Method for measuring the thermal stability of a succinic acid crystal intended for the production of polymers

Abstract

A method for measuring the thermal stability of succinic acid includes the following steps: 1) preparing a succinic acid crystal powder having less than 1% residual water content; 2) placing 10 g of the crystal powder in an oven at 220 C. for 2 hours; 3) pulverizing and sieving the crystal powder processed in this way, such that the particle size distribution thereof is between 0 and 10%, and preferably between 4 and 6% for particles larger than 500 m in size, between 20 and 40%, and preferably between 25 and 35% for particles between 200 and 500 m in size, between 50 and 75% and preferably between 55 and 70% for particles smaller than 200 m in size; and 4) measuring the color, in a spectrocolorimeter, of the pulverized and sieved powder and determining the average value of the yellow (index b).

Claims

1. A method for measuring the heat stability of succinic acid, the method comprising the following steps: 1) preparing a crystalline powder of succinic acid having a residual water content of less than 1%, 2) placing a sample of said crystalline powder in an oven at 220 C. for 2 h, 3) milling and sieving the crystalline powder thus treated, in such a way that its particle size distribution is: between 0 and 10%, for particles with a size greater than 500 m, between 20 and 40%, for particles with a size between 200 and 500 m, between 50 and 75%, for particles with a size less than 200 m, 4) measuring the colour of the milled and sieved crystalline powder in a spectrocolorimeter and determining a mean yellow value (index b).

2. A method for preparing a succinic acid polymer, comprising the following steps: (1) preparing a crystalline powder of succinic acid having a residual water content of less than 1%; (2) placing a sample of said crystalline powder in an oven at 220 C. for 2 h; (3) milling and sieving the crystalline powder thus treated, in such a way that its particle size distribution is: between 0 and 10% for particles with a size greater than 500 m, between 20 and 40% for particles with a size between 200 and 500 m, and between 50 and 75% for particles with a size less than 200 m; (4) measuring the color of the milled and sieved crystalline powder in a spectrocolorimeter and determining a mean yellow value (index b); (5) selecting a succinic acid having a mean yellow value of less than or equal to 2 as a raw material for the succinic acid polymer, and preparing the polymer with the selected succinic acid.

3. The method of claim 2, comprising: (3) milling and sieving the crystalline powder thus treated, in such a way that its particle size distribution is between 4 and 6% for particles with a size greater than 500 m, between 25 and 35% for particles with a size between 200 and 500 m, and between 55 and 70% for particles with a size less than 200 m.

4. The method of claim 2, wherein the succinic acid polymer is poly(butylene succinate).

Description

EXAMPLE 1

Fermentative Production, Extraction and Purification of Succinic Acid

(1) A first series of fermentations with a recombinant E. coli (in this case the strain SBS550MG-pHL413 described in Sanchez et al., Metabolic Engineering, 7 (2005) 229-239, and in documents U.S. Pat. No. 7,223,567 and US 2005/0042736) is carried out.

(2) A second series of fermentations is also carried out, with a recombinant strain of S. cerevisiae (in this case that described in patent application WO 2009/065778).

(3) The extraction of the succinates and the purification of the succinic acid are carried out from these fermentation media by the succession of the following steps:

(4) Removal of Insoluble Organic Impurities (Biomass and Cell Debris)

(5) The removal is carried out by tangential filtration with tangential flow on a membrane having a pore diameter of 100 nm, at between 40 and 80 C. (ceramic membrane with a channel diameter of 3.5 mm).

(6) The temperature is maintained preferentially at 60 C. with a transmembrane pressure of 1 bar and with diafiltration with 20% of demineralized water.

(7) Under these conditions, the flow is approximately 90 L/h/m.sup.2 and the permeate obtained is clear and bright. The permeate still contains more than 6000 ppm of soluble organic impurities, in this case soluble organic nitrogen.

(8) Removal of Soluble Organic Impurities (Residual Soluble Proteins)

(9) This removal consists in adsorbing the organic nitrogen on activated carbon or in denaturing it before removing it by filtration. In the case of denaturing, the procedure may be thermal or osmotic.

(10) A contact zone at 80 C. is used which allows the proteins to be flocculated for 10-15 min. The solution is then filtered through a filter with a pore diameter of 0.22 m.

(11) This step of removing organic nitrogen becomes optional when the microfiltration is carried out at 80 C. This is because, in that case, there is simultaneous denaturing of the organic nitrogen and retention on the microfiltration membrane.

(12) Chelation and Acidification

(13) The maximum admissible concentration for feeding to bipolar electrodialysis (BED) in terms of divalent cations (Ca.sup.2+, Mg.sup.2+, etc.) is 5 ppm. This is because the divalent cations present will react with the hydroxyl ions on the water electrolysis membranes to form salts of very low solubility, which crystallize in the membranes and make them permeable.

(14) In order to protect the integrity of the BED membranes, a chelation step is carried out in order to reach the safety threshold of 5 ppm.

(15) This step consists in complexing the divalent cations using aminophosphonic functions (Purolite S940, Amberlite IRC747, etc.) or diacetic functions (Purolite S930, Lewatit TP208) grafted onto cation exchange resin.

(16) In order to do this, the solution is supplied at 60 C., at a flow rate of 2 BV/h. Under these conditions, the volume of solution treated may reach 30 to 40 times the resin bed volume.

(17) Once it has been freed of these divalent cations, the solution can be acidified by BED. The module used is a stack from the company Eurodia: EUR6.

(18) This stack consists of two types of membranes: bipolar membranes, which allow the electrolysis of water (H.sub.2OH.sup.++OH.sup.) and the acidification of the succinates, cationic membranes, which allow the selective transfer of the monovalent cations.

(19) The electrolysis and the transfer of the cations are carried out by virtue of a potential difference which is applied to the system by a generator.

(20) The succinate salt is then acidified over time by the H.sup.+ ions released by the electrolysis of the water. In the same way, the monovalent cations are alkalinized by the hydroxyl ions after migration through the cationic membranes, to give a base again which can be recycled to the fermentation.

(21) The flow of cations transferred is approximately 22 eq/h/m.sup.2 for a conversion rate of 90%, which equates to a final pH of approximately 3.5.

(22) The acidification is finished on strong cationic resin (Purolite C150). This is because the last equivalents are very difficult to transfer, and hence the energy consumption of the operation is greatly increased (limitation mainly due to the osmotic pressure).

(23) The treatment is preferentially carried out at 40 C. and at 2 BV/h. The volume of solution treated is then approximately 10-15 BV.

(24) The pH of the solution after treatment on cationic resin is equal to 2 and contains succinic acid in its low-solubility form (free acid).

(25) Crystallization

(26) The acidified solution is concentrated by evaporation of water on a Wiegand falling-film evaporator. The concentration factor is about 5 to 10 depending on the initial concentration of succinic acid.

(27) The factor here is equal to 8, in order to obtain supersaturation.

(28) The concentration of the solution is then 420 g/L at 80 C., which corresponds to the supersaturation of the solution.

(29) The solution is then cooled by direct contact from 80 to 20 C. at a rate of 5 C./h.

(30) Crystallization begins spontaneously from the beginning of cooling, but seeding may be carried out in order to better control the physical properties of the crystallized succinic acid.

(31) After separation on a Rousselet centrifuge and washing with one volume of demineralized water per cake volume, the crystals are dried.

(32) At this step, the crystallization yield is >85% for a succinic acid purity of 99.7%/dry basis.

(33) Redissolution

(34) The redissolution of the crystals is carried out in demineralized water at 60 C. rather than at 20 C., in order to reduce the consumption of demineralized water.

(35) For the same purpose, the mother liquor and/or the water from washing the highly pure crystals can also be recycled at this step. The overall yield of succinic acid is then also optimized.

(36) Finishing Treatment

(37) This step consists of a decolorizing treatment and a demineralization step. The decolorizing can be carried out with activated carbon or by ozonation.

(38) Treatment with activated carbon has the major advantage of fixing the orotic acid, a nitrogenous impurity which has a very low solubility and which crystallizes with the succinic acid.

(39) In batch mode, the amount of Norit SX.sup.+ activated carbon is 1% in relation to the succinic acid. After 1 h of reaction at 60 C., the solution is filtered on a 3 m candle filter.

(40) The solution is then treated for demineralization at 2 BV/h at 60 C.

(41) Carried out successively on Purolite C150 strong cationic resin and then Daion WA30 weak anionic resin, the treatment allows removal of the traces of inorganic cations and anions, on the one hand, and of fumaric acid, on the other hand. Furthermore, the weak anionic resin allows additional decolorizing.

(42) Following treatment on weak anionic resin, the fumaric acid content may be reduced by a factor of 20 and the residual colouring is zero.

(43) Crystallization

(44) The solution which has purified can then be crystallized under the same conditions as the previous step.

(45) The yield of this crystallization step is also >85%. The mother liquor and the washing water are recycled to the dissolution of the technical-grade crystals. The overall yield of this step may thus approach 100%. The purity of the crystals is more than 99.8%.

EXAMPLE 2

Colorimetric Measurement of the Succinic Acid Produced by Fermentation of Recombinant Microorganisms

(46) Three successive fermentations were carried out for the E. coli strain (tests A to C); 6 for the S. cerevisiae strain (tests D to I) and the succinic acid was extracted and purified under the conditions of example 1.

(47) The nine samples of crystalline powder of succinic acid are then analyzed according to the method in accordance with the invention.

(48) 10 g of each of these samples were placed in an oven at 220 C. for 2 h 15. The powder recovered was milled and analysed in a spectrocolorimeter in accordance with the method of the invention.

(49) Table 1 below gives the values of the indices L, a and b as obtained.

(50) TABLE-US-00001 TABLE 1 Samples L a b Chemical 98.0 0.00 1.3 succinic acid A 91.1 0.99 6.2 B 97.3 0.19 1.8 C 97.1 0.14 1.9 D 96.9 0.00 2.1 E 96.5 0.29 2.3 F 97.4 0.16 1.7 G 97.3 0.14 1.7 H 98.1 0.11 1.1 I 97.3 0.23 1.9

(51) Compared with the reference chemical succinic acid sample (sold by the company Sigma), it appears here that only sample H would have the required colorimetric quality.

(52) In order to demonstrate this, polymerization tests with these various qualities were undertaken.

(53) Samples of PBS are synthesized via a two-step polycondensation reaction, in a 7.5 l stainless steel reactor equipped with a heating system, a mechanical stirrer, a distillation column, a vacuum line and a nitrogen gas inlet.

(54) 1889 g (16 mol of succinic acid) and 1513.7 g (16.8 mol) of 1,4 butanediol (BDO) are charged to the reactor.

(55) The reaction mixture is then heated at 225 C. at 2 bar of nitrogen pressure and stirred at a constant speed of 150 rpm.

(56) A mixture of water and tetrahydrofuran (1 to 2.5 mol % of THF) produced from the esterification and from the cyclization of the BDO are removed from the reactor by the distillation column.

(57) The degree of esterification is estimated via the amount of distillate collected.

(58) In a second step, the pressure is reduced to 0.7 mbar over the course of 120 minutes.

(59) The reaction is catalyzed by 3.914 g of Ti(OBu).sub.4 (Ti=200 ppm), which are added to the reactor during the step of decompression (approximately 20 mbar) in order to prevent as much as possible any contact with the residual water.

(60) The pressure conditions are maintained for 3.8 hours.

(61) The polymer is removed from the reactor and immersed in water. Approximately 15 mg of PBS granules are obtained after granulation.

(62) The degree of colouring of the polymer is then determined on polymerized sheets having the same thickness (of about 3 mm) using a Dyk Gardner TCS II spectrophotometer (observation angle of 10, illuminant D65).

(63) The results are also expressed by the indices L, a and b, and more particularly by the measurement of the yellow index (abbreviation: YI).

(64) The YI is in fact calculated using the following equation (ASTM D1925):

(65) $\mathrm{YI}=\frac{100\left(1.28X_{\mathrm{CIE}}-1.06Z_{\mathrm{CIE}}\right)}{Y_{\mathrm{CIE}}}$

(66) Table 2 below gives the YI of the polymers produced from two different samples of biobased succinic acid of known index b, on either side of the threshold for the value of the index b established hereinabove, i.e. index b=2.

(67) TABLE-US-00002 TABLE 2 Index PBS prepared from: b YI 2 batches of succinic 1.3 7.8 acid produced 1.3 6.1 chemically H 1.1 6.5 F 1.7 9.8 D 2.1 11

(68) A PBS is considered to be correct by those skilled in the art if its YI is <10.

(69) These results make it possible to confirm the good quality of the succinic acid H, and to show that it is possible to go up to an index b value of 1.7 for producing an acceptable BPS colorimetric quality.

EXAMPLE 3

Confirmation of the Threshold Value of the Index b of the Biobased Succinic Acid: Enrichment of a Reference Succinic Acid with Various Impurities of Defined Nature and Amount

(70) The impurities capable of acting as colouration precursors conventionally identified in fermentation media are in particular residual substances such as carbohydrates, inorganic or organic nitrogen, inorganic sulphur and some organic acids coproduced by these strains.

(71) In order to test the robustness of the crystalline succinic acid qualification method of the invention, and the predicted use that could be made of said method, a succinic acid of chemical origin (reference index b=1.3) or biobased origin (succinic acid corresponding to batch H of example 1reference index b=1.1) is artificially enriched with impurities representative of what is found in said fermentation media before carrying out the treatment in an oven at 200 C.

(72) The contaminants selected are the following (tested at the amounts indicated): carbohydrate: glucose at concentrations of about 100 ppm; organic nitrogen: valine at a concentration of 200 ppm for succinic acid of chemical origin; 1000 ppm for the biobased succinic acid; sulphur: Na.sub.2SO.sub.4 at the concentration of 200 ppm for the succinic acid of chemical origin; 1000 ppm for the biobased succinic acid; organic acids: fumaric acid, malic acid at the concentration of 1000 ppm, tested only with the succinic acid of chemical origin; sulphur-comprising organic acid:orotic acid at the concentration of 1000 ppm, tested only with the succinic acid of chemical origin; sulphur and nitrogen: ammonium sulphate at 1200 ppm, tested only with the biobased succinic acid.

(73) The results obtained are given in Table 3 below.

(74) TABLE-US-00003 TABLE 3 (b Samples Index b b.ref) Chemical SA + 100 ppm of glucose 3.3 2 Tests H + 100 ppm of glucose 3.2 2.1 Chemical SA + 200 ppm of valine 1.5 0.2 Tests H + 1000 ppm of valine 1.3 0.2 Chemical SA + 100 ppm of glucose + 200 ppm of >5 >4 valine Chemical SA + 200 ppm of Na.sub.2SO.sub.4 1.3 0 Tests H + 1000 ppm Na.sub.2SO.sub.4 1.2 0.1 Chemical SA + 1000 ppm of fumaric acid 1.7 0.5 Chemical SA + 1000 ppm of malic acid 1.5 0.2 Chemical SA + 1000 ppm of orotic acid 2 0.7 Tests H + 1200 ppm of ammonium sulphate 2 0.9

(75) These results demonstrate: The importance of residual glucose alone in the heat stability of the biobased succinic acid; the nitrogenous contaminants cause only a slight modification of the heat stability of the succinic acid; the combination of the nitrogenous and sulphur-comprising contaminants amplifies the colouration phenomenon.

(76) In this respect, the applicant company goes against a technical prejudice which claims that only the nitrogenous and sulphur-comprising impurities must be controlled (cf. patent EP 1 882 712 mentioned hereinabove); the succinic acid derivatives (malic acid and fumaric acid) and also the sulphur (Na.sub.2SO.sub.4) have no more significant impact than the colouration of the chemical succinic acid taken as reference, the nitrogenous contaminants cause only a slight modification of the heat stability of the succinic acid.

(77) In order to complete this work, PBS was produced from various batches of chemical succinic acid enriched in impurities (those above and other samples prepared specially for this second series of tests).

(78) Table 4 below gives the results of YI measured for each of the polymers produced.

(79) TABLE-US-00004 TABLE 4 Yellow index index bof the of the PBS produced with N (ppm) S (ppm) monomer PBS a first reference chemical 0 0 1.3 7.8 succinic acid a second reference chemical 0 0 1.3 6.1 succinic acid +1000 ppm of fumaric acid 0 0 1.7 9.8 +1000 ppm of malic acid 0 0 1.5 4.6 +1000 ppm of orotic acid 179 0 2 15.7 +200 ppm of Na.sub.2SO.sub.4 0 45 1.3 2.8 +200 ppm of valine 24 0 1.5 3.9 +20 ppm of glucose 0 0 2.5 11.3 +100 ppm of glucose 0 0 3.3 18.1 +100 ppm of glucose + 24 0 4 15.5 200 ppm of valine

(80) These first results confirm the influence of the glucose and orotic acid impurities on the quality of the polymer, as on that of the monomer.

(81) Moreover, if the curve plotting the values of the index b of the monomer as a function of the YI of the polymer is produced, and a PBS of correct colouration when its YI is 10 is always taken as reference, a threshold value for the index b of about 2 is found.