JPH11285395A - Production of polyol by reuse of microbial cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polyol in an industrially advantageous way by repeatedly culturing a polyol-productive microorganism and, from the 2nd time on, culturing it in e.g. a low-nutrient medium to collect the resulting cultured product so as to mitigate the burden of spawn culture and microbial cell disposal. SOLUTION: This polyol is obtained in an industrially advantageous way through mitigating the burden of spawn culture and microbial cell disposal, by the following procedure: a polyol-productive-microorganism [e.g. Trichosporonoides megatiliensis SN-G42 (FERM BP-1430)] is cultured in the 1st medium containing a carbon source, the microbial cells are separated and recovered from the medium after culture and the polyol thus produced is collected; subsequently, the microbial cells thus recovered are cultured in a low- nutrient medium containing a carbon source alone as the nutrient ingredient or a low-nutrient medium where the nutrient ingredients other than the carbon source are low in concentration compared to the case with the 1st medium, and the resulting microbial cells are separated and recovered from the medium after culture and the resultant polyol thus produced is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a polyol by fermentation of a microorganism.

[0002]

BACKGROUND OF THE INVENTION Polyol-producing microorganisms include:
It is known that microorganisms belonging to the genus Torulopsis (JP-B-47-8589 and JP-B-63-9833) produce glycerin, and Candida, Saccharomyces or Torulopsis (JP-B 47-20394).
Or a microorganism belonging to the genus Bretanomyces (Japanese Patent Publication No. 63-9834), the genus Pichia (Japanese Patent Publication No. 57-26114), or the genus Rhonela (Japanese Patent Application Laid-Open No. 2-69186). Are known to produce arabitol, and are of the genus Moniliella (JP-A-60-110295), the genus Aureobasidium (JP-B-61-31091), or the genus Trichosporonoides (WO97 / 26323). It is known that microorganisms belonging to the above-mentioned publications produce erythritol. In addition, many other microorganisms having mannitol, sorbitol, and xylitol producing ability are known.

[0003] Batch fermentation and continuous fermentation are known as fermentation methods for producing a target substance using microorganisms. The continuous fermentation is an operation method in view of stability when microorganisms are used continuously. Batch fermentation is generally used as a method for producing polyols on an industrial scale.

[0004] In the batch fermentation method, a microorganism is preliminarily cultured on a small-scale as an inoculum for main culture, and the obtained inoculum is
A large-scale fermenter containing a medium containing a raw material substrate for main culture is inoculated and cultured. The culture is terminated when the concentration of the target substance, polyol, reaches the highest point or when the raw material substrate is consumed, the microbial cells are separated from the culture solution, and the polyol is purified from the culture solution after the microbial cell separation. And get. The microbial cells isolated at this time have been disposed of.

[0005] In a method for producing polyols, particularly erythritol, a method for continuously using microbial cells is disclosed in Japanese Patent Application Laid-Open No. Hei 5-137585, in which the nitrogen content in the cells of erythritol-producing bacteria is 2.5-4. A method for producing erythritol by continuous culturing while maintaining 5% is disclosed. JP-A-8-23989 discloses a method for producing an oligosaccharide containing a high concentration of panose by contacting a microorganism belonging to the genus Aureobasidium with a sugar solution containing maltose as a main component. However, there is also disclosed a method for repeatedly producing oligosaccharides by bringing microbial cells separated from a culture solution after the main culture into contact with a sugar solution again. At this time, erythritol as a by-product other than the oligosaccharide was 10%.
% Is produced.

[0006]

In the fermentative production of a microorganism by batch fermentation, a fermentation time for the microbial cells is required in order to vigorously produce the target substance, and an energy source for the microbial cells to proliferate. Therefore, there is a problem that the time and the raw material substrate are wasted more than simply producing the target substance.

[0007] In addition, microbial cells that are produced in large quantities at the end of culturing can be sterilized even though they still have sufficient production capacity.
Although inactivated and disposed of, disposal costs are economically disadvantageous. On the other hand, in the batch fermentation, a seed bacterium necessary for the next fermentation production is newly prepared each time, and the cultivation of the seed bacterium in accordance with the production plan of the main culture is a burden on production management. The continuous fermentation method can be said to be an effective method to solve the above points, but since the production equipment does not always match the equipment for batch fermentation, it is necessary to install dedicated auxiliary equipment, and currently performing batch fermentation If this is the case, the installation of new auxiliary equipment for continuous fermentation is costly. Therefore, there is a demand for the development of a production method that improves the productivity of polyols by effectively utilizing the currently used batch fermentation apparatus.

Accordingly, an object of the present invention is to reduce the burden on the production process of seed culture in batch culture, reduce the disposal of microbial cells generated in large quantities, and minimize the installation of new equipment to minimize industrial production. Another object of the present invention is to provide a method for producing a high-concentration polyol.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned object, and as a result, the microbial cells separated and recovered from the medium in which the polyol fermentation has been completed are converted to nutrients using only a carbon source. Low nutrient medium, or when the nutrients other than the carbon source are cultured in a low nutrient medium with a low concentration compared to the first medium, it is found that the polyol is produced at a high concentration again, By repeating the step of separating and recovering and the step of culturing in a low nutrient medium, it has been found that microbial cells can be reused and a polyol can be efficiently produced, and the present invention has been completed.

That is, the gist of the present invention is to provide a method for producing a polyol by fermentation of microorganisms, wherein a microorganism having a polyol-producing ability is cultured in a first culture medium containing a carbon source, and microbial cells are separated and recovered from the culture medium. And the step of collecting the produced polyol, using the microbial cells separated and recovered as nutrients in a low nutrient medium containing only a carbon source or a low nutrient in which the nutrients other than the carbon source have a lower concentration than the first medium. The step of culturing in a medium, separating and collecting microbial cells from the medium,
The present invention resides in a method for producing a polyol, wherein the steps of collecting the produced polyol are sequentially performed. Furthermore, the method for producing a polyol, wherein the step of separating and recovering the microbial cells and the step of collecting the produced polyol, and the step of culturing the separated and recovered microbial cells in a low nutrient medium are repeated. In the manufacturing method.

According to a preferred embodiment of the present invention, Trichosporonoides megatiliensis SN-G42 (FER
MBP-1430), in which erythritol is produced by aerobic batch fermentation, the microorganisms are separated and recovered from the medium after erythritol production by filtration or centrifugation, and the produced erythritol is collected.
The isolated and recovered microbial cells are cultured again in a low nutrient medium containing only glucose as a nutrient or a low nutrient medium containing no more than 25% of nutrients other than glucose as compared with the first medium to produce erythritol again. A method for producing erythritol by reusing microbial cells collected by collection is provided.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The microorganism used in the present invention is not particularly limited as long as it is a microorganism having a polyol-producing ability. For example, microorganisms having an erythritol-producing ability include those described in JP-A-63-963.
Microorganisms belonging to the genus Aureobasidium described in JP-A-831; JP-A-9-154589 or WO
A microorganism belonging to the genus Trichosporonoides described in 97/26323; a microorganism belonging to the genus Moniliella described in JP-A-60-110295 or JP-A-9-154589; JP-A-9-154590 A microorganism belonging to the genus Yerobia, Ustilago, Filobadium or Trigonopsis described in the publication; a microorganism belonging to the genus Trignopsis or Candida described in JP-B-47-41549;
No. 1071; microorganisms belonging to the genera Candida, Trullopsis, Hansenula, Pichia or Debaryomyces; Biotechnology Letters (Bio)
technology. lett. , 7 (4), 119-23
4 (1985)), a microorganism belonging to the genus Hansenia spora, Saccharomyces, Chlorella, Chryveromyces or Torulaspora; a microorganism belonging to the genus Aspergillus (SA Barer, J. Che).
m. Soc. , 2538-2586 (1958)); Experimental Mycology (Gaby. E.,
Experimental Mycology, 4,1
60-170 (1980)); microorganisms belonging to the genus Eupenicillium, Monascus, Penicillium, Verticillium; microorganisms belonging to the genus Tigopihia (currently classified into the genus Yamadajima) (Suzuki, Fermentation and Industrial, 35 (6), (1977); a microorganism belonging to the genus Rhodotorula (J. Gen, Microbiol., 18, 2).
69 (1985)).

[0013] Examples of microorganisms having glycerin-producing ability include JP-B-47-8589 and JP-B-63-9.
And a microorganism belonging to the genus Torulopsis described in JP-A-833. Examples of the microorganism having an arabitol producing ability include Japanese Patent Publication No. 47-20394 or Japanese Patent Publication No. Sho 5-20.
A microorganism belonging to the genus Candida, Saccharomyces or Torulopsis described in JP-A-3-6231; a microorganism belonging to the genus Bretanomyces described in JP-B-63-9834; Pichia described in JP-B-57-26114 A microorganism belonging to the genus; a microorganism belonging to the genus Rhonera described in JP-A-2-69186; and a microorganism having an ability to produce mannitol, sorbitol, and xylitol.

Preferred microorganisms used in the present invention include Trichosporonoides megatiliensis strain SN-G42 (FERM) described in Japanese Patent Publication No. 4-635.
BP-1430, former name: Aureobasidium SN
-G42 strain).

The nutrient components of the medium for culturing microorganisms in the present invention generally comprise a carbon source, a nitrogen source, inorganic salts and the like. Examples of the carbon source include sugars such as glucose and fructose, and saccharified solutions containing these sugars such as saccharified starch solution, sweet potato molasses, and sugar beet molasses. Preferably, glucose is used. The sugar concentration in the medium is 20-60% in terms of solid content,
Preferably, it is used in the range of 30 to 40%.

As a nitrogen source, an inorganic nitrogen compound or an organic nitrogen compound which can be used by microorganisms is used. For example, yeast extract, peptone, corn steep liquor, ammonia, ammonium salts, urea, various amino acids, thiamine, biotin and the like are used. Is done. As the inorganic salts, inorganic salts such as phosphoric acid, magnesium, potassium, calcium, iron and the like are used. Further, in addition to the nutrient components described above, components that assist the growth of microorganisms such as vitamins can be added to the medium.

When culturing in a liquid medium, it is preferable to add an antifoaming agent, a pH adjusting agent and a buffer. As an antifoaming agent, an inorganic or organic antifoaming agent is used, for example, polyvinyl alcohol, sorbitan fatty acid ester, or a silicon-based compound.As the pH adjusting agent, sodium hydroxide, potassium hydroxide, sulfuric acid, Examples include hydrochloric acid and nitric acid, and examples of the buffer include sodium lactate and various phosphates.

In the present invention, the medium for the seed culture for the main culture and the medium for the first main culture inoculating the seed with the necessary nutrients such as the above-mentioned carbon source, nitrogen source and inorganic salts are contained. However, when the concentration of the nutrient in the medium of the second and subsequent main cultures is the same as the concentration of the nutrient in the medium of the first main culture, the amount of polyol produced is reduced. In the culture medium, the concentration of the nutrient component is made lower than that of the medium of the first main culture, or a medium containing only a carbon source such as glucose is preferably used. By using a low nutrient medium for the second and subsequent culture media, the production amount of the polyol can be equal to or greater than that of the first main culture.

According to the present invention, in the first main culture, two activities of the microorganism are required, that is, growth of the microorganism and polyol production. However, in the second and subsequent main cultures, the two main activities are separated from the first main culture. Since the recovered microbial cells are reused, it is not necessary to multiply the microbial cells, and it is preferable to concentrate the microbial cells on the polyol production activity. The present invention is preferably carried out by aerobic fermentation. However, when the medium nutrients of the second and subsequent main cultures are the same as the medium nutrients of the first main culture, the microbial cells grow, As a result, the production amount of the polyol decreases.

The reason for the decrease in the production amount is that the amount of microbial cells in the medium becomes excessive due to the growth of microbial cells, the amount of oxygen in the medium becomes insufficient, and the medium conditions shift from aerobic to anaerobic. It is considered that the production of the polyol of the gaseous reaction is suppressed. Therefore, as a method of preventing a decrease in the amount of production, the aerobic state of the second and subsequent main cultures is set to a higher aerobic state as compared with the first main culture, and excess microbial cells are eliminated. The growth of the microbial cells is maintained by maintaining aerobicity in the medium or limiting the amount of the nutrient components of the main culture of the second and subsequent main cultures compared to the nutrient components of the main culture of the first main culture. A method for suppressing this is conceivable.

The method of the present invention for limiting the amount of nutrient added is economically preferable because it reduces the cost of the culture medium as compared with the method for maintaining aerobicity. The extent to which the concentration of nutrients other than the carbon source is reduced may be adjusted so that the microorganisms inoculated in the culture medium do not grow beyond the amount of cells suitable for producing polyols. Although it depends on the type of nutrient required by the microorganism, it can be determined by a simple test as shown in Examples of the present invention. For example, Trichosporonoides megatiliensis SN-G42 (FERMBP-143)
In the fermentative production of erythritol using 0),
Nutrient components other than the carbon source are 25% of the concentration of the first culture medium.
It may be adjusted as follows. INDUSTRIAL APPLICABILITY The present invention is applicable to all methods for producing fermentation products using microorganisms, and is an effective method particularly for producing fermentation products such as polyols produced by aerobic fermentation.

As a method for separating and recovering microbial cells,
Examples include a method using an apparatus capable of rapidly separating a culture solution and microbial cells, and a diatomaceous earth filtration method, a microfiltration method, and a ultrafiltration method using a filter press using an organic membrane separator, a ceramic separator, and the like. , A ceramic filtration method, a centrifugal separation method using a batch type centrifuge, a continuous centrifuge, or the like. In addition, the culture solution after the production of the polyol is allowed to stand for 4 to 6 hours to precipitate the microbial cells, and a sedimentation method of sucking and recovering the microbial cells by a pump can be used. And the use of centrifugation methods. Further, it is preferable that the separated and collected microbial cells be stored in a cool place to prevent drying.

The whole amount of the separated and recovered microbial cells can be added to the next medium. However, the number of reuses of the microbial cells increases, and when the polyol-producing ability of the microbial cells decreases, or If it is difficult to transfer the total amount of the recovered microbial cells to the fermenter for the next main culture, the amount of microbial cells to be added to the fermenter for the next main culture should be smaller than the optimum microbial cell amount for polyol production. Microbial cells having high polyol-producing ability may be adjusted to an optimal amount for polyol production by reducing the amount and growing in a medium. In this case, it is necessary to add nutrients necessary for growth in addition to the carbon source to the medium, but when the amount of the nutrients is adjusted to the same concentration as in the first main culture, the microbial cells become excessive. Therefore, it is necessary to adjust the nutrient component to a lower concentration as compared with the concentration of the first main culture so that the amount of microbial cells becomes an optimal amount for polyol production.

In the method for producing the polyol of the present invention, first, a microorganism having a polyol-producing ability is cultured on a small scale to prepare a seed culture for the main culture, and then nutrient components necessary for the growth of microbial cells and the production of the polyol. Is inoculated into a large-scale fermenter equipped with a liquid medium containing, and cultured under aeration and stirring conditions. In the cultivation, when the polyol concentration reaches the highest point, the aeration and stirring are stopped to terminate the cultivation, the microbial cells are separated and collected from the culture solution, and the culture solution after the microbial cell separation is purified to obtain a polyol.

In the second and subsequent main cultures, the separated and recovered microbial cells are added to a liquid medium having a lower concentration of nutrients than the first main culture or, preferably, only a carbon source such as glucose. Then, the culture is started again to produce the polyol. After that, the process of separating and recovering the microbial cells from the culture solution after the production of the polyol without adding the inoculum for main culture, and adding the separated and recovered microbial cells to the newly prepared low-nutrient liquid medium The process of producing the polyol is repeated, and the polyol is produced by reusing the microbial cells.

Optimum conditions for culturing, such as pH during culturing, culturing temperature, and aeration in aerobic fermentation, vary depending on the microorganism used, but it is not necessary to change the conditions for each batch culturing. In addition, the culture time requires a time for microbial cells to proliferate in batch culture, but the present invention does not require that time, and is therefore shortened. The cultivation may be carried out by appropriately measuring the concentration of the polyol during the culturing by a known method such as high performance liquid chromatography or thin layer chromatography, and stopping when the polyol concentration reaches the highest point or when the carbon source is consumed.

The method of stopping the culture is not particularly limited as long as the method does not damage the microbial cells, and includes adjusting the aeration rate, lowering the culture temperature, and the like. The culture is performed under aerobic conditions. If so, the culture can be stopped by stopping the aeration.

[0028]

EXAMPLES Examples of the present invention will be specifically described below, but the present invention is not limited to these examples. <Example 1> (1) Seed culture In a 500 ml Erlenmeyer flask, 40 ml of a medium containing 30% of glucose (W / V) and 1% of yeast extract as nutrient components was put into a cotton cap, and sterilized in an autoclave at 120 ° C for 20 minutes. , Trichosporonoides megachiliensis SN-
G42 (FERM BP-1430) was inoculated. This was subjected to rotary culture at a temperature of 35 ° C. and a stirring speed of 220 rpm for 3 days to obtain a seed culture solution. (2) First fermentation Contains 40% of glucose and 1% of yeast extract as nutrients, and further contains an antifoaming agent (ADEKANOL LG, manufactured by Asahi Denka Kogyo KK)
109) 3 L of a medium containing 300 ppm was placed in a 7 L fermenter (Model MD-C, manufactured by Iwashya Co., Ltd.), sterilized, and a seed culture solution prepared in advance was added. The stirring speed was 680 rpm, the air supply amount was 1.5 L / min. Culture was performed at a temperature of 35 ° C. for 4 days, and the culture was terminated when glucose was consumed.

(3) Second fermentation From the culture solution after the first fermentation, a centrifuge (PS-18IV, manufactured by Tommy Seiko) under the condition of centrifugal force x 5000 G
And inoculate the obtained microbial cells into a 7-L fermenter containing 3 L of a medium containing only 40% of glycol as a nutrient, and culturing under the same conditions as in the first fermentation. Then, glucose was consumed in three days, and the culture was terminated. The erythritol production in the first and second fermentations was measured by high performance liquid chromatography, and the erythritol concentration was 1 in the first fermentation.
68 g / L, and the second fermentation was 200 g / L. Also, the fermentation days were shortened in the second fermentation, and the entire amount of microbial cells separated in the first fermentation was reused,
Compared to the case where conventional batch fermentation was performed twice, the amount of microbial cells to be discarded was reduced to half.

Example 2 (1) Seed Culture 50 mL of a medium containing 30% of glucose (W / V) and 1% of yeast extract as nutrients was put into a 500 mL Erlenmeyer flask, and the cotton plug was inserted. After sterilization for 3 minutes, Trichosporonoides megachiliensis SN-
G42 (FERM BP-1430) was inoculated. This was subjected to rotary culture at a temperature of 35 ° C. and a stirring speed of 220 rpm for 3 days to obtain a seed culture solution. (2) First fermentation Contains 40% of glucose and 1% of yeast extract as nutrients, and further contains an antifoaming agent (ADEKANOL LG, manufactured by Asahi Denka Kogyo KK)
109) 3 L of a medium containing 300 ppm was put into a 7-L fermenter (manufactured by Iwashia Corporation, model MD-C), sterilized, and a seed culture solution prepared in advance was added. The stirring speed was 680 rpm, the air supply amount was 1.5 L / min. The cells were cultured at a temperature of 35 ° C. for 3 days.

(3) Second fermentation 300 mL of the first fermentation end liquid was centrifuged under the conditions of centrifugal force x 5000 G (PS-18IV, manufactured by Tommy Seiko).
To isolate the cells, and the amount of wet cells per 300 mL of fermentation broth was determined. A 500 mL volume containing 13 mL of a second culture to which 1%, 0.5% or 0.25% of a yeast extract which is a nutrient such as 40% glucose and a nitrogen source, a vitamin source, and a mineral source is added as a nutrient component. The wet cells previously isolated are inoculated aseptically into an Erlenmeyer flask so as to have the same cell concentration as that of the medium at the end of the first fermentation, and cultured at 35 ° C. and a stirring speed of 220 rpm for 2 days. did. In the culture in an Erlenmeyer flask, an appropriate amount of sodium lactate was added in addition to the nutrient components to prevent a decrease in pH. The erythritol production in the first fermentation and the second fermentation was measured by high performance liquid chromatography, and the erythritol concentration was 140 g / L in the first fermentation. The results of the second fermentation are shown below.

[0032]

According to the present invention, since the microbial cells are repeatedly reused, the seed culture step essential for batch fermentation can be omitted, the culture time can be shortened, and the amount of waste cells generated can be reduced. Can also be reduced. In addition, the present invention does not require a special apparatus, and in the second and subsequent cultures, the amount of nutrients to be added to the medium is reduced, so that it is possible to provide an industrially advantageous method for producing a polyol in terms of cost.

Claims (10)

[Claims] In a method for producing a polyol by fermentation of microorganisms, a step of culturing a microorganism having a polyol-producing ability in a first medium containing a carbon source, separating and recovering microbial cells from the cultured medium, The step of collecting the produced polyol, the nutrient components other than the carbon source in the low nutrient medium having a low concentration compared to the first nutrient medium or a low nutrient medium using only the microbial cells separated and recovered as nutrients A method for producing a polyol, comprising sequentially performing a step of culturing, a step of separating and recovering microbial cells from the medium after the culturing, and a step of collecting the produced polyol. 2. A method for producing a polyol by fermentation of microorganisms, wherein a microorganism having a polyol-producing ability is cultured in a first culture medium containing a carbon source, and microbial cells are separated and recovered from the culture medium after the culture. The step of collecting the produced polyol, the nutrient components other than the carbon source in the low nutrient medium having a low concentration compared to the first nutrient medium or a low nutrient medium using only the microbial cells separated and recovered as nutrients The step of culturing, separating and recovering the microbial cells from the culture medium after culture, and the step of collecting the produced polyol in order to produce the polyol, the microorganism cells were separated and recovered from the culture medium after culture and produced. Collecting a polyol,
A process for producing a polyol, comprising repeating the step of culturing the separated and recovered microbial cells in a low nutrient medium. 3. In the step of culturing the separated and recovered microbial cells in a low nutrient medium, the concentration of nutrient components other than the carbon source in the low nutrient medium is determined by determining the amount of microbial cells suitable for producing a polyol. No.1 so that it does not grow beyond body weight
The method according to claim 1, wherein the concentration is adjusted to be lower than that in the first culture. 4. In the step of culturing the separated and recovered microbial cells in a low nutrient medium, the concentration of nutrient components other than the carbon source in the low nutrient medium is reduced to 25% of the concentration of the first medium.
The method according to any one of claims 1 to 3, wherein the method is prepared as follows. 5. The method according to claim 1, wherein the method for producing a polyol by fermentation of a microorganism is aerobic fermentation. 6. The process according to claim 1, wherein the microorganism is a microorganism having an ability to produce erythritol, and the main component of the polyol to be produced is erythritol. Method. 7. The method according to any one of claims 1 to 6, wherein the microorganism is a microorganism belonging to the genus Trichosporonoides. 8. The microorganism is Trichosporonoides megatiliensis SN-G42 (FERM BP-1430).
The method according to claim 1, wherein: 9. The production method according to claim 1, wherein the carbon source is glucose. 10. The method for separating and collecting microbial cells by diatomaceous earth filtration using a filter press, microfiltration, ultrafiltration, ceramic filtration or centrifugation. The method according to claim 1.