RU2574783C2 - Method of obtaining lactic acid, method of obtaining lactide and methods of obtaining polylactic acid - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology. Claimed are methods of obtaining lactic acid, obtaining lactide and obtaining polylactic acid. Continuous fermentation of microorganism, possessing ability of lactic acid fermentation, is realised. After that, fermentation medium is filtered through membrane with average pore diameter from 0.01 mcm to 1 mcm with difference of trans-membrane pressure from 0.1 to 20 kPa. Filtrate is collected with preservation of non-filtered liquid in culture medium or return of non-filtered liquid into culture medium and addition of raw material for fermentation to culture medium. Obtained filtrate is filtered through nano-filtration membrane. Distillation of obtained filtrate is realised at pressure from 10 Pa to 30 kPa and temperature from 25°C to 200°C to extract lactic acid. Lactic acid, obtained by claimed method, is catalytically converted into lactide or into polylactic acid by direct dehydration polycondensation.

EFFECT: inventions make it possible to obtain lactic acid with optic purity to 99% and output to 60-92% and obtain polylactic acid with improved thermal stability, mechanical strength and colour.

10 cl, 4 dwg, 6 tbl, 25 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to a method for producing lactic acid, in which a microorganism having the capacity of lactic acid fermentation is cultured, and lactic acid produced in the obtained fermentation medium is separated, and a method for producing polylactic acid from lactic acid obtained by the specified method. The present invention also relates to lactic acid and polylactic acid obtained by these methods.

BACKGROUND OF THE INVENTION

Lactic acid is widely used for food, pharmaceuticals and the like, and is also widely used in industry as a monomer material for the production of polylactic acid, which is a biodegradable plastic, so that its consumption increases. As you know, lactic acid is produced by fermentation of microorganisms that convert carbohydrate-containing substrates based on glucose into lactic acid.

In order to obtain lactic acid as a raw material for polylactic acid, a highly productive method for producing lactic acid is required since a large amount of lactic acid is needed. To increase lactic acid productivity, a high yield with respect to sugar consumption during microbial fermentation is required, as well as a high rate of lactic acid formation per unit time per unit volume, and Patent Document 1 discloses a method for increasing productivity using a porous membrane culturing device.

Polylactic acid can be produced by polymerization with the opening of a ring of lactide, which is a cyclic dimer of lactic acid, or by direct polymerization of the feedstock in the form of lactic acid. In the lactide method, lactic acid is first oligomerized and then depolymerized to separate the resulting lactide, which is then subjected to ring-opening polymerization in the presence of a catalyst. In this method, the polymerization process is complex and therefore requires a lot of labor and expense. Since impurities in the lactic acid feedstock can be removed by the lactide separation process in this process, relatively low quality lactic acid can be used. However, since impurities in the lactic acid feedstock, such as inorganic ions, cause a decrease in the yield of lactide to be separated, the feedstock in the form of lactic acid should be relatively free of impurities. In the direct polymerization process, on the other hand, the feedstock in the form of lactic acid undergoes direct dehydration polycondensation in the presence of a catalyst. In this method, a simplification of the process can be expected compared to the lactide method, however, impurities that inhibit the polymerization should be removed from the feedstock in the form of lactic acid in order to provide a high-quality feedstock in the form of lactic acid. Thus, the efficiency of lactic acid purification affects the increase in lactide and polylactic acid productivity.

The production of lactic acid by microbial fermentation is carried out by adding an alkaline agent to the culture medium to maintain an optimum pH for microbial fermentation, and examples of the alkaline agent that is added to the culture medium include calcium hydroxide. In cases where calcium hydroxide was used, lactic acid produced by microbial fermentation is present in the culture medium as calcium lactate. By adding an acid agent (e.g., sulfuric acid) to the culture medium after completion of cultivation, a solution of free lactic acid can be obtained, however, a calcium salt (e.g., calcium sulfate) is formed as an impurity.

As a method for separating lactic acid by removing simultaneously produced calcium salt, in cases where an insoluble calcium salt such as calcium sulfate precipitates, a filtering method is used through quality filter paper or the like, however, small amounts of the calcium salt dissolved in the solution cannot be removed and remain in a solution containing lactic acid. Therefore, in cases where this filtrate containing lactic acid, for example, is concentrated at a later stage of purification, calcium salts and other soluble inorganic salts are precipitated from a solution containing free lactic acid, which was problematic. It is known that if a solution containing lactic acid, from which inorganic ions have not been sufficiently removed, is heated in a process such as distillation, inorganic ions contribute to racemization and oligomerization of lactic acid.

Examples of a method for removing small amounts of inorganic ions from a solution containing lactic acid include methods using ion exchange resins (for example, see Patent Document 2). However, in order to maintain the implementation of the ion exchange of the ion exchange resin, the ion exchange resin must be periodically restored. In addition, since the regeneration of the ion exchange resin is carried out by using large amounts of an aqueous solution of sodium hydroxide and an aqueous solution of hydrochloric acid, a large amount of waste liquid is released during the regeneration, so that it takes a lot of money to dispose of the waste liquid, which is problematic. Also, the regeneration of the ion exchange resin leads to a decrease in the rate of regeneration of the ion exchange resin, as well as to weakening of the ion exchange, leading to a decrease in the rate of removal of inorganic acids, which is problematic.

In addition, methods are also known for removing small amounts of inorganic ion components, such as a method for removing calcium components from a solution containing lactic acid using an electrodialyzer with a bipolar membrane (for example, see Patent Document 3). However, the bipolar membrane used in these methods is expensive, and the removal efficiency of inorganic salts, such as calcium salts, was not necessarily high, which is problematic.

Methods have also been disclosed for removing inorganic salts from a solution containing lactic acid using a nanofiltration membrane (for example, see Patent Documents 4-6). However, the stage of recovery of lactic acid by distillation, the effect of distillation on the yield of lactic acid and the possibility of using the obtained lactic acid to obtain polylactic acid on an industrial scale by direct polymerization have not been disclosed.

Also, in Patent Documents 7-10, the fact was disclosed that the amount of specific impurities must be less than certain levels to obtain high molecular weight polylactic acid, however, the effects of impurities on thermal stability, mechanical strength and tint, which are important factors for the suitability of polylactic acids, were not disclosed. for chemical processing.

LEVEL DOCUMENTS

Patent documents

[Patent Document 1] WO2007 / 097260

[Patent Document 2] Japanese Translated PCT Patent Application Laid-open No. 2001-506274

[Patent Document 3] JP 2005-270025 A

[Patent Document 4] US 5503750

[Patent Document 5] US 5681728

[Patent Document 6] US 2004/0033573

[Patent Document 7] JP 6-279577 A

[Patent Document 8] JP 7-133344 A

[Patent Document 9] JP 8-188642 A

[Patent Document 10] JP 9-31170 A

SUMMARY OF THE INVENTION

OBJECTS SOLVED BY THE INVENTION

One of the objectives of the present invention is the provision of a method for producing lactic acid with high productivity, which can be used to obtain polylactic acid on an industrial scale by direct polymerization and can be used for highly productive synthesis of lactide; and methods for producing lactide and polylactic acid using lactic acid. The present invention also aims to obtain polylactic acid having excellent thermal stability, mechanical strength and color, and to obtain lactic acid in which the amount of specific impurities does not exceed a certain amount, as well as lactide and polylactic acid obtained using lactic acid as a feedstock.

MEANS FOR SOLVING TASKS

The authors of the present invention carried out intensive studies to solve the above problems, and found that culturing a microorganism having the ability to produce lactic acid in a continuous cultivation device using a porous membrane makes it possible to obtain lactic acid in the filtrate in high yield and high productivity, and by directing the obtained filtrate to the nanofiltration stage and the distillation stage, it is possible to obtain lactic acid, which is applicable for ryamoy polymerization and enables high-yield synthesis of lactide. The authors of the present invention also found that when using lactic acid, in which the amount of specific impurities does not exceed certain levels, as a starting material for polylactic acid, lactide with excellent color and polylactic acid with excellent thermal stability and mechanical stability can be obtained in high yield. strength and color, thus creating the present invention.

That is, the present invention includes the following objects (1) to (15).

(1) A method for producing lactic acid, comprising the following Steps (A) to (C):

(A) a continuous fermentation step, wherein the fermentation culture medium of a microorganism having the capacity of lactic acid fermentation is filtered through a porous membrane having an average pore size of at least 0.01 μm and less than 1 μm with a transmembrane pressure difference in the range from 0.1 to 20 kPa and the filtrate is collected while maintaining unfiltered fluid in the culture medium, or returning unfiltered fluid to the culture medium, and adding feedstock to the culture medium;

(B) a step of filtering the filtrate obtained in Step (A) through a nanofiltration membrane; and

(C) a step for distilling the filtrate obtained in Step (B) under a pressure of not less than 1 Pa and not more than atmospheric pressure, at not less than 25 ° C and not more than 200 ° C, to extract lactic acid.

(2) A method for producing lactic acid according to (1), wherein the pH of the filtrate obtained in Step (A) is adjusted to not less than 2 and not more than 4.5, after which the filtrate is provided for Step (B).

(3) A method for producing lactic acid according to (1) or (2), wherein Step (A) is a continuous fermentation step in the presence of a calcium salt, and a solution containing lactic acid obtained after Step (D) in which the calcium component in the filtrate obtained in Stage (A), is removed as insoluble sulfate, sent to Stage (B).

(4) A method for producing lactic acid in accordance with any one of (1) to (3), wherein the ratio of the rate of penetration of magnesium sulfate relative to the rate of penetration of citric acid through the nanofiltration membrane is not less than 3 at a working pressure of 0.5 MPa, the temperature of the feed liquid 25 ° C and a concentration in the feed fluid of 1000 ppm (ppm, parts per million).

(5) A method for producing lactic acid according to any one of (1) to (4), wherein the rate of penetration of magnesium sulfate through the nanofiltration membrane is not more than 1.5% at an operating pressure of 0.5 MPa, a temperature of the feed liquid 25 ° C and a concentration in the feed liquid of 1000 ppm

(6) A method for producing lactic acid according to any one of (1) to (5), wherein the membrane material of the nanofiltration membrane comprises polyamide.

(7) A method for producing lactic acid according to (6), wherein the polyamide comprises a cross-linked piperazine polyamide as a main component and further includes a forming component represented by chemical formula 1:

Chemical Formula 1

(where R is —H or —CH ₃ ; and n is an integer from 0 to 3).

(8) A method for producing lactide, where the lactic acid obtained by the method in accordance with any of (1) to (7) is used as a feedstock.

(9) A method for producing polylactic acid, wherein the lactide obtained by the method according to (8) is polymerized.

(10) A method for producing polylactic acid, wherein the lactic acid obtained by the process according to any one of (1) to (7) is polymerized by direct dehydration polycondensation.

(11) Lactic acid, a 90% aqueous solution of which contains methanol in a concentration of not more than 70 ppm, pyruvic acid in a concentration of not more than 500 ppm, furfural in a concentration of not more than 15 ppm. , 5-hydroxymethylfurfural in a concentration of not more than 15 ppm, methyl lactate in a concentration of not more than 600 ppm, acetic acid in a concentration of not more than 500 ppm and 2-hydroxybutyric acid in a concentration of not more than 500 ppm.

(12) Lactic acid according to (11), having an optical purity of not less than 90%.

(13) Lactide obtained using lactic acid in accordance with (11) or (12) as a feedstock.

(14) Polylactic acid obtained using lactic acid in accordance with (11) or (12) or lactide in accordance with (13) as a feedstock.

(15) Polylactic acid obtained by direct dehydration polycondensation of lactic acid in accordance with (11) or (12) as a feedstock.

EFFECT OF THE INVENTION

In accordance with the present invention, it is possible to produce high-quality lactic acid and the productivity of polylactic acid, which is a biodegradable general-purpose plastic, can be improved. In addition, when using lactic acid, in which the amount of specific impurities does not exceed a certain amount, polylactic acid having excellent thermal stability, mechanical strength and color can be obtained as a feedstock for polylactic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing an embodiment of a continuous cultivation apparatus used in the present invention.

FIG. 2 is a diagram showing the concentration of accumulated lactic acid and the rate of production of lactic acid during continuous cultivation carried out in Example 1 of the present invention.

FIG. 3 is a schematic diagram showing an embodiment of a nanofiltration membrane separation apparatus used in the present invention.

FIG. 4 is a schematic diagram illustrating an embodiment of a cross section of a cell into which a nanofiltration membrane was placed in a nanofiltration membrane separation apparatus used in the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in more detail.

Lactic Acid Production Method

The method for producing lactic acid of the present invention includes Steps (A) to (C) below:

(A) A continuous cultivation step, wherein the culture medium for fermenting a microorganism having the ability of lactic acid fermentation is filtered through a porous membrane having an average pore size of not less than 0.01 μm and less than 1 μm with a transmembrane pressure difference in the range of 0.1 up to 20 kPa, and the filtrate is collected, while maintaining unfiltered fluid in the culture medium or returning unfiltered fluid to the culture medium, and adding raw materials for fermentation to the culture medium;

(B) a step of filtering the filtrate obtained in Step (A) through a nanofiltration membrane; and

(C) The distillation step of the solution obtained in Step (B) at a pressure of not less than 1 Pa and not more than atmospheric pressure, at not less than 25 ° C and not more than 200 ° C, to extract lactic acid.

Now will be described a microorganism having the ability of lactic acid fermentation, which is used in Stage (A). A microorganism having the capacity of lactic acid fermentation is not limited, provided that it can produce lactic acid, and preferably a lactic acid bacterium or a microorganism that has been artificially given the ability of lactic acid fermentation or whose lactic acid fermentation capacity has been increased, can be used.

Lactic acid bacteria can be defined here as a prokaryotic microorganism that produces lactic acid with a yield of not less than 50% relative to the glucose consumed. Preferred examples of the lactic acid bacteria include those which belong to the genus Lactobacillus, the genus Pediococcus, genus Tetragenococcus, genus Carnobacterium, the genus Vagococcus, the genus Leuconostoc, the genus Oenococcus, genus Atopobium, Streptococcus genus, genus Enterococcus, genus Lactococcus, genus Bacillus, and genus Sporolactobacillus. By selecting among them, a lactic acid bacterium exhibiting a high yield of lactic acid relative to sugar consumption, the bacterium can preferably be used to produce lactic acid. In addition, by selecting a lactic acid bacterium exhibiting a high yield of L-lactic acid or D-lactic acid relative to sugar consumption, the bacterium can preferably be used to produce lactic acid having high optical purity.

Examples of a lactic acid bacterium exhibiting a high yield of L-lactic acid with respect to sugar intake include Lactobacillus yamanashiensis, Lactobacillus animalis, Lactobacillus agilis, Lactobacillus aviaries, Lactobacillus casei, Lactobacillacusususususususususususususususususususacusususbacusususbacusususbacusususususususi casei , Pediococcus dextrinicus and Lactococcus lactis , which can be selected and used to produce L-lactic acid.

Examples of a lactic acid bacterium exhibiting a high yield of D-lactic acid with respect to sugar intake include Sporolactobacillus laebolacticus, Sporolactobacillus inulinus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus delbruekii and Lactococcus lactis , which can be used for the preparation of lactic acid and lactis .

Examples of a microorganism that has been artificially given lactic acid fermentation capacity or whose lactic acid fermentation capacity has been increased include microorganisms obtained by known chemical mutagenesis and microorganisms that have been introduced with the lactate dehydrogenase gene (which may hereinafter be referred to as LDH) to impart or increase the fermentation capacity lactic acid. Preferred examples of this include recombinant microorganisms in which LDH has been incorporated intracellularly to increase the capacity of lactic acid fermentation.

Preferred examples of a host of recombinant microorganisms include prokaryotic cells such as E. coli and lactic acid bacteria; and eukaryotic cells, such as yeast; and the host is most preferably yeast. The yeast is preferably of the genus Saccharomyces , and the yeast is more preferably Saccharomyces cerevisiae .

The LDH gene used in the present invention is not limited, provided that it encodes a protein having the activity of converting reduced nicotinamide adenine dinucleotide (NADH) and pyruvic acid to oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) and lactic acid. For example, you can use the L-LDH gene obtained from a lactic acid bacterium showing a high yield of L-lactic acid relative to sugar intake, or the D-LDH gene obtained from a lactic acid bacterium showing a high yield of L-lactic acid relative to sugar intake. In addition, preferred examples of the L-LDH gene include genes derived from eukaryotes, such as a cow, a human and a frog, and the L-LDH gene, more preferably derived from Xenopus laevis . Examples of microorganisms into which the frog derived L-LDH gene is introduced include recombinant yeast disclosed in JP 2008-029329 A.

Examples of the LDH gene used in the present invention also include variants derived from genetic polymorphisms and mutagenesis. The term "genetic polymorphism" means a partial modification of the sequence of the bases of a gene due to a natural mutation that has occurred in the gene. The term "mutagenesis" means the artificial introduction of a mutation into a gene. Examples of the mutagenesis method include a method using a directed mutagenesis kit (Mutan-K (manufactured by TAKARA BIO INC.)) And a method using a random mutagenesis kit (BD Diversify PCR Random Mutagenesis (manufactured by CLONTECH)). The LDH used in the present invention may contain deletion (s) and / or insert (s) in part of its base sequence, provided that it encodes a protein having the activity of converting NADH and pyruvic acid to NAD ⁺ and lactic acid.

The porous membrane used in Stage (A) will now be described. The porous membrane used as the separating membrane is preferably less prone to clogging by a microorganism having the ability of lactic acid fermentation, and has the property of stably supporting filtering for a long time. Therefore, for the porous membrane used in the present invention, it is important to have an average pore size of not less than 0.01 μm and less than 1 μm. The porous membrane of the present invention has a separation and permeability performance suitable for the properties and use of the fluid to be treated, and, taking into account clogging parameters, permeability and dirt resistance, which affects the separation performance, the porous membrane is preferably a porous membrane a layer of resin. As a porous membrane having a porous resin layer, a membrane with a porous resin layer on the surface of the porous substrate material, a layer that acts as a separating functional layer, is preferred. The porous backing material supports the porous resin layer to strengthen the porous membrane.

The porous support material consists of organic material, inorganic material and / or the like, and preferably organic fiber is used. Preferred examples of the porous support material include woven materials and non-woven materials prepared using organic fibers such as cellulose fibers, cellulose triacetate, polyester fibers, polypropylene fibers and polyethylene fibers, among which non-woven materials are preferably used, since their density can be relatively easily controlled. They can be easy to manufacture and are not expensive.

The organic polymer membrane can suitably be used as a porous resin layer, which acts as a separating functional layer, as mentioned above. Examples of the material of the organic polymer membrane include polyethylene resins, polypropylene resins, polyvinyl chloride resins, polyvinylidene difluoride resins, polysulfone resins, polyethersulfone resins, polyacrylonitrile resins, polyolefin resins, cellulose resins, and triacetyl cellulose resins, and the material may be a base resin, components. Here, the main component means that the component is contained in an amount of not less than 50% by weight, preferably not less than 60% by weight. Among them, the most preferred examples of the material of the porous membrane include materials that can be easily formed by solutions and are excellent in physical strength and chemical resistance, such as polyvinyl chloride resins, polyvinylidene difluoride resins, polysulfone resins, polyethersulfone resins, polyacrylonitrile resins, polyolefin resins among which polyvinylidene difluoride resins and resins containing them as main components are preferably used.

Here, as the polyvinylidene difluoride resin, a vinylidene fluoride homopolymer is preferably used, and in addition to the vinylidene fluoride homopolymer, a copolymer with vinyl monomers capable of copolymerizing with vinylidene fluoride is also preferably used. Examples of vinyl monomers capable of copolymerization with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene and fluorotrichloroethane.

Examples of polyolefin resins include polyethylene, polypropylene, chlorinated polyethylene and chlorinated polypropylene, and chlorinated polyethylene is preferably used.

A brief overview of the preparation method of the porous membrane used in the present invention will now be presented. Firstly, on the surface of the aforementioned porous substrate material, a coating of an initial solution containing the aforementioned resin and solvent is formed upon soaking in the initial solution of the porous substrate material. After that, only the coated surface of the porous substrate material having the coating is brought into contact with a coagulation bath containing a precipitant for thickening the resin, forming a porous resin layer on the surface of the porous substrate material. The precipitant may also be contained in the initial solution. The temperature of the initial solution is usually preferably selected in the range from 15 to 120 ° C, taking into account the film-forming properties.

A pore-forming substance can be added to the initial solution. The pore-forming substance is extracted by immersion in a coagulation bath to make the resin layer porous. Adding a pore-forming substance allows you to adjust the average pore size. The pore-forming substance preferably has high solubility in a coagulation bath. Examples of a pore-forming substance that can be used include inorganic salts such as calcium chloride and calcium carbonate. Additional examples of a pore-forming substance that can be used include polyoxyalkylenes such as polyethylene glycol and polypropylene glycol; water-soluble macromolecular compounds such as polyvinyl alcohol, polyvinyl butyral and polyacrylic acid; and glycerin.

The solvent dissolves the resin. This acts on the resin and the pore-forming substance to stimulate their formation of a porous layer of resin. Examples of solvents that can be used include N-methylpyrrolididone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone and methyl ethyl ketone. Among them, NMP, DMAc, DMF and DMSO may preferably be used, in which the resins show high solubility.

In addition, a precipitant can also be added to the initial solution. A precipitant is a liquid that does not dissolve the resin. The precipitant has the action of controlling the rate of coagulation of the resin in order to control the pore size. Examples of a precipitant that can be used include water and alcohols such as methanol and ethanol. Among them, water and methanol are preferred in terms of cost. The precipitant may also be a mixture of the above substances.

As mentioned above, the porous membrane used in the present invention is preferably a porous membrane formed by a porous substrate material and a porous resin layer. The porous substrate material can either be impregnated with a layer of porous resin or not impregnated with a layer of porous resin, which is selected depending on the use. The average thickness of the porous substrate material is preferably selected in the range from 50 μm to 3000 μm. In cases where the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably selected in the range of 200 μm to 5000 μm, and the thickness of the membrane is preferably selected in the range of 20 μm to 2000 μm. Fabric or knitwear produced by forming an organic fiber or inorganic fiber into a cylindrical shape may be contained in a hollow fiber.

The porous membrane used in the present invention can be converted into an element of the separation membrane by combination with a substrate. The shape of the separating membrane element having the porous membrane is not limited, and the separating membrane element where the supporting plate is used as a substrate and the porous membrane used in the present invention is placed on at least one side of the supporting plate is one preferred embodiment a separation membrane element having a porous membrane used in the present invention. In cases where it is difficult to provide a large area of the membrane in this embodiment, placing porous membranes on both sides of the support plate to increase permeability is also a preferred embodiment.

The average pore size of the porous membrane used in the present invention is not less than 0.01 μm and less than 1 μm. With an average pore size of the porous membrane within this range, it is possible to achieve high blocking characteristics that prevent the cells from seeping and settling, and high permeability, as well as a lesser chance of clogging, so that the permeability can be maintained with high accuracy and reproducibility for a long time. The average pore size of the porous membrane is preferably not more than 0.4 μm, and the process can more preferably be performed with an average pore size of less than 0.2 μm. In cases where the average pore size is too small, the permeability may decrease, so the average pore size in the present invention is not less than 0.01 μm, preferably not less than 0.02, more preferably not less than 0.04 μm. Here, the average pore size can be determined by measuring the diameters of all the pores that can be observed within the region of 9.2 μm × 10.4 μm using a scanning electron microscope at a magnification of 10000x, and averaging the measured values.

Further, the standard deviation of the average pore size is preferably not more than 0.1 μm. In addition, in cases where the standard deviation of the average pore size is small, that is, in cases where the pore sizes are uniform, a homogeneous filtrate is more likely to be obtained, and therefore, a simpler control of the fermentation process is possible, so that the standard deviation of the average size the pore is preferably as small as possible.

The standard deviation σ of the average pore size is calculated in accordance with Equation 1 below, where N represents the number of pores observed within the aforementioned region of 9.2 μm × 10.4 μm, Xk represents the respective measured diameters, and X (ave) represents the average of the sizes since

Equation 1

For the porous membrane used in the present invention, permeability with respect to the culture medium is one of its important properties. The permeability coefficient of pure water for a porous membrane can be used as an indicator of permeability before use. In the present invention, the permeability coefficient of pure water for a porous membrane is preferably not less than 2 × 10 ^-9 m ³ / m ² / s / Pa in the case when the penetration is determined using purified water at a temperature of 25 ° C, prepared by filtration through reverse osmosis membrane, with a main height of 1 m, and in cases where the permeability coefficient of pure water for a porous membrane is from 2 × 10 ^-9 m ³ / m ² / s / Pa to 6 × 10 ^-7 m ³ / m ² / s / Pa, it is possible to achieve a penetration value that is actually sufficient .

The surface roughness of the membrane used in the present invention is a factor that affects the clogging of the separation membrane, and preferably, in cases where the surface roughness of the membrane is not more than 0.1 μm, the separation coefficient and membrane resistance of the separation membrane can be suppressed accordingly. , and therefore, continuous fermentation can be performed at a lower transmembrane pressure difference. Therefore, since clogging can be suppressed to ensure stable continuous fermentation, the roughness of the membrane surface should preferably be as small as possible.

In addition, in cases where the roughness of the membrane surface is small, the shear force arising on the membrane surface during the filtration of the microorganism can be expected to be small, and therefore, the destruction of the microorganism can be suppressed, and clogging of the porous membrane can be suppressed. Therefore, it is assumed that stable filtration is possible for a long time.

Here, the roughness of the membrane surface can be determined using the following atomic force microscope (AFM) under the following conditions.

Device

Atomic Force Microscope (Nanoscope IIIa, manufactured by Digital Instruments)

Conditions

Research: SiN Console (manufactured by Digital Instruments)

Scanning method: Contact method (measurement in air)

Underwater detection method (underwater measurement)

Scanning Area: 10 μm × 10 μm, 25 μm × 25 μm (measurement in air)

5 μm × 5 μm, 10 μm × 10 μm (underwater measurement)

Scan Resolution: 512 × 512

Sample Preparation

After the measurement, the membrane sample was soaked in ethanol at room temperature for 15 minutes and then soaked in water after reverse osmosis for 24 hours, followed by washing and drying in air.

The roughness of the membrane surface (d _rough ) is calculated in accordance with the following Equation 2 using the aforementioned AFM, based on the heights of the corresponding points in the direction of the Z axis.

Equation 2

d _rough : Surface roughness (μm)

Z _n : Height in the direction of the Z axis (μm)

: Средняя высота (мкм) в сканированной области

: Average height (μm) in the scanned area

N : Number of measured samples

The difference in transmembrane pressure during filtration of a microorganism through a porous membrane in Step (A) of the present invention may be such that the membrane is not rapidly clogged by the microorganism and the components of the medium, and it is important to perform filtration with a transmembrane pressure difference in the range from 0.1 to 20 kPa . The transmembrane pressure difference is preferably in the range from 0.1 to 10 kPa, more preferably in the range from 0.1 to 5 kPa, even more preferably in the range from 0.1 to 2 kPa. In cases where the difference in transmembrane pressure is not within the above range, clogging by the microorganism and the components of the medium can occur quickly and the amount of penetration can decrease, causing a problem during the continuous fermentation process.

In terms of the driving force of the filtration, a siphon can be used using the difference in fluid levels (difference in hydraulic head) between the fermentation culture medium and the porous membrane filtrate to obtain a difference in transmembrane pressure in the porous membrane. Also, as a driving force for the filtration, a suction pump can be placed on the filtration side of the porous membrane, or a pressure pump can be placed on the side of the fermentation culture medium of the porous membrane. The difference in transmembrane pressure can be controlled by changing the difference in liquid levels between the fermentation culture medium and the filtrate from the porous membrane. Also, in cases where the pump is used to obtain the transmembrane pressure difference, the transmembrane pressure difference can be controlled by the suction pressure, and in addition, the transmembrane pressure difference can be controlled by the gas or liquid pressure, which is used to direct the pressure from the fermentation culture medium. In cases where such pressure control is carried out, the difference between the pressure from the fermentation culture medium and the pressure from the filtration side of the porous membrane corresponds to the transmembrane pressure difference, and can be used to control the difference in transmembrane pressure.

The continuous fermentation apparatus used in Step (A) is not limited provided that the above conditions are met, and preferred examples of this include the examples disclosed in FIG. 1 and FIG. 2 in W02007 / 097260. Furthermore, the porous membrane element for filtering the fermentation culture medium is not limited provided that the above conditions are satisfied, and preferred examples of this include the examples disclosed in FIG. 3 and FIG. 4 in W02007 / 097260.

The fermentation feed used in the present invention is not limited so long as it stimulates the growth of a cultured microorganism having the capacity of lactic acid fermentation to ensure satisfactory production of the lactic acid of interest. Preferred examples of fermentation feedstocks include conventional liquid media containing carbon sources, nitrogen sources, inorganic salts and, where appropriate, organic micronutrients such as amino acids and vitamins, when necessary. Examples of carbon sources include sugar, such as glucose, sucrose, fructose, galactose and lactose; hydrolyzed starch solutions containing such sugars; sweet potato molasses; sugar beet molasses; high quality molasses; as well as organic acids such as acetic acid; alcohols such as ethanol; and glycerin. Examples of nitrogen sources include gaseous ammonia, ammonia, ammonium salts, urea, and nitric acid salts; and other additional organic nitrogen sources used, such as oilcake, soy hydrolyzate fluids, casein hydrolysates, other amino acids, vitamins, liquid corn extracts, yeast or yeast extracts, meat extracts, peptides such as peptones, and cells of various fermentable microorganisms and hydrolysates of this. Examples of inorganic salts that may be added as appropriate include phosphoric acid salts, magnesium salts, calcium salts, iron salts and manganese salts. In cases where the lactic acid fermentation microorganism used in the present invention requires specific nutrients for its growth, nutrients can be added as preparations or natural products containing them. You can also add anti-foaming agent if necessary. In the present invention, the culture medium means a liquid obtained by the growth of a microorganism having the ability of lactic acid fermentation in the fermentation feedstock, and the composition of the fermentation feedstock also added may be different, as appropriate, from the fermentation feedstock used at the beginning of the cultivation, so that lactic acid productivity increases.

In the continuous cultivation process in Step (A), cultivation of cells grown in a confined space, or cultivation with recharge can be performed in the initial phase of cultivation to increase the concentration of the microorganism, after which continuous cultivation (extraction) can be started, or the cells can be seeded in high concentration and subjected to continuous cultivation from the beginning of cultivation. You can start supplying the environment with the feedstock and extracting the culture at appropriate times. The timing of the start of supplying the medium with the feedstock and the timing of the start of crop extraction are not necessarily the same. Media supply of the feedstock and crop recovery can be performed either continuously or intermittently. The nutrients necessary for cell growth, as described above, can be added to the feedstock medium to ensure continuous cell growth. The concentration of the microorganism in the culture medium is preferably kept high in a range that does not cause a high rate of death of the microorganism due to the environment of the culture medium, which is unsuitable for the growth of the microorganism, in terms of achieving effective productivity. For example, maintaining a concentration of not less than 5 g / l in terms of dry weight, you can get good economic efficiency.

Further, if necessary, the microorganism can be removed from the fermenter. For example, since in cases where the concentration of the microorganism in the fermenter is too high and a clogging of the porous membrane is likely to occur, removal can be carried out to avoid clogging. In addition, since the production characteristics for lactic acid can vary depending on the concentration of the microorganism in the fermenter, the removal of the microorganism can be carried out using the production characteristics as an indicator to maintain the production characteristics.

The process of continuous cultivation by allowing the growth of fresh cells with the ability of lactic acid fermentation, usually performed preferably in a separate fermenter, taking into account the control culture. However, the number of fermenters is not limited, provided that continuous cultivation is performed to obtain the product, while allowing cell growth. Many fermenters can be used, for example, due to the low productivity of each fermenter. In this case, high productivity of the fermentation product can be obtained even by continuous culture, using many fermenters connected in parallel or in series via tubes.

Filtration through the nanofiltration membrane in Step (B) will now be described.

The nanofiltration membrane used in the present invention is also called a nanofilter (nanofiltration membrane, NF membrane), and is usually defined as “a membrane that allows monovalent ions to penetrate but blocks divalent ions.” The membrane is believed to have micro-voids measuring approximately a few nanometers, and is used primarily to block small particles, molecules, ions and salts in water.

The term “filtration using a nanofiltration membrane” means that the filtrate from Step (A) is filtered through a nanofiltration membrane to block or separate inorganic salts dissolved or precipitated as solid particles, allowing the lactic acid solution to penetrate as the filtrate. Here, inorganic salts include any form of inorganic salts contained in the culture medium, such as those dissolved in the filtrate of Stage (A), and as precipitated or precipitated in the filtrate of Stage (A).

In Stage (B), it is preferable to bring the pH of the filtrate of Stage (A) to not less than 2.0 and not more than 4.5. It is known that substances ionized in a solution are more likely to be removed or blocked by a nanofiltration membrane than non-ionized substances. Therefore, adjusting the pH of the filtrate from Step (A) to no more than 4.5, the ratio of lactic acid dissociated into lactic acid ions in the filtrate becomes small, and this allows more efficient penetration of lactic acid. In addition, in cases where the pH is less than 2.0, nanofiltration may be impaired. Also, since the pKa of lactic acid is 3.86, in cases where the pH does not exceed 3.86, lactic acid that has not been dissociated into lactic acid ions and hydrogen ions is contained in the filtrate from Stage (A) in greater quantity, so that lactic acid can be allowed to efficiently penetrate the nanofiltration membrane, which is more preferred. The regulation of the pH of the filtrate from Stage (A) can be performed either during microbial fermentation or after Stage (A). Also, the pH can be adjusted by adding an inorganic or organic acid in cases where the pH of the filtrate needs to be made more acidic, or by adding an alkaline substance such as calcium hydroxide, or aqueous ammonia in cases where the pH needs to be made more alkaline.

The filtrate from Step (A) for use with the nanofiltration membrane in Step (B) is preferably prepared by adding an alkaline substance to the culture medium in Step (A) to maintain the optimum pH for microbial fermentation, and passing the resulting culture medium through a porous membrane. The microorganism is usually cultivated at a pH of 4-8, a temperature of from 20 to 40 ° C. The alkaline substance that is added is not limited, and a basic calcium salt is preferably added.

In cases where Step (A) is a continuous fermentation step in the presence of a calcium salt, Step (D) in which the calcium component in the filtrate in Step (A) is removed as insoluble sulfate may be introduced before Step (B). More specifically, for example, Step (D) is performed by adding sulfuric acid to the filtrate of Step (A) and precipitating / filtering the calcium component in the filtrate of Step (A) as calcium sulfate, which is insoluble sulfate. By allowing the filtrate (separated liquid containing lactic acid) to pass through the nanofiltration membrane from Step (B), the calcium component can be more efficiently removed or blocked. Examples of basic calcium salts include calcium hydroxide, calcium carbonate, calcium phosphate, calcium oxide and calcium acetate, and the basic calcium salts are preferably calcium hydroxide. In cases where the calcium component in the filtrate of Step (A) is precipitated / filtered as insoluble sulfate, if the number of equivalents of sulfuric acid added to the culture medium exceeds the number of equivalents of calcium (number of equivalents of sulfuric acid> number of equivalents of calcium), the excess of sulfuric acid is partially penetrates the nanofiltration membrane. If this follows the exposure of the filtrate from Step (B) under heating conditions, such as concentration or distillation, the permeated sulfuric acid may act as a catalyst to stimulate oligomerization of lactic acid, resulting in a decrease in the yield upon distillation. Therefore, in cases where the calcium component in the filtrate from Stage (A) is precipitated / filtered as insoluble sulfate, the number of equivalents of added sulfuric acid is preferably no more than the number of equivalents of the calcium component in the filtrate from Stage (A). In cases where the number of equivalents of added sulfuric acid is adjusted based on pH, the pH is preferably not less than 2.0, since in this case the number of equivalents of sulfuric acid does not exceed the number of equivalents of the calcium component.

As a step preceding the aforementioned Step (D), Step (E) can be introduced in which organic acids other than lactic acid are removed from the filtrate from Step (A) by removing calcium lactate crystals. More specifically, the pH is adjusted by adding basic calcium to the filtrate from Step (A), and the resulting filtrate is filtered through a nanofiltration membrane equivalent to that used in Step (B), thereby collecting an aqueous solution containing calcium lactate from the supply side and removing organic acids, including acetic acid, from the filtrate.

In Stage (E), the pH of the filtrate from Stage (A) is preferably adjusted to not less than 6 and not more than 11. Since nanofiltration membranes have properties in which substances ionized (dissociated) in solution are more likely to be blocked than non-ionized ones ( undissociated) substances, then when adjusting the pH of the culture medium to not less than 6, the ratio of lactic acid dissociated into ions in the culture medium (dissociated lactic acid / undissociated lactic acid) becomes higher than the ratio of acetic acid dissociated into ions (dissociated acetic acid / undissociated acetic acid). In this case, an aqueous solution containing calcium lactate can be efficiently collected from the supply side, and organic acids, with the exception of lactic acid, can be effectively separated from the filtrate. In addition, in cases where the pH of the culture medium exceeds 11, there is an adverse effect on the duration of the nanofiltration membrane, which is not preferred.

In Stage (E), organic acids, with the exception of lactic acid, separated from the filtrate side of the nanofiltration membrane, are organic acids obtained from the filtrate from Stage (A) or from fermentation feedstocks, and acetic acid is preferably separated in the present invention.

Examples of basic calcium, preferably added in Stage (E) to adjust the pH of the filtrate from Stage (A), include calcium hydroxide, calcium carbonate, calcium phosphate, calcium oxide and calcium acetate in the form of solid particles and aqueous solutions, and the basic calcium is preferably hydroxide calcium. In cases where an aqueous solution is added, the concentration of basic calcium is not limited, and the basic calcium to be added may be in the form of a liquid solution having a concentration in excess of saturating solubility.

Examples of a method for evaluating the degree of removal, blocking, and filtering of inorganic salts dissolved or precipitated as solid particles by the nanofiltration membrane used in the present invention include an evaluation method that calculates the removal rate (blocking rate) of inorganic ions, but the method is not limited thereto. The blocking rate (removal rate) of the inorganic salt can be calculated in accordance with Equation 3 by measuring the concentration of inorganic salt contained in the liquid of the feedstock (culture medium) (concentration of inorganic salt in the liquid of the feedstock) and the concentration of inorganic salt contained in the filtrate (solution lactic acid) (concentration of inorganic salt in the filtrate), by analysis, presented by ion chromatography.

Inorganic salt removal rate (%) = (1- (inorganic salt concentration in the filtrate / inorganic salt concentration in the feed liquid)) × 100 .. (Equation 3)

The membrane separation efficiency of the nanofiltration membrane used in Step (B) is not limited, and the ratio of the rate of penetration of magnesium sulfate relative to the rate of penetration of citric acid through the nanofiltration membrane is preferably not less than 3 at an operating pressure of 0.5 MPa, a feed temperature of 25 ° C. C and the concentration in the liquid of the feedstock 1000 ppm In cases where the ratio of the rate of penetration of magnesium sulfate relative to the rate of penetration of citric acid through the nanofiltration membrane under the above conditions is not less than 3, inorganic salts contained in the filtrate of Stage (A) can be removed and lactic acid can be allowed to penetrate efficiently, which is preferred . Here, the penetration rate of magnesium sulfate can be calculated in accordance with Equation 4 by determining the concentration of magnesium sulfate contained in the feed liquid (concentration of magnesium sulfate in the feed fluid) and the concentration of magnesium sulfate contained in the filtrate (concentration of magnesium sulfate in the filtrate) by analysis represented by ion chromatography. Similarly, the penetration rate of citric acid can be calculated by replacing the concentration of magnesium sulfate in Equation 4 with the concentration of citric acid and determining the concentration of citric acid contained in the feed liquid (concentration of citric acid in the feed liquid) and the concentration of citric acid contained in the filtrate (concentration of citric acid in the filtrate) by analysis provided by high performance liquid chromatography.

The penetration rate of magnesium sulfate (%) = (concentration of magnesium sulfate in the filtrate) / (concentration of magnesium sulfate in the liquid of the feedstock) × 100 .. (Equation 4)

In addition, the penetration rate of magnesium sulfate is preferably not more than 1.5% at a working pressure of 0.5 MPa, a temperature of the liquid feed 25 ° C and a concentration in the liquid feed 1000 ppm. In cases where the penetration rate of magnesium sulfate through the nanofiltration membrane exceeds 1.5% under the aforementioned conditions, the concentration of the lactic acid solution passing through the nanofiltration membrane may precipitate inorganic salts, and the distillation process is likely to cause racemization and oligomerization due to the effects of the penetrated inorganic salts, and can reduce the yield during distillation. More preferably, the penetration rate of magnesium sulfate through the nanofiltration membrane is not more than 1.0%.

In addition, nanofiltration membranes having a sodium chloride removal rate (500 mg / L) of not less than 45% are preferably used. From the point of view of the penetration efficiency of the nanofiltration membrane, a nanofiltration membrane is preferably used for which the flow rate of penetration of sodium chloride (500 mg / l) per unit area of the membrane (m ³ / m ² / day) at a filtration pressure of 0.3 MPa is not less than 0 5 and not more than 0.8. The flow rate of penetration per unit area of the membrane (membrane flow of penetration) can be estimated by calculating in accordance with Equation 5 by determining the amount of permeated fluid, the collection time of the permeated fluid and the membrane region.

Membrane penetration flow (m ³ / m ² / day) = amount of fluid passed through / membrane area / collection time .. (Equation 5)

Examples of nanofiltration membrane material that can be used in the present invention include macromolecular materials such as cellulose acetate polymers, polyamides, polyesters, polyimides and vinyl polymers. The membrane is not limited to a membrane formed by only one of the materials, and may be a membrane comprising multiple membrane materials. From the point of view of the membrane structure, the membrane can be either an asymmetric membrane that has a dense layer on at least one side of the membrane and micropores having pore sizes that gradually increase in the direction from the dense layer to the inside of the membrane or the other side of the membrane, or a composite membrane, which has a very thin functional layer formed by another material on a dense layer of an asymmetric membrane. Examples of a composite membrane that can be used include the complex membrane described in JP 62-201606 A, which has a nanofilter consisting of a polyamide functional layer on a support membrane including polysulfone as the membrane material.

Among them, a composite membrane having a functional layer consisting of polyamide is preferable, since it is resistant to high pressure, high permeability, and high removal efficiency, which makes it a membrane with high potential. To maintain wear resistance against working pressure, high permeability and high blocking efficiency, a membrane having a structure in which polyamide is used as a functional layer supported by a substrate comprising a porous membrane and non-woven material is suitable. Furthermore, a composite nanofiltration membrane having, on a substrate, a functional layer of a crosslinked polyamide obtained by the polycondensation reaction between a polyfunctional amine and a polyfunctional acid halide is suitable as a polyamide semipermeable membrane.

In a nanofiltration membrane having a functional layer composed of polyamides, preferred examples of the carboxylic acid component of the monomers constituting the polyamide include aromatic carboxylic acids, such as trimesic acid, benzophenone tetracarboxylic acid, trimellitic acid, pyromellitic acid, isophthalic acid, terephthalichenylcarboxylic acid, terephthalphonic acid, acid and pyridinecarboxylic acid. Due to the solubility of the film-forming solvents, trimesic acid, isophthalic acid and terephthalic acid and mixtures thereof are more preferred.

Preferred examples of the amine component of the monomers constituting the polyamide include primary diamines having an aromatic ring, such as m-phenylenediamine, p-phenylenediamine, benzidine, methylene bis-dianiline, 4,4'-diaminobiphenyl ether, dianisidine, 3,3 ', 4-triaminobiphenyl ether, 3.3 ', 4,4'-tetraaminobiphenyl ether, 3,3'-dioxibenzidine, 1,8-naphthalenediamine, m (p) -monomethylphenylenediamine, 3,3'-monomethylamino-4,4'- diaminobiphenyl ether, 4, N, N '- (4-aminobenzoyl) -p (m) -phenylenediamine-2,2'-bis (4-aminophenylbenzimidazole), 2,2'-bis (4-aminophenylbenzooxazole) and 2.2 '- uc (4-minofenilbenzotiazol); and secondary diamines such as piperazine, piperidine and derivatives thereof. Among them, a nanofiltration membrane is preferably used, having a functional layer consisting of a cross-linked polyamide comprising piperazine or piperidine as monomers, since it is resistant to high temperature and chemical resistance in addition to pressure and wear resistance. The polyamide more preferably contains, as a main component, a cross-linked piperazine polyamide or a cross-linked piperidine polyamide and further comprises forming components represented by chemical Formula (1), even more preferably contains a cross-linked piperazine polyamide as a main component and further comprises a forming component represented by chemical Formula (1). In addition, in chemical Formula (1), preferably n = 3. Examples of a nanofiltration membrane having a polyamide functional layer containing cross-linked piperazine polyamide as a main component and further containing a forming component represented by Chemical Formula (1) include those described in JP 62-201606 A, and specific examples include UTC60 manufactured by TORAY INDUSTRIES, INC., Which is a cross-linked piperazine polyamide semipermeable membrane having a functional layer consisting of a polyamide containing a cross-linked piperazine polyamide in As the main component and additionally containing the forming component represented by the chemical Formula (1), where n = 3.

A nanofiltration membrane is typically used as a spiral wound membrane element, and a nanofiltration membrane used in the present invention is also preferably used as a spiral wound membrane element. Specific preferred examples of the nanofiltration membrane element include GEsepa, which is a cellulose acetate nanofiltration membrane manufactured by GE Osmonics; NF99 and NF99HF, which are nanofiltration membranes having a functional layer consisting of a polyamide manufactured by Alfa-Laval; NF-45, NF-90, NF-200 and NF-400, which are nanofiltration membranes having a functional layer consisting of cross-linked piperazine polyamide, manufactured by Filmtec Corporation; and SU-210, SU-220, SU-600, and SU-610, which are nanofiltration membrane modules manufactured by TORAY INDUSTRIES, INC., having UTC60 produced by the same manufacturer, which has a functional layer consisting of a polyamide containing cross-linked piperazine polyamide as the main component and also containing the forming component represented by chemical Formula (1). The nanofiltration membrane element is more preferably NF99 or NF99HF, which are nanofiltration membranes having a functional layer consisting of a polyamide manufactured by Alfa-Laval; NF-45, NF-90, NF-200 or NF-400, which are nanofiltration membranes having a functional layer consisting of crosslinked piperazine polyamide manufactured by Filmtec; or SU-210, SU-220, SU-600, or SU-610, which are nanofiltration membrane modules manufactured by TORAY INDUSTRIES, INC., having UTC60 manufactured by the same manufacturer, which has a functional layer consisting of a polyamide containing cross-linked piperazine polyamide as the main component and also containing the forming component represented by chemical Formula (1). Even more preferred is the nanofiltration membrane element SU-210, SU-220, SU-600 or SU-610, which are nanofiltration membrane modules manufactured by TORAY INDUSTRIES, INC., Having UTC60 made by the same manufacturer, which has a functional layer consisting of from a polyamide containing cross-linked piperazine polyamide as the main component and also containing the forming component represented by chemical Formula (1).

Filtration through the nanofiltration membrane in Stage (B) can be performed under pressure, and the filtration pressure is preferably in the range from 0.1 MPa to 8 MPa. In cases where the filtration pressure is less than 0.1 MPa, the membrane penetration rate can be reduced, while in cases where the filtration pressure is more than 8 MPa, the membrane can be damaged. In cases where the membrane is used at a filtration pressure in the range of 0.5 MPa to 7 MPa, the membrane permeation flux is high so that the lactic acid solution can be allowed to penetrate efficiently and the possibility of membrane damage is small, which is more preferred. The membrane is particularly preferably used at filtration pressure in the range from 1 MPa to 6 MPa.

The concentration of lactic acid in Stage (B) is not limited, and in cases where the concentration is high, the concentration of lactic acid contained in the filtrate in Stage (B) is also high, so that the length of time required for the concentration can be reduced, which is preferred due to cost reduction.

The concentration of inorganic salts in Stage (B) is not limited and can be no less than saturating solubility. Thus, in cases where the concentration of inorganic salt does not exceed saturating solubility, the inorganic salt is dissolved in the culture medium, and in cases where the concentration of inorganic salt is not less than saturating solubility, the inorganic salt partially precipitates. However, in Stage (B), both inorganic salts dissolved in the filtrate from Stage (A) and inorganic salts precipitated or precipitated in the filtrate of Stage (A) can be removed or blocked so that lactic acid can be filtered without limiting the concentration of inorganic salts .

The permeability of lactic acid through the nanofiltration membrane after separation of the lactic acid contained in the filtrate of Step (A) of the above method can be estimated by calculating the penetration rate of lactic acid. The penetration rate of lactic acid can be calculated in accordance with Equation 6 by measuring the concentration of lactic acid contained in the liquid feed (culture medium) (the concentration of lactic acid in the liquid feed) and the concentration of lactic acid contained in the filtrate (solution containing lactic acid ) (concentration of lactic acid in the filtrate), by analysis, represented by high performance liquid chromatography.

The penetration rate of lactic acid (%) = (concentration of lactic acid in the filtrate / concentration of lactic acid in the liquid of the feedstock) × 100 .. (Equation 6)

In the method for producing lactic acid of the present invention, the filtrate from Step (B) is further distilled in Step (C) to produce high purity lactic acid. The distillation step is carried out at a reduced pressure of not less than 1 Pa and not more than atmospheric pressure (normal pressure, approximately 101 kPa). In cases where the step is carried out under reduced pressure of not less than 10 Pa and not more than 30 kPa, the distillation temperature may be lower, which is more preferred. The distillation temperature in cases where the stage is performed at a reduced pressure is not less than 20 ° C and does not exceed 200 ° C, but in cases where the distillation is performed at a temperature of not less than 180 ° C, lactic acid racemization may be caused by impurities. Therefore, the distillation of lactic acid can preferably be carried out at a temperature of not less than 50 ° C and not more than 180 ° C, more preferably not less than 60 ° C and not more than 150 ° C.

Before subjecting Stage (C), the filtrate from Stage (B) can be concentrated once using a concentrator such as an evaporator, or the filtrate from Stage (B) can also be subjected to Stage (F), where the concentration of lactic acid is increased by filtration through a reverse osmosis membrane. In terms of reducing the energy for the concentration, Stage (F) is preferably used to increase the concentration of lactic acid by filtration through a reverse osmosis membrane. A reverse osmosis membrane here means a filter for removing ions and / or molecules with a low molecular weight, using as a driving force a pressure difference greater than the osmotic pressure of the liquid being treated, and examples of this that can be used include cellulose membranes, such as those made from acetate cellulose and membranes prepared by polycondensation of a multifunctional amine compound and a multifunctional acid halide to provide a separating functional layer made of polyamide on a microporous membrane of a substrate. In order to reduce dirt, i.e. contamination, on the surface of the reverse osmosis membrane, it is also preferable to use a slightly contaminated reverse osmosis membrane, which serves mainly for wastewater treatment, a reverse osmosis membrane, which is prepared by covering the separating surface of the functional layer made of polyamide with an aqueous solution a compound having at least one reactive group reactive with an acid halide group, allowing, t kim, the acid halide group remaining on the surface of the separating functional layer to create covalent bonds with the reactive groups. Since most of the divalent calcium ions have been removed in Step (B) of the present invention, stable membrane concentrations can be achieved without salt deposits on the surface of the reverse osmosis membrane.

Examples of the reverse osmosis membrane preferably used in the present invention include complex membranes having a cellulose acetate polymer as a functional layer (hereinafter referred to as cellulose acetate reverse osmosis membranes) and complex membranes having a polyamide functional layer (hereinafter referred to as polyamide reverse osmosis membranes). Here, examples of a cellulose acetate polymer include polymers prepared with cellulose ethers of organic acids, such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate and cellulose butyrate, which can be used solely as a mixture or as a mixed ether. Examples of the polyamide include linear polymers and crosslinked polymers formed by aliphatic and / or aromatic diamine monomers.

Examples of the shape of the membrane that can be used as appropriate include a flat membrane, a spirally twisted membrane, and a hollow fiber membrane.

Specific examples of the reverse osmosis membrane used in the present invention include polyamide reverse osmosis membrane modules manufactured by TORAY INDUSTRIES, INC., Such as low pressure type modules SU-710, SU-720, SU-720F, SU-710L, SU-720L, SU -720LF, SU-720R, SU-710P and SU-720P, as well as high pressure type modules SU-810, SU-820, SU-820L and SU-820FA, containing UTC70 as a reverse osmosis membrane; cellulose acetate reverse reverse membranes manufactured by the same manufacturer, SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100 and SC-8200; NTR-759HR, NTR-729HF, NTR-70SWC, ES10-D, ES20-D, ES20-U, ES15-D, ES15-U and LF10-D, manufactured by Nitto Denko Corporation; RO98pHt, RO99, HR98PP and CE4040C-30D, manufactured by Alfa-Laval; GE Sepa manufactured by GE; and BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040 and SW30HRLE-404, manufactured by FilmTec Corporation.

Lactic acid

The present inventors have found that lactic acid obtained by the aforementioned method for producing lactic acid contains only small amounts of impurities, and therefore, the quality of lactic acid is high enough for use in the preparation of polylactic acid by direct polymerization. In addition, the present inventors have determined the ranges of impurities with which you can get high-quality lactide (feedstock for polylactic acid) and polylactic acid, thereby completing the present invention. A first feature of the lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, methanol in a concentration of not more than 70 ppm, preferably not more than 65 ppm. more preferably not more than 50 ppm, even more preferably not more than 30 ppm The methanol content in a 90% aqueous lactic acid solution can be determined by gas chromatography (GC). In cases where lactic acid contains, in a 90% aqueous solution of lactic acid, methanol is more than 70 ppm, polylactic acid obtained by direct dehydration polycondensation of lactic acid has a low weight average molecular weight and low mechanical strength, which is not preferred. In addition, in cases where lactic acid having a methanol content of more than 70 ppm was used, the synthetic yield of lactide was reduced, which is not preferred.

A second feature of the lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, pyruvic acid in a concentration of not more than 500 ppm, preferably not more than 400 ppm. ., more preferably not more than 300 ppm. The content of pyruvic acid in a 90% aqueous lactic acid solution can be determined by high performance liquid chromatography (HPLC). In cases where lactic acid contains, in a 90% aqueous solution of lactic acid, pyruvic acid is more than 500 ppm, polylactic acid produced by polymerization of lactic acid produces an undesirable color. The color of polylactic acid can be estimated based on the degree of staining, and, as an indicator of the degree of staining, you can use the number of color units APHA. The APHA color unit number (Hazen color number) is a value calculated in accordance with the measurement method JISK0071-1 (established on October 20, 1998). Also, in cases where lactic acid having a pyruvic acid content of more than 500 ppm was used, the synthetic yield of lactide decreased and the number of APHA color units increased, which is not preferred.

A third feature of the lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, furfural at a concentration of not more than 15 ppm, preferably not more than 10 ppm. more preferably not more than 5 ppm. The furfural content in a 90% aqueous lactic acid solution can be determined by high performance liquid chromatography (HPLC). In cases where lactic acid has a furfural content of more than 10 ppm in a 90% aqueous solution of lactic acid, polylactic acid produced by polymerization of lactic acid produces undesirable color and thermal stability. The thermal stability of polylactic acid can be estimated based on the thermal rate of mass loss. Also, in cases where lactic acid containing furfural greater than 15 ppm has been used, the number of APHA color units in the resulting lactide increases, which is not preferred.

A fourth feature of the lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, 5-hydroxymethyl furfural at a concentration of not more than 15 ppm, preferably not more than 10 ppm ., more preferably not more than 5 ppm. The content of 5-hydroxymethylfurfural in a 90% aqueous lactic acid solution can be measured by high performance liquid chromatography (HPLC). Polylactic acid obtained by polymerization of lactic acid having a content of more than 10 ppm in a 90% aqueous solution of lactic acid, 5-hydroxymethylfurfural has undesirable color and thermal stability. Also, in cases where lactic acid having a 5-hydroxymethylfurfural content of more than 15 ppm has been used, the number of APHA color units increases in the resulting lactide, which is not preferred.

A fifth feature of lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, methyl lactate in a concentration of not more than 600 ppm, preferably not more than 400 ppm, more preferably not more than 100 ppm. The methyl lactate content in a 90% aqueous lactic acid solution can be determined by gas chromatography (GC). In cases where lactic acid contains, in a 90% aqueous solution of lactic acid, methyl lactate more than 600 ppm, polylactic acid obtained by direct dehydration polycondensation of lactic acid has a low weight average molecular weight and low mechanical strength, which is not preferred. In addition, in cases where lactic acid having a methyl lactate content of more than 600 ppm was used, the number of APHA color units increases in the resulting lactide, which is not preferred.

A sixth feature of lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, acetic acid in a concentration of not more than 500 ppm, preferably not more than 400 ppm. more preferably not more than 300 ppm. The content of acetic acid in a 90% aqueous solution of lactic acid can be determined by high performance liquid chromatography (HPLC). Polylactic acid obtained by polymerization of lactic acid having a content of more than 500 ppm in a 90% aqueous solution of lactic acid, acetic acid has undesirable thermal stability. Also, in cases where lactic acid having an acetic acid content of more than 500 ppm has been used, the synthetic yield of lactide is reduced, which is not preferred.

A seventh feature of lactic acid of the present invention is that lactic acid contains, as an impurity in a 90% aqueous solution of lactic acid, 2-hydroxybutyric acid in a concentration of not more than 500 ppm, preferably not more than 300 m. d., more preferably not more than 200 ppm. The content of 2-hydroxybutyric acid in a 90% aqueous lactic acid solution can be determined by high performance liquid chromatography (HPLC). Polylactic acid obtained by polymerization of lactic acid, containing, in a 90% aqueous solution of lactic acid, 2-hydroxybutyric acid greater than 500 ppm, has undesirable thermal stability. In addition, in cases where lactic acid having a 2-hydroxybutyric acid content of more than 500 ppm has been used, the synthetic yield of lactide is reduced, which is not preferred.

The lactic acid of the present invention can be either one of the (L) -form or (D) -form, or a mixture of the (L) -form and (D) -form. In cases where lactic acid is a mixture, the optical purity, which indicates the content of isomers of the (L) -form or (D) -form, is preferably not less than 90%, since the melting point of the obtained polylactic acid in this case is high. The optical purity is more preferably not less than 95%, even more preferably not less than 99%, most preferably not less than 99.9%.

Also included in the present invention are lactides produced by using the lactic acid of the present invention as a raw material, and a method for producing lactide; and polylactic acid produced using the lactic acid of the present invention as a feedstock, and a method for producing polylactic acid.

Lactide

The lactide of the present invention includes L, L-lactide, D, D-lactide and D, L-lactide, which are composed of L-lactic acid and / or D-lactic acid. The lactide is preferably an L, L-lactide or D, D-lactide.

The method for producing lactide is not limited, and a conventional method can be used in which lactic acid is heated under reduced pressure to obtain lactic acid oligomers, which are then depolymerized by heating in the presence of a catalyst under reduced pressure to cause transformation into lactide. The catalyst used for the depolymerization of lactic acid oligomers is not limited, and is usually a catalyst based on a metal selected from, or containing a metal compound from Group IA metals, Group IIIA, Group IVA, Group IIB, Group IVB and Group VA in the periodic table.

Examples of a Group IA metal catalyst include alkali metal hydroxides (e.g. sodium hydroxide, potassium hydroxide and lithium hydroxide), weak acid alkali salts (e.g. sodium lactate, sodium acetate, sodium carbonate, sodium octylate, stearate sodium, potassium lactate, potassium acetate, potassium carbonate and potassium octylate) and alkali metal alkoxides (e.g. sodium methoxide, potassium methoxide, sodium ethylate and potassium ethylate).

Examples of a metal based catalyst belonging to Group IIIA include aluminum ethylate, aluminum isopropylate, alumina, and aluminum chloride.

Examples of a Group IVA metal based catalyst include organotin catalysts (tin lactate, tin tartrate, tin dicaprylate, tin dilaurate, tin dipalmitate, tin distearate, tin dioleate, tin α-naphthoate, tin β-naphthoate, and tin tin naphthoate) as well as tin powder, tin oxide and tin halides.

Examples of a Group IIB metal based catalyst include zinc powder, zinc halides, zinc oxide and organozinc compounds.

Examples of a metal catalyst of Group IVB include titanium compounds such as tetrapropyl titanate; and zirconium compounds such as zirconium isopropylate.

Examples of the Group VA metal-based catalyst include antimony compounds such as antimony trioxide; and bismuth compounds such as bismuth (III) oxide.

Among them, a catalyst containing tin or a tin compound is preferred due to activity, and tin octylate is particularly preferred.

The amount of catalyst to be used is from about 0.01 to 20% by weight, preferably from about 0.05 to 15% by weight, more preferably from about 0.1 to 10% by weight, relative to lactic acid oligomers .

The depolymerization reaction can be performed using a conventional vessel for vertical reactions, or can be performed using a molecular distillation device. Examples of a molecular distillation device include devices such as a vertical still cube, a top-down film type, and a centrifuge type. Devices such as top-down film and centrifugal type are continuous process devices and are widely used in industry. A centrifugal type molecular distillation device uses a method in which a film of vaporized substance is distributed over a hot surface using centrifugal forces, and a downward film type molecular distillation device uses a method in which the vaporized substance is allowed to flow down a hot surface to form a thin film of evaporated material.

The depolymerization temperature is set from 160 to 300 ° C, preferably from 180 to 260 ° C, more preferably from 190 to 250 ° C. In cases where the temperature is lower than 160 ° C, distillation of the lactide is difficult and a significantly higher degree of vacuum is required. On the other hand, in cases where the temperature is higher than 300 ° C, racemization and staining are likely to occur.

The pressure inside the depolymerization device does not exceed the vapor pressure of the lactide at the depolymerization temperature and is usually about 1-50 torr. A lower pressure is preferable since in this case the heating temperature may be low. More specifically, the pressure is preferably 1-20 torr, more preferably 1-10 torr, even more preferably 1-5 torr.

The residence time in the depolymerization device is preferably as short as possible due to the prevention of racemization, and is usually not more than 1 hour. The use of a molecular distillation device is preferable since in this case the time can be no more than 10 minutes, preferably no more than 3 minutes, more preferably no more than 1 minute.

The lactide produced by the method of producing lactide can be removed outside the depolymerization reaction system as a vapor and collected. Lactide collection can simply be done using a capacitor attached to the depolymerization device.

Polylactic Acid

The polylactic acid of the present invention includes a homopolymer of L-lactic acid or D-lactic acid units; a polylactic acid block copolymer formed by a segment consisting of poly-L-lactic acid units and a segment consisting of poly-D-lactic acid units; and a copolymer with monomers other than lactic acid. In cases where polylactic acid is a copolymer, examples of monomer units other than lactic acid include glycol compounds such as ethylene glycol, propylene glycol, butanediol, heptanediol, hexanediol, octanediol, nonanediol, decanediol, 1,4-cyclohexanedimethanol, neopentyl glycol pentaerythritol, bisphenol A, polyethylene glycol, polypropylene glycol and polytetramethylene glycol; dibasic acids such as oxalic acid, adipic acid, sebacic acid, azelaic acid, dodecandioic acid, malonic acid, glutaric acid, cyclohexanedicarboxylic acid, terephthalic acid, isophthalic acid, phthalic acid, naphthalenedicarboxylic acid, bis (p-carboxy phenyl) acid, diphenyl ether of dicarboxylic acid, sodium sulfoisophthalic acid and tetrabutylphosphonium isophthalic acid; hydroxycarboxylic acids such as glycolic acid, hydroxypropionic acid, hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid and hydroxybenzoic acid; and lactones such as caprolactone, valerolactone, propiolactone, undecalactone and 1,5-oxepan-2-one. The degree of copolymerization of the other copolymerization components described above is preferably from 0 to 30 mol%, more preferably from 0 to 10 mol%, relative to the total monomer components.

The method for producing polylactic acid is not limited, and a conventional method for producing polylactic acid can be used. Specific examples of the known process include a two-stage lactide process, wherein the lactide, which is a cyclic dimer, is first produced using lactic acid as a feedstock, followed by ring opening polymerization; and a direct polymerization process, consisting of a single step, where the feedstock undergoes direct dehydration polycondensation in a solvent. Any of these methods may be used.

In the lactide method and the direct polymerization method, the length of time required for the polymerization can be reduced by using a catalyst for the polymerization reaction. Examples of the catalyst include metals such as tin, zinc, lead, titanium, bismuth, zirconium, germanium, antimony and aluminum, and derivatives thereof. Derivatives are preferably metal alkoxides, carboxylates, carbonates, oxides and halides. Specific examples include tin chloride, tin acetate, tin octylate, zinc chloride, lead oxide, lead carbonate, titanium chloride, alkoxy titanium, germanium oxide and zirconium oxide. Among these, tin compounds are preferred, and tin acetate and tin octylate are more preferred.

The polymerization reaction can be carried out in the presence of the aforementioned catalyst, usually at a temperature of from 100 to 200 ° C, although the temperature varies depending on the type of catalyst. In addition, to remove water produced by the polymerization reaction, the polymerization reaction is preferably carried out under reduced pressure, and the pressure is preferably not more than 7 kPa, more preferably not more than 1.5 kPa.

For a polymerization reaction, a compound having two or more hydroxyl groups or amino groups in a molecule can be used as a polymerization initiator. Examples of a compound used as a polymerization initiator that has two or more hydroxyl groups or amino groups in the molecule include polyols such as ethylene glycol, propylene glycol, butanediol, hexanediol, octanediol, neopentyl glycol, diethylene glycol, triethylene glycol, polyethylene glycol, polypropylene glycol, polypropylene glycol, polypropylene glycol, polypropylene glycol, polypropylene glycol pentaerythritol, dipentaerythritol, tripentaerythritol, sorbitol, poly (vinyl alcohol), poly (hydroxyethyl methacrylate) and poly (hydroxypropyl methacrylate); and polyvalent amines such as ethylenediamine, propylene diamine, butanediamine, hexanediamine, diethylenetriamine and melamine; among which polyols are more preferred.

The amount of polymerization initiator added is not limited and is preferably from 0.001 to 5 parts by weight, more preferably from 0.01 to 3 parts by weight relative to 100 parts by weight of the feedstock used (L-lactic acid, D-lactic acid, L, L -lactide or D, D-lactide).

In cases where polylactic acid is produced by the direct polymerization process, the lactic acid used as feedstock must be very pure, and the lactic acid of the present invention can be adequately used in the direct polymerization process. The solvent used for the direct polymerization process is not limited, provided that it does not adversely affect the polymerization, and may be water or an organic solvent. Examples of the organic solvent include aromatic hydrocarbons. Examples of aromatic hydrocarbons include toluene, xylene, naphthalene, chlorobenzene and diphenyl ether. In cases where polylactic acid is produced by the direct polymerization method, the polymerization is carried out by removing the water produced by the polymerization reaction outside the system. The removal method outside the system is preferably polymerization under reduced pressure, and the pressure is preferably not more than 7 kPa, more preferably not more than 1.5 kPa.

The polylactic acid of the present invention is characterized by a weight average molecular weight of not less than 120,000, a heat loss rate of less than 6.5% under a nitrogen atmosphere at a constant temperature of 200 ° C with a heating time of 20 minutes, and the number of APHA color units is not more than 15. V cases where the average molecular weight of polylactic acid is not less than 120,000, preferably not less than 140,000, polylactic acid has excellent mechanical strength; in cases where the rate of heat loss of mass is less than 6.5%, preferably not more than 6.0%, polylactic acid has excellent thermal stability; and in cases where APHA is not more than 15, preferably not more than 10, polylactic acid has excellent color; so that the polylactic acid of the present invention, which satisfies these physical properties, is suitable for various applications, such as fibers, films and molded products.

EXAMPLES

The present invention will now be described in more detail, however, the present invention is not limited to the Examples below.

In the present invention, Saccharomyces cerevisiae was used as a microorganism capable of fermenting L-lactic acid, into the chromosome of which the L-lactate dehydrogenase gene (L-LDH gene) derived from Xenopus laevis having the base sequence shown in SEQ ID NO was introduced: one.

Reference Example 1: Preparation of a yeast strain having the ability to ferment L-lactic acid

The strain derived from strain B3 described in JP 2008-029329 A was used as a yeast strain having the ability to ferment L-lactic acid. The production method is described below.

In strain B3, the L-LDH gene shown in SEQ ID NO: 1 was inserted at the SED1 locus. For insertion into the SED1 locus, PCR was performed using pTRS102 described in JP 2008-029329 as a template for amplification, and oligonucleotides (SEQ ID NOs: 2 and 3) as a set of primers for amplification of the PCR fragment of 1.3 kb containing the L-LDH gene obtained from Xenopus laevis, and the sequence of the terminator TDH3. Here, SEQ ID NO: 2 was designed so that a sequence corresponding to the 60 bp sequence above the initiation codon of the SED1 gene was added.

Then, using PCR using plasmid pRS423 as a template for amplification and oligonucleotides (SEQ ID NOs: 4 and 5) as a set of primers, a PCR fragment of approximately 1.3 kb containing the HIS3 gene, which is yeast selective marker. Here, SEQ ID NO: 5 was designed so that a sequence corresponding to the 60 bp sequence downstream of the termination codon of the SED1 gene was added.

Each DNA fragment was separated by electrophoresis on a 1% agarose gel and purified in accordance with the usual method. PCR was performed using a mixture of the obtained two types of fragments of approximately 1.3 kb as a template for amplification, and oligonucleotides (SEQ ID NO: 2 and 5) as a set of primers for amplification of the PCR fragment of approximately 2, 6 kb, in which the L-LDH gene derived from Xenopus laevis, the TDH3 terminator and the HIS3 gene, and the sequences corresponding to the 60 bp sequences above and below the SED1 gene, were added to the 5'- end and 3'-end, respectively.

The above DNA fragment was separated by electrophoresis on a 1% agarose gel and purified in accordance with the usual method. Strain B3 was transformed with a DNA fragment and grown on histidine-free medium to select transformants in which the L-LDH gene derived from Xenopus laevis was introduced into the chromosome below the SED1 gene promoter.

Confirmation of the fact that the transformant thus obtained is a yeast having the L-LDH gene from Xenopus laevis introduced into the chromosome below the promoter of the SED1 gene, was carried out as follows. A genomic transformant DNA was prepared using the Dr. Genomic DNA Extraction Kit. GenTLE (manufactured by TAKARA BIO INC.), And then PCR was performed using ready-made genomic DNA as an amplification matrix and oligonucleotides (SEQ ID NOs: 6 and 7) as a primer set to confirm receipt of an amplified DNA fragment of approximately 2.9 t .p.p. In the untransformed strain, the amplified DNA fragment of approximately 1.4 kb is obtained by the above-mentioned PCR. A transformant having the Xenopus laevis derived from the genome of L-LDH, introduced into the chromosome below SED1 gene promoter, hereinafter referred to as strain B4.

Then, the yeast strain SW015 described in JP 2008-48726 A carrying a temperature-sensitive mutation in the pdc5 gene was crossed with strain B4, obtained as described above, to obtain diploid cells. Diploid cells were cultured in the medium for the formation of asks in order to enable the asks to form. Asks were pulled apart using a micromanipulator to obtain the corresponding haploid cells, which were then checked for auxotrophy. Among the obtained haploid cells, a strain carrying the L-LDH gene from Xenopus laevis , which was inserted at the PDC1, SED1, and TDH3 loci, and a temperature-sensitive mutation in the PDC5 gene (lack of viability at 34 ° C) were selected, and the corresponding mating types were selected MATa and MATα. Among the yeast strains obtained, a strain having a MATa mating type was designated as a SU014-8A strain, and a strain having a MATα mating type was designated as a SU014-3B strain.

Then, lysine auxotrophy of strain SU014-8A was restored. Using the BY4741 genomic DNA manufactured by Funakoshi Corporation as a template and oligonucleotides (SEQ ID NOs: 8 and 9) as a set of primers, PCR was performed to amplify the PCR fragment of approximately 2 kb corresponding to the first half LYS2 gene. The above PCR fragment was separated by electrophoresis on a 1% agarose gel and purified in accordance with a conventional method. Strain SU014-8A was transformed with a PCR fragment to neutralize the amber mutation in the LYS2 gene. When cultivating the resultant in a medium without lysine, a transformant having a restored ability to synthesize lysine was selected.

Confirmation of the fact that the transformant thus obtained is a yeast in which the amber mutation in the LYS2 gene was neutralized, was performed as follows. First, the resulting transformant was crossed with strain 20GY77 having the wild-type LYS2 gene to obtain diploid cells. Diploid cells were cultured in the medium for the formation of asks in order to enable the asks to form. Asks were pulled apart using a micromanipulator to obtain the corresponding haploid cells, which were then checked for auxotrophy. It was confirmed that all obtained haploid cells had the ability to synthesize lysine. In cases where the ability to synthesize lysine was restored without neutralizing the mutation in LYS2, cells that did not have the ability to synthesize lysine are formed among haploid cells obtained as described above. The strain obtained by restoring the ability of strain SU014-8A to synthesize lysine is hereinafter referred to as HI001.

After that, the leucine auxotrophy of strain SU014-3B was restored. Using pRS425 as a template and oligonucleotides (SEQ ID NO: 10 and 11) as a set of primers, PCR was performed to amplify the PCR fragment of the LEU2 gene in approximately 2 kb. The above PCR fragment was separated by electrophoresis on a 1% agarose gel and purified in accordance with a conventional method. Strain SU014-3B was transformed with a PCR fragment to neutralize the LEU2 gene mutation. By culturing the resultant in a leucine-free medium, a transformant having a reduced ability to synthesize leucine was selected.

Confirmation of the fact that the transformant thus obtained is yeast in which the mutation in the LEU2 gene was neutralized, was carried out as follows. First, the obtained transformant was crossed with strain 20GY7 having the wild-type LEU2 gene to obtain diploid cells. Diploid cells were cultured in the medium for the formation of asks in order to enable the asks to form. Asks were pulled apart using a micromanipulator to obtain the corresponding haploid cells, which were then tested for auxotrophy. It was confirmed that all obtained haploid cells had the ability to synthesize leucine. In cases where the ability to synthesize leucine was restored without neutralizing the mutation in LEU2, cells that did not have the ability to synthesize lysine are formed among haploid cells obtained as described above. The strain obtained by restoring the ability of strain SU014-3B to synthesize lysine is hereinafter referred to as HI002.

Then, the thus obtained strain HI001 and strain HI002 were crossed with each other to obtain a diploid prototrophic strain that does not have auxotrophy. The resulting strain is hereinafter referred to as strain HI003.

The lactic acid level was confirmed by determining the amount of lactic acid using HPLC under the following conditions.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)

Mobile phase: 5 mM p-toluenesulfonic acid (flow rate: 0.8 ml / min)

Solution for reaction: 5 mM p-toluenesulfonic acid, 20 mM bis-Tris, 0.1 mM EDTA 2Na (flow rate: 0.8 ml / min)

Detection Method: electrical conductivity

Temperature: 45 ° C

The optical purity of L-lactic acid or D-lactic acid was measured using HPLC under the following conditions.

Column: TSK-gel Enantio LI (manufactured by Tosoh Corporation)

Mobile phase: 1 mm aqueous solution of copper sulfate

Flow rate: 1.0 ml / min

Detection Method: UV 254 nm

Temperature: 30 ° C

The optical purity of lactic acid was calculated by the following equation.

Optical purity (%) = 100 × (L-D) or (D-L) / (L + D)

(where L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid).

Reference Example 2: Preparation of L-Lactic Acid by Batch Culture

Using strain HI003 prepared in Reference Example 1 and a sugar starting material medium (70 g / L Yutosei (manufactured by MUSO Co., Ltd.)), a periodic culture check was performed. The medium was autoclaved (121 ° C, 15 minutes) before use. The concentration of the lactic acid, which is the product, was evaluated using the HPLC described in Reference Example 1, and the glucose concentration was determined using the Wako C Glucose Test (manufactured by Wako Pure Chemical Industries, Ltd.). The operating conditions of the batch cultivation apparatus of Reference Example 2 were as shown below:

Fermenter volume (amount of lactic acid fermentation medium), 2 (l); temperature regulation, 32 (° C); volume of ventilation for the fermenter, 0.1 (l / min); stirring speed for the fermenter, 200 (revolutions per minute); pH adjustment, adjustment to pH 5 with 1N calcium hydroxide.

Firstly, strain HI003 was cultured in 5 ml of raw sugar medium in vitro overnight with shaking (preliminary preculture). The pre-culture medium was inoculated with 100 ml of a new sugar feed medium and cultured in a 500 ml Sakaguchi flask for 24 hours with shaking (pre-culture). Temperature control and pH control were performed and fermentation cultivation was performed. As a result of periodic cultivation for 50 hours, the concentration of accumulated lactic acid was 45-49 g / l, and the optical purity was 99.9% for L-lactic acid.

Reference Example 3: Continuous Fermentation Process

Using strain HI003 prepared in Reference Example 1, lactic acid was continuously fermented using the culture apparatus shown in FIG. 1. The filtrate was removed from the membrane separation vessel using a Masterflex pump. The medium used was a feedstock medium with sugar (70 g / l Yutosei (manufactured by MUSO Co., Ltd.), 1.5 g / l ammonium sulfate). Before using this medium, the sugar feed was autoclaved at 121 ° C for 15 minutes at high pressure (2 atm). A molded article made of stainless steel and polysulfone resin was used as a member of the porous membrane element, and a hollow fiber membrane prepared by the method described in Reference Example 13 in W02007 / 097260 was used as the porous membrane. Operational modes were as follows.

Cultivation reactor volume: 20 (L)

The volume of the culture medium in the reactor for cultivation: 15 (l)

Used porous membrane: PVDF filtration membrane

The area of effective filtration of the membrane separation element: 2800 cm ²

Temperature control: 32 (° C)

Ventilation volume for the cultivation reactor: air, 1 (l / min)

Cultivation reactor agitation speed: 800 (rpm)

PH adjustment: adjust to pH 5 with 5N calcium hydroxide

Sterilization: 121 ° C, 0.2 MPa, 20 min, for the entire culture reactor, including the porous membrane element, and the medium used

The rate of removal of the culture medium: 0.16 m ³ / m ² / d

The removal of the culture medium using a Masterflex pump was started 50 hours after the start of cultivation, and the cultivation lasted up to 500 hours after the start of cultivation. The results of measuring the concentration of lactic acid, which is the product, and the rate of production of lactic acid are shown in FIG. 2. The concentration of lactic acid was determined by the method described in Reference example 1, and the rate of production of lactic acid was calculated using Equation 7 below.

The rate of production of lactic acid (g / l / h)

= concentration of lactic acid accumulated in the removed fluid (g / l) × rate of removal of the fermentation fluid (l / h) / operational volume of the fluid of the device (l) .. (Equation 7)

As a result, the difference in transmembrane pressure did not exceed 1 kPa, and a stable process was possible, without causing clogging of the membrane. The average rate of production of lactic acid over a period of time from 50 hours to 500 hours after the start of cultivation was 6 g / l / hour. Lactic acid obtained from the porous membrane filtrate for a period of time from 400 hours to 500 hours after the start of cultivation was used in the following examples (lactic acid concentration: 52 g / l, optical purity L-lactic acid: 99.9%).

Reference Example 4: Assessment of Magnesium Sulfate Permeability through a Nanofiltration Membrane

To 10 L of ultrapure water was added 10 g of magnesium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and the resulting mixture was stirred at 25 ° C for 1 hour to prepare an aqueous solution with 1000 ppm. magnesium sulfate. Then, 10 L of the prepared aqueous solution of magnesium sulfate was fed into the feed tank 13 of the membrane filtration device shown in FIG. 3. As a 90φ nanofiltration membrane, indicated by 19 in FIG. 4, a nanofiltration membrane from crosslinked piperazine polyamide "UTC60" (nanofiltration membrane 1, manufactured by TORAY INDUSTRIES, INC.), A nanofiltration membrane from crosslinked piperazine polyamide "NF-400" (nanofiltration membrane 2, manufactured by Nanofiltration Corporation Nanofiltration Film Corporation) nanofiltration membrane 3, manufactured by Alfa-Laval) or cellulose acetate nanofiltration membrane "GEsepa" (nanofiltration membrane 4, manufactured by GE Osmonics) were placed in a cell made of stainless steel Ali (SUS316). The temperature of the feed liquid was adjusted to 25 ° C, and the pressure of the high pressure pump 15 was adjusted to 0.5 MPa to collect the filtrate 16. The concentrations of magnesium sulfate contained in the feed liquid in the tank 13 and the filtrate 16 were analyzed by ion chromatography (manufactured by Dionex Corporation ) under the following conditions, thus calculating the penetration rate of magnesium sulfate.

Anion: column (AS4A-SC (manufactured by Dionex Corporation)), eluent (1.8 mM sodium carbonate / 1.7 mm sodium bicarbonate), temperature (35 ° C).

Cation: column (CS12A (manufactured by Dionex Corporation)), eluent (20 mM methanesulfonic acid), temperature (35 ° C).

The results are shown in table 1.

Table 1 Trademark (Manufacturer Name) Membrane material Filtration Pressure (MPa) The concentration of magnesium sulfate in the raw fluid (ppm) The concentration of magnesium sulfate in the filtrate (ppm) Magnesium sulfate penetration rate
(%) Nanofiltration membrane 1 UTC60 (TORAY INDUSTRIES, INC.) Crosslinked piperazine polyamide 0.5 1000 2 0.2 Nanofiltration membrane 2 NF-400 (Filmtec Corporation) Crosslinked piperazine polyamide 0.5 1000 twenty 2 Nanofiltration membrane 3 NF99 (Alfa-Laval) Polyamide 0.5 1000 twenty 2 Nanofiltration membrane 4 GEsepa (GE Osmonics) Cellulose acetate 0.5 1000 thirty 3

Reference Example 5: Assessment of Permeability of Citric Acid Through a Nanofiltration Membrane

To 10 L of ultrapure water was added 10 g of citric acid (manufactured by Wako Pure Chemical Industries, Ltd.), and the resulting mixture was stirred at 25 ° C for 1 hour to prepare an aqueous solution with 1000 ppm. citric acid. Then, the filtrate from nanofiltration membranes 1-4 was collected under the same conditions as in Reference Example 3. The concentrations of citric acid contained in the feed liquid in tank 13 and filtrate 16 were analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation) under the following conditions, thus calculating the penetration rate of citric acid and the penetration rate of citric acid / penetration rate of magnesium sulfate.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation); mobile phase: 5 mM p-toluenesulfonic acid (flow rate: 0.8 ml / min); reaction solution: 5 mM p-toluenesulfonic acid, 20 mM bis-Tris, 0.1 mM EDTA 2Na (flow rate: 0.8 ml / min); detection method: electrical conductivity; temperature: 45 ° C.

The results are shown in table 2.

table 2 Trademark
(Manufacturer Name) Membrane material Filtration Pressure (MPa) The concentration of citric acid in
raw
liquids
(ppm) The concentration of citric acid in the filtrate (ppm) Citric Acid Penetration Rate
(%) The rate of penetration of citric acid /
magnesium sulfate penetration rate Nanofiltration membrane 1 UTC60 (TORAY INDUSTRIES, INC.) Crosslinked piperazine polyamide 0.5 1000 180 eighteen 70 Nanofiltration membrane 2 NF-400 (Filmtec Corporation) Crosslinked piperazine polyamide 0.5 1000 140 fourteen 7 Nanofiltration membrane 3 NF99 (Alfa-Laval) Polyamide 0.5 1000 160 16 8 Nanofiltration membrane 4 GEsepa (GE Osmonics) Cellulose acetate 0.5 1000 60 6 2

Reference Examples 6-12: Verification of Lactic Acid Penetration of a Fermentation Culture Medium through a Nanofiltration Membrane

From the culture medium (2 L) obtained as in Reference Example 2, the cells were removed by centrifugation, and then concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the culture medium to a pH of 1.9 (Reference Example 6 ), 2.0 (Reference example 7), 2.2 (Reference example 8), 2.6 (Reference examples 9-11) or 4.0 (Reference example 12), followed by stirring the resulting mixture for 1 hour at 25 ° C, thus converting calcium lactate in the culture medium into lactic acid and calcium sulfate. Then the precipitated calcium sulfate was separated by filtration of the precipitate using No.2 quality filter paper (manufactured by ADVANTEC) by suction filtration, and 2 L of filtrate was collected.

Then, 2 L of each filtrate obtained as described above was poured into the raw material reservoir 13 of the membrane filtration apparatus shown in FIG. 3. As a 90φ nanofiltration membrane, indicated by 19 in FIG. 4, each of the nanofiltration membranes 1-4 was placed in a cell made of stainless steel (SUS316). In each case, the pressure of the high-pressure pump 15 was adjusted to 4 MPa to collect the filtrate 16. The concentration of sulfate ions and calcium ions contained in the feed liquid in tank 13 and filtrate 16 were analyzed by ion chromatography (performed by Dionex Corporation) under the same conditions as in Reference Example 4, and the concentration of lactic acid was analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation) under the same conditions as Reference Example 1. The results are shown in Table 3.

Table 3 Nanofiltration membrane pH The flow of penetration through the membrane (m ³ / m ² / d) Calcium ion concentration Sulfate ion concentration Lactic acid concentration Raw Fluid (mg / L) Filtrate (mg / l) Removal Rate (%) Raw Fluid (mg / L) Filtrate (mg / l) Removal Rate (%) Raw Fluid (mg / L) Filtrate (mg / l) Removal Rate (%) Reference example 5 one 1.9 2.54 557 0.8 99.9 1678 428 74.5 45 22.9 50.9 Reference Example 6 one 2 2.54 557 0.8 99.9 1678 428.3 74.5 45 22.9 50.9 Reference example 7 one 2.2 2.5 811 0.8 99.9 1165 133 88.6 48 24.5 51 Reference example 8 one 2.6 2.5 1497 1.3 99.9 918 24 97.4 48 24.9 51.9 Reference example 9 2 2.6 2.12 1497 4.8 99.7 918 26.8 97.1 48 21,2 44,2 Reference Example 10 3 2.6 2.08 1497 6.7 99.6 918 24.3 97.4 48 21.9 45.6 Reference Example 11 four 2.6 1.48 1497 2.1 99.9 918 1.8 99.8 48 21 43.8 Reference Example 12 one four 2,51 4909 2.9 99.9 402 7.9 98 49 20.9 42.7

As shown in table 3, it was found that calcium sulfate was removed with high efficiency at all pH. It was also shown that the rate of penetration of lactic acid and the flow of penetration through the membrane were the highest for nanofiltration membrane 1.

Examples 1-5: Examples for the production of lactic acid

Concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to 200 L of the porous membrane filtrate obtained in Reference Example 3 to a pH of 1.9 (Example 1), 2.0 (Example 2), 2 , 2 (Example 3), 2.6 (Example 4) or 4.0 (Example 5), followed by stirring the resulting mixture for 1 hour at 25 ° C, thus converting calcium lactate in the culture medium into lactic acid and sulfate calcium. Then the precipitated calcium sulfate was separated by suction filtration using quality filter paper by suction filtration, and 200 L of each of the filtrates were collected.

Then, 200 L of each filtrate obtained in the above Examples was poured into the feed tank 13 of the membrane filtration device shown in FIG. 3. A 4-inch nanofiltration module 2 (UTC60, manufactured by TORAY INDUSTRIES, INC.) Of the nanofiltration membrane 1, which showed the highest penetration rates of lactic acid in Reference Examples 6-12, was placed in a special tank, and the process was carried out by adjusting the pressure in the pump high pressure 15 to 4 MPa to collect the filtrate for each pH. The concentrations of sulfuric acid ions and calcium ions contained in the feed liquid in tank 13 and filtrate 16 were analyzed by ion chromatography (produced by Dionex Corporation) under the same conditions as in Reference Example 4, and the concentration of lactic acid was analyzed by high performance liquid chromatography (produced Shimadzu Corporation) under the same conditions as in Reference Example 1. As a result, it was shown that calcium sulfate was removed with high efficiency at all pHs, as in Reference Examples 6-12.

Thereafter, 100 L of each of the nanofiltration membrane filtrates was concentrated by distillation of water using an instantaneous evaporator (manufactured by TOKYO RIKAKIKAI) under reduced pressure (50 hPa). No precipitation of calcium sulfate was observed at this time.

After that, distillation was carried out under a reduced pressure of 133 Pa at 130 ° C. To confirm the racemization of distilled lactic acid, optical purity was determined by high performance liquid chromatography before and after distillation. The results are shown in table 4.

The obtained purified lactic acid was used to verify direct polymerization in Example 6 and to verify the synthesis of lactide in Example 7, and lactic acid before distillation was used to verify direct polymerization in Comparative Example 2 and to verify the synthesis of lactide in Comparative Example 3.

Comparative Example 1: Lactic Acid Production Example

Concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to 200 L of the membrane filtrate obtained as in Reference Example 3 to a pH of 2.0, after which the resulting mixture was stirred for 1 hour at 25 ° C, transforming thus calcium lactate in the culture medium into lactic acid and calcium sulfate. Then the precipitated calcium sulfate was separated by filtration of precipitates, using quality filter paper, suction filtration, and 200 L of filtrate was collected. The concentration of calcium sulfate contained in the filtrate was analyzed by ion chromatography, and it was shown that the concentration is 549 mg / L. Thus, it was shown that calcium sulfate was not sufficiently removed.

After that, 100 l of the filtrate was concentrated by distillation of water using an instantaneous evaporator under reduced pressure (50 hPa), and this caused the precipitation of calcium sulfate, which was not removed by the above-described high-quality filter paper. Then, distillation was carried out under a reduced pressure of 133 Pa at 130 ° C. To confirm the racemization of distilled lactic acid, the optical purity of lactic acid was determined before and after distillation by high performance liquid chromatography under the same conditions as in Reference Example 1. As a result, a decrease in optical purity was observed. In addition, partially oligomerized lactic acid was observed in the distillation residue, and the distillation yield was reduced to 30%. These results are shown in table 4, together with the results of purification through a nanofiltration membrane.

Table 4 Optical purity (%) Distillation yield (%) Before distillation After distillation Example 1 99.9 99.9 60 Example 2 99.9 99.9 81 Example 3 99.9 99.9 84 Example 4 99.9 99.9 88 Example 5 99.9 99.9 92 Comparative Example 1 99.9 94.0 thirty

Example 6: Verification of direct polymerization for lactic acid

In a reaction tank having a stirrer, 150 g of the lactic acid obtained in Example 2 was heated at 800 Pa at 160 ° C. for 3.5 hours to obtain oligomers. Then, 0.12 g of tin (II) acetate (manufactured by Kanto Chemical Co., Ltd.) and 0.33 g of methanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were added to the oligomers, and the resulting mixture was heated at 500 Pa at 180 ° C for 7 hours to obtain a prepolymer. The prepolymer was then crystallized by heating in an oven at 120 ° C for 2 hours. The resulting prepolymer was triturated using a hammer mill and sieved through a sieve to obtain a powdery mass having an average particle size of 0.1 mm. In the solid phase polymerization step, 150 g of the prepolymer were taken and placed in a furnace to which an oil rotary pump was connected, thereby performing a vacuum heat treatment. The pressure was set at 50 Pa and the heating temperature was set: 140 ° C for 10 hours; 150 ° C for 10 hours; and 160 ° C for 20 hours. The resulting polylactic acid was subjected to a weight average molecular weight analysis using GPC (manufactured by Tosoh Corporation), a melting point analysis using DSC (manufactured by SII NanoTechnology Inc.) and a thermal mass loss rate analysis using TG (manufactured by SII NanoTechnology Inc.).

Analysis of the weight average molecular weight of polylactic acid

The weight average molecular weight (Mw) of the polylactic acid produced by polymerization is the weight average molecular weight for standard polymethyl methacrylate as determined by gel permeation chromatography (GPC). GPC determination was performed using the HLC8320GPC (manufactured by Tosoh Corporation) as a GPC system and two sequential TSK-GEL SuperHM-M columns (manufactured by Tosoh Corporation). Detection was performed using a differential refractometer. In terms of conditions for determination, the flow rate was 0.35 ml / min, hexafluoroisopropyl alcohol was used as a solvent, and 0.02 ml of a solution with a sample concentration of 1 mg / ml was injected.

Analysis of the melting point of polylactic acid

The melting point of polylactic acid produced by polymerization is the value determined using a DSC7020 differential scanning calorimeter (manufactured by SII NanoTechnology Inc.), and the determination was carried out using 10 mg of the sample, under nitrogen atmosphere at a heating rate of 20 ° C / min.

Analysis of the rate of heat loss of mass of polylactic acid

The rate of heat loss of mass of polylactic acid produced by polymerization was determined using a TG / DTA7200 thermogravimetric differential thermal analyzer (manufactured by SII NanoTechnology Inc.). The determination was performed using 10 mg of the sample, in a nitrogen atmosphere at a constant temperature of 200 ° C for a heating time of 20 minutes.

Analysis of the degree of staining of polylactic acid

0.5 g of polylactic acid produced by polymerization was dissolved in 9.5 g of chloroform, and the degree of staining was analyzed using a colorimeter (manufactured by NIPPON DENSHOKU INDUSTRIES CO., LTD.) As the number of APHA staining units.

Polylactic acid obtained by direct polymerization of lactic acid had a weight average molecular weight of 155,000, a melting point of 165 ° C, a heat loss rate of 5%, and an APHA stain of 10.

Comparative Example 2: Verification of the direct polymerization of lactic acid

Lactic acid before distillation in Example 2 was concentrated by rotary evaporation to 90% by weight to obtain 150 g of concentrated lactic acid. Direct polymerization was carried out under the same conditions as in Example 6. The polylactic acid obtained by direct polymerization had a weight average molecular weight of 85,000, a melting point of 160 ° C, a heat loss rate of 15%, and a degree of coloring of APHA 50. Thus, the quality lactic acid was lower compared with the quality of lactic acid obtained in example 6, for all points of assessment.

Example 7: Verification of the synthesis of lactide

In a reaction tank having a stirrer, 150 g of the lactic acid obtained in Example 2 was concentrated by heating at atmospheric pressure at 135 ° C. for 30 minutes. Then, under reduced pressure (4500-6500 Pa), the temperature of the liquid was gradually increased to 135 ° C (20 minutes), 150 ° C (20 minutes) and 160 ° C (20 minutes) to obtain oligomers. Thereafter, 0.75 g of tin (II) octylate (Nacalai Tesque) was added to the oligomers and simple distillation was performed under reduced pressure (1000-2000 Pa) at 200 ° C for 2 hours to distill the lactide. To prevent clogging of the pipes, the condenser temperature was set to 110 ° C. The lactide fraction was obtained in an amount of 92.3 g. The lactide yield was 85.4% with respect to the initial L-lactic acid.

Chemical analysis of lactide

The chemical purity of the synthesized lactide (LL-lactide to reduced lactide ratio) was analyzed by gas chromatography GC2010 (manufactured by Shimadzu Corporation). The RT BDEXM capillary column (manufactured by RESTEK) was used as the column, and the determination conditions were: carrier gas flow rate (He), 69.2 ml / min; temperature of the evaporation chamber, 230 ° C; column temperature, 150 ° C; detector temperature (FID), 230 ° C; and a separation ratio of 50. The chemical purity of LL-lactide was calculated from the ratio of peak regions of LL-lactide, DD-lactide, and DL-lactide.

Analysis of the degree of staining of lactide

6 g of synthesized lactide was completely dissolved in 20 g of acetone, and the degree of staining was analyzed using a colorimeter (manufactured by NIPPON DENSHOKU INDUSTRIES CO., LTD.) As the number of APHA staining units.

As a result, the resulting lactide had a chemical purity of 96.2% and a degree of staining of APHA 2.

Comparative Example 3: Test for the synthesis of lactide

Lactic acid before distillation in Example 2 was concentrated by rotary evaporation to 90% by weight to obtain 150 g of concentrated lactic acid. Lactide was synthesized under the same conditions as in Example 7. Lactide was obtained in an amount of 79.1 g with a yield of 73.2% and had a chemical purity of 93.1% and a degree of staining of APHA 12. Thus, both the yield and the quality of lactic acid was worse than those for lactic acid obtained in example 7.

Example 8: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

50 g of the lactide obtained in Example 7 and 0.05 g of stearyl alcohol were placed in a reaction tank having a stirrer, and the atmosphere in the system was replaced with nitrogen, after which the resulting mixture was heated to 190 ° C until the lactide was dissolved. Then, 0.025 g of tin (II) octylate was added thereto, and polymerization was carried out at 190 ° C. for 2 hours. The obtained polylactic acid was analyzed by its weight average molecular weight, melting point, rate of heat loss of mass and degree of staining by the methods described in example 6. The weight average molecular weight was 135,000; melting point was 165 ° C; the rate of thermal mass loss was 5.1%; and the degree of staining was APHA 5.

Comparative Example 4: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerizing lactide in accordance with the same procedure as in Example 8, except that 50 g of the lactide obtained in Comparative Example 3 were used. The obtained polylactic acid had a weight average molecular weight of 109,000, melting point 162 ° C, the mass loss rate of 6.3% and the degree of staining of APHA 11. Thus, the quality of polylactic acid was worse than the quality of polylactic acid obtained in example 8 for all points of assessment.

Example 9: Analysis of impurities in lactic acid

Three liters of the filtrate obtained in the same manner as in Example 3 was filtered through an SU-610 nanofiltration membrane module (manufactured by TORAY INDUSTRIES, INC.) At a working pressure of 2.0 MPa to remove impurities. The aqueous lactic acid solution that passed through the nanofiltration membrane module was concentrated using an SU-810 reverse osmosis membrane module (manufactured by TORAY INDUSTRIES, INC.), And also concentrated by distillation of water using a rotary evaporator (manufactured by TOKYO RIKAKIKAI) under reduced pressure (50 hPa ), thus obtaining an 80% aqueous solution of lactic acid. Thereafter, distillation was carried out under a reduced pressure of 133 Pa at 133 ° C. to obtain 500 g of lactic acid.

Analysis of impurities in lactic acid

To the lactic acid obtained as described above, purified water was added to prepare a 90% aqueous solution of lactic acid. The impurities contained were analyzed by HPLC (high performance liquid chromatography) or GC (gas chromatography) under the following conditions. The results of the analysis are shown in table 5.

Analysis of acetic acid, pyruvic acid and 2-hydroxybutyric acid by HPLC

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation); mobile phase: 5 mM p-toluenesulfonic acid (flow rate: 0.8 ml / min); reaction solution: 5 mM p-toluenesulfonic acid, 20 mM bis-Tris, 0.1 mM EDTA 2Na (flow rate: 0.8 ml / min); detection method: electrical conductivity; temperature: 45 ° C.

Analysis of furfural and HMF using HPLC

Column: Synergie HydroRP (manufactured by Phenomenex, Inc.); mobile phase: 5% aqueous acetonitrile solution (flow rate 1.0 ml / min); detection method: UV (283 nm); temperature: 40 ° C.

Analysis of methanol and methyl lactate using the GC method

Column: DB-5 (0.25 mm × 30 m., Manufactured by J&W); column temperature: 50 ° C to 250 ° C (8 ° C / min); inlet temperature: 250 ° C; carrier gas: helium; carrier pressure: 65 kPa.

As shown in table 5, the concentration of all impurities, with the exception of acetic acid and pyruvic acid, was 0 ppm. (below the limit of sensitivity).

Table 5 Impurity Name Content in 90% Lactic Acid Methanol 0 ppm Acetic acid 200 ppm Pyruvic acid 200 ppm 2-hydroxybutyric acid 0 ppm Furfural 0 ppm 5-hydroxymethylfurfural 0 ppm Methyl lactate 0 ppm

Example 10: Verification of the direct polymerization of lactic acid and evaluation of the physical properties of polylactic acid

In a reaction tank having a stirrer, 150 g of the 90% aqueous lactic acid solution of Example 9 was heated at 800 Pa at 160 ° C. for 3.5 hours to obtain oligomers. Then, 0.12 g of tin (II) acetate (manufactured by Kanto Chemical Co., Ltd.) and 0.33 g of methanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were added to the oligomers, and the resulting mixture was heated at 500 Pa at 180 ° C for 7 hours to obtain a prepolymer. The prepolymer was then crystallized by heating in an oven at 120 ° C for 2 hours. The resulting prepolymers were ground using a hammer mill and sieved through a sieve to obtain a ground mass having an average particle size of 0.1 mm. At the solid-state polymerization stage, 150 g of the prepolymer were taken and placed in a furnace to which an oil rotary pump was connected, thus performing a vacuum heat treatment. The pressure was set at 50 Pa and the heating temperature was set at: 140 ° C for 10 hours; 150 ° C for 10 hours; and 160 ° C for 20 hours. The weight average molecular weight, melting point, rate of thermal mass loss, and the degree of staining of the obtained polylactic acid were analyzed by the methods described in example 6.

Example 11: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid obtained in example 9 to the lactic acid obtained in example 9, so that their concentrations were brought to 30 ppm methanol, 100 ppm. formic acid, 200 ppm pyruvic acid, 100 ppm 2-hydroxybutyric acid, 3 ppm furfural, 2 ppm 5-hydroxymethylfurfural and 100 ppm. methylalactate.

Example 12: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentration was brought to 65 ppm methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 5: Analysis of impurities in lactic acid and evaluation of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentrations were brought to 100 ppm methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 6: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentration was brought to 65 ppm methanol, 100 ppm. formic acid, 300 ppm acetic acid, 600 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 7: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentration was brought to 65 ppm methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 25 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 8: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentration was brought to 65 ppm methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 25 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 9: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentration was brought to 65 ppm methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 650 ppm. methyl lactate.

Comparative Example 10: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid the acid obtained in example 9 to the lactic acid obtained in example 9, so that their concentration was brought to 70 ppm methanol, 750 ppm 2-hydroxybutyric acid and 500 ppm methyl lactate.

Comparative Example 11: Analysis of impurities in lactic acid, verification of direct polymerization of lactic acid and assessment of the physical properties of polylactic acid

Polylactic acid was produced by polymerization and analyzed in the same manner as in Example 10, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in a 90% aqueous solution of lactic acid acid obtained in example 9 to lactic acid obtained in example 9, so that their concentration was brought to 600 ppm acetic acid and 300 ppm pyruvic acid.

Weight average molecular weights, melting points, heat loss rates, and APHA staining rates of polylactic acids obtained in Examples 10-12 and Comparative Examples 5-11 are shown in Table 6. In Examples 10-12, polylactic acids having excellent physical properties were obtained. by weight average molecular weight, rate of thermal mass loss and degree of staining. However, in Comparative Example 5, the weight average molecular weight was small, and therefore the mechanical strength was low, and the rate of heat loss of the mass was high, and therefore the thermal stability was low; in Comparative Examples 6-9, the rate of thermal mass loss was high and the degree of staining was high; and in Comparative Examples 10 and 11, the rate of thermal mass loss was high. From these results it is clear that in cases where the amounts of impurities in lactic acid are no more than predetermined values, it is possible to obtain polylactic acid having excellent thermal stability, mechanical strength and color.

Example 13: Analysis of impurities in lactic acid and assessment of the physical properties of lactide

In a reaction tank having a stirrer, 150 g of lactic acid obtained in Example 9 was concentrated by heating at 135 ° C. at atmospheric pressure for 30 minutes. Then, under reduced pressure (4500-6500 Pa), the liquid temperature was gradually increased to 135 ° C (20 minutes), 150 ° C (20 minutes) and 160 ° C (20 minutes) to obtain oligomers. Thereafter, 0.75 g of tin (II) octylate (Nacalai Tesque) was added to the oligomers, and simple distillation was carried out under reduced pressure (1000-2000 Pa) at 200 ° C for 2 hours to distill the lactide. To avoid clogging of the pipes, the condenser temperature was set to 110 ° C. The lactide fraction was obtained in an amount of 93.3 g. The yield of lactide was 87.2% with respect to the initial L-lactic acid.

Example 14: Analysis of impurities in lactic acid and assessment of the physical properties of lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 30 ppm. methanol, 100 ppm. formic acid, 200 ppm pyruvic acid, 100 ppm 2-hydroxybutyric acid, 3 ppm furfural, 2 ppm 5-hydroxymethylfurfural and 100 ppm. methyl lactate.

Example 15: Analysis of impurities in lactic acid and assessment of the physical properties of lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 65 ppm. methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 12: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 100 ppm. methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 13: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 65 ppm. methanol, 100 ppm. formic acid, 300 ppm acetic acid, 600 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 14: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 65 ppm. methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 25 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 15: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 65 ppm. methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 25 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 350 ppm. methyl lactate.

Comparative Example 16: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 65 ppm. methanol, 100 ppm. formic acid, 300 ppm acetic acid, 300 ppm pyruvic acid, 150 ppm 2-hydroxybutyric acid, 5 ppm furfural, 5 ppm 5-hydroxymethylfurfural and 650 ppm. methyl lactate.

Comparative Example 17: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in example 9, so that their concentrations were brought to 70 ppm. methanol, 750 ppm 2-hydroxybutyric acid and 500 ppm methyl lactate.

Comparative Example 18: Analysis of Impurities in Lactic Acid and Assessment of the Physical Properties of Lactide

Lactide was synthesized in the same manner as in example 13, except that 150 g of an aqueous solution of lactic acid was used, which was prepared by adding the appropriate components, along with the impurities contained in the 90% aqueous solution of lactic acid obtained in example 9, to the lactic acid obtained in Example 9, so that their concentrations were brought to 600 ppm. acetic acid and 300 ppm pyruvic acid.

The yield and degree of staining of APHA of the lactides obtained in Examples 13-15 and Comparative Examples 12-18 are shown in Table 6. In Examples 13-15, excellent results were obtained for the yield of lactide and degree of staining. However, in Comparative Examples 12-18, the yield was less than 80%; and in Comparative Examples 13-15, which contained a large amount of pyruvic acid, furfural, and the like, the degree of staining was high. From these results, it was found that in cases where the amounts of impurities in lactic acid are no more than predetermined values, lactide can be obtained showing a high yield and a low degree of staining.

Example 16: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

50 g of the lactide obtained in Example 13 and 0.05 g of stearyl alcohol were added to the reaction tank having a stirrer, and the atmosphere in the system was replaced with nitrogen, followed by heating the resulting mixture to 190 ° C until the lactide was dissolved. Then, 0.025 g of tin (II) octylate was added as a catalyst, and polymerization was performed at 190 ° C. for 2 hours. The resulting polylactic acid was analyzed by its weight average molecular weight, melting point, heat loss rate and degree of staining using the methods described in example 6.

Example 17: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 14 were used.

Example 18: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 15 were used.

Comparative Example 19: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 12 were used.

Comparative Example 20: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 13 were used.

Comparative Example 21: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 14 were used.

Comparative Example 22: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 15 were used.

Comparative Example 23: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 16 were used.

Comparative Example 24: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 17 were used.

Comparative Example 25: Obtaining polylactic acid by polymerization using lactide as a feedstock and assessing the physical properties of polylactic acid

Polylactic acid was produced by polymerization of lactide and analyzed in accordance with the same procedure as in example 16, except that 50 g of the lactide obtained in example 18 were used.

Weight average molecular weights, melting points, heat loss rates, and APHA staining rates of polylactic acids obtained in Examples 16-18 and Comparative Examples 19-25 are shown in Table 6. In Examples 16-18, polylactic acids having excellent physical properties were obtained. by weight average molecular weight, rate of thermal mass loss and degree of staining. However, in Comparative Examples 19 and 20, the weight average molecular weight was small. Also, in Comparative Examples 20-22, the weight loss rate was high and the degree of staining was high; and in Comparative Examples 23-25, the rate of mass loss was high.

Table 6 Lactic acid Example 10 Example 11 Example 12 Comp. example 5 Comp. example 6 Comp. example 7 Comp. example 8 Comp. example 9 Comp. example 10 Comp. example 11 Impurity Content (ppm) Methanol 0 thirty 65 one hundred 65 65 65 65 70 0 Acetic acid 200 200 300 300 300 300 300 300 0 600 Pyruvic acid 200 200 300 300 600 300 300 300 200 200 2-hydroxybutyric acid 0 one hundred 150 150 150 150 150 150 750 0 Furfural 0 3 5 5 5 5 25 5 0 0 5-hydroxymethylfurfural 0 2 5 5 5 25 5 5 0 0 Methyl lactate 0 one hundred 350 350 350 350 350 650 500 0 Estimated Direct Polymerization Results Weight average molecular weight (Mw) 175,000 156000 145000 115,000 128,000 153000 144,000 118,000 149000 161000 Melting Point (ºC) 167 165 164 163 164 165 163 162 162 165 Mass loss rate (%) 4.9 5.5 5.9 6.7 8.5 8.6 7.9 6.5 6.5 6.7 Degree of Staining (APHA) 7 10 fifteen 16 25 40 twenty 25 7 8 Lactide Example 13 Example 14 Example 15 Comp. example 12 Comp. example 13 Comp. example 14 Comp. example 15 Comp. example 16 Comp. example 17 Comp. example 18 Lactide score Exit (%) 85 81 80 78 73 75 76 75 77 75 Degree of Staining (APHA) 2 2 6 8 12 10 10 10 6 6 Polymerization of polylactic acid from lactide Example 16 Example 17 Example 18 Comp. example 19 Comp. example 20 Comp. example 21 Comp. example 22 Comp. example 23 Comp. example 24 Comp. example 25

Polymerization Assessment Results Weight average molecular weight (Mw) 135000 133000 122000 118,000 109,000 1224000 123000 130,000 129,000 131000 Melting Point (ºC) 165 164 162 162 162 163 162 163 162 163 Mass loss rate (%) 5.1 5.3 6.2 6.0 6.3 7.4 7.0 7.2 7.5 7.3 Degree of Staining (APHA) 5 6 8 8 eleven fourteen 12 9 8 9

APPLICABILITY IN OPERATING CONDITIONS

Lactic acid obtained by the method of the present invention for the production of lactic acid can be suitably used for food and pharmaceuticals, and as a monomer material for polylactic acid, which is a general purpose plastic degradable by microorganisms. In addition, polylactic acid obtained using lactic acid as a raw material has excellent thermal stability, mechanical strength and color, so that polylactic acid is suitable for various applications such as fibers, films and molded products.

DESCRIPTION OF SYMBOLS

1. Fermentation reactor

2. The tank for membrane separation

3. The element of the separating membrane

4. Gas supply device

5. Stirrer

6. The control device of the hydraulic differential pressure

7. Pump feeding culture medium

8. pH feed pump

9. pH Detecting / Monitoring Device

10. Pump for circulating fermentation liquid

11. Level sensor

12. Temperature regulator

13. Raw material reservoir

14. A cell equipped with a nanofiltration membrane or reverse osmosis membrane

15. High pressure pump

16. The flow of membrane filtrate

17. The flow of membrane concentrate

18. The flow of culture medium supplied by a high pressure pump

19. Nanofiltration membrane

20. Support plate

Claims (10)

1. A method of producing lactic acid, comprising the following stages (A) to (C):
(A) a continuous cultivation step, wherein the fermentation culture medium of a microorganism having lactic acid fermentation is filtered through a porous membrane having an average pore size of not less than 0.01 μm and less than 1 μm, with a transmembrane pressure difference in the range of 0.1 up to 20 kPa, and the filtrate is collected while maintaining unfiltered liquid in the culture medium or returning unfiltered liquid to the culture medium and adding raw materials for fermentation to the culture medium;
(B) a step of filtering the filtrate obtained in step (A) through a nanofiltration membrane; and
(C) a step for distilling the filtrate obtained in step (B) under a pressure of not less than 10 Pa and not more than 30 kPa, at not less than 50 ° C and not more than 180 ° C to recover lactic acid. 2. The method of producing lactic acid according to claim 1, where the pH of the filtrate obtained in the indicated step (A) is adjusted to not less than 2 and not more than 4.5, followed by the filtrate being directed to the indicated step (B). 3. The method for producing lactic acid according to claim 1, wherein said step (A) is a step of continuous fermentation in the presence of a calcium salt, comprising after step (A) an additional step (D), in which the calcium component in the filtrate obtained in step ( A) is removed as insoluble sulfate, and the solution containing lactic acid obtained after stage (D) is sent to the indicated stage (B). 4. The method of producing lactic acid according to claim 1, where in stage (B) the ratio of the rate of penetration of magnesium sulfate relative to the rate of penetration of citric acid through the specified nanofiltration membrane is not less than 3 at a working pressure of 0.5 MPa, the temperature of the raw liquid 25 ° C and a concentration in the feed fluid of 1000 ppm 5. The method of producing lactic acid according to claim 1, where in stage (B) the penetration rate of magnesium sulfate through said nanofiltration membrane is not more than 1.5% at an operating pressure of 0.5 MPa, a temperature of the raw liquid 25 ° C and a concentration of feed liquid 1000 ppm 6. The method for producing lactic acid according to claim 1, wherein the membrane material of said nanofiltration membrane comprises polyamide. 7. The method for producing lactic acid according to claim 6, wherein said polyamide comprises cross-linked piperazine polyamide as a main component and further includes a forming component represented by chemical formula 1:

where R is —H or —CH ₃ and n is an integer from 0 to 3. 8. The method of obtaining lactide, comprising the following stages:
(A) a continuous cultivation step, wherein the fermentation culture medium of a microorganism having lactic acid fermentation is filtered through a porous membrane having an average pore size of not less than 0.01 μm and less than 1 μm, with a transmembrane pressure difference in the range of 0.1 up to 20 kPa, and the filtrate is collected while maintaining unfiltered liquid in the culture medium or returning unfiltered liquid to the culture medium and adding raw materials for fermentation to the culture medium;
(B) a step of filtering the filtrate obtained in step (A) through a nanofiltration membrane;
(C) a step for distilling the filtrate obtained in step (B) under a pressure of not less than 10 Pa and not more than 30 kPa, at not less than 50 ° C and not more than 180 ° C to produce lactic acid; and
the subsequent stage of obtaining lactide, where the lactic acid obtained in stage (C) is used as a feedstock. 9. A method of producing polylactic acid, comprising the following stages:
(A) a stage of continuous cultivation, where the fermentation culture medium of a microorganism having the ability of lactic acid fermentation is filtered through a porous membrane having an average pore size of not less than 0.01 μm and less than 1 μm, with a transmembrane pressure difference in the range of 0.1 up to 20 kPa, and the filtrate is collected while maintaining unfiltered liquid in the culture medium or returning unfiltered liquid to the culture medium and adding raw materials for fermentation to the culture medium;
(B) a step of filtering the filtrate obtained in step (A) through a nanofiltration membrane;
(C) a step for distilling the filtrate obtained in step (B) under a pressure of not less than 10 Pa and not more than 30 kPa, at not less than 50 ° C and not more than 180 ° C to produce lactic acid;
followed by obtaining lactide from lactic acid obtained in stage (C), and polymerizing it to obtain polylactic acid. 10. A method of producing polylactic acid, comprising the following stages:
(A) a continuous cultivation step, wherein the fermentation culture medium of a microorganism having lactic acid fermentation is filtered through a porous membrane having an average pore size of not less than 0.01 μm and less than 1 μm, with a transmembrane pressure difference in the range of 0.1 up to 20 kPa, and the filtrate is collected while maintaining unfiltered liquid in the culture medium or returning unfiltered liquid to the culture medium and adding raw materials for fermentation to the culture medium;
(B) a step of filtering the filtrate obtained in step (A) through a nanofiltration membrane;
(C) a step for distilling the filtrate obtained in step (B) under a pressure of not less than 10 Pa and not more than 30 kPa, at not less than 50 ° C and not more than 180 ° C to produce lactic acid; and
the subsequent stage of obtaining polylactic acid, where the lactic acid obtained in stage (C) is polymerized by direct dehydration polycondensation.

Images