Novel bioplastic

Abstract

The invention relates to novel bioplastics. Disclosed is a method for producing a PHA polymer by using a two-step process using a bacterium. In a first step, bacteria are grown under heterotrophic conditions and exponential growth conditions using an organic substance as a carbon source. In a second step, the bacteria are then cultured under autotrophic conditions in an atmosphere of H2, CO2 and O2 wherein the O2 content is less than 10% (v / v) and the pressure is greater than 1 barg. As a result, PHA production with unique properties and high rates is possible.

Description

[0001] This application is a divisional application of the patent application with application number 202080089760.8, application date December 18, 2020, and invention name “New Bioplastic”. Technical Field

[0002] The present invention relates to a method for producing polyhydroxyalkanoate (PHA) using wild-type bacteria and extracting the produced PHA in an efficient manner.

[0003] PHA is a general term for a range of different biodegradable polymers consisting of polyesters of 3-hydroxyalkanoic acids. These polymers are of interest due to their wide range of applications and the fact that they are completely biodegradable and therefore have little or no long-term waste problems.

[0004] PHAs are generally classified as short-chain PHAs (sclPHA), medium-chain PHAs (mclPHA), or long-chain PHAs (lclPHA), depending on the number of carbon atoms in their constituent monomers. SclPHA contains C3-C5 monomers, and mclPHA contains C6-C 14 monomers, and lclPHA containing more than 14 carbons (>C l4 ) monomers. This variation in monomer chain length results in polymers with different properties, where sclPHA and lclPHA both have different properties, sclPHA has high crystallinity and is generally rigid and brittle, while lclPHA is sticky and very difficult to handle. The properties of sclPHA and lclPHA limit their range of applications. However, since sclPHA is more readily available, there is a need for sclPHA with improved properties.

[0005] The PHA structure can vary in two ways. First, PHA can vary according to the structure of the side groups, which are typically connected to carbon atoms with (R)-stereochemistry. The side groups form the side chains of hydroxyalkanoic acid and do not contribute to the PHA-carbon backbone. Secondly, PHA can vary according to the number and type of its repeating units. For example, PHA can be a homopolymer, a copolymer or a terpolymer. These changes in the PHA structure can lead to changes in its physical properties. These physical properties make PHA useful in many products that may have commercial value.

[0006] The stereochemistry in the monomers can be only R or S, or both types of monomers can be present. This further affects the properties of the polymer. Usually when produced by biological

[0007] Several types of PHAs make PHA a versatile family of polymers with properties that can be tuned by molecular design. For example, short chain length scl-PHA is divided into P3HB, generally referred to as polyhydroxybutyrate (PHB), P4HB, (pentanoic acid copolymer) PHBV, PHBH, P3HB4HB, and medium chain length mcl-PHA is PHBH (hexanoic acid copolymer), PHBO (octanoic acid copolymer), PHBD (dodecanoic acid copolymer).

[0008] The chemical structure of PHA can be described as a polymer chain formed by the repetition of the following units:

[0009]

[0010] Wherein R is an alkyl or alkenyl group of variable length, and m and n are integers. In some polymers, R and m are assumed to have the following values:

[0011] PHB: R=CH3, m=1

[0012] PHBV: R = CH3 or CH3-CH2-, m = 1

[0013] P4HB: R=H, m=2

[0014] P3HB4HB: for m=2, R=H, or for m=1, R=CH3

[0015] For mclPHA, the length of the alkyl chain of R can vary, for example, for polyhydroxyhexanoate PHBH, R is CH3-CH2-CH2- and m is equal to 2.

[0016] Due to their structure, PHA monomer units contain chiral carbon atoms. The polymer can therefore contain monomers that differ in their configuration. Due to the enzymatic pathway, PHAs synthesized by organisms usually contain only monomers in the R configuration.

[0017] In addition to plants and other organisms, bacteria are also very useful for the production of PHAs. In recent years, many efforts have been made to use genetically modified or unmodified bacteria in order to produce PHAs also on an industrial scale.

[0018] Schlegel et al. Nature 1961, 191, 463-465 “Formation and utilization of poly-β-hydroxybutyric acid by Knallgas bacteria (hydrogenomonas)” found that PHA (ie PHB) can be produced under certain conditions, especially using an atmosphere containing CO2, H2 and O2.

[0019] Ayaaki Ishizaki and Kenji Tanaka, Journal of fermentation and bioengineering 1990, 69(3), 170-174 "Batch Culture of Alcaligenes eutrophusATCC 17697T using recycled gas closed circuit culture system", Ayaaki Ishizaki and Kenji Tanaka, Journal of Fermentation and bioengineering 1991, 71(4) 254-257 "Production of Poly-b-Hydroxybutyric Acid from Carbon Dioxide by Alcaligeneseutrophus ATCC17697T" and Toshihiro Takeshita et al. J. Fac. Agr. Kyushu Univ. 1993,38(1-2), 55-64, Studies on Dissolved Hydrogen Behavior in Autotrophic Cultureof Alcaligenes autrophus ATCC 17697T" discloses the synthesis of PHB in bacteria under autotrophic conditions. One disadvantage of the conditions is the requirement for a ratio of hydrogen and oxygen that is explosive (oxygen>6%). This limits the use of these conditions.

[0020] 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 Alcaligeneseutrophus ATCC 17697 T" discloses two-stage heterotrophic-autotrophic growth, wherein the amount of fructose and O2 in the autotrophic stage is 2-3%. However, the use of organic substrates reduces the PHA storage efficiency of the bacteria.

[0021] The use of CO2 as a carbon source makes these processes very valuable for the environmentally friendly production of plastics that are even biodegradable.

[0022] US 5,942,597 and WO 97 / 07229 A1 describe the extraction of PHA from oil-containing plants using solvent mixtures.

[0023] Among other PHA types, most PHBs are currently successfully produced on an industrial scale by processes using glucose as a raw material. PHBs obtained by industrial processes are highly crystalline because their chemical structure consists of optically pure single monomer units (high stereoregularity). However, the introduction of chain irregularities from comonomers or the insertion of, for example, 4B with 3B or other stereoregularities is very important to achieve improved flexibility and, therefore, better performance and processability for most general plastic applications.

[0024] The procedures proposed in the prior art are not suitable for industrial scale processing. In addition, the extraction of PHA produced from bacteria is also difficult. They also require long reaction times. Summary of the invention

[0025] It is therefore an object of the present invention to provide a process for the production and preferably further purification of PHA in bacteria, more preferably on an industrial scale.

[0026] This object is achieved by the invention as claimed in the independent claim. Advantageous embodiments are described in the dependent claims.

[0027] The object of the present invention is also achieved by a method. Hereinafter, the various steps of the method will be described in more detail. The steps do not necessarily have to be performed in the order given in the text. In addition, other steps not explicitly described may be part of the method.

[0028] The object of the present invention is achieved by a method for producing PHA, which comprises the following steps:

[0029] a) growing the bacteria under heterotrophic conditions in a culture medium;

[0030] b) Cultivating the bacteria under autotrophic conditions in an atmosphere of CO2, H2 and O2, wherein the amount of O2 is below 10% (v / v) and the pressure is at least 1 barg.

[0031] Bacteria that can be used in the present invention include any bacteria that can produce PHA, preferably bacteria that naturally produce PHA. Wild-type bacteria are preferred. Such bacteria are not genetically modified. By using wild types, the process must meet less stringent regulations.

[0032] In one embodiment, the bacteria are Pelomonas saccharophila (formerly also known as Pseudomonas saccharophila), Azomonas lata (formerly also known as Alcaligenes latus), and R. Eutropha (also known as Cupriavidus necator). These are non-pathogenic Gram-negative bacteria that can be found in soil and water. Their facultative chemolithoautotrophic metabolism allows them to grow on organic compounds, or to use H2 and CO2 as reducing agents and carbon sources, respectively, under nutrient limitation and in the presence of oxygen. Depending on the availability of nutrients, these two modes may also coexist. If used in the process of the present invention without any comonomer, these bacteria produce PHB.

[0033] In a preferred embodiment, the bacterium is a wild bacterium selected from the group consisting of Copperworms, even more preferably the stream Copperworms H16, which is a non-pathogenic Gram-negative stream. Other preferred streams are those available from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under the DSM numbers DSM-428, DSM-531, DSM-11098, DSM-3102, DSM-529, DSM-545.

[0034] In addition, wild bacteria selected from Preudosomas Putida or Pseudomonas aeruginosa may be used.

[0035] In the first step, the bacteria are grown under heterotrophic conditions. These are conditions that utilize organic compounds as carbon and energy sources. In preferred embodiments under these conditions, the bacteria show exponential growth.

[0036] In a preferred embodiment, the bacteria are cultured using conditions that are not limiting for nutrients, especially nitrogen, carbon or phosphorus.

[0037] Typically an ambient atmosphere is used.

[0038] In a preferred embodiment, this step is carried out at ambient pressure or at pressure resulting solely from the reaction conditions, such as heating in a closed vessel.

[0039] The culture medium used in step a) is an aqueous culture medium.

[0040] In a preferred embodiment, the culture medium used in the first step comprises at least one ammonium salt as nitrogen source, preferably ammonium sulfate.

[0041] As carbon source, different organic substances can be used. This can be a sugar such as sucrose, fructose or glucose, a polyol such as glycerol, an organic acid or a salt or ester thereof, such as acetic acid or malate or ethyl acetate. The carbon source is preferably soluble in the culture medium.

[0042] In a preferred embodiment, the culture medium used in the first step comprises at least one phosphate as a phosphorus source, preferably an ammonium, sodium or potassium salt of phosphate, in particular their monobasic form can be used, more preferably H2PO4 salt, more preferably (NH4)H2PO4, KH2PO4 and / or NaH2PO4. The salt can be used as a hydrate.

[0043] Ammonium hydroxide, citric acid, and / or sulfuric acid can be used to adjust the pH.

[0044] The culture medium may contain further salts and additives, such as magnesium salts, iron salts, vitamins or trace elements such as Zn, B, Co, Cu, Ni, Mo or Mn.

[0045] The pH of the culture medium is preferably 4.5 to 7.5, more preferably 6.4 to 7.1.

[0046] In a preferred embodiment, the amount of C at the beginning of step a) is between 2 and 50 g / l, preferably between 5 and 20 g / l.

[0047] In a preferred embodiment, the amount of N at the beginning of step a) is between 0.1 and 5 g / l, preferably between 0.1 and 2.5 g / l.

[0048] In a preferred embodiment, the amount of P at the beginning of step a) is between 0.05 and 5 g / l, preferably between 0.1 and 3.5 g / l.

[0049] In a preferred embodiment, the amount of carbon source at the beginning of step a) is 5 to 50 g / l, preferably 10 to 50 g / l. Said values ​​depend on the molar mass of the carbon source and can be adapted to the desired C content.

[0050] In a preferred embodiment, contents of 5 to 20 g / l of C, 0.1 to 2.5 g / l of N, and 0.1 to 3.5 g / l of P are preferred.

[0051] In a preferred embodiment, the bacteria are added in a pre-prepared inoculum.

[0052] For the inoculum, contents of 5 to 30 g / l C, 0.1 to 1.5 g / l N and 0.2 to 5 g / l P are preferred.

[0053] The values ​​at the start of step a) are those after addition of the inoculum.

[0054] Preferred sources of C, N and / or P for the inoculum are the same as mentioned for the culture medium of step a).

[0055] In a preferred embodiment, the following amounts are present at the start of step a):

[0056] Carbon source (glycerol, sucrose or glucose or fructose) 15 to 70 g / l, (NH4)2SO4 1 to 5 g / l, KH2PO4 0.5 to 20 g / l, citric acid x1H2O 0.1-3 g / l and NaH2PO4 0 to 2 g / l.

[0057] Other Mg or Ca salts may be present. In addition, trace element solutions are added, including Zn, Mn, B, Co, Cu, Ni and Mo.

[0058] In a preferred embodiment, at the start of step a), the reactor is filled with a volume of 5 to 20% of its total volume.

[0059] Step a) is preferably carried out at a temperature between 20 and 40°C, more preferably between 29 and 35°C.

[0060] The reactor may need to be stirred.

[0061] During the growth of the bacteria, the growth nutrients present are consumed.In a preferred embodiment, at least some nutrients are fed into the culture medium so that these nutrients are preferably kept within the ranges mentioned above.

[0062] The feed can be added to the space of the culture medium inside the reactor. Preferably the culture medium is not removed during the cultivation.

[0063] In a preferred embodiment, the feed solution is added at a rate between 0.1 and 5% / h, preferably 0.2 to 1%, calculated from the total volume of the reactor. The feed rate can be adjusted based on the content of the feed and / or the culture conditions, especially the pressure used.

[0064] The feed solution preferably comprises at least one carbon source.

[0065] In another embodiment, the feed solution comprises at least one carbon source, at least one nitrogen source.

[0066] In another embodiment, the feed solution comprises at least one carbon source, at least one nitrogen source and at least one phosphorus source. Preferred sources are the same as mentioned for the culture medium of step a).

[0067] In a preferred embodiment, the carbon source is the same as the carbon source of the starting conditions.

[0068] In a preferred embodiment, the feed solution comprises a carbon content of 100 to 500 g / l, preferably 150 to 300 g / l.

[0069] Preferably, the feed solution further comprises a N content of 1 to 10 g / l, preferably 1 to 5 g / l.

[0070] Preferably, the feed solution further comprises a P content of 0.5 to 15 g / l, preferably 1 to 10 g / l.

[0071] Preferably, the feed comprises the contents of C, N and P as mentioned before or each at their preferred values.

[0072] The feed solution may contain other Ca and / or Mg salts, and acids such as citric acid.The feed may also contain a trace element solution.

[0073] In a preferred embodiment, the feed comprises 150 to 300 g / l C, 1 to 5 g / l N, 1 to 10 g / l P.

[0074] Preferred sources of C, N and / or P for the feed are the same as mentioned for the culture medium of step a).

[0075] In a preferred embodiment, the feed comprises the following ingredients:

[0076] Carbon source (glucose or sucrose or glycerol) 150 to 300 g / l C;

[0077] (NH4)2SO4 and / or (NH4)H2PO4, in an amount of 1 to 5 g / l N;

[0078] KH2PO4 0 to 15 g / l;

[0079] NaH2PO4 0 to 15 g / l;

[0080] Bacterial growth continues until a cell density of at least 10 (measured by OD at 600 nm) is reached, preferably at least 15, more preferably at least 20. Relevant growth points may also be related to other measurements such as time or composition.

[0081] Step a) is generally run for at least 5 hours up to 40 hours, preferably 10 hours up to 25 hours.

[0082] In the next step, the bacteria are grown under autotrophic conditions. Under such conditions, CO2 is used as a carbon source for the bacteria.

[0083] In this step, the culture medium is contacted with an atmosphere comprising H2, CO2 and O2. In order to minimize the risk of explosion, the content of O2 is less than 10% (v / v). In a preferred embodiment, the content of CO2 is between 2% and 25% (v / v). In a preferred embodiment, the content of H2 is between 50% and 92% (v / v).

[0084] In a preferred embodiment, the O2 content is less than 8% (v / v), preferably less than 6% (v / v).

[0085] In a preferred embodiment, the O2 content is less than 8% (v / v), the CO2 content is between 2% and 25% (v / v), and the H2 content is between 50% and 92% (v / v).

[0086] In a preferred embodiment, the O2 content is less than 6% (v / v), the CO2 content is between 5% and 20% (v / v), and the H2 content is between 50% and 92% (v / v).

[0087] In a preferred embodiment, the nitrogen content of the atmosphere, if present, is less than 1% (v / v).Some small amount of nitrogen may be introduced into the reaction vessel by not degassing the solution fed into the reactor.

[0088] The source of such an atmosphere may be syngas.

[0089] If precursors of other monomers are added, the amount of CO2 is preferably between 1 and 25% (v / v) of CO2, preferably between 2% and 25% (v / v), more preferably 2 to 20% (v / v).

[0090] The ratios mentioned are the ratios present at the start of step b). Since gases are used during fermentation, it may be necessary to adjust the amounts to the previous ranges during fermentation. In a more preferred embodiment, the ratios are maintained within these ranges during step b).

[0091] The pressure in step b) is at least 1 barg or gauge pressure. In a preferred embodiment, the pressure is at least 2 barg, more preferably at least 3 barg.

[0092] In a preferred embodiment the pressure ranges from 2 to 20 barg, preferably from 3 to 20 barg, more preferably from 3 to 10 barg.The pressure is measured under culture conditions.

[0093] During step b) the pressure is preferably maintained within these ranges. In a preferred embodiment the pressure during the entire step b) is at least 1 barg.

[0094] Based on a standard atmospheric pressure of 1.013 bar, 1 barg as used in this application corresponds to an absolute pressure of 2.013 bar. All other ranges are adjusted accordingly.

[0095] By using increased pressure, the growth of the PHA is accelerated and the duration of step b) is shortened. Surprisingly, the increased pressure also results in a PHA with different properties compared to the PHA obtained without increased pressure.

[0096] Preferably, the content of different gases is measured by partial pressure.

[0097] Preferably, the pressure remains constant during the duration of step b). More preferably, the composition and pressure of the atmosphere remain constant during the duration of step b).

[0098] In step b), no further nutrients are added to the reactor.

[0099] The pH in step b) is preferably between 6.5 and 7.5, more preferably between 6.8 and 7.0. The pH can be adjusted using acids and / or bases, preferably sulfuric acid and / or ammonium hydroxide.

[0100] As culture medium, the same culture medium as in step a) can be used, but without any nitrogen source or carbon source.

[0101] In step b), the cultivation is preferably run under conditions at least lacking nitrogen. The nitrogen source limits the accumulation of biomass. This leads to the accumulation of PHA.

[0102] In a preferred embodiment, the cell dispersion at the end of step a) is directly used in step b) as the starting medium.

[0103] The temperature in step b) is preferably between 20 and 45°C, more preferably between 25 and 35°C.

[0104] It may be necessary to stir the reactor during the reaction.

[0105] In a further embodiment of the invention, precursors of other monomers are added at step b) to obtain a copolymer of PHB. In a preferred embodiment, these are salts of organic acids, preferably sodium or potassium salts. Examples of such salts are the corresponding salts of propionic acid, butyric acid, valeric acid or caproic acid, depending on the length of the desired side chain. These precursors are generally added in an amount to obtain a unit content of 5% to 30%, preferably 5% to 20% (in molar ratio) in the produced PHA. These precursors are preferably added to an amount of up to 1 to 20 g / L, preferably 2 to 10 g / l.

[0106] The reaction in step b) is run until a certain amount of PHA is formed, typically until a content of 50 to 90 wt.-% PHA calculated on the dry weight of the whole biomass is formed.

[0107] Under these conditions, step b) may be run until a final cell density of greater than 50 g / L, preferably greater than 100 g / L is reached.

[0108] It is also preferred to stop the reaction before the Mw of the PHA and / or PHB begins to decrease due to side reactions.

[0109] The duration of the culture is generally 20 to 80 hours, preferably 30 to 60 hours, more preferably 30 to 50 hours.

[0110] Step b) can be carried out in the same reactor as step a) or in a different reactor, preferably in a different reactor.

[0111] If necessary, further purification steps, such as filtration or centrifugation, are performed between the two steps.

[0112] In a preferred embodiment, the cells are separated from the culture medium prior to the subsequent purification step.

[0113] The cells may also be washed with alcohol (eg, methanol and / or ethanol).

[0114] PHA is formed inside the bacteria. For extraction, several methods are possible.

[0115] In one embodiment, the cells are first disrupted by mechanical stress such as increased atmospheric pressure.The PHA is then extracted using a solvent for PHA, preferably a polar organic solvent, more preferably acetone or chloroform, especially acetone.

[0116] In a preferred embodiment, other solvents having a higher boiling point than the solvent used for PHA are also added. For example, oils or alkanes having 10 to 14 carbon atoms. The other solvents are preferably used in a weight ratio of 1:20 to 1:4 compared to the solvent used for PHA.

[0117] If the solvent for PHA is removed from the mixture, the PHA can be recovered as high purity flakes. Preferably, the solvent for PHA is removed by heating. The solvent for PHA can be reused for further extraction.

[0118] Cell lysis can also be combined with the extraction step, as acetone also causes cell lysis. In this embodiment, acetone and optionally other solvents are added to the isolated cells. The amount of solvents (especially acetone) and other solvents used for PHA is preferably used in excess compared to the weight of the isolated cells. Preferably, it is in an amount of at least 2 times the weight of the isolated cells, more preferably at least 5 times the weight.

[0119] In a preferred embodiment, the solvent for PHA and other solvents are added, the mixture is then mixed and the non-aqueous phase is separated.

[0120] The produced PHA is obtained as flakes during the removal of the solvent for the PHA, especially acetone.

[0121] The PHA polymer produced by this process has a narrow molecular weight distribution.

[0122] Unexpectedly, the PHA polymers produced are less crystalline polymers compared to PHA produced under ambient conditions. This makes, for example, PHB polymers less brittle than typical PHB polymers. They also exhibit lower moduli than these polymers.

[0123] In a preferred embodiment, the amorphous content of the polymer is between 25% and 50%, preferably 30% and 40% (measured by solid state NMR).

[0124] Comonomers are often used to obtain less crystalline polymers, which are generally smoother and more elastic. For example, pure PHB produced according to the present invention shows some properties similar to PHBV produced by conventional procedures and a content of 10 mol% comonomer. Also for PHBV produced by the process of the present invention, a smaller amount of comonomer is required to obtain the same properties.

[0125] Another object of the present invention is the PHA polymer produced by the process of the present invention.

[0126] Polymers can be used in various products depending on their properties. They can also be blended with other polymers.

[0127] Another object of the present invention is a molded article, pellet or masterbatch comprising or formed from a PHA polymer as described above.

[0128] The molded articles can be produced in any manner, for example by extrusion, casting, injection molding, pressing, sintering, calendaring, film blowing, melt spinning, compression molding and / or thermoforming, for components in automotive construction, transport and / or communications, components for industrial equipment, machine and plant construction, household appliances, containers, devices for medical technology, components for electrical or electronic products. The invention therefore also relates to the use of the polymer material according to the invention for the aforementioned purposes.

[0129] The polymers can be used to produce coating materials, foils, films, laminates, fibers, molded parts, molded articles, injection molded articles, such as bottles or fibers, extrudates, containers, packaging materials, coating materials, granules, beads, microbeads and drug dispensers.

[0130] The polymer can be formed into any product known from previous products. Conventional additives and other polymers can also be added.

[0131] The present invention includes the following aspects / embodiments / features in any order and / or in any combination:

[0132] 1. A method for producing PHA, comprising the following steps:

[0133] a) growing the bacteria under heterotrophic conditions in a culture medium;

[0134] b) cultivating the bacteria under autotrophic conditions in an atmosphere of CO2, H2 and O2, wherein the amount of O2 is below 10% (v / v) and the pressure is at least 1 barg.

[0135] 2. The method of any preceding or following embodiment / feature / aspect, wherein the bacterium is a wild-type bacterium.

[0136] 3. The method of any preceding or following embodiment / feature / aspect, wherein the bacterium is Cupriavidus necrotus.

[0137] 4. The method of any preceding or following embodiment / feature / aspect, wherein, in step a), the carbon source is selected from a sugar, a polyol or an organic acid or a salt or ester thereof.

[0138] 5. The method of any preceding or following embodiment / feature / aspect, wherein, in step a), the bacteria are grown under exponential growth conditions.

[0139] 6. The method of any preceding or following embodiment / feature / aspect, wherein the pressure in step b) is at least 2 barg.

[0140] 7. The method of any preceding or following embodiment / feature / aspect, wherein the pressure in step b) is in the range of 2 to 20 barg.

[0141] 8. The method of any preceding or following embodiment / feature / aspect, wherein the content of CO2 in step b) is between 2% and 25% (v / v).

[0142] 9. The method of any preceding or following embodiment / feature / aspect, wherein the content of H2 in step b) is between 50% and 92% (v / v).

[0143] 10. A PHA polymer produced by the process of any preceding or following embodiment / feature / aspect.

[0144] 11. A molded article, pellet or masterbatch comprising a PHA polymer of any preceding or following embodiment / feature / aspect.

[0145] 12. Use of the PHA polymer of any preceding or following embodiment / feature / aspect for producing coatings, foils, films, laminates, fibers, molded parts, molded articles, injection molded articles, extrudates, containers, packaging materials, coatings, particles, beads, microbeads and drug dispensers. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1 : According to the present invention a) PHB (commercial) and b) PHB_CO2 13 C Profile fitting of the methylene resonance at 4 ms in CPMAS NMR;

[0147] Figure 2 : PHB_CO2 (larger gray dots) and PHB_comm (commercial) (small black squares) samples 13 C VCT curve. Example

[0148] NaH2PO4 was used as the dihydrate.

[0149] Example A

[0150] Seed medium A was prepared with: glucose 20 g / l, (NH4)2SO4 4 g / l, MgSO4x4H2O 1.2 g / l, KH2PO4 4 g / l, citric acidx1H2O 1.86 g / l and trace element solution 10 ml / l (ZnSO4x7H2O 0.10 g, MnCl2x4H2O0.03 g, H3BO3 0.30 g, CoCl2x6H2O 0.20 g, CuCl2x2H2O 0.01 g, NiCl2x6H2O 0.02 g, Na2MoO4x2H2O0.03 g and distilled water 1000.00 ml).

[0151] To obtain the inoculum, bacteria (Cupria necrotica H16) were added to the seed medium and the inoculum was added to the reactor.

[0152] The feed solution was added to the reactor under growth conditions. For chemolithotropic growth, the following feed solution was used, which contained glucose 660 g / l, (NH4)2SO4 12 g / l, NaH2PO4 12 g / l, MgSO4x7H2O 3.6 g / l, KH2PO4 12 g / l, citric acid 30 g / l, trace element solution 15 ml / l.

[0153] This solution was fed to the reactor at a rate of 3-5 ml / h with stirring until reaching an OD of 20. Typically this was before a reaction time of 21 h.

[0154] For autolithotropic growth, the reaction mixture is either placed into a new reactor or remains in the same reactor.

[0155] For the inorganic autotrophic growth, CO2, H2 and O2 were fed to the reactor at a total pressure of 3 barg (H2: 80%, 2.4 barg; CO2: 17%, 0.5 barg; O2: 3%, 0.1 barg).

[0156] The reactor was stirred and the fermentation was run until an OD>200 was reached. Typically the reaction time was at least 50 hours, for example after 92 hours and in this example an OD of 342.67 g / l was reached).

[0157] The fermentation was then stopped and PHA (PHB) was extracted.

[0158] Example 1B

[0159] Seed medium B was prepared with: sucrose 20 g / l, (NH4)2SO4 2 g / l, MgSO4x4H2O 1.0 g / l, KH2PO4 0.6 g / l, citric acidx1H2O 0.11 g / l, NaH2PO4 1.43 g / l, CaCl2x2H2O 0.1 g / l and trace element solution 3 ml / l (ZnSO4x7H2O 0.10 g, MnCl2x4H2O 0.03 g, H3BO3 0.30 g, CoCl2x6H2O 0.20 g, CuCl2x2H2O 0.01 g, NiCl2x6H2O 0.02 g, Na2MoO4x2H2O 0.03 g and distilled water 1000.00 ml).

[0160] The seed medium was inoculated with bacteria (Cupria necrotica H16) and the inoculum was added to the reactor.

[0161] The feed solution was added to the reactor under growth conditions. For chemolithotrophic growth, the following feed solution was used, which contained sucrose 600 g / l, (NH4)2SO4 14 g / l, NaH2PO4 7.69 g / l, MgSO4x7H2O 4.5 g / l, KH2PO4 2 g / l, citric acid 0.22 g / l, trace element solution 15 ml / l.

[0162] This solution was fed to the reactor at a rate of 3-5 ml / h with stirring until reaching an OD of 20. Typically this was before a reaction time of 21 h.

[0163] For the inorganic autotrophic growth, CO2, H2 and O2 were fed to the reactor at a total pressure of 3.1 barg (H2: 81%, 2.5 barg; CO2: 16%, 0.5 barg; O2: 3%, 0.1 barg). Propionic acid was added stepwise to a total amount of 4 g / l.

[0164] The reactor was stirred and the fermentation was run until OD>200 was reached. Typically the reaction time is at least 50 hours, for example after 53 hours, and in this example an OD of 220 g / l was reached). This resulted in 75.2 g / l of product. PHBV was obtained from these cells. (Elastic modulus 0.95 GPa, Tm=167°C, V content about 6%, Eta=4.3 g / l).

[0165] Example 1C

[0166] Seed medium C was prepared with: glycerol 50 g / l, (NH4)2SO4 4 g / l, MgSO4x4H2O 1.2 g / l, KH2PO4 13.3 g / l, citric acidx1H2O 1.85 g / l and trace element solution 10 ml / l (ZnSO4x7H2O 0.10 g, MnCl2x4H2O 0.03 g, H3BO3 0.30 g, CoCl2x6H2O 0.20 g, CuCl2x2H2O 0.01 g, NiCl2x6H2O 0.02 g, Na2MoO4x2H2O 0.03 g and distilled water 1000.00 ml).

[0167] Bacteria (Cupria necrotica H16) were added to the seed medium to obtain an inoculum, and the inoculum was added to the reactor.

[0168] The feed solution was added to the reactor under growth conditions. For chemolithotrophic growth, the following feed solution was used, which contained glycerol 500 g / l, (NH4)2SO4 12 g / l, NaH2PO4 12 g / l, MgSO4x7H2O 3.6 g / l, KH2PO4 12 g / l and trace element solution 15 ml / l.

[0169] This solution was fed to the reactor at a rate of 3-5 ml / h with stirring until reaching an OD of 20. Typically this was before a reaction time of 21 h.

[0170] For the inorganic autotrophic growth, CO2, H2 and O2 were fed to the reactor at 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). Propionic acid was added stepwise to an amount of 6 g / l.

[0171] The reactor was stirred and the fermentation was run until OD>200 was reached. Typically the reaction time was at least 50 hours. PHBV was obtained from these cells.

[0172] Comparative Example 1D (atmospheric pressure)

[0173] Example A was repeated, wherein the conditions in stage 2 were similar to those in L. Garcia-Gonzalez et al., Catalysis Today 2015, 257, 237-245 “Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO2 using a two-stage cultivation system”. Seed medium A was prepared with: glucose 20 g / l, (NH4)2SO4 4 g / l, MgSO4x4H2O 1.2 g / l, KH2PO4 4 g / l, citric acidx1H2O 1.86 g / l and trace element solution 10 ml / l (ZnSO4x7H2O 0.10 g, MnCl2x4H2O 0.03 g, H3BO3 0.30 g, CoCl2x6H2O 0.20 g, CuCl2x2H2O 0.01 g, NiCl2x6H2O 0.02 g, Na2MoO4x2H2O 0.03 g and distilled water 1000.00 ml).

[0174] To obtain the inoculum, bacteria (Cupria necrotica H16) were added to the seed medium and the inoculum was added to the reactor.

[0175] The feed solution was added to the reactor under growth conditions. For chemolithotrophic growth, the following feed solution was used, which contained glucose 660 g / l, (NH4)2SO4 12 g / l, NaH2PO4 12 g / l, MgSO4x7H2O 3.6 g / l, KH2PO4 12 g / l, citric acid 30 g / l and trace element solution 15 ml / l.

[0176] This solution was fed to the reactor at a rate of 3-5 ml / h with stirring until reaching an OD of 20. Typically this was before a reaction time of 21 h.

[0177] For autolithotropic growth, the reaction mixture is either placed into a new reactor or remains in the same reactor.

[0178] For the inorganic autotrophic growth, CO2, H2 and O2 were fed to the reactor at atmospheric pressure, wherein the composition of the gas mixture was H2: 84%, CO2: 13%, O2: 3%.

[0179] The reactor was stirred and the fermentation was run until an OD>200 was reached. Typically the reaction time was at least 150 hours, for example after 184 hours, and in this example an OD of 230 g / l was reached.

[0180] The fermentation was then stopped and the PHB was extracted.

[0181] Material properties

[0182] The PHB produced by the described process exhibits thermal properties similar to those of PHB produced by standard chemical methods. The molecular weight is within the range of commercially available PHBs with narrow MWD. ​​However, the polymer is less brittle and more amorphous than typical PHBs. PHB has better tensile properties (elongation at break > 20%), lower modulus (approximately 1000 MPa compared to > 2000 MPa), better clarity and lower T than pure PBH. g (-8°C). PHB produced under similar conditions but without increased pressure was crystalline and similar to commercial PHB.

[0183] Table 1 compares some properties of the PHB of the present invention with those of the PHB reference and PHBH produced by bioprocess:

[0184] Table 1:

[0185]

[0186] Although the melting temperature is the same as expected for pure PHB, the modulus and tensile properties are more similar to those of PHBH copolymers.

[0187] DSC measurements

[0188] A Mettler DSC 30 scanner was used. The tests were performed using a controlled nitrogen flow. The samples were subjected to the following thermal cycle: two heating steps from -100°C to 200°C interspersed with a cooling step from 200°C to -100°C. The heating / cooling rate was 10°C / min.

[0189] From the DSC thermogram, the melting point (T m1 ), the crystallization temperature under cooling (Tc), and the melting point in the second heating scan (T m2 The integration of the peak allows to estimate the first (△H m1 ) and the second time (△H m2 ) heating scan and cooling (△H c ) conditions. Comparing the thermogram with the thermogram of the reference PHB, it can be observed that the shape of the curves is very similar, but the melting and crystallization temperatures of the analyzed PHB are higher. Table 2 shows the measured values. Despite the instrumental differences, the values ​​of comparative example 1D correspond to those in the literature.

[0190] Table 2:

[0191]

[0192] Molecular weight

[0193] The molecular weight is measured indirectly by the intrinsic viscosity, where the relationship between viscosity and molecular weight is given by the Mark-Houwink expression. Table 3 shows the measured values ​​(a=0.78, k=0.000118):

[0194] Table 3:

[0195]

[0196] Nuclear Magnetic Resonance (NMR)

[0197] The identification of PHA powder was performed by solid-state NMR analysis ( 13 C CPMAS NMR, Figure 1 ). This was performed using a Bruker 400WB spectrometer operating at a proton frequency of 400.13 MHz. NMR spectra were acquired with a cp pulse sequence under the following conditions: 13 C frequency: 100.48 MHz, π / 2 pulse 3.5 μs, decoupling length 5.9 μs, recycle delay: 4 s, 128 scans; contact time 2 ms. The sample was loaded into a 4 mm zirconia rotor and spun at 10 kHz under air flow. Adamantane was used as an external secondary reference. The spectrum of PHB from CO2 according to the present invention is superimposable with the spectrum of commercial PHB.

[0198] In the NMR spectrum, both methyl and methylene signals are represented by sharp peaks together with a broad shoulder on the right. Thus, these shoulders demonstrate different chain packing in the solid state. In the case of other polymers, the presence of this type of shoulder is usually attributed to an amorphous component. The overlap of the spectra of the two samples highlights the very small difference in the intensity of the above-mentioned shoulder. The amorphous component in the PHB sample from the process of the present invention is higher.

[0199] Further experiments with NMR kinetics (measuring magnetization as a function of contact time) showed a higher amorphous content of 35.9%, compared to the expected 21.8% for the reference PHB sample (distribution fit at 42 ppm and 43.6 ppm). This shows that the packing of the polymer chains is different in the PHB produced by the present invention. Figure 1 The results show that a) PHB reference and b) PHB of the present invention 13 Distribution fitting of the methylene resonance at 4 ms in C-CPMAS NMR.

[0200] For all samples, the PHB according to the present invention showed higher amorphous content compared to the commercial PHB or the sample from Comparative Example ID (Table 4).

[0201] Table 4

[0202]

[0203] Finally, the trend of the magnetization intensity (peak area) as a function of contact time was evaluated ( Figure 2 ), the behavior can be related to the chain mobility at the molecular level. The normalized curves of the four resonances (C=O (upper left), CH (upper right), CH2 (lower left), and CH3 (lower right)) are shown in Figure 2 : PHB_CO2 shows a homogeneous trend; in contrast, PHB_comm (commercial) appears to consist of multiple domains with different mobilities.

[0204] For both materials, the magnetization increases fast when the rigid material reaches a plateau, indicating a very long decay typical of polymers. The CO region does not show significant differences between the two samples. The second step of growth indicates the presence of a second, inhomogeneously distributed, very mobile component. It can be assumed that the two samples are different mixtures of enantiomers.

[0205] Uniaxial tensile test

[0206] The test was performed using an Instron tensile tester model 4250 equipped with a 100N load cell. The test was performed at a crosshead speed equal to 1 mm / min. The specimens for the test had been prepared by cutting a film of the PHB under study. The film was obtained by dissolving the polymer into a Petri dish using chloroform and then evaporating the solvent. Five specimens were tested.

[0207] The results in terms of elastic modulus, fracture stress and fracture strain are summarized in Table 5.

[0208] Table 5:

[0209]

Claims

1. A method for producing PHA, comprising the following steps: a) growing the bacteria under heterotrophic conditions in a culture medium; b) Cultivating the bacteria under autotrophic conditions in an atmosphere of CO2, H2 and O2, wherein the amount of O2 is below 10% (v / v) and the pressure is at least 1 barg.

2. The method according to claim 1, wherein the bacteria are wild-type bacteria.

3. The method according to one of claims 1 or 2, wherein the bacterium is Cupriavidus necrotus.

4. The method according to claim 1, wherein: In step a), the carbon source is selected from sugars, polyols or organic acids or salts or esters thereof.

5. The method according to claim 1, wherein: In step a), the bacteria are grown under exponential growth conditions.

6. The method according to claim 1, wherein: The pressure in step b) is at least 2 barg.

7. The method according to claim 1, wherein: The pressure in step b) is in the range of 2 to 20 barg.

8. The method according to claim 1, wherein: The content of CO2 in step b) is between 2% and 25% (v / v).

9. The method according to claim 1, wherein: The content of H2 in step b) is between 50% and 92% (v / v).

10. PHA polymer produced by the process according to one of claims 1 to 9.

11. A molded article, pellet or masterbatch comprising the PHA polymer according to claim 10.

12. Use of the PHA polymer according to claim 10 for producing coating materials, foils, films, laminates, fibers, molded parts, molded articles, injection molded articles, extrudates, containers, packaging materials, coating materials, granules, beads, microbeads and drug dispensers.

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