JP5262011B2 - Lactic acid production method and production apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing lactic acid by removing a calcium component and sulfuric acid in a lactic acid solution originated from microorganism culture. <P>SOLUTION: The method for producing the lactic acid includes removing the calcium component and the sulfuric acid by separating the calcium component and the sulfuric acid in the lactic acid solution originated from the microorganism culture by a nanofiltration membrane. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a method for separating and producing lactic acid produced in a culture solution by fermentation culture of microorganisms. Specifically, the present invention relates to a lactic acid production method and a production apparatus for obtaining a lactic acid solution by removing calcium components remaining in a microorganism culture solution with a nanofiltration membrane.

  Lactic acid is widely applied to industrial uses as a monomer raw material for biodegradable plastics in addition to uses such as foods and pharmaceuticals, and the demand is increasing. 2-Hydroxypropionic acid, i.e., lactic acid, is known to be produced by fermentation by a microorganism, and the microorganism converts a substrate containing a carbohydrate typified by glucose into lactic acid. Lactic acid is classified into (L) -form and (D) -form optical isomers depending on the configuration of the hydroxyl group bonded to the carbon at the carbonyl α-position. According to microbial fermentation, (L) -form or (D) -form lactic acid is selectively selected by appropriately selecting microorganisms, or a mixture of (L) -form and (D) -form (racemic form). Of lactic acid.

The production of lactic acid by microbial fermentation is generally carried out while maintaining an optimum pH for microbial fermentation by adding an alkaline substance to the culture solution. Many lactic acid is an acidic substance produced by microbial fermentation, in order to alkaline material is added, the culture is present as a lactate. In this case, free lactic acid can be obtained by adding an acidic substance to the culture solution after the end of fermentation.

  Specifically, calcium hydroxide is often used as an alkaline substance added to the culture solution. In this case, lactic acid produced by microbial fermentation exists as calcium lactate in the culture solution. Thereafter, a free lactic acid solution can be obtained by adding an acidic substance (for example, sulfuric acid) to the culture solution after completion of the culture, but a calcium salt (for example, calcium sulfate) is by-produced.

  As a method of removing the calcium salt generated here and separating lactic acid, a method of filtering with a qualitative filter paper or the like is used when the calcium salt is hardly soluble, such as calcium sulfate. However, in this method, the calcium salt precipitated as a solid is removed, but a trace amount of calcium salt dissolved in the solution is not removed and remains in the lactic acid solution. Therefore, when the filtrate containing this lactic acid is subjected to a concentration operation in a subsequent purification step, for example, there is a problem that a calcium salt precipitates (precipitates) again in the free lactic acid solution. In addition, it is known that when a lactic acid solution is heated by an operation such as distillation in a state where calcium ions are not sufficiently removed, racemization and oligomerization of lactic acid proceed due to the influence of calcium ions. Therefore, a method for effectively removing a trace amount of calcium component (calcium salt) remaining in the lactic acid solution is required.

  As a method for removing a trace amount of calcium component from a lactic acid solution, a method using an ion exchange resin is disclosed (for example, see Patent Document 1). However, in order to maintain the ion exchange performance of the ion exchange resin, it is necessary to periodically regenerate the ion exchange resin. In addition, regeneration of the ion exchange resin is performed using a large amount of sodium chloride aqueous solution. However, with the regeneration, a large amount of waste liquid is discharged, and there is a problem that the waste liquid treatment is costly. Further, when the ion exchange resin is repeatedly regenerated, there is a problem that the regeneration rate of the ion exchange resin is lowered and the ion exchange performance is lowered and the removal rate of the calcium salt is lowered.

A method of removing a trace amount of calcium component from a lactic acid solution by a bipolar membrane using an electrodialyzer is also known (see, for example, Patent Document 2). However, the bipolar membrane used in this method is expensive and has a problem that the removal efficiency of the calcium salt is never high.
JP 2001-506274 A (Claims) Japanese Patent Laying-Open No. 2005-270025 (Claims)

The invention, object that looks as though it was described above, i.e., in the case of producing lactic acid by microbial fermentation in the presence of calcium salts, and solve the problem of effectively removing a calcium component in the culture medium, efficiently lactate The object is to provide a method of manufacturing.

  As a result of intensive studies to solve the above problems, the present inventors are able to remove the calcium component in the culture solution with high efficiency by filtering the microorganism fermentation culture solution using a nanofiltration membrane. As a result, the present invention has been completed.

That is, this invention is comprised from following (1)-(6).
(1) A method for separating and producing lactic acid produced in a culture solution by fermentation of microorganisms in the presence of a calcium salt, wherein the calcium component in the culture solution is added with sulfuric acid to form a hardly soluble sulfate salt. A step A for obtaining a separation liquid containing lactic acid by removing, a step B for obtaining a lactic acid solution by removing the calcium component by filtering the separation liquid containing lactic acid obtained in step A through a nanofiltration membrane, nanofiltration membranes methanol removal rate of 10% or less or one isopropyl alcohol removal rate of 20% to 50% and removal rate removal rate of 95% or less and sucrose 70% or more of citric acid of the B 95% It is the above, The manufacturing method of lactic acid characterized by the above-mentioned.
(2) The method for producing lactic acid according to (1), wherein the membrane material of the nanofiltration membrane contains a crosslinked piperazine polyamide as a main component and a constituent represented by Chemical Formula 1.

(In the formula, R represents —H or —CH ₃ , and n represents an integer of 0 to 3.)
(3) The lactic acid according to (1) or (2), further comprising a step C of distilling the lactic acid solution obtained in the step B at a pressure of 1 Pa or higher and an atmospheric pressure of 25 ° C. or higher and 200 ° C. or lower. Manufacturing method.
(4) The method for producing lactic acid according to (1) or (2), further comprising a step D in which the lactic acid solution obtained in the step B is further filtered through a reverse osmosis membrane to increase the lactic acid concentration.
(5) The method according to (4) , further comprising a step C of distilling the lactic acid solution obtained in the step B after the step D at a temperature of 25 ° C. or higher and 200 ° C. or lower under a pressure of 1 Pa or higher and atmospheric pressure or lower. Of producing lactic acid.
(6) A manufacturing apparatus of lactic acid used lactic acid produced in the culture solution by microbial fermentation in the presence of calcium salts when you manufactured separately, in manufacturing to separate the lactic acid Removes the calcium component in the culture solution as a poorly soluble sulfate by adding sulfuric acid to obtain a separation liquid containing lactic acid, and the separation liquid containing lactic acid obtained in step A is passed through a nanofiltration membrane. filtered, comprising the step B of obtaining a lactic acid solution to remove the calcium component, step nano methanol removal rate of the filtration membrane 10% or less or one of isopropyl alcohol 50% or less and citric acid at least 20% removal rate of the B An apparatus for producing lactic acid, wherein the removal rate of lactic acid is 70% to 95% and the sucrose removal rate is 95% or more.

  The method for producing lactic acid according to the present invention can effectively remove a calcium component dissolved in a fermentation broth or contained as a hardly soluble solid by a simple operation. Therefore, for example, in subsequent purification steps such as concentration operation and distillation operation, reprecipitation (precipitation) of calcium salt due to concentration, and racemization and oligomerization of lactic acid due to residual calcium ions can be suppressed.

  Hereinafter, the present invention will be described in more detail.

  The method for producing lactic acid of the present invention is a method for separating and producing lactic acid produced in a culture solution by fermentation of microorganisms in the presence of a calcium salt, and filtering the fermentation culture solution through a nanofiltration membrane. And the process B which removes the calcium component in a fermentation culture solution and obtains a lactic acid solution is included.

  The nanofiltration membrane used in Step B of the method for producing lactic acid of the present invention is also called a nanofiltration membrane or NF membrane, and “a membrane that transmits monovalent ions and blocks divalent ions”. It is a film generally defined as It is a membrane that is considered to have a minute gap of about several nanometers, and is mainly used to block minute particles, molecules, ions, salts, and the like in water. The inventors of the present invention have studied in detail the relationship between the fractionation performance of the nanofiltration membrane and the permeation performance of lactic acid, the inhibition performance of calcium ions and the inhibition performance of sulfate ions, and as a result, the nanofiltration used in the present invention It was found that the fractionation performance of the membrane is closely related to the permeation performance of lactic acid, the inhibition performance of calcium ions and the inhibition performance of sulfate ions.

Separation performance of nanofiltration membranes and reverse osmosis membranes is generally due to filtration tests of sodium chloride aqueous solution and magnesium sulfate that have the effect of charge repulsion and methanol, ethanol , isopropyl alcohol, acetone, formalin, urea, glucose, It is evaluated by a filtration test of an aqueous solution containing organic substances having different sizes such as sucrose and molecules in which molecules such as acetic acid, citric acid and ethylenediamine are adjusted to a non-dissociated state by adjusting pH. Separation performance is evaluated by the salt removal rate and the molecular weight cut-off curve, and the water permeability is evaluated by the amount of filtrate at that time. Separation performance and water permeation performance are contradictory, that is, if the membrane structure is densified in order to increase molecular blocking performance, the water permeation performance will be reduced, so it is not simply a matter of densifying the membrane structure on average. It is necessary to evaluate and grasp the blocking performance of molecules of various sizes, determine the sharpness (uniformity) of the required fraction curve, and make the molecular structure as high as possible in water permeability. Therefore, the design of the film structure is a difficult task.

Here nanofiltration membranes of the present invention, removal removal of methanol is 10% or less or One removal of the isopropyl alcohol is 20% or more 50% or less and the removal rate of citric acid 70% to 95% or less and sucrose When the rate is 95% or more, the inventors have found that the lactic acid permeability is high, and the calcium ion rejection rate and the sulfate ion rejection rate are increased. FIG. 1 shows a molecular weight cut-off curve of the nanofiltration membrane of the present invention.

After optimizing the molecular structure of the molecular diameter of methanol , isopropyl alcohol, citric acid, and sucrose by molecular orbital calculation at B3LYP / 6-31 + G (d, p) level, calculate the distance between each atom in the molecule The farthest interatomic distances are 0.28 nm, 0.43 nm, 0.74 nm, and 0.96 nm, respectively. On the other hand, the size of undissociated lactic acid is a 0.50 nm, Ri methanol removal rate of the size 0.28nm exceeds 10%, the 50% removal of the isopropyl alcohol in size 0.43nm Ri exceeds, the removal rate of citrate size 0.74nm exceeds 95% will be reduced transmittance and water permeability of the lactic acid, it was found that undesirable as nanofiltration membranes for the present invention.

Further, when the hydrated ion structure of calcium ion was determined by molecular dynamics calculation, the calcium ion had a structure in which 27 water molecules were hydrated, and the size of the hydrated ion was 1.2 nm. Revealed. This result of examining the rate of removal of calcium in mind, if the removal rate of sucrose 95% or more, or One removal of the isopropyl alcohol removal rate of 20% or more and citric acid 70% The present inventors have found that the removal rate of calcium ions and sulfate ions which are counter ions thereof is increased.

Thus, 95% or less as nanofiltration membranes methanol removal rate of 10% or less or One isopropyl alcohol removal rate of the of the present invention are removal rate of 20% to 50% and citric acid 70% 1 When the sucrose removal rate is 95% or more, the lactic acid permeability is preferably between about 40% and 60%, and the calcium ion removal rate is preferably close to 100%. It was found that lactic acid can be purified with high efficiency by designing a nanofiltration membrane so that the drawing performance falls within a specific range.

Here, the measurement conditions for the removal rate of methanol , isopropyl alcohol, citric acid, and sucrose are as follows. Using a flat membrane having a membrane area of about 30 cm ² , the raw water concentration was 1,000 mg / L, the raw water temperature was 25 ° C., and the raw water pressure was 0.75 MPa. The raw water pH was 6.5 for methanol , isopropyl alcohol, sucrose, and 2.8 for citric acid.
Removal rate (%) = (1− (permeate concentration / raw water concentration)) × 100 (Equation 1)
As the material for the nanofiltration membrane, polymer materials such as cellulose acetate polymer, polyamide, polyester, polyimide, vinyl polymer, polyacrylonitrile, sulfonated polysulfone, and the like can be used. In addition, the membrane structure has a dense layer on at least one side of the membrane, and on the asymmetric membrane having fine pores gradually increasing from the dense layer to the inside of the membrane or the other side, or on the dense layer of the asymmetric membrane. Either a composite film having a very thin functional layer formed of another material may be used.

  Among these, a composite film having a high-pressure resistance, high water permeability, and high solute removal performance and having an excellent potential and using a polyamide as a functional layer is preferable. In order to maintain durability against operating pressure, high water permeability, and blocking performance, a structure in which polyamide is used as a functional layer and is held by a support made of a porous membrane or nonwoven fabric is suitable. As the polyamide semipermeable membrane, a composite semipermeable membrane having a functional layer of a crosslinked polyamide obtained by polycondensation reaction of a polyfunctional amine and a polyfunctional acid halide on a support is suitable.

  In the nanofiltration membrane containing polyamide as a membrane material, preferable carboxylic acid components of monomers constituting the polyamide include, for example, trimesic acid, benzophenone tetracarboxylic acid, trimellitic acid, pyrometic acid, isophthalic acid, terephthalic acid, naphthalene Aromatic carboxylic acids such as dicarboxylic acid, diphenyl carboxylic acid, pyridine carboxylic acid and the like can be mentioned, but considering solubility in a film forming solvent, trimesic acid, isophthalic acid, terephthalic acid, and a mixture thereof are more preferable. A preferred amine component of the monomer constituting the polyamide is preferably an amine component mainly composed of a crosslinked piperazine polyamide and containing the component represented by the chemical formula 1, and examples thereof include piperazine and piperidine. Piperazine and piperidine are more preferably used because they have heat resistance and chemical resistance in addition to pressure resistance and durability. In Chemical Formula 1, those having n = 3 are more preferably used. Examples of the structural component represented by Chemical Formula 1 include those described in JP-A No. 62-201606.

  The nanofiltration membrane used in Step B of the method for producing lactic acid according to the present invention is not limited to the membrane composed of the above-mentioned one type of material, but is a membrane containing a plurality of membrane materials or a composite membrane combining a plurality of membranes. May be. For example, as described in JP-A-62-201606, a composite membrane in which a nanofiltration membrane made of the above material is formed as a separation functional layer on a support membrane made of polysulfone as a membrane material can be used.

  The nanofiltration membrane is generally used as a spiral membrane element, but the nanofiltration membrane used in the present invention is also preferably used as a spiral membrane element.

  In the step B of the method for producing lactic acid of the present invention, “filtering with a nanofiltration membrane” means filtering and dissolving a separation liquid containing lactic acid produced by fermentation culture of microorganisms through a nanofiltration membrane. This means that the calcium component is removed or blocked, and the lactic acid solution is permeated as a filtrate. Here, the calcium component includes any form of calcium ions, calcium salts, and compounds containing calcium contained in the culture solution, and those dissolved in the culture solution and precipitated in the culture solution. Or any that are precipitated and included.

  Examples of the method for evaluating the degree of removal or inhibition of the calcium component precipitated as dissolved or solid include a method of evaluating by calculating a calcium ion removal rate (blocking rate), but are limited to this method. It is not a thing. The calcium ion blocking rate (removal rate) is determined by analysis represented by ion chromatography. The calcium ion concentration contained in the raw water (separated liquid) (raw water calcium ion concentration) and the calcium contained in the permeated water (lactic acid solution). By measuring the ion concentration (permeated water calcium ion concentration), it can be calculated by Equation 2.

Calcium ion removal rate (%) = (1− (permeated water calcium ion concentration / raw water calcium ion concentration)) × 100 (Formula 2)
Examples of a method for evaluating the nanofiltration membrane permeability of an aqueous lactic acid solution include a method of calculating and evaluating lactic acid permeability, but the method is not limited to this method. Lactic acid permeability is determined by analysis represented by high performance liquid chromatography. Lactic acid concentration (raw water lactic acid concentration) contained in raw water (separate) and lactic acid concentration (permeated lactic acid concentration) contained in permeated water (lactic acid solution). ) Can be calculated by Equation 3.

Lactic acid permeability (%) = (permeated lactic acid concentration / raw water lactic acid concentration) × 100 (Formula 3)
As a method for evaluating the membrane unit area and the permeation flow rate (membrane permeation flux) per unit pressure, the permeated water amount, the time during which the permeated water amount was sampled, and the membrane area can be calculated by Equation 4.

Membrane permeation flux (m ³ / (m ² · day)) = permeate amount / (membrane area × water sampling time) (Equation 4)
In step B of the method for producing lactic acid according to the present invention, filtration of the microorganism culture solution through the nanofiltration membrane is performed by applying pressure. The filtration pressure is preferably used in the range of 0.1 MPa or more and 8 MPa or less, but if it is lower than 0.1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, it affects the damage of the membrane. When used in the following, the membrane permeation flux is high, so that the lactic acid solution can be efficiently permeated and is less likely to affect the membrane damage, and is preferably used at 1 MPa or more and 6 MPa or less. Particularly preferred.

In Step B of the method for producing lactic acid according to the present invention, the pH of the separation liquid used for the filtration through the nanofiltration membrane is preferably 4.5 or less. In the nanofiltration membrane, a substance ionized in a solution has a higher removal rate than a substance that is not ionized. Therefore, since the dissociation constant pKa of lactic acid is 3.86, when the pH is 3.86 or more, dissociation proceeds to lactic acid ions and hydrogen ions, and efficient lactic acid permeation cannot be performed. The upper limit of pH was examined from the viewpoint of lactic acid permeability. As a result, when the pH exceeds 4.5 , the lactic acid permeability becomes inefficient, so the pH of the separation liquid is 4.5 or less, preferably lactic acid. It is important that the dissociation constant pKa is 3.8 or less.

As the water permeation performance of the nanofiltration membrane used in Step B of the method for producing lactic acid of the present invention, the permeated water amount (m ³ / m ² / day) at a filtration pressure of 0.3 MPa with sodium chloride (500 mg / L). Is preferably 0.5 or more and 0.8 or less.

  The concentration of lactic acid in the lactic acid solution in the microorganism culture solution used for separation by the nanofiltration membrane is not particularly limited. However, if the concentration is high, the concentration of lactic acid contained in the permeate is also high, so the concentration time is shortened. Therefore, it is suitable for cost reduction, for example, 10 g / L or more and 100 g / L or less is preferable.

  In step B of the method for producing lactic acid according to the present invention, the concentration of the calcium component contained in the culture solution is preferably not more than the saturation solubility. That is, if the calcium component is less than or equal to the saturation solubility, it is dissolved in the culture solution, and precipitation within the nanofiltration membrane element does not occur, and stable separation operation can be continued. On the other hand, if the solubility is higher than the saturation solubility, a part of the calcium component is precipitated, so that it is difficult to stably separate lactic acid by the nanofiltration membrane. Therefore, the concentration of the calcium component is preferably not more than the saturation solubility, that is, not more than 2 g / L.

  In the method for producing lactic acid according to the present invention, the separation liquid containing lactic acid obtained after the step A of removing the calcium component in the culture solution as a hardly soluble sulfate is filtered through the nanofiltration membrane of the step B. Provide to process. Specifically, as step A, for example, sulfuric acid is added to the culture solution, and the calcium component in the culture solution is precipitated as hardly soluble calcium sulfate, and the precipitate is filtered through a gravity filter, a pressure filter, a filter leaf. Perform the step A of separating with a filter such as a filter, a vacuum continuous filter, a centrifuge, a microfiltration membrane, an ultrafiltration membrane, a filter cloth, a filter paper, etc., and the filtrate (separate containing lactic acid) ) Through the nanofiltration membrane of step B, the calcium component can be effectively removed or blocked.

  In the method for producing lactic acid of the present invention, high-purity lactic acid can be obtained by subjecting the lactic acid solution obtained by filtering the culture solution of step A through a nanofiltration membrane to step C of further distillation. The distillation step is preferably performed under a reduced pressure of 1 Pa or more and atmospheric pressure (normal pressure, about 101 kPa) or less. The distillation temperature when performing under reduced pressure is preferably 20 ° C. or more and 200 ° C. or less. However, when distillation is performed at 180 ° C. or more, lactic acid may be racemized due to the influence of impurities. If it is more than 180 degreeC, the distillation of lactic acid can be performed suitably. Before being subjected to this distillation step, the lactic acid solution that has permeated the nanofiltration membrane may be once concentrated using a concentrating device represented by an evaporator.

Moreover, it is also preferably employable to subject the lactic acid solution obtained in Step B to Step D to further increase the lactic acid concentration by filtration through a reverse osmosis membrane. Here, the reverse osmosis membrane is a filtration membrane that removes ions and low molecular weight molecules using a pressure difference equal to or higher than the osmotic pressure of water to be treated as a driving force. As the reverse osmosis membrane used here, for example, a cellulose separation functional layer is provided on a microporous support membrane by polycondensation of a cellulose type such as cellulose acetate or a polyfunctional amine compound and a polyfunctional acid halide. A membrane can be used. In order to suppress fouling on the reverse osmosis membrane surface, the surface of the polyamide separation functional layer is coated with an aqueous solution of a compound having at least one reactive group that reacts with an acid halide group and remains on the surface of the separation functional layer. A low fouling reverse osmosis membrane mainly for sewage treatment in which a covalent bond is formed between the acid halogen group to be reacted and the reactive group can also be preferably employed. Since most of the divalent calcium ions can be removed in the step B of the present invention , stable membrane concentration can be performed without generating scale on the reverse osmosis membrane surface.

  Hereinafter, lactic acid production by fermentation culture of microorganisms used in the method for producing lactic acid of the present invention will be described.

  The fermentation raw material used for lactic acid production by fermentation culture of microorganisms may be any material that promotes the growth of the microorganisms to be cultured and can produce lactic acid as the desired fermentation product, A normal liquid medium containing inorganic salts and organic micronutrients such as amino acids and vitamins as appropriate is preferable. Carbon sources include sugars such as glucose, sucrose, fructose, galactose, lactose, starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, and organic acids such as acetic acid, alcohols such as ethanol And glycerin are also used.

  Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, corn Steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added.

  When the microorganism used in the present invention requires a specific nutrient (for example, an amino acid) for growth, the nutrient is added as such or as a natural product containing it. Moreover, you may use an antifoamer as needed. In the present invention, the culture solution refers to a solution obtained as a result of growth of microorganisms or cultured cells as fermentation raw materials. You may change suitably the composition of the fermentation raw material added to a culture solution from the fermentation raw material composition at the time of a culture | cultivation start so that the productivity of the target lactic acid may become high.

  In the present invention, microorganisms are usually cultured in the range of pH 4-8 and temperature 20-40 ° C. The pH of the culture solution is adjusted to a predetermined value within the above range with an alkaline substance, specifically, a basic calcium salt. As the basic calcium salt, calcium hydroxide, calcium carbonate, calcium phosphate, calcium oxide, calcium acetate and the like are preferably used.

  In the culture of microorganisms, if it is necessary to increase the oxygen supply rate, add oxygen to the air to maintain the oxygen concentration at 21% or higher, or pressurize the culture, increase the stirring speed, increase the aeration rate, etc. Can be used.

  After cultivating microorganisms, batch culture or fed-batch culture is performed to increase the concentration of microorganisms, and then continuous culture (pulling) may be started. You can go. It is possible to supply the raw material culture solution and extract the culture from an appropriate time. The starting times of the supply of the raw material culture solution and the withdrawal of the culture are not necessarily the same. Further, the supply of the raw material culture solution and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for microbial cell growth as shown above to a raw material culture solution so that microbial cell growth may be performed continuously. The concentration of microorganisms or cultured cells in the culture solution should be maintained at a high level so that the environment of the culture solution is not suitable for the growth of microorganisms or cultured cells and the rate of death does not increase. As an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more.

  The continuous culture operation performed while growing fresh cells having fermentation production ability is usually preferably performed in a single fermenter in terms of culture management. However, the number of fermenters is not limited as long as it is a continuous culture method for producing a product while growing cells. A plurality of fermenters may be used because the capacity of the fermenter is small. In this case, high productivity of the fermented product can be obtained even if continuous fermentation is performed by connecting a plurality of fermenters in parallel or in series by piping.

  Although there is no restriction | limiting in particular about the microorganisms used by this invention, For example, yeasts, such as baker's yeast often used in fermentation industry, bacteria, such as colon_bacillus | E._coli and coryneform bacteria, filamentous fungi, actinomycetes, etc. are mentioned. Cultured cells such as animal cells and insect cells are also included in the microorganism used in the present invention. The microorganism to be used may be isolated from the natural environment, or may be partially modified by mutation or genetic recombination.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to a following example.

(Production of yeast strains capable of producing lactic acid)
Reference Example 1 Production of Yeast Strain Having Lactic Acid Production Capacity A yeast strain having a lactic acid production capacity was constructed as follows. Specifically, a yeast strain capable of producing L-lactic acid was constructed by linking a human-derived LDH gene downstream of the PDC1 promoter on the yeast genome. For polymerase chain reaction (PCR), La-Taq (manufactured by Takara Shuzo) or KOD-Plus-polymerase (manufactured by Toyobo Co., Ltd.) was used according to the attached instruction manual.

  After culturing and recovering human breast cancer cell line (MCF-7), total RNA was extracted using TRIZOL Reagent (Invitrogen), and SuperScript Choice System (Invitrogen) was used with the obtained total RNA as a template. CDNA was synthesized by reverse transcription reaction. Details of these operations followed the attached protocol. The obtained cDNA was used as an amplification template for subsequent PCR.

  The L-ldh gene was cloned by PCR using KOD-Plus-polymerase using the cDNA obtained by the above operation as an amplification template and the oligonucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 as a primer set. Each PCR amplified fragment was purified and the end was phosphorylated with T4 Polynucleotide Kinase (manufactured by TAKARA), and then ligated to a pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (manufactured by TAKARA). Escherichia coli DH5α was transformed with the ligation plasmid product, and plasmid DNA was collected to obtain a plasmid in which various L-ldh genes (SEQ ID NO: 3) were subcloned. The obtained pUC118 plasmid into which the L-ldh gene was inserted was digested with restriction enzymes XhoI and NotI, and each of the obtained DNA fragments was inserted into the XhoI / NotI cleavage site of the yeast expression vector pTRS11. In this way, a human-derived L-ldh gene expression plasmid pL-ldh5 (L-ldh gene) was obtained.

  By PCR using the plasmid pL-ldh5 containing the human-derived LDH gene as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 5 as primer sets, a 1.3-kb human-derived LDH gene and Saccharomyces cerevisiae derived A DNA fragment containing the terminator sequence of the TDH3 gene was amplified. In addition, a DNA fragment containing the TRP1 gene derived from Saccharomyces cerevisiae of 1.2 kb was amplified by PCR using the plasmid pRS424 as an amplification template and the oligonucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 7 as a primer set. Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method. A product obtained by the PCR method using a mixture of the 1.3 kb fragment and the 1.2 kb fragment obtained here as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 7 as a primer set is 1 2.5% agarose gel electrophoresis was carried out to prepare a 2.5 kb DNA fragment linked with human-derived LDH gene and TRP1 gene according to a conventional method. Saccharomyces cerevisiae strain NBRC10505 was transformed with this 2.5 kb DNA fragment in a conventional manner so as not to require tryptophan.

  Confirmation that the obtained transformed cells were cells in which the human-derived LDH gene was linked downstream of the PDC1 promoter on the yeast genome was performed as follows. First, genomic DNA of a transformed cell was prepared according to a conventional method, and a 0.7 kb amplified DNA fragment was obtained by PCR using the oligonucleotides represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a primer set. Confirmed by being. In addition, whether or not the transformed cells have lactic acid production ability is shown below that lactic acid is contained in the culture supernatant obtained by culturing the transformed cells in SC medium (METHODS IN Yeast GENETICS 2000 EDITION, CSHL PRESS). It confirmed by measuring the amount of lactic acid by HPLC method on conditions.

  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 Bistris, 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 was measured by the HPLC method under the following conditions.

  Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation), mobile phase: 1 mM copper sulfate aqueous solution, flow rate: 1.0 ml / min, detection method: UV254 nm, temperature: 30 ° C.

  Moreover, the optical purity of L-lactic acid is calculated by Formula 5. Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

Optical purity (%) = 100 × (LD) / (L + D) (Formula 5)
As a result of HPLC analysis, 4 g / L of L-lactic acid was detected, and D-lactic acid was below the detection limit. From the above examination, it was confirmed that this transformant has L-lactic acid production ability. The obtained transformed cells were used in subsequent examples as yeast strain SW-1.

Reference Example 2 Production of L-lactic acid by batch fermentation The most typical batch fermentation was performed as a fermentation form using microorganisms, and the lactic acid productivity was evaluated. A batch fermentation test was conducted using the lactic acid fermentation medium shown in Table 1. The medium was used after autoclaving (121 ° C., 15 minutes). The yeast SW-1 strain constructed in Reference Example 1 was used as a microorganism, and the concentration of lactic acid as a product was evaluated using the HPLC shown in Reference Example 1, and the glucose concentration was measured using a glucose test Wako. C (Wako Pure Chemical Industries) was used. The operating conditions of Reference Example 2 are shown below.

  Reaction tank capacity (lactic acid fermentation medium amount): 2 (L), temperature adjustment: 30 (° C.), reaction tank aeration rate: 0.2 (L / min), reaction tank stirring speed: 400 (rpm), pH adjustment: Adjust to pH 5 with 1N calcium hydroxide.

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). Temperature adjustment and pH adjustment were performed, and fermentation culture was performed. The amount of bacterial cell growth at this time was 15 in terms of absorbance at 600 nm. The results of batch fermentation are shown in Table 2.

Example 1
(Preparation of separation liquid to be separated by nanofiltration membrane)
Concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the culture solution (2 L) fermented with lactic acid in Reference Examples 1 and 2 until the pH reached 2.6, and then stirred at 25 ° C. for 1 hour, so that calcium lactate in the culture solution Converted to lactic acid and calcium sulfate. Subsequently, the precipitated calcium sulfate was filtered off with suction using qualitative filter paper No. 2 (manufactured by Advantech), and the precipitate was filtered off, and 2 L of filtrate was recovered.

(Separation experiment with nanofiltration membrane)
Next, 2 L of the filtrate obtained above was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. Nanofiltration membrane 7 35% removal of methanol is 1% or One isopropyl alcohol removal rate of and removal rate of the citric acid 86% and sucrose removal rates are 99.7% (piperazine polyamide semipermeable membrane UTC60 , Manufactured by Toray Industries, Inc.) was cut to a diameter of 64 mmφ and set in a stainless steel (SUS316) cell shown in FIG. 3, the pressure of the high-pressure pump 3 was adjusted to 4 MPa, and the permeated water 4 was recovered. The concentrations of sulfate ions and calcium ions contained in the raw water tank 1 and the permeated water 4 were analyzed by ion chromatography (manufactured by DIONEX), and the lactic acid concentration was analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation). The results are shown in Table 3.

  As shown in Table 3, it was found that sulfate ions and calcium ions were removed with high efficiency.

Example 2
(Distillation of lactic acid solution)
The permeate 1 L (lactic acid concentration: 24 g / L) separated from the nanofiltration membrane of Example 1 was concentrated by evaporating water under reduced pressure (50 hPa) using a rotary evaporator (manufactured by Tokyo Rika Co., Ltd.). At this time, precipitation of calcium sulfate was not observed.

  Subsequently, vacuum distillation was performed at 133 Pa and 130 ° C. In order to confirm the racemization of distilled lactic acid, the optical purity was measured by high performance liquid chromatography before and after the distillation, and no decrease in the optical purity was observed. Table 4 shows the results.

From the results of Examples and Comparative Examples, removal of methanol is 10% or less or One removal of the isopropyl alcohol is 20% or more 50% or less and the removal rate of citric acid 70% to 95% or less and sucrose It has been clarified that the nanofiltration membrane with a removal rate of 95% or more can remove calcium sulfate and sulfuric acid in the culture solution with high efficiency. That is, according to the present invention, the calcium component and sulfate ion in the lactic acid solution in the microorganism culture solution can be removed with high efficiency by the nanofiltration membrane, and the calcium component does not precipitate even when concentrated, and further subjected to the distillation step. It was revealed that racemization and oligomerization did not proceed.

It is a fractional molecular weight curve of the nanofiltration membrane of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram which shows one embodiment of the nanofiltration membrane manufacturing apparatus used by this invention. It is a schematic diagram which shows one embodiment of cell sectional drawing with which the nanofiltration membrane of the nanofiltration membrane manufacturing apparatus used by this invention was mounted | worn.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Raw water tank 2 Cell equipped with nanofiltration membrane 3 High pressure pump 4 Flow of membrane permeate 5 Flow of membrane concentrate 6 Flow of culture solution sent by high pressure pump 7 Nanofiltration membrane 8 Support plate

Claims (5)

A method for separating and producing lactic acid produced in a culture solution by fermentation of microorganisms in the presence of a calcium salt,
A step A of obtaining a separation liquid containing lactic acid by removing the calcium component in the culture liquid as a hardly soluble sulfate by adding sulfuric acid;
Including the step B of filtering the separation liquid containing lactic acid obtained in step A through a nanofiltration membrane to remove the calcium component to obtain a lactic acid solution;
At an operating pressure of 0.75 MPa, the methanol removal rate when passing through a methanol aqueous solution at 25 ° C., pH 6.5, and a concentration of 1,000 mg / L is 10% or less , and at an operating pressure of 0.75 MPa, 25 The removal rate of isopropyl alcohol is 20% or more and 50% or less when an aqueous isopropyl alcohol solution having a concentration of 1,000 mg / L is permeated at 25 ° C., pH 6.5, and an operating pressure of 0.75 MPa. 8. When the citric acid aqueous solution having a concentration of 1,000 mg / L is permeated, the citric acid removal rate is 70% to 95% , and the operating pressure is 0.75 MPa, 25 ° C., pH 6.5, production side of lactic acid sucrose removal rates is characterized by using a nanofiltration membrane of 95% or more in step B of when is transmitted through the sucrose aqueous solution with a concentration 1,000 mg / L . 2. The method for producing lactic acid according to claim 1, wherein the membrane material of the nanofiltration membrane contains a crosslinked piperazine polyamide as a main component and a constituent represented by Chemical Formula 1. 3.

(In the formula, R represents —H or —CH ₃ , and n represents an integer of 0 to 3.) The lactic acid solution obtained in the step B is further subjected to a pressure of 1 Pa or more and atmospheric pressure or less.
The manufacturing method of lactic acid of Claim 1 or 2 including the process C distilled at 25 degreeC or more and 200 degrees C or less.   The method for producing lactic acid according to claim 1 or 2, further comprising a step D in which the lactic acid solution obtained in the step B is further filtered through a reverse osmosis membrane to increase the lactic acid concentration.   The method for producing lactic acid according to claim 4, further comprising a step C of distilling at 25 ° C. or more and 200 ° C. or less under a pressure of 1 Pa or more and atmospheric pressure after the step D.

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