Novel co-biopolymers

Abstract

The present invention discloses a method for producing PHA- copolymer using bacteria, by using a two-step process. In the first step the bacteria are grown under heterotrophic conditions using an organic substance as carbon source and exponential growth conditions. In a second step the bacteria are then cultivated under autotrophic conditions under an atmosphere of H2, CO2 and O2, wherein the O2 content is less than 10 % (v / v) and the pressure is more than 1 barg and at least one precursor for further monomers is added before and / or during this step, wherein the precursor is selected from an organic substance comprising at least 3 carbon atoms and at least 2 oxygen atoms, wherein the organic substance in its hydrolyzed form comprises at least two of carbon atoms, which are part of a functional group selected from COOH, C=O, C-OH. By this the production of PHA-copolymers with unique properties and at a high rate is possible.

Description

[0001]CBCPT24013EPWO 05.06.2025Applicant: CO2BioClean GmbH Mergenthalerallee 73-75 65760 Eschborn Deutschland Novel co-biopolymers FIELD OF THE INVENTION The present invention relates to a method for producingpolyhydroxyalkanoate(PHA)-copolymers using a wild type bacte-ria and extracting the produced PHA in an efficient way. PHA is the general term for a range of diverse biodegrada- ble polymers that consist of polyesters of 3-hydroxyalkanoic acids. These polymers are of interest due to a broad range of applications and the fact that they are completely biodegrada- ble thus offering little or no long term waste issues. PHAs are generally classified as short chain length PHAs (sclPHAs), medium chain length PHAs (mclPHAs) or long chain length PHAs (lclPHAs), depending upon the number of carbon at- oms of the constituting monomers thereof. SclPHA comprises monomers of C3-C5, mclPHA comprises monomers of C6-C14, and lcl- PHA comprises monomers of more than 14 carbons (>Cl4). This variation in monomer chain length gives rise to different properties in the polymer, with sclPHAs and lclPHAs bothCBCPT24013EPWO 05.06.2025having different properties, sclPHAs having a high degree ofcrystallinity and being usually rigid and brittle, and lclPHAs being sticky and very difficult to handle. The properties of sclPHAs and lclPHAs limit the range of their applications. But since sclPHAs are easier obtainable there is need of sclPHAs with improved properties. PHA structures can vary in two ways. First, PHAs can vary according to the structure of the pendant groups, which are typically attached to a carbon atom having (R)-stereochemis- try. The pendant groups form the side chain of hydroxy alka- noic acid not contributing to the PHA-carbon backbone. Second, PHAs can vary according to the number and types of their re- peat units. For example, PHAs can be homopolymers, copolymers, or terpolymers. These variations in PHA structure can cause variations in their physical characteristics. These physical characteristics make PHAs useful for a number of products that may be commercially valuable. The stereochemistry in the monomers may be only R or S, or monomers of both types may be present. This further influences the properties of the polymer. The several types of PHAs make PHAs a versatile family of polymers with properties tuneable by molecular design. For ex- ample, short chain length scl-PHA are divided into P3HB, com- monly simply polyhydroxobutyrate (PHB), P4HB, (valeric acid copolymer) PHBV, PHBH, P3HB4HB, medium chain length mcl-PHA are PHBH (hexanoic acid copolymer), PHBO (octanoic acid copol-ymer) and PHBD (dodecanoic acid copolymer).The chemical structure of PHAs may be described as a poly- meric chain formed by repetitions of the following unit:CBCPT24013EPWO 05.06.2025 Where R is an alkyl or alkenyl group of variable length and m and n are integers, in some polymers R and m assuming the following values: PHB: R=CH3, m=1 PHBV: R=CH3or CH3-CH2-, m=1 P4HB: R=H, m=2 P3HB4HB: R=H for m=2 or R=CH3 for m=1 (PH3,4B)For mclPHA the length of the alkyl chain of R can be dif-ferent, e.g. for polyhydroxyhexanoate PHBH R is CH3-CH2-CH2-and m equals 2. PHA-copolymers comprise a mixture of several different mon- omer units. The term copolymer as used in this application may also comprise polymer with more than two different monomer units, e.g. three (terpolymers) or more. Due to their structure the PHA monomer units contain a chi- ral carbon atom. The polymer may therefore comprise monomers differing in their configuration. PHA synthesized by organisms usually only contains monomers in R-configuration due to the enzymatic pathway. Besides plants and other organisms, bacteria are very use-ful for producing PHA. In recent years many efforts were takento use genetically modified or unmodified bacteria (Wild type)in order to produce PHA also at an industrial scale.CBCPT24013EPWO 05.06.2025Schlegel et al. Nature 1961, 191, 463-465 “Formation and utilization of poly-^-hydroxybutyric acid by Knallgas bacteria (hydrogenomonas)” found that PHAs, namely PHB can be producedunder nutritional stress conditions, especially using an at-mosphere comprising CO2, H2and O2. Ayaaki Ishizaki and Kenji Tanaka Journal of fermentationand bioengineering 1990, 69(3), 170-174 “Batch Culture of Al- caligenes eutrophus ATCC 17697T using recycled gas closed cir- cuit culture system”, Ayaaki Ishizaki and Kenji Tanaka Journal of Fermentation and bioengineering 1991, 71(4) 254-257. “Pro- duction of Poly-b-Hydroxybutyric Acid from Carbon Dioxide byAlcaligenes eutrophus ATCC17697T” and Toshihiro Takeshita etal. J. Fac. Agr. Kyushu Univ. 1993, 38(1-2), 55-64. „Studies on Dissolved Hydrogen Behavior in Autotrophic Culture of Al- caligenes autrophus ATCC 17697T” disclose the synthesis of PHB in bacteria under autotrophic conditions. One drawback of the conditions is the requirement of ratio of hydrogen and oxygen, which is explosive (oxygen > 6 %). This limits the use of these conditions. Kenji Tanaka and Ayaaki Ishizaki Journal of fermentation and bioengineering, 1994, 77(4), 425-427 “Production of Poly- D-3-Hydroxybutyric Acid from Carbon Dioxide by a Two-Stage Culture Method Employing Alcaligenes eutrophus ATCC 17697 T”discloses a two-stage heterotrophic-autotrophic growth withfructose and O2in autotrophic stage in an amount of 2-3%. But the PHA storage efficiency of bacteria is decreased with or- ganic substrates. The use of CO2as carbon source makes these processes very valuable for the environmentally friendly production of plas- tics, which are even biodegradable.CBCPT24013EPWO 05.06.2025US 5,942,597 and WO 97 / 07229 A1 describe the extraction from PHA from oil plants using solvent mixtures. The applications WO 2021 / 130128 A1 and WO 2022 / 268899 A1 describe the production of PHA and PHA-copolymers using in- creased pressure. Among other PHA types, currently mostly PHB is successfully produced on industrial scale by process that make use of glu- cose as feedstock. PHB obtained by industrial process are highly crystalline due to their chemical structure composed by a single monomeric unit which is optically pure (high stere- oregularity). However, introducing chain irregularities fromcomonomer, or insertion, e.g. 4B vs. 3B or else stereoregular-ity is very important to achieve improved flexibility and thus better performances and processability for most general plas- tics applications. Usually, co-monomers are used to obtain less crystallinepolymers, which are usually smoother and more elastic. The procedures presented in the prior art are not suitablefor industrial scale processing. Also, the extraction of thePHA produced from the bacteria is difficult. They also requirelong reaction times. Also, the production of copolymers especially with longerchains is difficult and these monomers are only incorporated in a minor amount using biological processes. BRIEF SUMMARY OF THE INVENTIONCBCPT24013EPWO 05.06.2025It is, therefore, an object of the present invention toprovide a method for producing and preferably further purify-ing PHA-copolymers in bacteria, more preferably on an indus-trial scale. This aim is achieved by the inventions as claimed in the independent claims. Advantageous embodiments are described in the dependent claims. The object of the invention is also achieved by a method. In what follows, individual steps of a method will be de- scribed in more details. The steps do not necessarily have to be performed in the order given in the text. Also, further steps not explicitly stated may be part of the method. The object of the invention is achieved by a method forproducing PHA-copolymers comprising the following steps:a) growing bacteria under heterotrophic conditions in a me- dia; b) cultivating the bacteria under autotrophic conditionsunder an atmosphere of CO2, H2 and optional O2, wherein theamount on O2 is below 10 % (v / v) and pressure is at least 1barg, wherein at least one precursor for further monomers isadded before and / or during step b), wherein the at least oneprecursor is selected from an organic substance comprising atleast 3 carbon atoms and at least 2 oxygen atoms, wherein theorganic substance in its hydrolyzed form comprises at least two of carbon atoms are part of a functional group selected from COOH, C=O, C-OH. The bacteria that are useful in the present invention in- clude any bacteria that can produce PHAs, preferably bacteria which naturally produce PHAs. Wild type bacteria areCBCPT24013EPWO 05.06.2025preferred. Such bacteria are not genetically engineered. By using a wild type the process has to fulfil less strict regu- lations. In one embodiment, the bacterium is Pelomonas saccharophila (also formerly known as Pseudomonas saccharophila), Azomonaslata (also formerly known as Alcaligenes latus) and R. eu-tropha (also known as Cupriavidus necator). These are non-pathogenic, gram-negative bacteria, which can be found in soil and water. Their facultative chemolithoautotrophic metabolism allows them to grow either on organic compounds or using H2and CO2as reductive agent and carbon source, respectively, when submitted to a nutrient limitation and in presence of oxygen. Both modes can also be concomitant depending on the availabil- ity of nutrients. In a preferred embodiment the bacteria are the wild bacte-ria selected from Cupriavidus necator, even more preferablystream Cupriavidus necator H16, which is a non-pathogenic,gram-negative stream. Other preferred steams are the streamsavailable under the DSM numbers DSM -428, DSM-531, DSM-11098,DSM-3102, DSM-529 and DSM-545 at the DSMZ-German Collection ofMicroorganisms and Cell Cultures GmbH. Additionally, wild bacteria selected from PreudosomasPutida or Aeuruginosa can be used.In the first step the bacteria are grown under hetero- trophic conditions. These are conditions utilizing organic compounds as carbon and energy source. In a preferred embodi-ment under these conditions the bacteria show exponentialgrowth.CBCPT24013EPWO 05.06.2025In a preferred embodiment the bacteria are grown using con-ditions with no limitations regarding the nutrients, espe- cially nitrogen, carbon or phosphor. Usually, an ambient atmosphere is used.In a preferred embodiment the step is performed at ambient pressure or a pressure resulting only from the reaction condi- tions, e.g. heating in a closed vessel. The medium used for step a) is an aqueous medium. In a preferred embodiment the medium for the first step comprises at least one Ammonium salt as nitrogen source, pref- erably Ammonium sulphate. As carbon source different organic substances may be used. This may be sugars like sucrose, fructose or glucose, polyols like glycerol, organic acids or salts or esters thereof, asacetic acid or malate or ethyl acetate. The carbon source ispreferably soluble in the medium. In a preferred embodiment the molecular mass of each carbon source used is below 600 g / mol preferably below 500 g / mol, even more preferably below 400 g / mol. In a preferred embodiment the medium for the first step comprises at least one phosphate salt as phosphor source, preferably ammonium, sodium or potassium salts of phosphates, especially in their monobasic form may be used, more prefera- bly an H2PO4salt, more preferably (NH4)H2PO4, KH2PO4and / or NaH2PO4. The salts may be used a hydrate.CBCPT24013EPWO 05.06.2025Ammonium hydroxide, hydrochloric acid, citric acid and / orsulfuric acid may be used for adjusting the pH. The medium may comprise further salts and additives, like magnesium salts, iron salts, calcium salts, vitamins, yeastextract or trace elements like Zn, B, Co, Cu, Ni, Mo or Mn.The pH of the medium is preferably 4.5 to 7.5, more prefer- ably 6.4 to 7.1. In a preferred embodiment the amount of C at the beginningof step a) is between 2 and 50 g / l, preferably between 5 and30 g / l.In a preferred embodiment the amount of N at the beginningof step a) is between 0.1 and 5 g / l, preferably 0.1 and 4 g / l.In a preferred embodiment the amount of P at the beginningof step a) is between 0.05 and 5 g / l, preferably 0.1 and 3.5g / l. In a preferred embodiment at the beginning of step a) the amount of carbon source is 5 to 50 g / l, preferably 10 to 50g / l. The values depend on the molar mass of the carbon sourceand may be adapted to the content of C needed. In a preferred embodiment a content of 5 to 30 g / l C, 0.1 to 4 g / l N and 0.1 to 3.5 g / l P is preferred. In a preferred embodiment the bacteria are to this solutionto obtain an inoculum.CBCPT24013EPWO 05.06.2025The bacteria may also be added in an inoculum to this mix- ture. For the inoculum a content of 5 to 30 g / l C, 0.1 to 1.5g / l N and 0.2 to 5 g / l P is preferred.The values for the beginning of step a) refer to the values after adding the inoculum. The preferred sources for C, N and / or P for inoculum are the same as mentioned for medium for step a). In a preferred embodiment at the beginning of step a) the following amounts are present: Carbon source (glycerol, sucrose or glucose or fructose) 15to 70 g / l, (NH4)2SO4 1 to 5 g / l, KH2PO4 0.5 to 20 g / l, Citricacid x 1 H2O 0.1-3 g / l and NaH2PO4 0 to 2 g / l.Further salts of Mg or Ca may be present. Additionally, a trace element solution is added, comprising Zn, Mn, B, Co, Cu, Ni and Mo. In a preferred embodiment at the beginning of step a) the reactor is filled at a volume of 5 to 20 % of its total vol- ume. Step a) is preferably run at a temperature between 20 and 40 °C, more preferably at a temperature between 29 to 35 °C. It may be necessary to stir the reactor. During the growth of the bacteria the growth nutrients pre- sent are consumed. In a preferred embodiment at least some nu- trients are fed into the medium in order to keep these nutri- ents preferably in the ranges as mentioned above.CBCPT24013EPWO 05.06.2025The feeding may add to the volume of the medium inside the reactor. Preferably no medium is removed during cultivation. In a preferred embodiment the feeding solution is added at a rate between 0.1 to 5 % / h calculated from the total volume of the reactor, preferably 0.2 to 1 %. The feeding rate can be adapted based on the content of the feed and / or the cultiva- tion conditions, especially the pressure used. The feeding solution preferably at least comprises at least one carbon source. In another embodiment the feeding solution comprises at least one carbon source, at least one nitrogen source. In another embodiment the feeding solution comprises at least one carbon source, at least one nitrogen source and at least one phosphor source. The preferred sources are the same as mentioned for medium for step a) In another embodiment the carbon source is identical withthe carbon source of the starting conditions. In a preferred embodiment the feeding solution comprises a carbon content of 100 to 500 g / l, preferably 150 to 300 g / l. In an embodiment of the invention the feeding solution fur-ther comprises a content of N of 1 to 10 g / l, preferably 1 to5 g / l. In an embodiment of the invention the feeding solution fur-ther comprises a content of P of 0.5 to 15 g / l, preferably 1to 10 g / l.CBCPT24013EPWO 05.06.2025Preferably the feed comprises the content of C or C, N andP as previously mentioned or each in their preferred values.The feed solution may comprise further salts of Ca and / or Mg, as well as acids like citric acid. The feed may also com- prise a trace element solution. In a preferred embodiment the feed comprises 150 to 500g / l, preferably 150 to 300 g / l of C. The preferred sources for C, N and / or P for the feed are the same as mentioned for medium for step a) In a preferred embodiment the feed comprises the following ingredients: Carbon source (Glucose or sucrose or glycerol) 150 to 500g / l, preferably 150 to 300 g / l of C.In a preferred embodiment the feed only comprises a carbon source. The growth of the bacteria is continued until a cell den-sity of at least 10 (measured by OD at 600 nm), preferably atleast 20, more preferably at least 30 is reached, even morepreferably at least 60 is reached. The relevant growth pointmay also be related to other measurements, like time or compo- sition. Step a) is usually run for at least 5 hours up to 50 hours,preferably 20 hours up to 35 hours.CBCPT24013EPWO 05.06.2025In the next step the bacteria are grown under autotrophic conditions, except that additionally at least one precursor offurther monomers is added before and / or during this step. Un-der such conditions CO2 and the carbon source are used as car-bon source for the bacteria. These conditions can be seen as mixotrophic, since more than one carbon source is used. The amount of carbon source and the precursor added in sucha total amount so that the polymer produced will contain car-bon from CO2 and the added carbon source and further monomersderived from the precursors. By this at least a part of the polymer produced will bind the CO2from the used atmosphere. In a preferred embodiment the carbon source and precursorare added to an amount for up to 90 mol.-% of the carbon atomsof the polymer produced. These values are the calculated val- ues, this means that if the polymer produced comprises 100 moles of carbon atoms, only 90 moles of carbon source is added during the process. In an even more preferred embodiment, the amount is between 5 mol.% and 90 mol.-%, even more preferred 5 mol.-% and 70 mol.-%, even more preferred 10 mol.-% and 50 mol.-%. In a preferred embodiment the carbon source is added in an amount of 10 mol.-% to 40 mol.-%. In this step the media is contacted with an atmosphere com- prising H2, CO2and optional O2. In order to minimize the riskof explosion the content of O2 is less than 10 % (v / v). In apreferred embodiment the content of CO2 is between 2 % and 40 %(v / v). In a preferred embodiment the content of H2 is 50 % to96 % (v / v) or 50 % to 92 % (v / v).CBCPT24013EPWO 05.06.2025In a preferred embodiment the content of O2is less than 8 % (v / v), preferably less than 6 % (v / v), even more preferablyless than 5 % (v / v), especially preferred less than 3.1 %(v / v). In a preferred embodiment the content of O2is less than 8% (v / v), CO2 is between 2 % and 40 % (v / v) and H2 is between 50% and 92 % (v / v) or between 50 % and 96 % (v / v), preferablythe content of O2 is less than 4 % (v / v), CO2 is between 4 %and 40 % (v / v) and H2 is between 50 % and 92 % (v / v) or between50 % and 96 % (v / v). Surprisingly it has been found that the addition of atleast a precursor in the second step allows an efficient pro-duction of PHB-copolymers with further modifications. It also allows the use of less H2in the process. In a preferred embodiment the amount of H2 is between 50 %(v / v) and 85 % (v / v), even more preferred between 50 % and 82% (v / v). In a preferred embodiment the amount of H2 is between 50 %(v / v) and 85 % (v / v), while the amount of CO2 is more than 10 %(v / v), even more preferred the amount of H2 is between 50 % and82 % (v / v) while the amount of CO2 is more than 15 % (v / v). Thevalues adapt if O2is also present in the system in the amounts mentioned above. In a preferred embodiment of the invention the amount ofCO2 is above 10 % (v / v), while the amount of H2 is less than 85% (v / v), preferably the amount of CO2 is above 15 % (v / v),while the amount of H2 is less than 82 % (v / v).CBCPT24013EPWO 05.06.2025In a preferred embodiment of the invention the amount of H2is between 50 % (v / v) and 85 % (v / v), while the amount of CO2is from 10 % to 50 % (v / v), even more preferred the amount ofH2 is between 50 % and 82 % (v / v) while the amount of CO2 isfrom 15 % to 50 % (v / v), preferably 15 % to 45 % (v / v). Thevalues adapt if O2is also present in the system in the amounts mentioned above. Preferably the range of CO2is adapted based on the amount of O2present. O2is present preferably in anamount less than 5 % (v / v), preferably less than 3.1 % (v / v).Such an amount is sufficiently low to increase the safety of the process. In a preferred embodiment the content of nitrogen in theatmosphere is less than 6 % (v / v), preferably less than 1 %(v / v) if present. Some minor amounts of nitrogen may be car-ried into the reaction vessel by not degassing the solutions fed into the reactor. In another embodiment of the invention, especially if more than one precursor is used or if the process is run at more than 3.5 barg the amount of hydrogen is increased and theamount of CO2 is reduced. This leads to a better fermentationand a cleaner product. In this case for the preferred embodiment the amount of H2is between 80 % (v / v) and 96 % (v / v), even more preferred be- tween 85 % and 96 % (v / v), even more preferred between 87 % and 96 % (v / v). In a preferred embodiment the amount of H2is between 80 % (v / v) and 94 % (v / v), while the amount of CO2is more than 4 % (v / v), even more preferred the amount of H2is between 85 % and 96 % (v / v) while the amount of CO2is more than 4 % (v / v). TheCBCPT24013EPWO 05.06.2025values adapt if O2is also present in the system in the amounts mentioned above. In a preferred embodiment of the invention the amount ofCO2 is above 4 % (v / v), while the amount of H2 is less than 96% (v / v), preferably the amount of CO2 is above 4 % (v / v), whilethe amount of H2 is less than 95 % (v / v).In a preferred embodiment of the invention the amount of H2is between 80 % (v / v) and 96 % (v / v), while the amount of CO2is from 4 % to 18 % (v / v), even more preferred the amount of H2is between 85 % and 96 % (v / v) while the amount of CO2 is from5 % to 10 % (v / v), preferably 5 % to 8 % (v / v). The valuesadapt if O2is also present in the system in the amounts men- tioned above. Preferably the range of CO2is adapted based on the amount of O2present. O2is present preferably in an amountless than 5 % (v / v), preferably less than 3.1 % (v / v). Such anamount is sufficiently low to increase the safety of the pro- cess. In a preferred embodiment the content of nitrogen in theatmosphere is less than 6 % (v / v), preferably less than 1 %(v / v) if present. Some minor amounts of nitrogen may be car- ried into the reaction vessel by not degassing the solutions fed into the reactor. The source for such an atmosphere may be synthesis gas. The ratios mentioned are the ratios present at the begin- ning of step b). Since the gases are used during the fermenta- tion it may be necessary to adjust the amounts to the previous ranges during the fermentation. In a more preferred embodi- ment, the ratios are kept within these ranges during step b).CBCPT24013EPWO 05.06.2025The pressure in step b) is at least 1 barg or gauge pres- sure. In a preferred embodiment the pressure is at least 2 barg, more preferably at least 3 barg. In a preferred embodiment the pressure ranges from 2 to 20barg, preferably 3 to 20 barg, more preferably 3 to 10 barg.The pressure is measured under cultivation conditions. The pressure is the preferably kept in these ranges duringstep b). In a preferred embodiment the pressure during wholestep b) is at least 1 barg. Based on the standard atmospheric pressure of 1.013 bar 1 barg as used in the present application corresponds to 2.013 bar absolute pressure. All other ranges are adapted accord- ingly. Surprisingly the increased pressure led to PHA-copolymerwith different properties compared to PHA-copolymer obtainedwithout increased pressure. Preferably the content of the different gases is measured by the partial pressures. Preferably the pressure is kept constant for the duration of step b). More preferably the atmosphere composition and pressure are kept constant for the duration of the step b). In another embodiment of the invention at least one carbonsource is fed into the system in step b). By this the amountof H2needed can be reduced, preferably the amount of H2and O2needed can be reduced.CBCPT24013EPWO 05.06.2025The carbon source can also be combined with carbon sources as described previously. This may be sugars like sucrose, fructose or glucose, polyols like glycerol, organic acids or salts or esters thereof, as acetic acid or malate or ethyl ac- etate. In a preferred embodiment no carbon source is added to step b) if the process is run under increased pressure, preferablyan increased pressure of more than 1 barg.In order to produce copolymers precursors for further mono-mers are added in step b). These are typically salts of or-ganic acids, preferably sodium or potassium salts. Examples for such salts are the corresponding salts of propanoic acid, butanoic acid, pentanoic acid or hexanoic acid, depending on the length of the side chain needed. These salts are not as good as the precursors of the present invention. The carbon source is preferably soluble in the medium. It was now found that by adding a specific type of precur-sor a wider range of copolymers can be produced. Those precur- sors are better soluble and / or less toxic for the bacteria. These include, but are not limited to, diols, diacids, hy- droxy acids, or mixtures thereof, or their dehydrogenatedforms (e.g. cyclic lactones).The at least one precursor is selected from an organic sub-stance comprising at least 3 carbon atoms and at least 2 oxy-gen atoms, wherein the organic substance in its hydrolyzed form comprises at least two of carbon atoms are part of afunctional group selected from COOH, C=O, C-OH. The at leastone precursor is preferably soluble in the medium. In anotherembodiment these are salts of organic acids, preferably sodiumCBCPT24013EPWO 05.06.2025or potassium salts. The carbon atoms are part of a functionalgroup if they are the carbon atom in the corresponding COOH, C=O or C-OH groups. In a preferred embodiment two of the carbon atoms in the hydrolyzed form are part of a functional group selected from COOH, C=O or C-OH. The hydrolyzed form is the form of the substance, in whichall ether or ester bonds are hydrolyzed to a carboxylic acidgroup and a hydroxyl group, e.g. gamma butyrolactone corre-sponds to gamma hydroxobutyric acid or ether group into twohydroxyl groups. If the hydrolyzation results in more than onemolecule, at least two carbon atoms with the functional groups COOH, C=O, C-OH are present in one of the molecules. In a preferred embodiment the carbon atoms within the mole- cule, which are part of the functional group selected from COOH, C=O, C-OH are separated by at least one carbon atom not linked to oxygen, preferably by at least one CH2-group. In a preferred embodiment the molecular mass of the atleast one precursor is below 400 g / mol, preferably below 300g / mol, even more preferably below 200 g / mol. In a preferred embodiment the organic substance comprises 3to 12 carbon atoms, preferably 3 to 8 carbon atoms, even morepreferably 3 to 6 carbon atoms.The presence of the multiple polar groups increases the solubility of the substance so that the substance can be added in a much higher amount. This increases the inclusion of the resulting monomer in the PHA-copolymer.CBCPT24013EPWO 05.06.2025In a preferred embodiment each of the organic substance iseither a linear or cyclic molecule, preferably a linear mole- cule or a cyclic ester. In a preferred embodiment each of the organic molecule in its hydroylzed form comprises a COOH group and a C-OH group, a COOH group and a COOH group, a C-OH group and a C-OH group, a COOH group and a C=O group, a C=O group and a C-OH group, a C=O group and a C=O group, preferably a COOH group and a C-OH group, a COOH group and a COOH group, a C-OH group and a C-OH group, a COOH group and a C=O group. If the COOH group in the precursor is used as an ester itis preferably an alkyl ester with an alkyl group of 1 to 4 carbon atoms. If the C-OH group in the precursor is used as an ether itis preferably an alkyl ether of the organic molecule with an alkyl group of 1 to 4 carbon atoms. In preferred embodiment the organic substance corresponds already to the hydrolyzed form if it is not a cyclic ester. In this case the precursor is an organic substance already com- prising the at least two carbon atoms, which are part of the functional group selected from COOH, C=O, C-OH, or the organic substance comprises an R-COOR-group in which the R groups form a ring, preferably a 4, 5, 6 or 7 membered ring. Examples for such compounds are hydroxylated organic acids, which may also be used as ester or cyclized form, like Hydrox-obutyricacid like beta-hydroxybutyric acid (cyclic ester isbeta-butyrolactone), gamma-hydroxybutyric acid (cyclic esteris gamma-butyrolactone), valerolactones like beta-valerlolac- tones, gamma-valerlolactones, organic acids with Keto groupsCBCPT24013EPWO 05.06.2025like pyruvic acid, acetoacetic adid, levulinic acid, Polyols like 1,4-butandiols, 1,4-pentanediol, 1,6-hexanediol, 1,3-pro- pandiol, organic acids with at least two carboxylic acid groups like succinic acid. Levulinic acids can be used to produce PHBV, but it can beadded in much higher levels than propionic acid. It is lesstoxic for the bacteria than propionic acid or valeric acid. Gamma-hydroxobutyric acid or gamma-butyrolactone, succinicacid and 1,4-butanediol can be used to form 4B to producePH3,4B. Adifference between a carbon source and the precursor isthat the precursor is specific for a type of monomer.The precursors are chosen to produce a co-polymer dependingon the carbon source and the precursors used. In a preferred embodiment only at least one precursor isfed in step b). In step b) no further nutrients are then fedinto the reactor. The addition of the precursor and / or carbon source can be in different point in time during and / or before step b). It is preferred that it is added after step a). The precursor and / or carbon source can be added in a sepa- rate step before the pressure is applied. It is also possible that it is added during step b). It is also possible that the feeding starts before applying the atmosphere and ends during step b). The addition can be continuous or in one or more por- tions. It is important that the respective amount of carbon source is added in total.CBCPT24013EPWO 05.06.2025The addition of the precursor and / or carbon source can be performed in a different reactor or the same reactor of the cultivating step. It is also possible that it is added to a different reactor, which is connected with the cultivating re- actor. In a preferred embodiment the precursor and / or carbon source is added at a rate between 0.1 to 5 % / h calculated from the total volume of the reactor, preferably 0.2 to 1 %. The feeding rate can be adapted based on the content of the feed and / or the cultivation conditions, especially the pres- sure used. In preferred embodiment each of the at least one precursor is added in an amount of 0.1 to 15 g / g % (weight % precursors / main carbon source), preferably 0.1 to 10 g / g%. The main carbon source is the carbon source added in the feed in stepa) or the combined carbon source added in step a) and b). Thecontent of seed culture is not taken into account. The amountis the amount in g as dissolved in the added feed. In a preferred embodiment of the invention at least one precursor is added in an amount of 0.1 to 30 mol%, preferably 1 to 25 mol%, more preferably 1 to 20 mol% (mol / amount ofmol of the main carbon source in %).Preferably at least 80 mol % of the amount of each precur- sors added is included into the PHA-copolymer. In a preferred embodiment one precursor or a mixture of 2, 3 or 4 precursors is used. The precursors can each be added in different amount to adjust the properties of the resultingCBCPT24013EPWO 05.06.2025copolymer. It is also possible to use different precursorsleading to the same monomer in the final PBH-copolymer. In a preferred embodiment of the invention a mixture of 2 precursors is used. In a preferred embodiment at least one precursor is chosen from levulinic acid, succinic acid, gamma-butyrolactone or 1,4-butanediol. A preferred combination of precursors is levulinic acid andgamma-butyrolactone or levulinic acid and succinic acid.Surprisingly it has been found, that especially when using two precursors in a specific molar ratio between 1,2:1 and 3:1, preferably 1,5:1 to 2,5:1, more preferably 1,8:1 to 2,2:1, even more preferably 2:1, a highly modified terpolymer with very good yield can be obtained. In a preferred embodi- ment the precursor with the higher amount is a precursor for a valeric acid monomer, e.g. levulinic acid. In a preferred embodiment the precursor of the lower amount is a precursor for 4B-monomers, e.g. succinic acid, 1,4-budan- tiol or gamma-butyrolactone, preferably 1,4-butandiol and suc- cinic acid, even more preferred succinic acid. Surprisingly it has also been found that with succinic acid or 1,4-butandiol as only precursor a PH3,4B-copolymer withvery special properties can be obtained especially using theautolithotropic growth. Especially with succinic acid the ob- tained polymer shows unique crystallization properties.CBCPT24013EPWO 05.06.2025The pH in step b) is preferably 6.5 to 7.5, more preferably6.8 to 7.0. The pH may be adjusted using acid and / or bases,preferably sulfuric acid and / or ammonium hydroxide. As a medium the same medium as step a) may be used, but without any nitrogen source. In step b) the cultivation is preferably running under atleast nitrogen deficient conditions. The nitrogen source lim- its the biomass accumulation. This leads to PHA accumulation. In a preferred embodiment the cell dispersion at the end of step a) is directly used for step b) as starting medium. The temperature in step b) is preferably between 20 °C and 45 °C, more preferably between 25 °C and 35 °C. It may be necessary to stir the reactor during the reac- tion. The reaction in step b) is running until the amount of PHAis formed, usually until a content of 50 % to 90 % of PHA by weight calculated from the dry weight of the whole biomass is formed. Under these conditions it is possible to run step b) until final cell densities of more than 50 g / L, preferably more than100 g / L is reached.The reaction is also preferably stopped before the Mw of the PHA and / or PHB starts decreasing due to side reactions. The duration of the cultivation is usually 10 to 100 hours, preferably 20 to 80 hours, more preferably 20 to 60 hours.CBCPT24013EPWO 05.06.2025The reaction may also be stopped if a certain OD and / or cell dry weight (CDW) is reached. A preferred OD is an OD of more than 100, preferably more than 200. Step b) may be run in the same or a different reactor than step a), preferably a different reactor. If necessary further purification steps, like filtration or centrifugation are performed between the two steps. In a preferred embodiment the cells are separated from the medium before the next purification steps. It is also possible to wash the cells with an alcohol like methanol and / or ethanol. The PHA-copolymer is intracellular, i.e. formed inside thebacteria. For the extraction several methods are possible. In an embodiment of the invention the polymer is extractedfrom the cells using a solvent for the Polymer, preferably a polar organic solvent, more preferably acetone, chloroform, dimethyl carbonate or propylene carbonate, especially acetoneor dimethyl carbonate. Also, a mixture of solvents may beused. It may be necessary to induce cell lysis before or dur-ing extraction. This can be achieved for example by mechanical stress and / or induced by the solvent for the polymer. In an embodiment the cells are first broken by mechanicalstress, e.g. increased atmosphere pressure. The polymer is then extracted using a solvent for the polymer, preferably apolar organic solvent, more preferably acetone, chloroform,dimethyl carbonate or propylene carbonate, especially acetoneCBCPT24013EPWO 05.06.2025or dimethyl carbonate. Also, a mixture of solvents may beused. In a preferred embodiment a further solvent with a higherboiling point than the solvent for polymer is also added. Forexample, an oil or an alkane with 10 to 14 carbon atoms. Thefurther solvent is preferably used in a ratio of 1:20 to 1:4 by weight compared to the solvent for the polymer. If from this mixture the solvent for the polymer is removedthe polymer can be recovered as flakes or powder of high pu-rity. Preferably the solvent for polymer is removed by heat.The solvent for polymer may be reused for a further extrac-tion. It is also possible that the cell lysis is combined with the extraction step, since acetone also leads to cell lysis. In this embodiment the acetone and optionally the further sol- vent is added to the separated cells. The amount of solvent for PHA-copolymers, especially acetone, and further solvent is preferably used in an excess compared to the weight of the separated cells. Preferably in an amount of at least 2 times the weight, more preferably at least 5 times the weight of the separated cells. It is also possible that the cell lysis is separated fromthe extraction step, by using a homogenizer for cell lysis. Inthis embodiment the dimethyl carbonate and optionally the fur- ther solvent is added to the separated cells. The amount of solvent for PHA, especially dimethyl carbonate, and further solvent is preferably used in an excess compared to the weight of the separated cells. Preferably in an amount of at least 2 times the weight, more preferably at least 5 times the weight of the separated cells.CBCPT24013EPWO 05.06.2025In a preferred embodiment solvent for PHA-copolymer and thefurther solvent is added and the mixture is then mixed and the non-aqueous phase is separated. The PHA-copolymer produced is obtained as flakes during theremoval of the solvent for the polymer, especially acetone ordimethyl carbonate. The PHA-copolymer produced by the present process has anarrow molecular weight distribution and a very high contentof the one or more comonomers. In another embodiment of the invention step b) is performedunder heterotrophic conditions under atmospheric pressure. Inthis embodiment a carbon source is added during step b). The carbon source is preferably a carbon source as de- scribed for step a) or b), preferably glucose, sucrose or glycerol, preferably glucose. The carbon source is added together or separate with the further precursors during this step. The feed of the carbon source may contain 150 to 500 g / l, preferably 150 to 300 g / l of C. Also in this embodiment further precursors as described for step b) are added. In preferred embodiment in this embodiment each of the at least one precursor is added in an amount of 0.1 to 30 g / g % (weight % precursors / main carbon source), preferably 0,5 to20 g / g%. The main carbon source is the combined carbon sourceCBCPT24013EPWO 05.06.2025added in step a) and b). The content of seed culture is not taken into account. The amount is the amount in g as dissolved in the added feed. In a preferred embodiment in this embodiment each of the at least one precursor is added in an amount of 0.1 to 30 mol%, preferably 1 to 25 mol%, more preferably 1 to 20 mol% (mol / amount of mol of the main carbon source). Beside the addition of carbon source in step b) and the lack of pressure the process is run as described for the pro- cess previously. Also the product can be extracted the same way. Another object of the invention is a PHA-copolymer produced by the process of the present invention, preferably comprising at least three different monomers. Another object of the invention is a PHA-copolymer compris- ing one further comonomer with a content of 10 to 50 mol%, preferably 15 to 50 mol%. Preferably the comonomer is 3V or 4B. Even more preferably the polymer is produced using the au-totrophic process. This is calculated as molar percentage ofthe monomers of the polymer. Another object of the invention is a PH3,4B-copolmyer with a 4B content of less than 40 mol%, preferably between 5 and 40 mol%, preferably 10 to 40 mol%. Another object of the invention is a PHA-copolymer compris- ing at least three different monomers chosen from 3B, 3V and 4B. Such a copolymer can be produced using levulinic acid andgamma-butyrolactone, 1,4-butanediol or succinic acid as fur-ther precursors added. Preferably the terpolymer comprises 4BCBCPT24013EPWO 05.06.2025with at least 5 mol%. Even more preferably the comonomers 3V and 4B are each present in an amount between 5 to 30 mol%. In a preferred embodiment the 4B monomer is derived from 1,4-butanediol or succinic acid, preferably succinic acid. Es- pecially succinic acid leads to polymers with beneficial prop- erties. In a preferred embodiment the PHA-polymer produced has a ^Hc2(measured using DSC, during 2ndheating) of less than 5 J / g, preferably less than 2 J / g. Preferably the PHA-polymer is a PHA-copolymer with only one further monomer and with a ^Hc2(measured using DSC, during 2ndheating) of less than 5 J / g, preferably less than 2 J / g, evenmore preferred less than 1 J / g. In a preferred embodiment no ^Hc2is measured. Such polymers show almost or even no cold crystallization. The polymer can be used for various products depending on its properties. It may also be blended with other polymers. Another object of the invention is a moulded article, gran- ulate or a master batch comprising or formed from a PHA-copol- ymer as described previously. The moulded articles can thereby be produced in any way, for example by extrusion, casting, injection moulding, press- ing, sintering, calendering, film-blowing, melt-spinning, com- pression moulding and / or thermoforming, for components in au- tomobile construction, transport and / or communications, compo-nents for industrial equipment, machine- and plant construc-tion, household appliances, containers, devices for medical technology, components for electrics or electronics. Hence,CBCPT24013EPWO 05.06.2025the invention likewise relates to the use of a polymer mate- rial according to the invention for the previously mentioned purposes. The polymer may be used for the production of coating mate-rials, foils, films, laminates, fibers, moulded parts, mouldedarticles, injection moulded articles, e.g. bottles or fibers,extrudates, containers, packaging materials, coating materi- als, particles, beads, micro beads and medicine dispensers. Preferably the coating material may be produced by spray drying, more preferably by spray drying directly from the pol-ymer solution. The polymer solution is sprayed over the mate-rials to be coated at the evaporation temperature of the sol- vent of the polymer solution in order to coat the material forming a solid polymer coat on the surface. Preferably the fibers may be produced by spinning, more preferably by spinning directly from the polymer solution. Preferably a non-woven fabric can be produced by spinning, more preferably by spinning directly from the polymer solu-tion. For this the polymer solution is pumped through a sieveand deposited on a surface forming the non-woven fabric. The polymer can be formed to any product as known from theprevious products. It is also possible to add usual additives and other polymers. Figures Fig. 1: Non-woven fabric produced from BD AUTO (a) size comparison, b) enlarged view c) microscopic picture);CBCPT24013EPWO 05.06.2025Fig. 2: Non-woven fabric produced from LA AUTO (a) size comparison, b) enlarged view c) microscopic picture). Fig. 3: Non-woven fabric produced from LA SA AUTO (a) size comparison, b) enlarged view c) microscopic picture). Fig. 4: Non-woven fabric produced from LA GLU (a) size com- parison, b) enlarged view c) microscopic picture). Fig. 5: Non-woven fabric produced from GBL LA GLU (a) size comparison, b) enlarged view c) microscopic picture). Examples NaH2PO4is used as dihydrate. Comparative Example A A seed culture medium A was prepared with Glucose 15 g / l,(NH4)2SO4 1 g / l, MgSO4x 4H2O 0.2g / l g / l, KH2PO4 1.5 g / l, Na2HPO43.55 g / l, yeast extract 1 g / l and Trace elements solution 10ml / l (ZnSO4 x 7 H2O 2.26 g, MnSO4 x 1 H2O 0.34 g, CaCl2 x 2H2O 2g, CuSO4x 5 H2O 1 g, FeSO4x 7 H2O 10 g, (NH4)6Mo7O24x 4 H2O0.106 g, Na2B4O70.122 g, HCl 37% 10 ml and distilled water1000.00 ml). To obtain the inoculum the bacteria (Cupriavidus necator H16) is added to the seed culture medium and the inoculum is added to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth, the following feed solu- tion was used comprising Glucose 500 g / l, without any co-sub- strate.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 15 ml / h until an OD of 50 was reached. Usually,this is before a reaction time of 28-30 h. For autolithotropic growth the reaction mixture stays in the same reactor. For autolithotropic growth CO2, H2and O2are fed to the re-actor with a total pressure of 3 barg (H2: 80%, 2.4 barg; CO2:17 %, 0.5 barg; O2: 3%, 0.1 barg). The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50 hours, e.g. after 68 hours an CDW of 65.9 g / L (cell dry weight) is reacted in the present example. The fermentation is then stopped and the PHA is extracted. Example 1B A seed culture medium B was prepared with Sucrose 20 g / l, (NH4)2SO42 g / l, MgSO4x 4H2O 1.0 g / l, KH2PO40.6 g / l, Citric acid x 1 H2O 0.11 g / l, NaH2PO41.43 g / l, CaCl2x 2 H2O 0.1 g / l and Trace elements solution 3 ml / l (ZnSO4x 7 H2O 0.10 g, MnCl2x 4 H2O 0.03 g, H3BO30.30 g, CoCl2 x 6 H2O 0.20 g, CuCl2 x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and dis- tilled water 1000.00 ml). The seed culture medium was inoculated with bacteria (Cu-priavidus necator H16) and the inoculum is added to a reactor.Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 500 g / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 5 ml / min until an OD of 50 was reached. Usually,this is before a reaction time of 28-30 h. For autolithotropic growth CO2, H2and O2is fed to the re- actor with a total pressure of 3.1 barg (H2: 81%, 2.5 barg; CO2: 16 %, 0.5 barg; O2: 3%, 0.1 barg). Levulinic acid to anamount of 3 g / g % in total is added stepwise.The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50 hours, e.g. after 54 hours and CDW of 80.5 g / l is reacted inthe present example. This leads to 64.9 g / l product. Fromthese cells, PHBV is obtained. (Elastic modulus 0.95 GPa, Tm = 167 °C, Content of V approx. 6%, Eta= 4.3 g / l). Example 1C A seed culture medium C was prepared with Glycerol 50 g / l, (NH4)2SO44 g / l, MgSO4x 4H2O 1.2 g / l, KH2PO413.3 g / l, Citric acid x 1 H2O 1.85 g / l and Trace elements solution 10 ml / l(ZnSO4 x 7 H2O 0.10 g, MnCl2 x 4 H2O 0.03 g, H3BO3 0.30 g, CoCl2x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To the seed culture medium the bacteria (Cupriavidus ne-cator H16) is added to obtain the inoculum and the inoculum isadded to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 500 g / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 5 ml / h until an OD of 50 was reached. Usually,this is before a reaction time of 28-30 h. For autolithotropic growth CO2, H2,and O2are fed to the reactor with a total pressure of 3.1 barg (H2: 80.6%, 2.5 barg; CO2: 16.1 %, 0.5 barg; O2: 3.2%, 0.1 barg). Levulinic acid of 6 g / g % is added stepwise. The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50hours. e.g. after 54 hours and CDW of 80.5 g / l is reacted inthe present example. This leads to 65.4 g / l product. From these cells, PHBV is obtained. Example 1D A seed culture medium C was prepared with Glycerol 50 g / l, (NH4)2SO44 g / l, MgSO4x 4H2O 1.2 g / l, KH2PO413.3 g / l, Citric acid x 1 H2O 1.85 g / l and Trace elements solution 10 ml / l (ZnSO4x 7 H2O 0.10 g, MnCl2x 4 H2O 0.03 g, H3BO30.30 g, CoCl2x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To the seed culture medium the bacteria (Cupriavidus ne-cator H16) is added to obtain the inoculum and the inoculum isadded to a reactor. Under growing conditions, a feed solution is added to thereactor. For chemolithotropic growth the following feed solu-tion was used comprising Glucose 500 g / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 5 ml / h until an OD of 50 was reached. Usually,this is before a reaction time of 28-30 h. For autolithotropic growth CO2, H2,and O2are fed to the reactor with a total pressure of 3.1 barg (H2: 80.6%, 2.5 barg; CO2: 16.1 %, 0.5 barg; O2: 3.2%, 0.1 barg). Levulinic acid of 4 g / g % and 1,4 butanediol 1.5 g / g % is added stepwise. The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50hours. e.g. after 54 hours and CDW of 80.5 g / l is reacted inthe present example. This leads to 61.7 g / l product. From these cells, PHBV is obtained. Example 1E A seed culture medium C was prepared with Glycerol 50 g / l, (NH4)2SO44 g / l, MgSO4x 4H2O 1.2 g / l, KH2PO413.3 g / l, Citric acid x 1 H2O 1.85 g / l and Trace elements solution 10 ml / l(ZnSO4 x 7 H2O 0.10 g, MnCl2 x 4 H2O 0.03 g, H3BO3 0.30 g, CoCl2x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To the seed culture medium the bacteria (Cupriavidus ne-cator H16) is added to obtain the inoculum and the inoculum isadded to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 500 g / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 5 ml / h until an OD of 50 was reached. Usually,this is before a reaction time of 28-30 h. For autolithotropic growth CO2, H2,and O2are fed to the reactor with a total pressure of 3.1 barg (H2: 80.6%, 2.5 barg; CO2: 16.1 %, 0.5 barg; O2: 3.2%, 0.1 barg). Levulinic acid of 4 g / g % and gamma-butyrolactone 0.5 g / g % is added stepwise (g / g% are weight % precursors / main carbon source). The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50hours. e.g. after 54 hours and CDW of 80.5 g / l is reacted inthe present example. This leads to 61.7 g / l product. Fromthese cells, PH3,4BV (poly-3HB-co-3HV-co-4HB) is obtained. Example 1F A seed culture medium C was prepared with Glycerol 50 g / l, (NH4)2SO44 g / l, MgSO4x 4H2O 1.2 g / l, KH2PO413.3 g / l, Citric acid x 1 H2O 1.85 g / l and Trace elements solution 10 ml / l(ZnSO4 x 7 H2O 0.10 g, MnCl2 x 4 H2O 0.03 g, H3BO3 0.30 g, CoCl2x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To the seed culture medium the bacteria (Cupriavidus ne-cator H16) is added to obtain the inoculum and the inoculum isadded to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 500 g / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 5 ml / h until an OD of 50 was reached. Usually,this is before a reaction time of 28-30 h. For autolithotropic growth CO2, H2,and O2are fed to the reactor with a total pressure of 3.1 barg (H2: 80.6%, 2.5 barg; CO2: 16.1 %, 0.5 barg; O2: 3.2%, 0.1 barg). Levulinic acid of 4g / g % and succinic acid of 0.7 g / g % is added stepwise (g / g%are weight % precursors / main carbon source). The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50hours, e.g. after 54 hours and CDW of 80.5 g / l is reacted inthe present example. This leads to 61.7 g / l product. From these cells, PH3,4BV (poly-3HB-co-3HV-co-4HB) is obtained. Comparative 1D A seed culture medium A was prepared with Glucose 20 g / l, (NH4)2SO44 g / l, MgSO4x 4H2O 1.2 g / l, KH2PO44 g / l, Citric acid x 1 H2O 1.86 g / l and Trace elements solution 10 ml / l (ZnSO4x 7 H2O 0.10 g, MnCl2x 4 H2O 0.03 g, H3BO30.30 g, CoCl2x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To obtain the inoculum the bacteria (Cupriavidus necator H16) is added to the seed culture medium and the inoculum is added to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 660 g / l, (NH4)2SO412 g / l, NaH2PO412 g / l, MgSO4x 7H2O 3.6 g / l, KH2PO412 g / l, Citric acid 30 g / l, Trace element solution 15 ml / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 5 ml / h until an OD of 20 is reached. Usually thisis before a reaction time of 21 h. For autolithotropic growth the reaction mixture is eitherput into a new reactor or stays in the same reactor. For autolithotropic growth CO2, H2and O2is fed to the re- actor at atmospheric pressure with composition of the gas mix-ture H2: 84%, CO2: 13 %, O2: 3%.The reactor is stirred and the fermentation is run until anOD >200 is reached. Usually, the reaction time is at least 150hours, e.g. after 184 hours and OD of 230g / l is reacted in the present example. The fermentation is then stopped and the PHA is extracted with Dimethylcarbonate as solvent. The properties of the different polymers are shown in table 1: Table 1:Property PHBPHBV PHBV PH3,4BV PHB Comparative Compara- Example Example Example 1D tive 1 A 1B 1C 1D Density1.25 1.25 1.25 1.25 1.25[g / cm] (v1183)CBCPT24013EPWO 05.06.2025Molecular745 k 652.3 k 728 k 713.6 k 571 kweight[g / mol] (GPC) Melting178 164 162 145 179point [°C] (11357-3) Crystal-67 71 70 69 75linity % (NMR) Although the melting temperature is the same as expected for pure PHB, the modulus and the tensile properties are more similar to those of the PHBH copolymers. Example 2 (autotrophic) A seed culture medium C was prepared with Glycerol 50 g / l,(NH4)2SO4 4 g / l, MgSO4x 4 H2O 1.2 g / l, KH2PO4 13.3 g / l, Citricacid x 1 H2O 1.85 g / l and Trace elements solution 10 ml / l (ZnSO4 x 7 H2O 0.10 g, MnCl2 x 4 H2O 0.03 g, H3BO30.30 g, CoCl2 x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To the seed culture medium the bacteria (Cupriavidus ne- cator H16) is added to obtain the inoculum and the inoculum is added to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 500 g / l.CBCPT24013EPWO 05.06.2025This solution was fed to the reactor under stirring at arate of 3 – 30 ml / min until an OD of at least 40, preferably40 to 50. The addition can be performed in intervals. Usually,this is before a reaction time of 24-30 h. For autolithotropic growth CO2, H2, and O2are fed to the reactor with a total pressure of 4 to 4.2 barg. The corre- sponding conditions are shown in table 2. The cosubstrate(s)is / are added to a corresponding concentration (See Tables 2and 3). The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50 hours. e.g. after 54 hours and CDW of at least 80, preferably 80-110 g / l, e.g. 81.5 g / l is reacted in the present example. This leads to 64-68 g / l e.g. 65.2 g / l product. From these cells, PHBV or PH3,4B or PH3,4BV is obtained. Table 2: Conditions used for Example 2 using Levulinic acid (LA), succinic acid (SA), 1,4-butandiol (BD) as concentrationin the reactor during step b); AUTO means autotrophic growth;CO2 / H2 / O2is the gas composition; PHBV is a commercial sample of PHBV. Sample CO2 / H2 / O2 (barG)LA SA BD (mol / L) (mol / L) (mol / L) LA 0.2 / 3.7 / 0.1 0.00414 0 AUTO 5 % / 92.5 % / 2.5 % mol / L 0.2 / 3.8 / 0.1 SA 0.00415 4,9 % / 92,7 % / 2,4 - 0 AUTO mol / L % PHBV 8% mol (SIGMANA NA NA NAALD- RICH) LA 0.2 / 3.7 / 0.1 0.0083 0 AUTO 5 % / 92,5 % / 2,5 % mol / L (2)CBCPT24013EPWO 05.06.2025BD 0.2 / 3.7 / 0.2 0.0083 AUTO 4,9 % / 90,2 % / 4,9 mol / L % LA SA 0.2 / 3.7 / 0.2 AUTO 4,8 % / 88,1 % / 4,9 0.00415 0.00208 % mol / L mol / L LA BD 0.2 / 3.7 / 0.2 0.00208 AUTO 4,8 % / 88,1 % / 4,9 0.00415 mol / L % mol / L Table 3: total concentration of the cosubstrate(s) in the reactor. Cosubstrate Total likely structure Sample (mmol / L) LA AUTO 4.14 PHBVSA AUTO 4.15 PH34BPHBV 8% mol (SIGMA ALD- PHBV NA RICH) BD AUTO 8.30 PH34BLA SA AUTO 6.23 PH34BVLA BD AUTO 6.23 PH34BVThe samples were tested by DSC measurement (Table 4 and Ta-ble 5). The monomer content of the polymers was calculated (Table 11). Table 4: Main thermal parameters as measured by DSC tests in the cooling scan. ^Hc1 likely structure Sample Tc1 (°C)(J / g) LA SA AUTO 77.3 38.6 PH34BVBD AUTO 83.7 59.0 PH34BLA AUTO 64.7 9.2 PHBVSA AUTO 94.5 57.8 PH34BPHBV 53.7 2.7 PHBVCBCPT24013EPWO 05.06.2025Table 5: Main thermal parameters as measured by DSC tests in the second heating scan Sample T (°^Hc2 ^Hm2 gC) Tc2 (°C)Tm2 (°C) (J / g) (J / g) LA SA AUTO 2.6 48.5 2.5 169.5 66.1BD AUTO 0.4 47.4 1.5 171.2 76.8LA AUTO -0.5 59.4 32.7 172.2 54.6SA AUTO 34.3 - - 169.6 64.6PHBV 0.0 62.9 51.4 163.1 65.4Table 11: Estimated monomer (3V and 4B) content in the pol-ymer using the Tm2 (x)=−80x2−40x+182 for calculating x=mol 4B inpolymer (BD AUTO, SA AUTO) and equation T 2m2 (y)=−30y −30y+182for calculating y= mol 3V. Cosub- Cosub- Cosub- Cosub- Cosub- strate strate strate strate strate Cosub- Cosub- 4B V feed 4B in- in- Sample strate strate mol% mmol / L feed serted serted typeV mol%mmol / L V pro 4B pro mol mol fed fed LA SA 3V and13 4.2 2.1 8.7 6.235.94 AUTO 4B BD AUTO 4B 0.00 21.6 0.0 8.32.6LA AUTO 3V 35.94 0.00 4.2 0.08.7SA AUTO 4B 0.00 19.44 0.0 4.24.7PHBV 3V 43.81 NA NA NA(Sigma) Considering that LA AUTO and SA AUTO have a cosubstrate in-serted versus mol fed of 8.7 and 4.7 respectively (Table 11),while calculating the ratio cosubstrate inserted versusCBCPT24013EPWO 05.06.2025cosubstrate fed and assuming 3B ratio is as for LA AUTO, the cosubstrate inserted to feed ratio of 4B for the terpolymers is higher than that of the SA AUTO, which means there is a non linear effect for the terpolymers used in the given ratio. Interestingly the sample with succinic acid (SA) shows no crystallisation in the second heating. The sample SA AUTO does not show cold crystallization but asharp crystallization during the cooling ramp, so it crystal- lizes better than all other samples. This was very surprising. The cold crystallization is in reality a slow post-crystalli- zation that changes the properties of the polymers after they are converted into end use form, e.g. shrink post injection, fracture if pipes. Additionally, the material has a Tgslightly above room temperature, making the film more transparent. Also, the sample with 1,4-butanediol has a very low ^Hc2during the second heating. It also seems that PH34BV has higher ^Hcduring cooling, so crystallizes more under cooling. Adding the further monomer to the V improves crystallization, especially in a molar rate 2:1(V : 4B) in the feed.The sample SA AUTO was also analyzed for intrinsic viscos- ity and molecular weight (Table 6). Table 6: Values of intrinsic viscosity and molecular weight for the analyzed samples Sample η [dl / g] Mw [Da] Mw [Da]CBCPT24013EPWO 05.06.2025a=0.82, a=0.78, K=7.7*10-5K=11.8*10-5dl / g dl / g SA AUTO 1.32 ±1.46*105± 1.56*105± 0.03 0.01*1050.01*105Example 3 (heterotrophic grow: GLU) A seed culture medium C was prepared with Glycerol 50 g / l, (NH4)2SO44 g / l, MgSO4x 4 H2O 1.2 g / l, KH2PO413.3 g / l, Citric acid x 1 H2O 1.85 g / l and Trace elements solution 10 ml / l (ZnSO4x 7 H2O 0.10 g, MnCl2x 4 H2O 0.03 g, H3BO30.30 g, CoCl2x 6 H2O 0.20 g, CuCl2x 2 H2O 0.01 g, NiCl2x 6 H2O 0.02 g, Na2MoO4x 2 H2O 0.03 g and distilled water 1000.00 ml). To the seed culture medium the bacteria (Cupriavidus ne- cator H16) is added to obtain the inoculum and the inoculum is added to a reactor. Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solu- tion was used comprising Glucose 400-500 g / l. This solution was fed to the reactor under stirring at arate of 3 – 30 ml / min until an OD of 50 was reached. The addi-tion can be performed in intervals. Usually, this is before areaction time of 24-30 h. For heterotrophic growth the glucose 400-500 g / l solutionwas fed to the reactor under stirring at a rate of 10 – 50ml / min together with cosubstrates, at a concentration as shown in the Tables 7 and 8.CBCPT24013EPWO 05.06.2025The reactor is stirred and the fermentation is run until an OD >200 is reached. Usually, the reaction time is at least 50hours. e.g. after 60-65 hours and CDW of at least 80, prefera-bly 70-85, e.g. 80.5 g / l is reacted in the present example.This leads to 60- 65 e.g. 64 g / l product. From these cells,PHBV or PH3,4B or PH3,4BV is obtained. Table 7: Conditions used for Example 3 using Levulinic acid (LA), succinic acid (SA), 1,4-butandiol (BD), gamma-butyrolac-tone (GBL), glucose (GLU) as concentration in reactor in stepb); GLU LA SA BD or Sample (mol / L) (mol / L) (mol / L) GBL*(mol / L) BD GLU0.13mol / L 0.014 mol / L 0 0.007 mol / LLA SA GLU0.13 0.014 0.007 mol / L 0LA mol / L mol / L LA GLU0.13 0.027 mol / L 0 0mol / L GBL GLU LA 0.13mol / L 0.014 mol / L 0 0.007* mol / L0.13 SA GLU 0.027 mol / L mol / L Table 8: total concentration of the carbon source cosub- strate(s) in the reactor added by the feed (including glucose) Cosubstrate Total likely structure Sample (mmol / L) BD GLU LA 21.0 PH34BVSA GLU LA 21.0 PH34BVLA GLU 27.0 PHBVGBL* GLU LA 21.0 PH34BVSA GLU 27.0 PH34BCBCPT24013EPWO 05.06.2025The samples were tested by DSC measurement (Table 9 and Ta- ble 10). The monomer content of the polymers was calculated (Table 12). Table 9: Main thermal parameters as measured by DSC tests in the cooling scan; PHBV is a commercial sample: likely Sample T (°^Hc1 c1 C)struc- (J / g) ture GBL LA GLU 63.2 13.3 PH34BVLA GLU - - PHBVSA GLU 89.2 58.3 PH34BBD GLU LA 81.8 54.1 PH34BVSA GLU LA 84.8 56.4 PH34BVPHBV 53.7 2.7 PHBVTable 10: Main thermal parameters as measured by DSC tests in the second heating scan Sample TTc2 g(°C)(°C) ^Hc2 (J / g) Tm2 (°C) ^Hm2 (J / g)GBL LA GLU- 60.5 22.2 173.2 37.11.3 / 21.2 LA GLU -3.8 67.6 26.1 173.4 27.1SA GLU 23.1 - - 169.5 59.4BD GLU LA 3.6 47.3 1.8 173.7 68.2SA GLU LA 0.2 44.5 1.9 168.7 66.5PHBV 0.0 62.9 51.4 163.1 65.4Table 12: Estimated monomer (3V) content in the polymerequation T 2m2 (y)=−30y −30y+182 for calculating y= mol 3V in LACBCPT24013EPWO 05.06.2025GLU, the ratio found is then used to calculcate the mol of 3B given the actual feed and the equation Tm2(x)=−80x2−40x+182 for calculating x=mol 4B in terpolymers with BD, GBL and SA: Cosub- Cosub- Co- Cosub- Cosub- strate strate sub- Cosub- Cosub- strate strate V in-4B in-Sample stra strate strate V feed4B feedserted serted te mol V% mol 4B% mmol / L mmol / L pro mol pro mol type fed fed GBL LA 4B 10.89 12.89 14 7 1.3 1.8GLU 3VLA GLU 3V 21.00 0.00 27 1.3 NASA GLU 4B 0.00 22.00 NA 1.2BD GLU 4B 10.89 10.89 14 7 1.3 1.6LA 3V SA GLU 4B 10.89 26.61 14 7 1.3 3.8LA 3V PHBV 0.0 NA NA NA NAConsidering that LA GLU and SA GLU have a cosubstrate in- serted versus mol fed of 1.3 and 1.2 respectively (Table 12),while calculating the ratio cosubstrate inserted versus cosub- strate fed and assuming 3B ratio is as for LA GLU, the cosub- strate inserted to feed ratio of 4B for the terpolymers ishigher than that of the SA GLU, which means there is a non linear effect for the terpolymers used in the given ratio. Comparing the LA GLU with BD GLU LA and SA GLU LA, it was ob-served that in particular SA GLU LA has about the same meltingparameters than LA GLU with 25% less total mol of co-sub-strate. The terpolymer is more effective than the copolymer. The addition of SA or BD improves crystallization and leads to higher ^Hc1 during cooling. Especially the samples SA GLU LAand BD GLU LA show a very low ^Hc2.SA GLU also shows cold crystallization as SA AUTO. This shows that this property depends on the type of precursor usedCBCPT24013EPWO 05.06.2025for the monomer. Succinic acid leads to a different polymer than 1,4-butanediol. Although the absolute amount of precursor used in the het- erotrophic process is much higher, the inclusion in the poly- mer seems to be similar to the heterotrophic process. The ad- vantage of the heterotopic process is therefore the use of much less precursor to achieve the same inclusion of the mono- mer in the polymer. Two samples were also analyzed for intrinsic viscosity andmolecular weight (Table 11). Table 11: Values of intrinsic viscosity and molecular weight for the analyzed samples Sample η [dl / g] Mw [Da]Mw[Da] a=0.82, a=0.78, K=7.7*10-5K=11.8*10-5dl / g dl / g 1.29 ± 1.42*105± 1.51*105± BD GLU LA0.02 0.01*1050.01*1050.94 ± 9.59*104± 9.99*104± SA GLU LA0.04 0.17*1040.14*104DSC measurements Mettler DSC 30 scanner was used. The specimen was subjected to a first heating from -30 to 200 °C, a cooling from 200 to - 30 °C and a second heating from -30 to 200 °C. The tests were carried out in a nitrogen atmosphere with a constant N2flux ofCBCPT24013EPWO 05.06.2025100 ml / min and the heating and cooling rates were fixed at10 °C / min.From the DSC thermograms the melting point (Tm1) of thepolymer, the crystallization temperature (Tc1) and under cool-ing conditions and crystallization temperature (Tc2) the melt- ing point (Tm2) and glass transition temperature (Tg) in the second heating scan were identified. The integration of thepeaks allowed to estimate the melting and crystallization en-thalpy under the first (^Hm1) and the second (^Hm2,^Hc2) heating scans and under cooling (^Hc1) conditions. Molecular weight The molecular weight was measured indirectly, by intrinsic viscosity, where relation between the viscosity and the molec- ular weight is given by the Mark-Houwink expression. Two dif- ferent parameters for PHB were used and are shown in tables 6 and 11. Uniaxial tensile tests The test was performed using an Instron tensile tester model 4250 equipped with a 100 N load cell. The test was car- ried out at a cross-head speed equal to 1 mm / min. The speci- mens for the test have been prepared by cutting a film of the studied PHB. The film was obtained from the dissolution of the polymer with chloroform into a Petri dish and the subsequentevaporation of the solvent. Five specimens were tested.Nuclear magnetic resonance (NMR)CBCPT24013EPWO 05.06.2025All the samples were analyzed with solid-state nuclear mag- netic resonance spectroscopy (Bruker 400Avance WB equipped with double channel 4mm CPMAS probe, Bruker Spa, Rheinstetten) at13C frequency of 100.46 MHz with both simple pulse and cross-polarized sequences at room temperature at 8 kHz of spinning speed to avoid signal overlap. All the results of profile fitting are considered acceptable with a confidence level >97%. In the NMR spectrum both methyl and methylene signals are represented by sharp peaks together with a right broad shoul- der. Thus, these shoulders are proof of a different chain packing in the solid state. The presence of this type of shoulder in the case of other polymers is usually attributed to an amorphous component. Non-woven fabric: The non-woven fiber was produced by spinning a polymer so- lution—prepared by dissolving the polymer in chloroform or di-methyl carbonate (DMC) using a pump that delivered the solu-tion into a spinneret head. The spinneret head was equipped with 13 holes, each with a diameter of 100^µm. The extruded fi-bers were collected on a X-mesh filter cloth with a pore sizeof 150^µm, submerged in a heated water bath maintained at a temperature between 30^°C and 70^°C. Figure 1 shows pictures from a non-woven fabric producedfrom BD AUTO (a) size comparison, b) enlarged view c) micro- scopic picture). Figure 2 shows pictures from a non-woven fabric producedfrom LA AUTO (a) size comparison, b) enlarged view c) micro- scopic picture).CBCPT24013EPWO 05.06.2025Figure 3 shows pictures from a non-woven fabric producedfrom LA SA AUTO (a) size comparison, b) enlarged view c) mi- croscopic picture). Figure 4 shows pictures from a non-woven fabric producedfrom LA GLU (a) size comparison, b) enlarged view c) micro- scopic picture). Figure 5 shows pictures from a non-woven fabric producedfrom GBL LA GLU (a) size comparison, b) enlarged view c) mi- croscopic picture). BD AUTO forms the best 3D network in the non-woven fabric using only one additional precursor. In the pure PHBV samples (LA GLU, LA AUTO) there are onlyvery little fibers formed for the non-woven fabric. In con- trast to this all PH34BV-samples (LA SA AUTO, GBL LA GLU) show better formation of fibers. It seems that the addition of 4B really improves the formation of the 3D network for spun non- woven fabrics. GBL LA GLU shows the best defined filament fi- bers.

Claims

CBCPT24013EPWO 05.06.2025CLAIMS1. Method for producing PHA-copolymers comprising the fol-lowing steps: a) growing bacteria under heterotrophic conditions ina media; b) cultivating the bacteria under autotrophic con-ditions under an atmosphere of CO2, H2and optional O2, wherein the amount of O2 is below 10 % (v / v) and pres-sure is at least 1 barg, wherein at least one precursor for further monomers is added before and / or during step b), wherein the at least one precursor is selected froman organic substance comprising at least 3 carbon atomsand at least 2 oxygen atoms, wherein the organic sub- stance in its hydrolyzed form comprises at least two of carbon atoms are part of a functional group selected from COOH, C=O, C-OH2. Method according to claim 1, wherein the bacterium is awild type bacterium.

3. Method according to one of the claims 1 or 2, whereinthe bacterium is Cupriavidus necator.

4. Method according to one of the claims 1 to 3, whereinthe organic substance comprises 4 to 12 carbon atoms.

5. Method according to one of the claims 1 to 4, wherein instep a) the bacteria are grown under exponential growth conditions.

6. Method according to one of the claims 1 to 5, whereinthe at least one precursor is selected from levulinicCBCPT24013EPWO 05.06.2025acid, gamma-butyrolactone, succinic acid and / or 1,4-bu- tanediol.

7. Method according to one of the claims 1 to 6, wherein atleast 2 different precursors are added in step b).

8. Method according to claim 7, wherein two different pre-cursors are added in a molar ratio between 1,2:1 to 3:1.

9. Method according to claim 8, wherein the precursor withthe higher ratio is levulinic acid.

10. Method according to one of the claims 1 to 9, whereinone precursor is succinic acid.

11. Method according to one of the claims 1 to 10, whereinthe pressure in step b) is at least 2 barg.

12. Method according to one of the claims 1 to 11, whereinthe pressure in step b) ranges from 2 to 20 barg.

13. Method according to one of the claims 1 to 12, whereincontent of CO2 in step b) is between 2 % and 40 % (v / v).

14. Method according to one of the claims 1 to 13, whereinthe content of H2 in step b) is between 50 % and 96 %(v / v).

15. Method according to one of the claims 1 to 10, whereinstep b) is performed under heterotropic conditions and under atmospheric pressure.CBCPT24013EPWO 05.06.202516. Method according to claim 15, wherein a carbon sourceis added in step b).

17. PHA-copolymer as produced by the process according toone of the claims 1 to 16.

18. PHA-copolymer comprising one further comonomer with acontent of 10 to 50 mol%, wherein the comonomer is se- lected from 3V or 4B.

19. PHA-copolymer comprising at least three different mono-mers, selected from 3B, 3V and 4B.

20. PHA-copolymer according to claim 20, wherein the ter-polymer comprises at least 5 mol% 4B.

21. Moulded article, granulate, fiber, non-woven fabric ormaster batch comprising the PHA-copolymer according to any one of the claims 17 to 20.

22. Use of the PHA-copolymer according to any one of theclaims 17 to 20 for the production of coating materials,foils, films, laminates, fibers, non-woven fabric, moulded parts, moulded articles, injection moulded arti-cles, extrudates, containers, packaging materials, coat- ing materials, particles, beads, micro beads and medi- cine dispensers.

23. Method for producing a coating material using a polymersolution of PHA-copolymer according to any one of claims 17 to 20.CBCPT24013EPWO 05.06.202524. Method for producing fibers using a polymer solution ofPHA-copolymer according to any one the of claims 17 to20.

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