NOVEL BIOPLASTICS

Abstract

A method for producing PHA polymer includes using bacteria in which the bacteria are grown under heterotrophic conditions using an organic substance as carbon source and exponential growth conditions. The bacteria are then cultivated under autotrophic conditions under an atmosphere of H.sub.2, CO.sub.2 and O.sub.2, wherein the O.sub.2 content is less than 10% (v/v) and the pressure is more than 1 barg.

Claims

1. Method for producing PHA comprising: growing bacteria under heterotrophic conditions in a media; and cultivating the bacteria under autotrophic conditions under an atmosphere of CO.sub.2, H.sub.2 and O.sub.2, wherein the amount on O.sub.2 is below 10% (v/v) and pressure is at least 1 barg.

2. Method according to claim 1, wherein the bacterium is a wild type bacterium.

3. Method according to claim 1, wherein the bacterium is Cupriavidus necator.

4. Method according to claim 1, wherein in said growing the carbon source comprises sugars, polyols, or organic acids or salts or esters thereof.

5. Method according to claim 1, wherein in said growing the bacteria are grown under exponential growth conditions.

6. Method according to claim 1, wherein the pressure in said cultivating is at least 2 barg.

7. Method according to claim 1, wherein the pressure in said cultivating ranges from 2 to 20 barg.

8. Method according to claim 1, wherein content of CO.sub.2 in said cultivating is between 2% and 25% (v/v).

9. Method according to claim 1, wherein the content of H.sub.2 in said cultivating is between 50% and 92% (v/v).

10. PHA polymer as produced by the process according to claim 1.

11. Moulded article, granulate or master batch comprising the PHA polymer according to claim 10.

12. Use of the PHA polymer according to claim 10 for the production of coating materials, foils, films, laminates, fibers, moulded parts, moulded articles, injection moulded articles, extrudates, containers, packaging materials, coating materials, particles, beads, micro beads and medicine dispensers.

13. A product comprising the PHA polymer according to claim 10, wherein said product comprises a coating material, foil, film, laminate, fiber, moulded part, moulded article, injection-moulded article, extrudate, container, packaging material, particle, bead, micro-bead or medicine dispenser.

Description

FIGURES

[0127] FIG. 1: Profile fitting of the methylene resonance in the .sup.13C CPMAS NMR at 4 ms of a) PHB (commercial) and b) PHB CO.sub.2 according to the invention;

[0128] FIG. 2: .sup.13C VCT curves of PHB CO.sub.2 (larger grey dots) and PHB_comm (small black squares) samples.

EXAMPLES

[0129] NaH.sub.2PO.sub.4 is used as dihydrate.

Example A

[0130] A seed culture medium A was prepared with Glucose 20 g/l, (NH.sub.4).sub.2SO.sub.4 4 g/l, MgSO.sub.4×4H.sub.2O 1.2 g/l, KH.sub.2PO.sub.4 4 g/l, Citric acid×1 H.sub.2O 1.86 g/l and Trace elements solution 10 ml/1 (ZnSO.sub.4×7 H.sub.2O 0.10 g, MnCl.sub.2×4 H.sub.2O 0.03 g, H.sub.3BO.sub.3 0.30 g, CoCl.sub.2×6 H.sub.2O 0.20 g, CuC.sub.12×2 H.sub.2O 0.01 g, NiCl.sub.2×6 H.sub.2O 0.02 g, Na.sub.2MoO.sub.4×2 H.sub.2O 0.03 g and distilled water 1000.00 ml).

[0131] To obtain the inoculum the bacteria (Cupriavidus necator H16) is added to the seed culture medium and the inoculum is added to a reactor.

[0132] Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solution was used comprising Glucose 660 g/l, (NH.sub.4).sub.2SO.sub.4 12 g/l, NaH.sub.2PO.sub.4 12 g/l, MgSO.sub.4×7H.sub.2O 3.6 g/l, KH.sub.2PO.sub.4 12 g/l, Citric acid 30 g/l, Trace element solution 15 ml/l.

[0133] This solution was fed to the reactor under stirring at a rate of 3-5 ml/h until an OD of 20 is reached. Usually this is before a reaction time of 21 h.

[0134] For autolithotropic growth the reaction mixture is either put into a new reactor or stays in the same reactor.

[0135] For autolithotropic growth CO.sub.2, H.sub.2 and O.sub.2 is fed to the reactor with a total pressure of 3 barg (H.sub.2: 80%, 2.4 barg; CO.sub.2: 17%, 0.5 barg; O.sub.2: 3%, 0.1 barg).

[0136] 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 92 hours and OD of 342.67 g/l is reacted in the present example).

[0137] The fermentation is then stopped and the PHA (PHB) is extracted.

Example 1B

[0138] A seed culture medium B was prepared with Sucrose 20 g/l, (NH.sub.4).sub.2SO.sub.4 2 g/l, MgSO.sub.4×4H.sub.2O 1.0 g/l, KH.sub.2PO.sub.4 0.6 g/l, Citric acid×1 H.sub.2O 0.11 g/l, NaH.sub.2PO.sub.4 1.43 g/l, CaCl.sub.2×2 H.sub.2O 0.1 g/l and Trace elements solution 3 ml/1 (ZnSO.sub.4×7 H.sub.2O 0.10 g, MnCl.sub.2×4 H.sub.2O 0.03 g, H.sub.3BO.sub.3 0.30 g, CoC.sub.12×6 H.sub.2O 0.20 g, CuC.sub.12×2 H.sub.2O 0.01 g, NiCl.sub.2×6 H.sub.2O 0.02 g, Na.sub.2MoO.sub.4×2 H.sub.2O 0.03 g and distilled water 1000.00 ml).

[0139] The seed culture medium was inoculated with bacteria (Cupriavidus necator H16) and the inoculum is added to a reactor.

[0140] Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solution was used comprising Sucrose 600 g/l, (NH.sub.4).sub.2SO.sub.4 14 g/l, NaH.sub.2PO.sub.4 7.69 g/l, MgSO.sub.4×7H.sub.2O 4.5 g/l, KH.sub.2PO.sub.4 2 g/l, Citric acid 0.22 g/l, Trace element solution 15 ml/l.

[0141] This solution was fed to the reactor under stirring at a rate of 3-5 ml/h until an OD of 20 is reached. Usually this is before a reaction time of 21 h.

[0142] For autolithotropic growth CO.sub.2, H.sub.2 and O.sub.2 is fed to the reactor with a total pressure of 3.1 barg (H.sub.2: 81%, 2.5 barg; CO.sub.2: 16%, 0.5 barg; O.sub.2: 3%, 0.1 barg). Propionic acid to an amount of 4 g/l in total is added stepwise.

[0143] 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 53 hours and OD of 220 g/l is reacted in the present example). This leads to 75.2 g/l product. From these cells PHBV is obtained. (Elastic modulus 0.95 GPa, Tm=167° C., Content of V approx. 6%, Eta=4.3 g/1).

Example 1C

[0144] A seed culture medium C was prepared with Glycerol 50 g/l, (NH.sub.4).sub.2SO.sub.4 4 g/l, MgSO.sub.4×4H.sub.2O 1.2 g/l, KH.sub.2PO.sub.4 13.3 g/l, Citric acid×1 H.sub.2O 1.85 g/l and Trace elements solution 10 ml/1 (ZnSO.sub.4×7 H.sub.2O 0.10 g, MnCl.sub.2×4 H.sub.2O 0.03 g, H.sub.3BO.sub.3 0.30 g, CoC.sub.12×6 H.sub.2O 0.20 g, CuC.sub.12×2 H.sub.2O 0.01 g, NiCl.sub.2×6 H.sub.2O 0.02 g, Na.sub.2MoO.sub.4×2 H.sub.2O 0.03 g and distilled water 1000.00 ml).

[0145] To the seed culture medium the bacteria (Cupriavidus necator H16) is added to obtain the inoculum and the inoculum is added to a reactor.

[0146] Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solution was used comprising Glycerol 500 g/l, (NH.sub.4).sub.2SO.sub.4 12 g/l, NaH.sub.2PO.sub.4 12 g/l, MgSO.sub.4×7H.sub.2O 3.6 g/l, KH.sub.2PO.sub.4 12 g/l and Trace element solution 15 ml/l.

[0147] This solution was fed to the reactor under stirring at a rate of 3-5 ml/h until an OD of 20 is reached. Usually this is before a reaction time of 21 h.

[0148] For autolithotropic growth CO.sub.2, H.sub.2 and O.sub.2 is fed to the reactor with a total pressure of 3.1 barg (H.sub.2: 80.6%, 2.5 barg; CO.sub.2: 16.1%, 0.5 barg; O.sub.2: 3.2%, 0.1 barg). Propionic acid to an amount of 6 g/l is added stepwise.

[0149] The reactor is stirred and the fermentation is run until an OD>200 is reached. Usually the reaction time is at least 50 hours. From these cells PHBV is obtained.

Comparative Example 1 D (Atmospheric Pressure)

[0150] Example A was repeated with conditions in phase 2 in analogue to L. Garcia-Gonzalez et al. Catalysis Today 2015, 257, 237-245 “Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO.sub.2 using a two-stage cultivation system”. A seed culture medium A was prepared with Glucose 20 g/l, (NH.sub.4).sub.2SO.sub.4 4 g/l, MgSO.sub.4×4H.sub.2O 1.2 g/l, KH.sub.2PO.sub.4 4 g/l, Citric acid×1 H.sub.2O 1.86 g/l and Trace elements solution 10 ml/1 (ZnSO.sub.4×7 H.sub.2O 0.10 g, MnCl.sub.2×4 H.sub.2O 0.03 g, H.sub.3BO.sub.3 0.30 g, CoC.sub.12×6 H.sub.2O 0.20 g, CuC.sub.12×2 H.sub.2O 0.01 g, NiCl.sub.2×6 H.sub.2O 0.02 g, Na.sub.2MoO.sub.4×2 H.sub.2O 0.03 g and distilled water 1000.00 ml).

[0151] To obtain the inoculum the bacteria (Cupriavidus necator H16) is added to the seed culture medium and the inoculum is added to a reactor.

[0152] Under growing conditions, a feed solution is added to the reactor. For chemolithotropic growth the following feed solution was used comprising Glucose 660 g/l, (NH.sub.4).sub.2SO.sub.4 12 g/l, NaH.sub.2PO.sub.4 12 g/l, MgSO.sub.4×7H.sub.2O 3.6 g/l, KH.sub.2PO.sub.4 12 g/l, Citric acid 30 g/l and Trace element solution 15 ml/l.

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

[0154] For autolithotropic growth the reaction mixture is either put into a new reactor or stays in the same reactor.

[0155] For autolithotropic growth CO.sub.2, H.sub.2 and O.sub.2 is fed to the reactor at atmospheric pressure with composition of the gas mixture H.sub.2: 84%, CO.sub.2: 13%, O.sub.2: 3%.

[0156] The reactor is stirred and the fermentation is run until an OD>200 is reached. Usually the reaction time is at least 150 hours, e.g. after 184 hours and OD of 230 g/1 is reached in the present example.

[0157] The fermentation is then stopped and the PHB is extracted.

[0158] Material Properties

[0159] The PHB produced by the described process shows similar thermal properties of PHB produced by standard chemical methods. The molecular weight is in the rage of commercially available PHBs with a narrow MWD. But the polymer is less fragile than typical PHB and more amorphous. The PHB has better tensile properties than pure PBH (elongation brake>20%), a lower modulus (approx. 1000 MPa, vs>2000 MPa), better transparency and a low T.sub.g (−8° C.). PHB produced under similar conditions but without increased pressure is crystalline and similar to commercial PHB.

[0160] Table 1 compares some properties of the PHB of the invention with a PHB Reference and PHBH produced by a biological process:

TABLE-US-00001 TABLE 1 PHBV PHB Example PHB PHBH comparative Property PHB 1C reference reference 1D Density 1.25 1.20 1.25 1.2 1.25 [g/cm.sup.3] Molecular 745k 712k 398k 500k 571k weight [g/mol] Melting 181 167 179 145 179 point [° C.] Glass T T.sub.g −8 −12 0 2 0 [° C.] Elongation 22 150 3 14 5 break [%] Tensile 1010 950 2500 1350 2450 modulus [MPa] Flexural 1300 1230 2800 1600 2780 modulus [MPa] Tensile 33 30 35 36 34 strength [MPa]

[0161] 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.

[0162] DSC Measurements

[0163] Mettler DSC 30 scanner was used. The tests were conducted with a controlled flow of nitrogen. The samples were subjected to the following thermal cycle: two heating steps from −100° C. to 200° C. interspersed by a cooling step from 200° C. to −100° C. The heating/cooling rate was 10° C./min.

[0164] From the DSC thermograms the melting point (T.sub.m1) of the polymer, the crystallization temperature (T.sub.c) under cooling conditions and the melting point in the second heating scan (T.sub.m2) were identified. The integration of the peaks allowed to estimate the melting enthalpy under the first (ΔH.sub.m1) and the second (ΔH.sub.m2) heating scans and under cooling (ΔH.sub.c) conditions. Comparing the thermograms with that of a reference PHB it is possible to ob-serve that the shape of the curves is quite similar but both melting and crystallization temperatures of the analysed PHB are higher. Table 2 shows the measured values. Despite instrumental differences the values for the comparative example 1D correspond to the values in the literature.

TABLE-US-00002 TABLE 2 T.sub.m1 ΔH.sub.m1 T.sub.c ΔH.sub.c T.sub.m1 ΔH.sub.m2 Sample [° C.] [J/g] [° C.] [J/g] [° C.] [J/g] PHB 179.9 84.7 89.2 63.1 181.5 83.2 PHB reference 176.8 92.3 78.0 63.0 178.7 92.8 Comparative 176.5 92.4 75.0 62.9 178.6 92.9 1D CO.sub.2 low P

[0165] Molecular Weight

[0166] The molecular weight was measured indirectly, by intrinsic viscosity, where relation between the viscosity and the molecular weight is given by the Mark-Houwink expression. Table 3 shows the measured values (a=0.78, k=0.000118):

TABLE-US-00003 TABLE 3 Sample η [dl/g] M.sub.w [Da] Reference PHB 2.75 ± 0.04 3.98*10.sup.5 ± 0.01*10.sup.5 PHB 4.50 ± 0.07 7.45*10.sup.5 ± 0.01*10.sup.5 PHBV 4.30 ± 0.06 7.12*10.sup.5 ± 0.01*10.sup.5 Comparative example 1D 3.45 ± 0.05 5.71*105 ± 0.01*105

[0167] Nuclear Magnetic Resonance (NMR)

[0168] The identification of the PHA's powders was performed through a solid state NMR analysis (.sup.13C CPMAS NMR, FIG. 1). This were carried out with a Bruker 400 WB spectrometer operating at a pro-ton frequency of 400.13 MHz. NMR spectra were acquired with cp pulse sequences under the following conditions: .sup.13C 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. Sample was packed in 4 mm zirconia rotor and spun at 10 kHz under air flow. Adamantane was used as external secondary reference. The spectrum of PHB from CO.sub.2 according to the invention is superimposable to the one of commercial PHB.

[0169] In the NMR spectrum both methyl and methylene signals are represented by sharp peaks together with a right broad shoulder. 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. The superimposition of the spectra of the two samples highlights a very small difference in the intensity of the above-discussed shoulders. The amorphous component is higher in the PHB sample from the inventive process.

[0170] Further Experiments of NMR dynamics, measuring magnetization as function of contact time show higher amorphous content 35.9% vs 21.8% expected from the reference PHB sample (profile fitting at 42 ppm and 43.6 ppm). This shows that the packing of the polymer chains is to be different in the PHB produced by the invention. FIG. 1 shows the profile fitting of the methylene resonance in the .sup.13C-CPMAS NMR at 4 ms of a) PHB reference and b) PHB of the invention.

[0171] For all samples the PHB according to the invention showed a higher amorphous content compared to the commercial PHB or the sample from the comparative example 1 D (Table 4).

TABLE-US-00004 TABLE 4 A % A % A % Comp. Example δ (ppm) PHB_CO.sub.2 PHB_referenceC 1 D Attrib. 43.6 64.1 78.2 76.1 Crystalline 42 35.9 21.8 23.9 Amorphous

[0172] Finally, evaluating the trend of the magnetization (peak area) as a function of contact time (FIG. 2) it is possible to relate the behaviour to chain mobility at the molecular level. The normalized curves for the four resonances (C═O (top left), CH (top right), CH.sub.2 (bottom left), CH.sub.3 (bottom right)) are presented in FIG. 2: PHB CO.sub.2 shows a homogeneous trend; instead the PHB_comm seems composed of multiple domains with different mobility.

[0173] For both materials, the magnetization increases fast as for rigid materials reaching a plateau that suggest a very long de-cay typical of polymers. The CO region does not show remarkable difference between the two samples. The second step of growth suggests the presence of a second very mobile component not ho-mogeneously distributed. One could hypothesize that the two samples are a different mixture of enantiomers.

[0174] Uniaxial Tensile Tests

[0175] The test was performed using an Instron tensile tester model 4250 equipped with a 100 N load cell. The test was carried out at a cross-head speed equal to 1 ram/min. The specimens 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 subsequent evaporation of the solvent. Five specimens were tested.

[0176] The results in terms of elastic modulus, stress at break and strain at break are summarized in Table 5.

TABLE-US-00005 TABLE 5 Elastic Stress at Strain at Sample modulus E [GPa] break [MPa] break [%] PHB 1.01 ± 0.07 16.6 ± 2.4 22 ± 5