JP2001029063A - Microalga capable of producing high-purity d-lactic acid and method and arrangement for producing d-lactic acid using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new microalga, Nannochloris sp. 26A4 capable of mass-producing D-lactic acid serving as a raw material for agrochemical and medicaments at a low cost without the need of further purification through producing the D-lactic acid of high optical purity from the intracellular starch of the above microalge. SOLUTION: This new microalga, Nannochlois sp. 26A4 belonging to the genus Nannochloris is capable of producing D-lactic acid with high optical purity from the intracellular starch. It is preferable that D-lactic acid is produced by converting the intracellular starch thereto and then separating the thus produced D-lactic acid from the fermentation liquid at a cold dark place followed by concentration, wherein the above conversion is conducted by the following process: the microalge is cultured, then collected and subsequently held in an anaerobic atmosphere; alternatively, the microalga is cultured autotrophically through photosynthesis, and the resultant microalga thus cultured and proliferated is brought into a form of concentrated slurry, which, in turn, is held in an anaerobic and dark atmosphere to effect fermentation; and furthermore, it is preferable that the D-lactic acid thus obtained is held at a cold dark place so as to maintain its optical purity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the production of D-lactic acid having an extremely high optical purity suitable for agricultural chemicals and pharmaceutical raw materials.

[0002]

2. Description of the Related Art Lactic acid has optical isomers, and there are D-form and L-form. Conventional lactic acid production is performed using fermentation using microorganisms collectively referred to as lactic acid bacteria and using biomass such as sucrose, glucose, and starch as raw materials. At this time, D-form, L-form or racemic lactic acid can be selectively obtained with a certain degree of purity by selecting a microorganism species to be used.

In order to specifically produce only D-lactic acid, for example, glucose is used as a raw material and Sporolactobacillus
A method of performing lactic acid fermentation by sp.
The optical purity of D-lactic acid thus obtained is about 97%. In the remaining 3%, L-lactic acid is present as an impurity.

[0004] From such D-lactic acid, even higher purity D-lactic acid
-To obtain lactic acid, a method of removing L-lactic acid by the action of L-lactic acid oxidase, or adding magnesium to lactic acid to precipitate magnesium lactate, and then extracting D-lactic acid with butanol Methods have been reported. However, these purification methods are not feasible due to the fact that the enzymes used are not commercially available at the bulk level and are expensive to obtain, or lactic acid other than D-lactic acid is easily mixed during butanol extraction. . On the other hand, as a method for separating and purifying optical isomers, a method using an optical resolution column has been reported. However, since the carrier for resolving optical isomers is very expensive, it is difficult to industrialize purification using an optical resolution column. For these reasons, the optical isomer purity of commercially available D-lactic acid is at most about 97% at most.

[0005]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide D-lactic acid having extremely high optical purity which can be used as a raw material for agricultural chemicals and pharmaceuticals without further purification. And Specifically, an object of the present invention is to provide a microalga capable of producing D-lactic acid with high optical purity, a D-lactic acid production method and a production apparatus using the microalga.

[0006]

Means for Solving the Problems Heretofore, the present inventors have found that microalgae containing starch produce lactic acid as a fermentation product under a dark anaerobic condition. (Japanese Patent Application Hei 7-31411)
9). This time, targeting these microalgae that produce lactic acid,
A diligent search for the productivity of D-lactic acid was performed. As a result, microalgae containing a large amount of starch and capable of converting intracellular starch into D-lactic acid with extremely high optical purity under dark anaerobic conditions,
Succeeded in separating from seawater in Sakido Town, Nagasaki Prefecture. The present invention has arrived at the base of such a discovery.

[0007] The inventors have found that the above-mentioned object has been solved and the object has been achieved by the following means. That is, (1) a microalga Nannochloris s belonging to the genus Nannochloris which produces D-lactic acid with high optical purity from intracellular starch.
p.26A4; (2) collecting the microalgae described in (1) after culturing,
By maintaining the anaerobic atmosphere, the starch in microalgae cells can be D-
(3) A method for producing D-lactic acid using the microalgae according to (1), wherein the microalgae is photoautotrophically cultured. Microalgae cultivation part, and a microalgae concentration part for forming a concentrated slurry of microalga grown and cultured, fermentation by maintaining the obtained concentrated slurry in an anaerobic and dark atmosphere, the inside of the microalgae A storage unit for converting the starch into D-lactic acid, a lactic acid separation and concentration unit for separating and concentrating the obtained D-lactic acid, and a storage unit for stably storing the separated and concentrated D-lactic acid. D-lactic acid production equipment provided;
It is.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Microalgae The microalga strain of the present invention is a microalga that produces high-purity D-lactic acid, and specifically, is a new strain of Nannochloris sp. 26A4 (Nannnochloris sp. 26A4) belonging to the genus Nannochloris. This strain is stored as a marine microalgae (Nannochloris sp. 26A4) in the Mitsubishi Heavy Industries Research Laboratory. Here, the term "high purity" refers to optically high purity and refers to D-lactic acid containing less than 3%, preferably 0.1% or less, of L-lactic acid as an impurity.

A method for isolating Nannochloris sp. Strain 26A4 from seawater is described below.

Obtained seawater from Sakido-cho, Nagasaki (about 100 mL)
Into a 300 mL Erlenmeyer flask, add nutrients and the like to the composition shown in Table 1, and set the fluorescent lamp to 1500 Lu.
x, gently shaking with continuous irradiation at 25 ° C.

[0011]

[Table 1]

After about three weeks, the presence of green microorganisms considered to be microalgae became visible. The culture solution (ie, cell suspension) in which the enrichment culture was performed was diluted 100-fold with seawater, and 0.5 ml of the diluted solution was dropped on an agar medium described below and applied to the agar surface. The agar medium is shown in Table 1.
1.5% by weight of purified agar was added to a medium having the composition shown in Table 2, sterilized by an autoclave, added to a petri dish, and allowed to cool and solidify. As a result of allowing the applied petri dish containing the agar medium to stand still under a fluorescent lamp at 1500 Lux and 25 ° C.,
After about 3 weeks, 15 green colonies appeared on the surface of the medium. Each colony was transplanted to a medium having each composition shown in Table 1, cultured and propagated, and then the ability of each cell to produce D-lactic acid was examined. The production capacity of D-lactic acid was determined by measuring the concentration of the produced lactic acid by gas chromatography, and the optical purity was confirmed by high performance liquid chromatography (HPLC) using an optical isomer separation column.

The most excellent D among the microalgae used in the test
-The microalgae having lactic acid-producing ability had the photosynthetic pigment components and morphological characteristics shown in Table 2 below.

[0014]

[Table 2]

From the above characteristics, the present microalgae was identified as Nannochloris, and the resulting algal strain was identified as Nannochloris sp.
No. 4 (Nannnochloris sp. 26A4) strain.

In the D-lactic acid production method of the present invention described below, the microalgae that can be used is the aforementioned Nannochloris sp.
In addition to the A4 strain, it is also possible to use the CCAP registration number 66/3 belonging to Tetraselmis sp., But since the D-lactic acid productivity is higher, Nannochloris sp. 26A4 strain Is particularly preferred.

The production of D-lactic acid using the present algal strain is basically based on the method of producing lactic acid by the present inventors (Japanese Patent Application No. 7-31).
The method can be carried out using the process shown in 4119), but preferably the following modified method is used.

2. The method for producing D-lactic acid will be described below with reference to FIG. In FIG.
The microalga culturing step 1 is a step of culturing the D-lactic acid-producing microalga of the present application photoautotrophically. In addition, this step can be performed using a flowing water type culture tank or the like having an open upper surface with a water depth of about 10 to 30 cm. In this step, the present microalgae can basically be cultured by supplying inorganic nutrients such as nitrogen and phosphorus from seawater and irradiating sunlight. The seawater used here is supplied to the culture tank after removing foreign substances such as microalgae predators in the seawater by filtration or the like.

Next, when the concentration of the microalgae in the culture solution becomes about 0.1 to 1.0 g (dry solid content) per liter of the culture solution, the culture is stopped. Perform In the microalga concentration step 2, in the case of a microalga having a high sedimentation property, the microalgae is once concentrated to about 1 to 5 w / v% (10 to 50 g per liter) by natural precipitation, and then centrifuged or a belt filter is used. To a solids content of 10 to 20 W / V%. here,
The concentration in this step is to make the D-lactic acid concentration in the subsequent stage advantageous, that is, to facilitate the handling as a slurry, and to enable dry stirring in the subsequent stage, and to further increase the D-lactic acid concentration. It is preferable to concentrate to a solid content as much as possible within a range having fluidity.

The microalgae slurry thus obtained is led to a holding step 3 capable of holding a dark and anaerobic atmosphere to produce D-lactic acid. The holding step 3 can be performed in an apparatus composed of a semi-closed container with a pH monitor equipped with a slow stirring means such as a slurry pump or a stirrer. Here, the “dark atmosphere” refers to a state where light is blocked to such an extent that photosynthesis is not performed. That is, it refers to an environmental condition obtained by cutting the incident light from the outside onto the surface of the microalga slurry by 90% or more, preferably 99.9% or more.
By introducing the slurry into the holding step 3 and holding it in a dark and anaerobic atmosphere while gently stirring, D-lactic acid is produced by fermentation of intracellular starch performed by the microalgae itself. During this time, if necessary, the pH of the slurry can be maintained at around neutral using an alkaline solution such as NaOH and an acid solution such as HCl. Alternatively, CaC
By adding O _3, near neutral pH, preferably it may be maintained at 6.5 to 8.0. However, depending on the concentration of the generated lactic acid, the decrease in pH is suppressed.
Is not essential. The residence time of the microalgae in the holding step 3 varies depending on the type of the microalgae, the holding conditions, and the like.
A range of 10 to 70 hours is common.

When the concentration of D-lactic acid in the slurry is 30 to 100
When the amount becomes about g / liter, the process is promptly led to the D-lactic acid separation / concentration step 4, where D-lactic acid is separated and concentrated. In the D-lactic acid concentration and separation step, by avoiding contact of the resulting D-lactic acid with a small amount of lactate oxidase or the like present in a fermentation broth containing D-lactic acid, D-lactic acid having a high optical purity is produced. -Concentrate D-lactic acid while preventing lactic acid from deteriorating.

In this D-lactic acid separation / concentration step 4, step 3
After adding calcium or the like to the microalgae slurry containing D-lactic acid obtained in the above and precipitating it as a lactate, dehydration with sulfuric acid is carried out for recovery, or a supercritical extraction method using a solvent such as propane or the like. And the like can be concentrated to a desired concentration of D-lactic acid. The D-lactic acid thus obtained is retained in the D-lactic acid storage step 5. In the D-lactic acid holding step 5, high optical purity can be maintained by storing D-lactic acid in a cool and dark place. The cool and dark place refers to an environment in which a temperature of 3 to 20 ° C., preferably 5 to 10 ° C. is maintained, and light incident on the lactic acid liquid surface from the outside can be cut by 90% or more, preferably 99.9% or more. When maintaining a high optical purity for a long period of time, a container holding D-lactic acid is placed in a negative active gas, for example,
It is preferable to fill with nitrogen, argon, helium, or the like.

The residue after separating D-lactic acid can be dried and then disposed of by incineration or the like, or it can be fermented and used as compost.

3. D-lactic acid production apparatus A further invention of the present application is an apparatus for implementing the above-described D-lactic acid production method of the present invention. The production of D-lactic acid according to the method of the present invention makes it possible to produce D-lactic acid with extremely high optical purity as compared with a conventional method using microalgae. In addition, the present apparatus can be maintained for a long time while maintaining the high purity of the obtained D-lactic acid.

A preferred example of the small ethanol production apparatus of the present invention will be described below with reference to FIG. A preferred apparatus of the present invention comprises a microalga culturing unit 1 for culturing microalgae,
Microalgae concentrating unit 2 for concentrating microalgae grown in the culture solution, holding unit 3 for producing D-lactic acid, and lactic acid separation / concentration unit 4 for separating and concentrating the obtained D-lactic acid When,
A D-lactic acid storage unit 5 for storing the separated and concentrated D-lactic acid while maintaining optically high purity.

The microalga culturing unit 1, the microalga concentrating unit 2, the holding unit 3, the lactic acid separating / concentrating unit 4, and the D-lactic acid storage unit 5
One container may be used, or each container may be independent.

The working steps of the present apparatus are as shown in FIG. First, the microalga is cultured in the microalga culturing unit 1, and the microalga grown by the culture is concentrated in the microalga concentrating unit 2 to form an alga body concentrated slurry, and subsequently, D-lactic acid is generated in the holding unit 3. . D-lactic acid production is performed in a dark anaerobic atmosphere. It is also possible to arrange a pH adjusting unit connected to the holding unit 3 (not shown in the figure).
The pH of the solvent in the D-lactic acid generation unit may be maintained near neutral by the H preparation unit. After the completion of D-lactic acid generation, D-lactic acid is separated and concentrated in the lactic acid separation / concentration unit 4.

The microalga culturing unit 1 of the present apparatus preferably has a water depth of about 10 to 30 cm, and is preferably a flowing water type culturing tank having an open upper surface, but is not limited thereto. The material is, for example, vinyl chloride, chlorosulfonated polyethylene, or the like.

The D-lactic acid generator 2 of the present apparatus is suitably a light-shielding container, for example, a container made of stainless steel or the like.

The holding section 3 is preferably composed of a semi-closed vessel equipped with a pH monitor provided with a slow stirring means such as a slurry pump or a stirrer. The pH adjusting section needs to be formed of a material suitable for storing the pH adjusting solution. For example, hard glass, polypropylene fluoride resin, polypropylene and the like are used.

The lactic acid separation / concentration unit 4 is preferably made of a material that can withstand heating, reduced pressure, etc., so as to be suitable for the concentration of D-lactic acid. For example, stainless steel, titanium and the like.

Further, the D-lactic acid storage part is preferably a container capable of shielding light and having excellent airtightness. For example, the storage part is made of stainless steel, titanium, or the like. It is preferable to apply a low-corrosive packing made of a material such as fluororesin or silicon rubber. Further, in the D-lactic acid storage part, it is possible to arrange a gas injection part and an exhaust part for injecting or discharging an inert gas, but it is necessary that the installation does not impair airtightness. .

[Examples] Hereinafter, the contents of the present invention will be described more specifically by using examples.

Example 1 Nannochloris sp.26A4 strain was cultured using a mixture of nutrients three times the composition of the modified ES medium shown in Table 1 in filtered seawater as the medium. 50 L of this medium and a culture of the Nannochloris sp.
g) in a flat transparent container, and about 15,000 with a white fluorescent lamp.
The cells were cultured at 25 ° C. for 5 days while continuously irradiating with 0 Lux and ventilating air (with 0.5% CO ₂ added) to obtain a culture solution containing 12 g (as dry solids) of alga bodies in 5 liters. Was.

In equilibrium with the above operation, this microalgae was washed with 40 (w)
/ V)% hydrolyzed with perchloric acid to convert the contained starch to glucose, and then converted to the amount of starch to determine the starch content. As a result, this microalgae was found to have a starch content of 40% per dry weight. Next, 5 liters of this culture was concentrated by a centrifugal sedimentation method, and Nannochloris sp. 26A
An algal cell slurry solution containing 12 g of the four algal cells was used as a raw material for producing D-lactic acid.

Example 2 The microalgae slurry obtained in Example 1 was transferred to a semi-closed container having a capacity of 0.1 liter, and nitrogen gas was injected into the slurry solution for a short time to remove the enzyme in the container. The mixture was shaken at 30 ° C. (65 reciprocations / minute) to generate D-lactic acid.
During this time, 0.1N NaOH and 0.1N HCl were added to maintain the pH of the slurry in the range of 6.5 to 8.0. The produced lactic acid was analyzed by gas chromatography, and the time-dependent change in D-lactic acid concentration was examined. As a result, 40 g / liter of lactic acid was obtained after about 3 days (FIG. 3). To this, calcium carbonate having a final concentration of 50 g / l was added to precipitate, and sulfuric acid was added thereto to precipitate calcium sulfate, and the precipitate was filtered through filter paper to obtain a lactic acid solution. In addition, the recovery rate of the obtained lactic acid was 95%.

Next, in order to examine the purity of the obtained optical isomer of lactic acid, analysis was carried out by liquid chromatography using optically active substance separation. In the analysis, the obtained lactic acid was appropriately diluted, and fine particles such as cell debris were removed with a filter, and subjected to optically active substance separation liquid chromatography. As a result, as shown in FIG. 4, no L-lactic acid was detected and only D-lactic acid was produced, and the concentration was 5100 μg / ml. Since the detection limit of this optically active substance separation liquid chromatography is 5 μg / ml or less, the optical purity of D-lactic acid was estimated to be at least 99.9%.

From the above, D-lactic acid having extremely high optical purity can be produced by the microalgae according to the present invention.

From this, it was proved that D-lactic acid having extremely high optical purity can be produced by the lactic acid production method of the present invention. Further, the obtained D-lactic acid was placed in a light-shielding bottle, supplemented with He as an inert gas, and maintained in a refrigerated container at 10 ° C. for about one year, but the optical purity of D-lactic acid after one year was 99%. .9
% Was kept. By replacing with an inert gas, D-
The high optical purity of lactic acid could be maintained for a long time.

[0040]

The microalga Nannochloris sp.26A of the present invention
By using the 4 strains, culture is performed using seawater, and the algal slurry obtained by concentrating the culture solution is maintained in a dark and anaerobic atmosphere while maintaining the pH at about neutral, and D with high optical purity is obtained. -It is possible to produce lactic acid.

As a result, the conventional D value of about 97% optical purity
The production of lactic acid can be replaced with a standard product with higher optical purity, and D-lactic acid with extremely high optical isomer purity, which is used as an agricultural or pharmaceutical raw material, can be produced in large quantities.

Further, the production method of the present invention can utilize seawater obtained in large quantities at low cost. Therefore, in areas where it is difficult to secure freshwater, degraded land that is difficult to develop can be used as a factory site. Thereby, it is possible to utilize the D-lactic acid production process on a larger scale by the microalgae.

Since carbon dioxide is fixed by the action of photosynthesis by sunlight and can be used effectively, it is very effective as a measure for reducing atmospheric carbon dioxide on a global scale.

[Brief description of the drawings]

FIG. 1 is a flow chart showing an outline of a process for producing D-lactic acid using a microalga Nannochloris sp. 26A4 strain of the present invention.

FIG. 2 is a flowchart illustrating a D-lactic acid production apparatus of the present invention.

FIG. 3 shows the microalga Nannochloris sp. 26A
The figure which shows the time-dependent change of the lactic acid produced | generated in the microalga slurry in the Example of D-lactic acid production using four strains.

FIG. 4 shows the microalga Nannochloris sp. 26A
The figure which shows the optical purity of D-lactic acid in D-lactic acid production using 4 strains.

Continuation of front page (72) Inventor Ryohei Ueda 1-8-1 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture F-term in the Basic Research Laboratory, Mitsubishi Heavy Industries, Ltd. 4B029 AA02 BB04 CC01 DA01 4B064 AD33 CA08 CC03 CC05 CD02 4B065 AA83X AC12 BA22 BC18 BC48 CA10

Claims (5)

[Claims] 1. A microalga Nannochloris sp. 26A4 belonging to the genus Nannochloris which produces D-lactic acid with high optical purity from intracellular starch. 2. A method for producing D-lactic acid, wherein the microalgae according to claim 1 is recovered after culturing, and is maintained in an anaerobic atmosphere to convert starch in microalgal cells into D-lactic acid. 3. The method for producing D-lactic acid using a microalgae according to claim 1, wherein the microalgae is photoautotrophically cultured, and the microalgae grown by the culture are converted into a concentrated slurry. Fermentation by maintaining the obtained concentrated slurry in an anaerobic and dark atmosphere, converting the starch in the microalgae into D-lactic acid, and converting the obtained D-lactic acid into a fermentation solution in a cool and dark place. A method for producing D-lactic acid, comprising separating and concentrating. 4. D-lactic acid obtained according to claim 2 or 3 is separated and concentrated in a cool dark place from a fermentation solution, and then maintained in a cool dark place to maintain optical purity. How to store lactic acid. 5. An apparatus for producing D-lactic acid using the microalgae according to claim 1, wherein the microalgae is cultivated in a photoautotrophic manner with a microalgae culturing unit. A microalgae enrichment unit for forming a concentrated slurry of the microalgae that has grown, and a fermentation by keeping the obtained concentrated slurry in an anaerobic and dark atmosphere to maintain starch in the microalgae into D-lactic acid A D-lactic acid producing apparatus comprising: a lactic acid separation / concentration unit for separating / concentrating the obtained D-lactic acid; and a storage unit for stably storing the separated / concentrated D-lactic acid.