Processes for recovering and purifying polyhydroxyalkanoates from cell cultures

Abstract

A process for recovering and purifying polyhydroxyalkanoates from a cell culture may include: (a) acidifying the culture to obtain a pH value less than or equal to 6, and submitting the culture to a cell fractionation treatment using high-pressure homogenization at a temperature greater than or equal to 10 C. and less than or equal to 80 C. to obtain a suspension; (b) basifying the suspension to obtain a pH value greater than or equal to 8; (c) diluting the suspension and submitting the diluted PHA suspension to tangential filtration to obtain a concentrated suspension as retentate and an aqueous phase as permeate; (d) submitting the concentrated suspension to bleaching; (e) diluting the suspension after the bleaching and submitting the diluted bleached suspension to tangential filtration to obtain a concentrated bleached suspension as retentate and an aqueous phase as permeate; and/or (f) submitting the concentrated bleached suspension to drying.

Claims

1. A process for recovering and purifying polyhydroxyalkanoates (PHA) from a cell culture, the process comprising: (a) acidifying the cell culture by adding an aqueous solution of an inorganic or organic acid selected from: sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, citric acid, or mixtures thereof, so as to obtain a pH value less than or equal to 6, and submitting the cell culture to a cell fractionation treatment using high-pressure homogenization at a temperature greater than or equal to 10 C. and less than or equal to 80 C., so as to obtain a PHA suspension; (b) basifying the PHA suspension thus obtained so as to obtain a pH value greater than or equal to 8; (c) diluting the PHA suspension and submitting the diluted PHA suspension to first tangential filtration so as to obtain a concentrated PHA suspension as first retentate and a first aqueous phase as first permeate; (d) submitting the concentrated PHA suspension to bleaching; (e) diluting the PHA suspension after the bleaching and submitting the diluted bleached suspension to second tangential filtration so as to obtain a concentrated bleached PHA suspension as second retentate and a second aqueous phase as second permeate; and (f) submitting the concentrated bleached PHA suspension to drying.

2. The process of claim 1, wherein the cell culture is submitted to a preliminary step of concentration.

3. The process of claim 2, wherein the preliminary step of concentration yields a cell concentration greater than or equal to 20 grams/liter (g/L) and less than or equal to 800 g/L.

4. The process of claim 1, wherein in step (a), the cell culture is acidified so as to obtain a pH value less than or equal to 5.

5. The process of claim 1, wherein a pressure greater than or equal to 500 bar and less than or equal to 2,000 bar is applied during the homogenization.

6. The process of claim 1, wherein in step (b), the PHA suspension is basified so as to obtain a pH value greater than or equal to 9.

7. The process of claim 1, wherein the PHA suspension thus basified is treated with at least one surfactant at a temperature greater than or equal to 10 C. and less than or equal to 80 C.

8. The process of claim 1, wherein after the treatment according to step (b), the PHA suspension is diluted so as to obtain a solid concentration greater than or equal to 10 grams/liter (g/L) and less than or equal to 500 g/L.

9. The process of claim 1, wherein in at least one of the tangential filtration steps, at least one ceramic or polymeric membrane is used, having a mean pore dimension greater than or equal to 0.05 microns (m) and less than or equal to 10 m.

10. The process of claim 1, wherein in at least one of the tangential filtration steps, the PHA suspension is fed through at least one tangential flow filter with a pressure greater than or equal to 1 bar and less than or equal to 10 bar.

11. The process of claim 1, wherein in at least one of the tangential filtration steps, flow speed greater than or equal to 2 m/sec and less than or equal to 10 meters/second is maintained through at least one tangential flow filter.

12. The process of claim 1, wherein the bleaching step (d) is carried out by adding oxidizing agent.

13. The process of claim 1, wherein the bleaching step (d) is carried out at a temperature greater than or equal to 10 C. and less than or equal to 60 C.

14. The process of claim 1, wherein after the treatment according to step (d), the bleached PHA suspension is diluted so as to obtain a solid concentration greater than or equal to 10 grams/liter (g/L) and less than or equal to 100 g/L.

15. The process of claim 1, wherein the concentrated bleached PHA suspension obtained from step (e) is further concentrated using orthogonal filtration and then directed to the drying step (f).

16. The process of claim 15, wherein at least one flocculating agent is added to the concentrated bleached PHA suspension, and a resulting combination is then fed to the orthogonal filtration step.

17. The process of claim 1, wherein a pressure greater than or equal to 500 bar and less than or equal to 1.500 bar is applied during the homogenization.

18. The process of claim 1, wherein the PHA suspension thus basified is treated with at least one surfactant at a temperature greater than or equal to 20 C. and less than or equal to 50 C.

19. The process of claim 1, wherein after the treatment according to step (b), the PHA suspension is diluted so as to obtain a solid concentration greater than or equal to 25 grams/liter (g/L) and less than or equal to 100 g/L.

20. The process of claim 1, wherein in at least one of the tangential filtration steps, at least one ceramic or polymeric membrane is used, having a mean pore dimension greater than or equal to 0.2 microns (m) and less than or equal to 5 m.

Description

EXAMPLE 1

(1) A fermentation process was carried out by a Ralstonia eutropha strain on an organic molasses-based substrate deriving from processing of sugar beet, so as to obtain a cell concentration equal to 80 g/L.

(2) The culture broth was concentrated by a tangential flow filter having a pore diameter (cut-off) equal to 1.2 m, so as to obtain a cell concentration equal to 150 g/L.

(3) The cell culture thus concentrated was added to a sulphuric acid solution (concentration 10% by weight), in an amount such as to obtain a pH value equal to about 4.5.

(4) The acidified culture was then introduced into a high pressure homogenizer (maximum pressure: 1500 bar), at room temperature. The homogenization process was carried out continuously with three consecutive passages inside the homogenizer.

(5) The PHA suspension thus obtained was then added with a sodium hydroxide solution (concentration 50% by weight), so as to obtain a pH value equal to about 10.0, and then with a sodium dodecyl sulphate aqueous solution (4 g of surfactant per liter of suspension), keeping the suspension at room temperature.

(6) The suspension thus treated was then diluted in ratio 1:4 with water, and then subjected to filtration through a tangential flow filter with a cut-off equal to 0.8 m, so as to obtain a solid concentration equal to 150 g/L.

(7) The concentrated PHA suspension thus obtained was then subjected to bleaching by addition of a hydrogen peroxide solution at 30% by weight, in ratio 1:8 with respect to the volume of the suspension.

(8) After diluting with water in ratio 1:3, the bleached suspension was subjected to a tangential filtration with a filter of the same type used after the basifying step (cut-off: 0.8 m). The retentate contained the granules of purified PHA, whereas the permeate was used for the aforementioned dilution steps.

(9) The retentate was subjected to drying through a spray dryer at 240 C., so as to obtain a PHA powder with a water content lower than 1% by weight.

EXAMPLE 2

(10) The same culture broth as in Example 1 was treated as follows for recovering and purifying the PHA.

(11) The culture broth was concentrated by a tangential flow filter having a cut-off equal to 0.4 m, so as to obtain a cell concentration equal to 250 g/L.

(12) The cell culture thus concentrated was added with a sulphuric acid solution (concentration 30% by weight), in an amount such as to obtain a pH value equal to about 4.5.

(13) La acidified culture was then introduced into a high pressure homogenizer (maximum pressure: 1000 bar), at room temperature. The process of homogenization was carried out continuously with two consecutive passages inside the homogenizer.

(14) The PHA suspension thus obtained was then added with a sodium hydroxide solution (concentration 30% by weight), so as to obtain a pH value equal to about 9.0, and then with a sodium dodecyl sulphate aqueous solution (5 g of surfactant per liter of suspension), keeping the suspension at room temperature.

(15) The suspension thus treated was then diluted in ratio 1:3 with water, and then subjected to filtration through a tangential flow filter having a cut-off that is equal to 0.4 m, so as to obtain a concentration of solids that is equal to 150 g/L.

(16) The concentrated PHA suspension thus obtained was then subjected to bleaching by addition of a hydrogen peroxide solution at 30% by weight, in ratio 1:4 with respect to the volume of the suspension.

(17) After diluting with water in ratio 1:3, the bleached suspension was subjected to tangential filtration with a filter of the same type used after the basifying step (cut-off: 0.8 m). The retentate contained the granules of purified PHA, whereas the permeate was used for the aforementioned dilution steps.

(18) The retentate was subjected to a further filtration operation through a candle filter, in which the suspension was introduced with a pressure equal to 4.0 bar, from which an aqueous permeate and a concentrated suspension were obtained, the latter being then subjected to drying by a fluid bed dryer at 180 C., so as to obtain a powder of PHA with a water content lower than 0.5% by weight.

EXAMPLE 3

(19) The example 1 was repeated in the same conditions starting from a cell suspension of 500 L which had a dry mass concentration of 77 g/L and a PHA content equal to 53 g/L. Table 1 shows the concentrations on a dry basis of the overall mass and of the PHA measured in the various process steps, that is, after the base treatment (treated cell suspension) and at the end of the process after bleaching and tangential filtration (final PHA suspension). The PHA obtained was characterised in terms of purity, molecular weight and yield.

(20) The molecular weight (Mw, mean ponderal molecular weight) was determined by using the device GPC-HPLC Breeze 2, Waters, provided with refractive index detectors and UV-VIS, with chromatographic column Mini Mix D (molecular weight range 200-400,000 Da). The calibration was carried out by using monodispersed polystyrene standards (Sigma Aldrich, Milano) having the following molecular weights: 2,440 Da, 13,700 Da, 29,300 Da, 50,400 Da, 105,600 Da and 370,000 Da. The flow velocity of the mobile phase was of 0.3 ml/min, whereas the concentration of the injected PHA chloroform solutions was of about 3 mg/ml.

(21) The purity of PHA was determined by an apparatus HPLC Shimadzu with chromatographic column Alltech OA-1000. The flow velocity was of 0.7 ml/min and the mobile phase was an aqueous solution brought to pH equal to 2 with sulphuric acid. Before analysis, the PHA suspension was dried so as to obtain a powder and then the PHA was depolymerised by a methanolysis process in methanol and sulphuric acid at 3%, obtaining a degradation of the polymer in its monomers.

(22) TABLE-US-00001 TABLE 1 Dry mass Concen- concen- tration Volume tration PHA Purity Yield Mw Step (L) (g/L) (g/L) (%) (%) (kDa) Initial cell 500 77.0 53.0 100 320 suspension Treated cell 372 65.0 59.1 83 297 suspension PHA final 285 73.2 72.5 99 78 278 suspension

(23) The data shown in Table 1 highlight that the PHA obtained had a purity of 99%, a yield of 78% and a molecular weight 278 kDa. The shown data are the result of experiments carried out on 20 samples, thus demonstrating that they are repeatable data.

EXAMPLE 4 (COMPARISON)

(24) Starting from the same cell suspension used in example 3, the recovery and purification process described in WO2011/045625, pages 6-8, was repeated. The PHA thus obtained was characterised as described in example 3. The data shown in Table 2 highlight that the obtained PHA had a purity of 95%, a yield of 59% and a molecular weight of 167 kDa. Also in this case, the data shown are the result of experiments carried out on 20 samples.

(25) From the comparison of the results obtained in example 3 and 4, it is evident how, by starting from the same cell suspension containing PHA, the method of the invention allows to obtain PHA with a greater degree of purity, a greater yield and a higher molecular weight.

(26) TABLE-US-00002 TABLE 2 Dry mass Concen- concen- tration Volume tration PHA Purity Yield Mw Step (L) (g/L) (g/L) (%) (%) (kDa) Initial cell 500 77.0 53.0 100 320 suspension Treated cell 387 52.9 46.6 68 187 suspension PHA final 300 54.7 52.1 95 59 167 suspension