains: a) Na₂HPO4 in an amount of 75.2 g/L of solution; b) K₂HPO₄ in an amount of 30 g/L of solution; c) NaCI in an amount of 5 g/L of solution; d) NH4CI in an amount of 5 g/L of solution.

23. M9 medium according to claim 20, characterized in that the pH value of the salt solution for M9 (x10) is in the range of 6.5 - 9.0, preferably 7.2.

24. M9 medium according to claim 19, characterized in that the trace salt solution for M9 (100x) contains: a) EDTA in an amount of 2 to 20 g/L of solution; b) FeCl₃-6H₂O in an amount of 0.2 to 1.5 g/L of solution; c) ZnCk in an amount of 0.02 to 0.2 g/L of solution; d) CUCI₂-2H₂O in an amount of 0.005 to 0.05 g/L of solution; e) C0CI₂-2H₂O in an amount of 0.002 to 0.02 g/L of solution; f) H₃BO₃ in an amount of 0.002 to 0.02 g/L of solution; g) MnCl₂-4H₂O in an amount of 0.0005 to 0.004 g/L of solution.

25. M9 medium according to claim 24, characterized in that the trace salt solution for M9 (100x) preferably contains: a) EDTA in an amount of 5 g/L of solution;

SUBSTITUTE SHEET (RULE 26) b) FeCl₃-6H₂O in an amount of 0.83 g/L of solution; c) ZnCl2 in an amount of 0.084 g/L of solution; d) CUCI₂-2H₂O in an amount of 0.013 g/L of solution; e) C0CI2-2H2C) in an amount of 0.010 g/L of solution; f) H₃BO₃ in an amount of 0.010 g/L of solution; g) MnCl₂-4H₂O in an amount of 0.0016 g/L of solution.

26. M9 medium according to claim 20, characterized in that the pH value of the trace salt solution for M9 (x100) is in the range of 6.5 - 9.0, preferably 8.2.

27. M9 medium according to claim 19, characterized in that it is for cultivating microorganisms producing poly-3-hydroxybutyrate.

28. GMS medium for inoculum cultivation, characterized in that the GMS medium contains a) K2HPO4 in an amount of 2 to 20 g/L of solution; b) K₂HPO₄ in an amount of 0.1 to 5 g/L of solution; c) NH4CI in an amount of 0.1 to 10 g/L of solution; d) FeNH4 citrate in an amount of 0.05 to 1 g/L of solution; e) Na citrate in an amount of 0.1 to 10 g/L of solution; f) K₂SO₄ in an amount of 0.1 to 5 g/L of solution; g) trace salts for GMS in an amount of 1 to 15 mL/L of solution;

29. GMS medium according to claim 28, characterized in that GMS medium preferably contains: a) K2HPO4 in an amount of 8 g/L of solution; b) K₂HPO₄ in an amount of 2 g/L of solution; c) NH4CI in an amount of 3 g/L of solution; d) FeNH4 citrate in an amount of 0.15 g/L of solution; e) Na citrate in an amount of 3 g/L of solution; f) K₂SO₄ in an amount of 0.9 g/L of solution; g) trace salts for GMS in an amount of 3 mL/L of solution.

30. GMS medium according to claim 28, characterized in that the following components are sterile added to the GMS medium: a) 5 mg/mL of tetracycline in an amount of 50 to 500 μL/50 mL of medium; b) 1 mg/mL of thiamine in an amount of 0.5 to 20 μL/50 mL of medium; c) 50% glucose in an amount of 0.1 to 10 mL/50 mL of medium; d) 50% MgSCU in an amount of 50 to 500 μL/50 mL of medium;

SUBSTITUTE SHEET (RULE 26) e) 1.68% CaCl₂ in an amount of 50 to 500 μL/50 mL of medium; f) 100 mg/mL of proline in an amount of 50 to 1000 μL/50 mL of medium.

31. GMS medium according to claim 30, characterized in that components are sterile added to the GMS medium, preferably in the following amounts: a) 5 mg/mL of tetracycline in an amount of 125 μL/50 mL of medium; b) 1 mg/mL of thiamine in an amount of 5 μL/50 mL of medium; c) 50% glucose in an amount of 500 mL/50 mL of medium; d) 50% MgSCUin an amount of 190 μL/50 mL of medium; e) 1 .68% CaCh in an amount of 190 μL/50 mL of medium; f) 100 mg/mL of proline in an amount of 300 μL/50 mL of medium.

32. GMS medium according to claim 28, characterized in that the trace salt solution for GMS (100x) contains: a) MgSO₄xH₂O in an amount of 0.1 to 5 g/L of medium; b) ZnSO₄x7H₂O in an amount of 0.1 to 10 g/L of medium; c) C0CI2X6H₂O in an amount of 0.1 to 10 g/L of medium; d) Na₂NMoO4x2H₂O in an amount of 0.1 to 10 g/L of medium; e) CaCl₂x6H₂O in an amount of 0.5 to 15 g/L of medium; f) CUSO₄X5H₂O in an amount of 0.1 to 10 g/L of medium; g) H₃BO₃ in an amount of 0.1 to 5 g/L of medium; h) 32% HCI in an amount of 50 to 300 mL/L of medium.

33. GMS medium according to claim 32, characterized in that the trace salt solution for GMS (100x) preferably contains: a) MgSO₄xH₂O in an amount of 1 g/L of medium; b) ZnSO₄x7H₂O in an amount of 2.78 g/L of medium; c) C0CI2X6H₂O in the amount of 1.86 g/L of medium; d) Na₂NMoG4x2H20 in an amount of 2 g/L of medium; e) CaCl₂x6H₂O in an amount of 4.5 g/L of medium; f) CUSO₄X5H₂O in an amount of 1.85 g/L of medium; g) H₃BO₃ in an amount of 0.5 g/L of medium; i) 32% HCI in an amount of 100 mL/L of medium.

34. The use of M9 medium with EDTAfor the cultivation of microorganisms producing poly-3-hydroxybutyrate with a controlled content of heavy metals in the polymer.

35. The use of GMS medium for the cultivation of microorganisms producing poly-3- hydroxybutyrate with a controlled content of heavy metals in the polymer.

SUBSTITUTE SHEET (RULE 26)

36. The use according to claim 34 or 35, characterized in that the high molecular weight poly-3-hydroxybutyrate is degraded to a low molecular weight polymer under controlled conditions.

SUBSTITUTE SHEET (RULE 26)