JP2804005B2 - Method for producing L-aspartic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-aspartic acid from fumaric acid and ammonia or ammonium fumarate by recycling mother liquor from a reacted medium to produce a large amount of mineral acid such as ammonium sulfate. The present invention relates to an improved method for preventing wastewater containing ammonium salts from being discharged.

[0002]

2. Description of the Related Art L-Aspartic acid is produced by contacting an enzyme-containing substance having aspartase activity with a substrate medium containing fumaric acid and ammonia or ammonium fumarate. Usually, to recover L-aspartic acid from the reacted medium, the pH of the reacted medium is adjusted to about pH 2.7, which is the isoelectric point of L-aspartic acid, using a mineral acid such as sulfuric acid. In this method, crystals of L-aspartic acid are precipitated by cooling, and the crystals are separated by filtration.

This method is used industrially because it uses an inexpensive mineral acid, has a high L-aspartic acid product recovery rate as crystals, and has a high purity of the L-aspartic acid obtained. Is a useful way. However, the loss of ammonia as a raw material is large,
There is a problem that a large amount of wastewater containing an ammonium salt of a mineral acid such as ammonium sulfate is discharged in a large amount. It is very difficult to remove ammonium salts from an aqueous solution in terms of wastewater treatment, and problems such as water pollution due to an increase in the concentration of ammonium salts containing ammonium salts have arisen in inland bays such as lakes and marshes and the Seto Inland Sea.

US Pat. No. 4,560,533 discloses that L-aspartic acid is crystallized by adding maleic acid to a reacted medium during the production of L-aspartic acid to make it acidic, and maleic acid in the filtrate is isomerized. A method for recycling a reaction solution has been proposed. However, in this method, maleic acid used for crystallization is isomerized to fumaric acid capable of acting aspartase using a catalyst containing bromine ions, and a step of removing the catalyst after the isomerization is included, The process is complicated.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a process for producing L-aspartic acid which is simple in process and does not discharge a large amount of ammonium salt.

[0006]

The present inventors have conducted intensive studies on a simple method for producing L-aspartic acid in which wastewater containing such a high concentration of ammonium salt is not discharged in large quantities. Further, when fumaric acid, which is a raw material of L-aspartic acid, is used for crystallization of L-aspartic acid in place of a mineral acid such as sulfuric acid, a salt between monoammonium L-aspartate and fumaric acid is formed. When the exchange occurs, L-aspartic acid and monoammonium fumarate are formed, L-aspartic acid having low solubility under acidic conditions can be separated as crystals, and the amount of fumaric acid added is L
-The use of aspartic acid and roughly equivalent amount increase the recovery rate, and the solubility of monoammonium fumarate is not so large at low temperature.However, the operations of salt exchange and separation and purification of crystals are carried out under heating to obtain fumaric acid. By operating under conditions where the solubility of the monoammonium salt is high, it has been found that contamination of the monoammonium fumarate into L-aspartic acid crystals can be reduced, and the present invention has been completed.

Accordingly, the present invention provides a method for reacting an enzyme-containing substance having aspartase activity with a substrate medium containing fumaric acid and ammonia and / or ammonium fumarate at a fumaric acid concentration of 15 to 25% by weight.
-To produce aspartic acid, then 0.85 to 1.2 relative to L-aspartic acid contained in the reacted medium
L-aspartic acid was precipitated by adding twice the molar amount of fumaric acid to the reacted medium, and the precipitated L-aspartic acid crystals were filtered and washed under a temperature condition of 40 ° C. or more to wash L-aspartic acid. Provided is a method for producing L-aspartic acid, which comprises collecting aspartic acid, and then adding ammonia to the mother liquor and washing liquid to reuse as a substrate medium.

[0008]

According to the present invention, in the production of L-aspartic acid, a large amount of wastewater containing ammonium salts is not discharged, and the mother liquor from the reacted medium is recycled. The L-aspartic acid dissolved in the L-aspartic acid can be recovered in the next cycle, so that the raw material substrate can be effectively used, and a method for producing L-aspartic acid which is more advantageous in terms of environment and economy than the conventional method can be provided.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the method of the present invention will be described, but the present invention is not limited to only such embodiments. Examples of the enzyme-containing substance having aspartase activity used in the present invention include Escherichia coli and Brevibacte which are known to have high aspartase activity.
cells of genus rium, or crushed cells obtained by sonication, trituration, freeze-thawing, enzymatic treatment, surfactant treatment, etc., and further obtained by conventional methods such as ammonium sulfate salting out and acetone precipitation. It is partially purified or purified by a conventional method such as a chromatography column, and any of them can be used for the reaction.

[0010] These aspartase activity-containing microbial cells, or their processed products or enzymes can be immobilized on a carrier and used. As a carrier for immobilization, a natural polymer such as cellulose, alginic acid, carrageenan, and mannan gel, or a suitable synthetic polymer such as an ion exchange resin, polyvinyl alcohol, and polyacrylamide can be used in a usual manner. By immobilization, separation of aspartase or an aspartase-containing substance from the product is facilitated, and the operation of circulating the mother liquor from the reacted medium can be performed more easily.

Further, it is also possible to use a reaction after previously inactivating enzyme activities other than aspartase activity which may hinder the reaction, such as a fumalase activity contained in an enzyme-containing substance having an aspartase activity used in the reaction. It is possible. For example, the enzyme-containing substance is preliminarily prepared in the presence of L-aspartic acid and ammonium ions in an alkaline range of 40 to 40.
By performing heat treatment at 60 ° C., the fumarase activity can be inactivated in advance.

It is selected from fumaric acid or fumarate used in the present invention, and may be a mixture thereof. As the ammonia used in the present invention, gaseous ammonia, an aqueous ammonia solution and the like can be used, but an aqueous ammonia solution is advantageous in handling. The concentration of the ammonia water is not particularly limited, but is industrially 1
0-35% by weight is preferred for use.

The amount of ammonia used is preferably 1.0 times or more and 3 times or less the mole of fumaric acid used in the reaction. If the amount of ammonia is less than 1 mole, the yield of L-aspartic acid is lowered, which is not preferable. On the other hand, if the amount of ammonia is 3 times or more, the stability of the enzyme or enzyme-containing substance involved in the reaction is likely to deteriorate, which is not preferable. If necessary, an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide can be used in combination within the above range of the amount of ammonia. The pH of the reaction solution is in the range of 5 to 10, preferably 7.0 to 7.0.
9.0, more preferably aspartase optimal
The pH is preferably adjusted to 8.0 to 9.0.

When fumaric acid and ammonia are mixed, the mixing may be carried out in any manner, but it is preferred that the entire amount of both is not mixed at once, but the other is gradually added to one total amount. Preferably, ammonia or aqueous ammonia is gradually added to the fumaric acid suspension. The reaction medium used for preparing the substrate medium in the method of the present invention is not particularly limited as long as it is aqueous, and the most typical one is water. Further, a buffer, for example, a common buffer such as a phosphate buffer may be used. The concentration of fumaric acid at the time of the reaction is preferably 15 to 25% by weight of an aqueous solution, preferably 1 to 25% by weight, in view of the solubility and productivity of the fumarate.
It is in the range of 5 to 20% by weight.

The substrate medium may further contain a manganese salt such as manganese chloride or manganese sulfate, a magnesium salt such as magnesium chloride or magnesium sulfate, or a divalent metal such as zinc salt, calcium salt, nickel salt, cobalt salt or iron salt. 0.1 to 50 mM salt, preferably 1 to 10
It is desirable to add at a concentration of mM. The mode of the reaction tank in the present invention is not particularly limited, but the reaction can be carried out in a conventionally known reaction tank such as a batch-type reactor or a column-type reactor. One or more reaction vessels may be used. The temperature at the time of the reaction is preferably about 20 ° C. as a lower limit because the reaction rate is lower at a lower temperature, and is preferably about 50 ° C. at a higher temperature because it causes inactivation of aspartase, and more preferably 25 to 40 ° C. It is good to carry out in the range of ° C.

After the reaction of fumaric acid and ammonia under the above conditions, fumaric acid is added to the obtained reacted medium to precipitate L-aspartic acid. Further, fumarate can be added within a range that does not hinder circulation use of the mother liquor. As the fumarate, ammonium fumarate, sodium fumarate, potassium fumarate and the like are used. Using the fumaric acid or the like, L-
By performing the precipitation of aspartic acid, the mother liquor can be recycled. When a mineral acid is used for precipitation, ammonium salt of the mineral acid accumulates, and a desalting step is required to recycle the mother liquor, which is not preferable. Here, fumaric acid added for the precipitation of L-aspartic acid may be a solid, or fumaric acid may be added as a slurry with water.

[0017] The amount of fumaric acid added to the reacted medium for the precipitation of L-aspartic acid is preferably approximately equivalent to the amount of L-aspartic acid present in the reacted medium. For example, L-aspartic acid can be precipitated by adding 0.85-1.2 times mol, preferably 0.85-1.1 times mol, of L-aspartic acid contained in the reacted medium. it can.

If the amount is less than this range, the amount of L-aspartic acid that can be separated as crystals decreases, and the recovery rate is undesirably low. On the other hand, if the amount is larger than this range, a large amount of fumarate is mixed into the crystals of L-aspartic acid, which is not preferable. When precipitating L-aspartic acid, it is preferable to gradually add fumaric acid to the reacted medium. According to this method, the precipitated L-aspartic acid crystals are large, which facilitates separation from the mother liquor by centrifugal filtration or the like, and facilitates handling of the crystals.

The reacted medium to which fumaric acid is added is 0 to 1
10 minutes to 4 hours at 00 ° C, preferably 20 to 80 ° C
For 30 to 120 minutes to complete salt exchange and crystallization. As a method for filtering the precipitated L-aspartic acid, a usual method such as suction filtration or centrifugal filtration can be used, but a method such as centrifugal filtration that can reduce the liquid content is more preferable. The filtration temperature is 40 to 70C, preferably 40 to 60C. If it is higher than this range, the solubility of L-aspartic acid will increase, and the recovery of L-aspartic acid will decrease. If it is lower than this range, the solubility of fumaric acid monoammonium salt will decrease, and fumaric acid will be added to L-aspartic acid crystals. It is not preferable because a large amount of salt is mixed.

The filtered crystals are washed with water. If the temperature of the washing water is low, a large amount of washing water is required to obtain a high-purity L-aspartic acid product, which is not preferable for reusing the washing liquid. By setting the temperature of the washing water to 40 to 70 ° C., preferably 40 to 60 ° C., high-purity L-aspartic acid crystals can be obtained with a small amount of washing water. The amount of washing water used is from 5 to 500% by weight, preferably from 10 to 200% by weight, based on the amount of L-aspartic acid crystals.
It is good to use in the range of weight%.

In this manner, in a very simple manner, L-containing a small amount, for example 0.1 to 3% by weight, preferably 0.1 to 1% by weight, of fumaric acid and / or fumarate is used.
Crystals of aspartic acid can be obtained. The size of the L-aspartic acid crystals thus obtained can be adjusted to a desired average size by changing the crystallization method.
It is also possible to obtain crystals in the range of 00 μm. This L
-Aspartic acid is very useful as industrial aspartic acid. This L-aspartic acid can be used for food additives, pharmaceuticals, etc. by repeating purification.

The mother liquor from which the crystals of L-aspartic acid have been separated is mixed with the above-mentioned washing solution, and ammonia is added to reuse the mother liquor as a substrate medium for the production of L-aspartic acid. If necessary, fumaric acid is added, or the mother liquor or the washing solution is concentrated, and the adjustment is appropriately performed. For example, if the number of moles of L-aspartic acid separated as crystals is larger than the number of moles of fumaric acid added for precipitating L-aspartic acid, L obtained by the subsequent reaction of fumaric acid and ammonia -As the amount of aspartic acid decreases,
By adding fumaric acid as needed, the amount of fumaric acid added when precipitating L-aspartic acid relative to the amount of L-aspartic acid contained in the reacted medium before precipitating L-aspartic acid is added. And the total amount of fumaric acid to be added is adjusted from 0.85 to 1.2 times mol.

If necessary, the volume is adjusted to the same volume as the initial substrate medium by concentrating the mother liquor,
After adjusting the ratio of ammonia to fumaric acid to be 1 to 3 moles, the reaction of fumaric acid with an enzyme-containing substance having aspartase activity of ammonia, precipitation of L-aspartic acid by addition of fumaric acid, L The mother liquor is circulated as a substrate medium by repeating the separation, washing and preparation of the mother liquor of the aspartic acid crystals; According to the invention, the mother liquor can be circulated more than ten times.

The addition of ammonia to the mother liquor is performed, for example, in consideration of the following points, in order to repeat the circulating use of the mother liquor and to maintain the aspartase activity of the microbial cells or the crushed product thereof for a long time. Is preferred. That is, the amount of ammonia contained in the mother liquor used for circulation is 1 to 3 times, preferably 1.times., The amount of fumaric acid contained in the mother liquor.
Ammonia is added so that it becomes 5-2.5 times mol.

In the present invention, fumaric acid and ammonia or ammonium fumarate are used in an amount such that the amount of ammonia becomes 1 to 3 times the mol of fumaric acid, and an enzyme-containing substance having aspartase activity is used in an amount of 20 to 50%. React at ° C. This reaction produces ammonium L-aspartate, and fumaric acid is added to the obtained reacted medium to precipitate L-aspartic acid. The precipitated L-aspartic acid is separated from the mother liquor under a temperature condition of 40 ° C. or more, and further washed to obtain L-aspartic acid containing a small amount of fumaric acid and / or a salt thereof. Aspartic acid is extremely useful as industrial aspartic acid. The mother liquor from which the L-aspartic acid crystals have been separated can be repeatedly used as a substrate medium for the production of L-aspartic acid together with the liquid from which the L-aspartic acid crystals have been washed. As a result, efficient use of raw materials and reduction of waste are achieved.

[0026]

Next, the present invention will be described by way of examples, but the present invention is not limited to only these examples.

Embodiment 1 FIG . 1 for 5L jar fermenters
20 g of fumaric acid, 1 g of potassium phosphate, 0.5 g of magnesium sulfate heptahydrate, 20 g of yeast extract and 20 g of corn steep liquor were dissolved in water per liter of water, and 3 L of medium whose pH was adjusted to 6.8 with ammonia was charged and sterilized. Thereafter, Escherichia coli (Escherichia col.), Which had been separately cultured in a 500-mL shake flask with 50 mL of the same medium as above, was cultured.
i) (ATCC 11303) was inoculated, and cultured with aeration and agitation at 37 ° C. At the time when the fumaric acid in the medium disappeared, acetic acid was added to the cell culture, the pH was adjusted to about 5, the mixture was left at 45 ° C. for 1 hour, and the culture was centrifuged to separate the cells. 3
The mixture was divided into 0 equal parts, frozen at -80 ° C and refrigerated.

100 g of fumaric acid and 0.125 g of magnesium sulfate heptahydrate were added to 300 ml of water, neutralized with 25% aqueous ammonia to adjust the pH to 8.3, and water was added to add 0.5 L of water. The substrate medium was used. One of the frozen microbial cells previously divided into 30 equal portions was added to this substrate medium, and reacted at 37 ° C. for 5 hours while gently shaking. As a result of analysis of the reacted medium, 99.0 mol% of L-
Ammonium aspartate was produced.

After the reacted medium was centrifuged to remove the cells, 110 g of fumaric acid was added, the mixture was heated to 60 ° C., kept for 30 minutes, and then cooled to 50 ° C. After cooling, the solution was filtered with a suction filter to obtain about 370 ml of a mother liquor. On the other hand, L
About 200 g of crystals containing aspartic acid were washed with about 500 ml of warm water at 50 ° C. and dried. When the weight and purity of the obtained crystal were examined, the weight was 105 g and the purity was 96.2.
% By weight (excluding water) (3.0% by weight of fumaric acid) (average crystal size: 250 μm).

Next, the mother liquor and the washing solution were combined, concentrated under reduced pressure, adjusted to pH 8.3 by adding 25% aqueous ammonia, and water was added to obtain 0.5 L of a substrate medium. One of the frozen cells was added to the substrate medium and reacted at 37 ° C. for 5 hours as described above. After removing the cells by centrifugation in the same manner as above, 110 g of fumaric acid was added, followed by heating and cooling.
The precipitated L-aspartic acid was filtered by suction, and about 0.5 L
The crystals were washed twice with warm water at 50 ° C., dried sufficiently after removing water. The weight of the obtained crystals was 114.5 g,
Purity is 99.0% by weight (excluding water) (fumaric acid 0.73
% By weight) (average crystal size: 150 μm).

Thereafter, the above-mentioned second operation was repeated, and the reaction between fumaric acid and ammonia was performed a total of 10 times (the number of circulations of the mother liquor was 9). The L-aspartic acid obtained by the operation 10 times gave a yield of 99.1 mol% with respect to the fumaric acid added for crystallization in the operation 9;
The purity was 99.1% by weight (excluding moisture) (0.61% by weight of fumaric acid) (average crystal size: 100 μm). The crystals obtained by the 10th repetition operation were added to 50 ° C warm water 3
Then, the mixture was thoroughly filtered by suction, dried to remove water, and dried three times. As a result, the purity of the crystals was 99.5% by weight (excluding water) (0.15% of fumaric acid). % By weight) (average crystal size: 90 μm).

Embodiment 2 FIG . As in Example 1, 0.5
One of the frozen cells was added to the L substrate medium to carry out the reaction. Analysis of the reacted medium revealed that 9%
9.1 mol% of L-aspartic acid was produced. After the reacted medium was centrifuged to remove the cells, 85 g of fumaric acid was added, and the mixture was heated to 60 ° C. and maintained for 30 minutes.
Cooled to 0 ° C. After cooling, suction-filtered with a suction filter,
About 370 ml of mother liquor was obtained. On the other hand, the crystals containing L-aspartic acid, which were separated by filtration, were washed with about 0.5 L of warm water at 50 ° C. and dried. The weight of the obtained crystals was 106 g, and the purity was 92.7% by weight (excluding water) ( Fumaric acid 6.1% by weight)
(Average crystal size: 200 μm).

Then, the mother liquor and the washing solution were combined, concentrated under reduced pressure, adjusted to pH 8.3 by adding 15 g of fumaric acid and 25% aqueous ammonia, and added with water to add 0.5 L of substrate. Medium. One of the frozen cells was added to the substrate medium and reacted at 37 ° C. for 5 hours as described above. After removing the cells by centrifugation in the same manner as above, 85 g of fumaric acid was added, followed by heating and cooling. The precipitated L-aspartic acid was subjected to suction filtration, and the crystals were washed twice with about 0.5 L of warm water at 50 ° C., sufficiently dried to remove water, and then dried. The weight of the obtained crystals was 113.5 g, and the purity was 99.0% by weight (excluding water) (0.73% by weight of fumaric acid) (average crystal size: 15%).
0 μm).

Comparative Example 1 As in Example 1, 0.5
One of the frozen cells was added to the L substrate medium to carry out the reaction. Analysis of the reacted medium revealed that 9%
9.0 mol% of L-aspartic acid was produced. After the reacted medium was centrifuged to remove the cells, 50 g of fumaric acid was added, and the mixture was heated to 60 ° C. and maintained for 30 minutes.
Cooled to 0 ° C. After cooling, suction-filtered with a suction filter,
About 400 ml of mother liquor was obtained. On the other hand, the crystals containing L-aspartic acid separated by filtration were washed with about 0.5 L of warm water at 50 ° C. and dried. The weight of the obtained crystals was 80.7 g, and the purity was 98.4% by weight (excluding water). ) (Fumaric acid 1.2% by weight) (average crystal size 200 μm).

Comparative Example 2 As in Example 1, 0.5
One of the frozen cells was added to the L substrate medium to carry out the reaction. Analysis of the reacted medium revealed that 9%
9.0 mol% of L-aspartic acid was produced. After the reacted medium was centrifuged to remove the cells, 85 g of fumaric acid was added, and the mixture was stirred at 30 ° C. for 30 minutes, followed by suction filtration with a suction filter to obtain about 350 ml of mother liquor. On the other hand, about 0.5 L of water at 30 ° C. was added to the filtered crystal containing L-aspartic acid, mixed well, suction filtered, and dried. The obtained crystal weighed 153.0 g, The purity was 60.3% by weight (excluding water) (33.2% by weight of fumaric acid) (average crystal size: 200 μm).

Embodiment 3 FIG . 75 g of fumaric acid and 0.125 g of magnesium sulfate heptahydrate were added to 300 ml of water.
After neutralization with 5% aqueous ammonia to adjust the pH to 8.3, water was added to obtain a 0.5 L substrate medium. One of the frozen microbial cells previously divided into 30 equal portions was added to this substrate medium, and reacted at 37 ° C. for 5 hours while gently shaking. Analysis of the reacted medium revealed that 99.1 mol% of L-aspartic acid was formed based on the charged fumaric acid.

After the reacted medium was centrifuged to remove the cells, 67.5 g of fumaric acid was added, heated to 60 ° C., kept for 30 minutes, and then cooled to 50 ° C. After cooling, the solution was suction-filtered with a suction filter to obtain about 410 ml of mother liquor. On the other hand, about 0.5 L of 50-
After washing and drying with warm water at ℃, the weight of the obtained crystals was 67.1 g, and the purity was 96.8% by weight (excluding water) (3.1% by weight of fumaric acid) (average crystal size: 200 μm). .

Next, the mother liquor and the washing solution were combined, concentrated under reduced pressure, adjusted to pH 8.3 with 7.5 g of fumaric acid and 25% aqueous ammonia, and added with water to 0.5 L. Substrate medium. One of the frozen cells was added to the substrate medium and reacted at 37 ° C. for 5 hours as described above. After removing the cells by centrifugation in the same manner as above, 67.5 g of fumaric acid was added, followed by heating and cooling. The precipitated L-aspartic acid was subjected to suction filtration, and the crystals were washed twice with about 0.5 L of warm water at 50 ° C., sufficiently dried to remove water, and then dried. The weight of the obtained crystals was 75.9 g, and the purity was 99.0% by weight (excluding water) (0.73% by weight of fumaric acid) (average crystal size: 15%).
0 μm).

Comparative Example 3 As in the third embodiment, 0.5
One of the frozen cells was added to the L substrate medium to carry out the reaction. Analysis of the reacted medium revealed that 9%
9.0 mol% of L-aspartic acid was produced. After the reacted medium was centrifuged to remove the cells, 52.5 g of fumaric acid was added, the mixture was heated to 60 ° C, kept for 30 minutes, and then cooled to 50 ° C. After cooling, the solution was suction-filtered with a suction filter to obtain about 450 ml of mother liquor. On the other hand, the crystals containing L-aspartic acid filtered off were washed with about 0.5 L of warm water at 50 ° C. and dried, and the weight of the obtained crystals was 46.2.
g, purity 98.1% by weight (excluding water) (fumaric acid 1.
6% by weight) (average crystal size: 200 μm). The results of the above Examples and Comparative Examples are summarized in the following table.

[0040]

[Table 1] As shown in the table, when the molar ratio of fumaric acid added was small, the recovery of L-aspartic acid that could be separated as crystals was low, but when the molar ratio was large, the recovery was high. When the purification operation was performed at 50 ° C., a value of 90% by weight or more was obtained under any conditions.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-7-308195 (JP, A) JP-A-8-33491 (JP, A) JP-B-43-8708 (JP, B1) JP-B-38- 6588 (JP, B1) US Patent No. 4560653 (US, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 13/00-13/24 CA (STN)

Claims (2)

(57) [Claims] The present invention relates to a method for producing L-form by reacting an enzyme-containing substance having aspartase activity on a substrate medium containing fumaric acid and ammonia and / or ammonium fumarate.
The reaction to produce aspartic acid is carried out at a fumaric acid concentration of 15
-25% by weight, and then the L-aspartic acid present in the reacted medium is added to the reacted medium by adding 0.85-1.2 times mol of fumaric acid to the reacted medium.
Aspartic acid is precipitated, and the L-aspartic acid crystals obtained by filtration and washing under a temperature condition of 40 ° C. or more are collected to obtain L-aspartic acid, and then ammonia is added to the mother liquor and the washing liquid. A method for producing L-aspartic acid, which is reused as a substrate medium. 2. Fumaric acid and / or its salt is 0.1 to
3% by weight, and the average size of the crystals is 50-
The method according to claim 1, wherein L-aspartic acid having a size of 500 μm is collected as a final product.