JP2013179868A - Culture method of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture method of microorganism, capable of performing subculture of a microorganism at proper timing without needing a complicated operation, and thus efficiently propagating the microorganism.SOLUTION: This culture method for microorganism for culturing a microorganism in a liquid medium includes: a step of measuring pH of a culture fluid with time, and subculturing the microorganism to another liquid medium after change in pH value reaches a plateau. Otherwise, the culture method of microorganism for culturing a microorganism in a liquid medium includes: a step of measuring pH of a culture fluid with time, and subculturing the microorganism to another liquid medium after the inclination of a pH change curve obtained by plotting pH value on a vertical axis and culture time on a horizontal axis changes from a negative value to zero.

Description

  The present invention relates to a method for culturing microorganisms, and more particularly, to a method for cultivating microorganisms that can carry out transplanting of microorganisms at an appropriate timing.

  In recent years, with the development of genome decoding, culture technology, breeding technology, and gene modification technology, it has become possible for microorganisms to produce various substances, such as industrial enzymes, pharmaceutical active ingredients, and compound intermediates using microorganisms. The production of useful substances is actively carried out. In order to increase the production amount of the target substance in the production of useful substances using microorganisms, it is required to increase the amount of microorganisms as much as possible. In addition, there are cases where microorganisms are cultivated for edible use, and there are cases where a large amount of cells are required to be used for decomposition and fermentation of organic compounds by microorganisms. It has been demanded.

  A liquid medium is mainly used for culturing microorganisms, and the cells are introduced into a liquid medium containing components essential for the growth of microorganisms such as a carbon source and a nitrogen source, and shaken and stirred while maintaining an appropriate temperature. Then, culture is performed (for example, Non-Patent Document 1). When culturing microorganisms in a liquid medium, the growth of microorganisms stops when nutrients in the medium are consumed and depleted. Therefore, after culturing to some extent, it becomes necessary to transplant to a new medium. In addition, when cultivating microorganisms on an industrial scale, a method of gradually increasing the amount of the culture solution and the amount of bacteria by several stages of seed culture and then transferring to the final main culture (main culture) Is generally used.

  Transplanting into a new medium is required to be performed at a stage where the bacterial cell concentration is as high as possible and the bacterial cell growth is active. That is, if the culture is advanced, the bacterial cell concentration increases, but if the culture is continued for a certain extent, the bacterial cell growth becomes inactive due to a lack of nutrients in the medium. It is known that when a cell is transferred to a new medium at a stage where the growth of the bacterial cells becomes inactive, the induction period is prolonged until adaptation to the new medium and active bacterial cell growth is observed again. Furthermore, there are microorganisms that change metabolic pathways when they fall into nutrient starvation, which can adversely affect the production of the target substance.

  As a method for transferring cells, for example, the culture solution is periodically sampled to measure the concentration of the cells, and from the relationship between the culture time and the change in the concentration of the cells, the growth of the cells is active and the concentration of the cells Judging the stage that entered the latter half of the high exponential growth period, planting the cells has been carried out. Further, after performing the test under the same culture conditions and confirming that the transition of the bacterial cell concentration with the passage of the culture time is the same every time, the transplantation may be performed after the passage of a certain culture time.

Methods in Yeast Genetics 2005: A Cold Spring Harbor Laboratory Course Manual

  However, the work of sampling the culture solution over time, measuring the cell concentration, and searching for the exponential growth period is very complicated, and the final reaching bacteria may vary depending on the product lot of the medium material used and the difference in the culture tank. It is necessary to consider the influence such as the change in the body concentration and the range of the bacterial cell concentration indicating the exponential growth phase. On the other hand, the method using time as an index cannot be employed if the culture conditions used in the past are different from the culture conditions. There was a problem of being affected by.

  SUMMARY OF THE INVENTION An object of the present invention is to provide a microorganism culturing method that can perform microbial inoculation at an appropriate timing without requiring complicated operations and can efficiently proliferate microorganisms.

  As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by measuring the pH of the microorganism culture solution over time, and have completed the present invention. That is, the present inventors have a period in which the pH change of the culture solution becomes a plateau in the later stage of the exponential growth period of the microorganism, and by transferring the microorganism after that period, We found that the growth of microorganisms in the country became active without delay.

The present invention includes the following [1] to [9] related to a microorganism culturing method completed based on the above findings.
[1] A method of culturing a microorganism in a liquid medium, wherein the pH of the culture solution is measured over time, and the microorganism is placed in another liquid medium after the change in pH value reaches a plateau. A method for culturing microorganisms, comprising a step of planting.
[2] A microorganism culturing method for culturing a microorganism in a liquid medium, wherein the pH of the culture solution is measured over time, and the slope of the pH change curve plotted with the pH value on the vertical axis and the culture time on the horizontal axis A method for culturing microorganisms, comprising a step of planting microorganisms in another liquid medium after the time point becomes zero.
[3] A microorganism culturing method for culturing a microorganism in a liquid medium, wherein the pH of the culture solution is measured over time, and the change of the pH value reaches the inflection point and the direction of the change is reversed. A method for culturing a microorganism, comprising the step of planting the microorganism in another liquid medium.
[4] The method of cultivating a microorganism according to [1], wherein the step of planting the microorganism in another liquid medium is performed from the time when the pH value changes to a plateau to the time when the pH changes to 0.05.
[5] The method for culturing a microorganism according to any one of [1] to [4], wherein the liquid medium is a rich medium.
[6] The method for culturing a microorganism according to any one of [1] to [5], wherein the culture temperature is 20 to 40 ° C.
[7] The microorganism is a genus Saccharomyces, a genus Shizosaccharomyces, a genus Hansenula, a genus Pichia, a genus Ogataea, a genus Candida, an Aspergillus A method of culturing a microorganism according to any one of [1] to [6], which is a filamentous fungus such as an genus (Aspergillus) or a genus Trichoderma, or Escherichiacoli.
[8] The method for culturing a microorganism according to any one of [1] to [7], wherein the microorganism is a yeast belonging to the genus Shizosaccharomyces or Escherichiacoli.
[9] The method for culturing a microorganism according to any one of [1] to [8], wherein the microorganism is the fission yeast Shizosaccharomyces pombe.

  According to the present invention, it is possible to provide a method for cultivating microorganisms that can perform mitosis of microorganisms at an appropriate timing without requiring complicated operations and can efficiently propagate the microorganisms.

PH of the liquid medium in Reference Example 1 is a graph showing changes in the dissolved oxygen concentration (DO) and exhaust CO _2. It is a graph showing the changes in OD ₆₆₀ and concentration of glucose in a liquid medium in Reference Example 1. It is a graph showing the changes in OD ₆₆₀ and concentration of glucose in a liquid medium in Reference Example 2. Is a graph showing the transition of the OD ₆₆₀ of the liquid medium after subcultured in Reference Example 2. It is a graph showing transition of pH and dissolved oxygen concentration (DO) of the liquid culture medium in Reference Example 3. It is a graph showing the changes in OD ₆₆₀ and concentration of glucose in a liquid medium in Reference Example 3. It is a graph showing transition of pH of a liquid culture medium in a reference example 4, and dissolved oxygen concentration (DO). It is a graph showing the changes in OD ₆₆₀ and concentration of glucose in a liquid medium in Reference Example 4. It is a graph showing transition of pH of a liquid culture medium in Reference Example 5, and dissolved oxygen concentration (DO). It is a graph showing the changes in OD ₆₆₀ and concentration of glucose in a liquid medium in Reference Example 5. It is a graph showing transition of pH of a liquid culture medium in a reference example 6, and dissolved oxygen concentration (DO). It is a graph showing the changes in OD ₆₀₀ and concentration of glucose in a liquid medium in Reference Example 6. 3 is a graph showing transition of OD ₆₆₀ , glucose concentration and pH of a liquid medium in lot 1-1 of Example 1. FIG. 3 is a graph showing transition of OD ₆₆₀ , glucose concentration and pH of a liquid medium in lot 1-2 of Example 1. FIG. Is a graph showing the transition of the OD ₆₆₀ of the liquid medium after subcultured in Example 1. _{OD 660} of the liquid medium in lots 2-1 of Example 2 is a graph showing the changes in glucose concentration and pH. _{OD 660} of the liquid medium in lots 2-2 of Example 2 is a graph showing the changes in glucose concentration and pH. Is a graph showing the transition of the OD ₆₆₀ of the liquid medium after subcultured in Example 2.

  The microorganism culturing method of the present invention is a microorganism culturing method in which a microorganism is cultured in a liquid medium, and the pH of the culture solution is measured over time. The method includes the step of planting in a liquid medium. When microorganisms are cultured in a liquid medium, the nutrient content in the medium is maximum and the cell concentration is minimum at the start of culture. And if culture | cultivation is continued, while the nutrient in a culture medium will be consumed, a microbial cell will proliferate. Along with this, the concentration of each component in the medium, for example, the concentration of carbon source such as glucose, the concentration of nitrogen source such as amino acid, the concentration of dissolved oxygen and the like also change. In analyzing various parameters that change during the culturing of microorganisms, the present inventors have found that the pH change has certain characteristics. That is, the pH of the culture solution decreases or increases from the beginning of the culture, but when the culture is continued to some extent, the pH decrease or increase stops. In the present specification, the time point at which the above-described pH decrease or increase stops is referred to as the time point when the pH change reaches a plateau.

  If the liquid medium for microorganisms is used for culturing for the purpose of cell growth, it usually contains essential components for growth such as carbon source, nitrogen source, vitamins and phosphorus, or components for accelerating growth. Including. If it is not a medium used for culturing microorganisms that grow under special environments such as strong acidity and strong alkalinity, in addition to these nutrients, ingredients that greatly affect the pH of the medium are added to make the medium a strong acid. There is no adjustment to the property or strong alkalinity. The change in pH of the culture solution is considered to originate from basic metabolic activities related to consumption of nutrients such as carbon sources and nitrogen sources by microorganisms. In the seed culture stage up to the main culture, it is common not to carry out pH control of the culture solution using a drug due to the complexity of the operation. For this reason, although the absolute value of pH varies depending on each medium and culture, it is considered that the movement of the pH change in which the pH decreases or rises and eventually reaches a plateau is common in culture using a culture medium for growing microorganisms. Moreover, when pH control of a culture solution is performed, it is thought that the time-dependent change of the addition amount of an alkali or an acid can be used as a parameter | index similarly.

  Cell growth generally enters an exponential growth phase in which cells grow exponentially after an induction phase. Then, it reaches the stationary phase where the bacterial cell growth stops. Since the growth rate of the bacterial cells is highest in the exponential growth phase, keeping the bacterial cells in the medium in the exponential growth phase leads to efficient bacterial growth. When the nutrients in the medium are consumed and depleted, the bacterial cell growth eventually enters the stationary phase. Therefore, before entering the stationary phase from the exponential growth phase, it is necessary to plant the culture solution to supply nutrients. On the other hand, considering the cost of the medium itself and the time and effort required for planting, it is more efficient to continue the culture with one medium until the bacterial cell concentration becomes as high as possible. In consideration of the above, it is most preferable to carry out the medium inoculation at the later stage of the exponential growth period in which the growth of the bacterial cells is active and the bacterial cell concentration is high.

As a result of further studies, the present inventors have found that the time point at which the pH change of the medium reaches a plateau falls in the late phase of the exponential growth phase in the cell growth phase. Using this correspondence, the pH is measured over time, and when the change in pH reaches a plateau, transplanting is performed to measure time-consuming OD (absorbance) of the culture solution. Even if it is not performed, the culture solution can be transplanted at the later stage of the exponential growth phase.
Hereinafter, the present invention will be described in more detail.

[plateau]
In the present invention, the time point when the pH change reaches a plateau refers to the time point when the pH drop or rise of the culture solution stops. The time point when the pH change stops is not a temporary stop of the pH change for a moment, but a stop of the pH change when viewed in a certain unit of time. This is a point in time at which a so-called minimum value is reached in a pH change curve in which changes with time of pH are plotted with the vertical axis representing the pH value and the horizontal axis representing the culture time. The minimum value in this case is a minimum value in a section of several hours, for example, a minimum value in a section of 8 hours, and not a minimum value in a short section of several minutes. Usually, only one point of the minimum value of pH appears between the start of culture and the growth of bacterial cells to the stationary phase. When the local minimum value appears several times in the interval of several hours from the start of the culture until the cell growth reaches the stationary phase, it is preferable to set the local minimum value that appears first as the time of transplanting.
The time point when the pH change reaches a plateau is also the time point when the slope becomes zero in the pH change curve in which the change over time in pH is plotted with the pH value on the vertical axis and the culture time on the horizontal axis.
After the change in the pH of the medium reaches a plateau, the pH of the culture solution usually changes in the opposite direction. The point in time when the direction of the pH change is reversed from the pH change through the plateau may be used as an index for planting. There may be a case where the pH hardly changes after the plateau is reached, but in this case as well, there is no inconvenience since the time when the pH change becomes a plateau may be used as an index.

  In the culture method of the present invention, transplanting is performed using as an index the time when the pH change of the culture solution reaches a plateau. Since the duration of the exponential growth phase varies depending on the culture temperature, microorganisms, medium type, and the like, it can be determined as appropriate according to the culture conditions as to how many hours from the point of time when the plateau is reached. In order to ensure that the planting can be carried out at the time when it falls later in the exponential growth phase, it is preferable that the time for carrying out the planting is not so long after the plateau is reached, from the time when the pH change becomes a plateau. It is preferable to carry out transplanting until a carbon source such as glucose is consumed. More specifically, the time for performing the transplanting can be defined as a range from the time when the pH change reaches a plateau until the pH changes by 0.05. In the study using fission yeast, the conditions for transplanting at the time when the pH changed 0.15 from the time when the pH change reached a plateau, compared to the conditions for transplanting in the above range, The rise of cell growth in the main culture was slightly delayed.

[PH measurement method]
In the present invention, the pH measurement method is not particularly limited, and a known method can be used. In order to measure the change in pH over time, it is preferable to use an apparatus capable of measuring in real time. The measurement of the pH of the medium is simple because it can be performed directly by using a known measuring instrument. For example, by using a pH electrode (product number: 405-DPAS-SC-K8S) from Metra-Toledo Co., Ltd. and attaching it to the culture tank so that the liquid junction at the tip of the pH electrode is immersed in the culture medium, Online real-time measurement is possible. On the other hand, when measuring the bacterial cell concentration in the culture solution by absorbance, the reliability of the absorbance is reduced when the bacterial cell concentration is high, so the medium containing the bacterial cells is sampled and diluted with water before using an absorptiometer. It is necessary to use and measure, and is complicated.

[Initial cell concentration]
At the start of seed culture, the cell concentration is not particularly limited, and can be appropriately determined for each culture in consideration of the purpose, target culture amount, and the like. By setting the cell concentration at the start of seed culture low, the number of stages of seed culture until the main culture can be reduced. Therefore, the culture is started with OD ₆₆₀ of 0.08 to 0.71. Preferably, it is more preferable to start the culture in a state of 0.18 to 0.38.
OD (Optical density) indicates optical density, and OD ₆₆₀ is the absorbance of light having a wavelength of ₆₆₀ nm, and is generally used as an indicator of yeast cell concentration. Similarly, OD ₆₀₀ indicates the absorbance of light having a wavelength of 600 nm, and is generally used as an indicator of the concentration of cells such as Escherichia coli. These measuring methods are, for example, using a U-1500 type ratio beam spectrophotometer (model: 118-0110) manufactured by Hitachi, Ltd., and putting a culture solution sampled from a culture tank into a plastic cell for a sample, and having an OD ₆₆₀ The value can be measured. If the cell concentration of the culture broth is high, dilute with water and measure. Furthermore, the measuring method of a microbial cell density | concentration is not specifically limited, Well-known methods other than the above can be used.

[Planting]
Transplanting is the addition of some or all of the medium in which the microorganisms are cultured to another medium that is not used. The composition of the culture medium before planting and the culture medium of the planting destination may be the same or different. Moreover, the culture scale before planting and the culture scale after planting may be the same or different. For example, it can be scaled up gradually by first culturing with a small amount of medium and repeating planting. As described above, the planting is preferably performed so that the OD ₆₆₀ of the medium after planting is 0.08 to 0.71 at the start of the culture, and is 0.18 to 0.38. Is more preferable.

[Culture medium]
As the medium used in the culture method of the present invention, any medium such as a synthetic medium, a semi-synthetic medium, and a natural medium can be used as long as it is used for the growth of microorganisms. A medium containing a carbon source and a nitrogen source is preferable. As the carbon source, glucose and sucrose are preferable. As the nitrogen source, amino acids and ammonium salts are preferable. Moreover, since the growth rate of microorganisms becomes high, a rich medium is more preferable. As the eutrophic medium, those containing yeast extract (yeast extra), proteolysates such as tryptone, peptone and polypeptone, and those derived from natural components such as wort are preferable. Specific examples of the eutrophic medium include YPD medium, YEL medium, LB medium, and the like. In addition to the nutrients described above, the medium may contain substances for promoting the growth of microorganisms such as vitamins, nucleic acids, metal salts, phosphorus, trace elements and the like. Furthermore, there are cases where microorganisms consume ethanol, acetic acid, etc. produced by metabolizing the carbon source as a carbon source.

[culture]
In the culture method of the present invention, the culture tank for culturing the microorganism is not particularly limited. The culture apparatus preferably has a function of keeping the temperature of the culture solution constant and stirring the culture solution. The optimal culture temperature varies depending on the type of microorganism, the purpose of culture, and the like, and thus the culture temperature can be appropriately determined, but is preferably 20 to 40 ° C, more preferably 25 to 37 ° C.

[Microorganisms]
The culture method of the present invention can be applied to any microorganism. In the present invention, a microorganism refers to a unicellular organism that can be cultured in a liquid medium. Preferably, yeasts such as Saccharomyces genus, Shizosaccharomyces genus, Hansenula genus, Pichia genus, Ogataea genus, Candida genus, Aspergillus genus, etc. These are microorganisms that have been industrially mass-cultured, such as filamentous fungi such as the genus Trichoderma and Escherichiacoli. The culture method of the present invention can also be applied to a transformant in which a gene encoding a heterologous protein is introduced into the microorganisms in the present specification.

  Hereinafter, the present invention will be described in more detail with reference examples and examples, but the present invention is not limited to these reference examples and examples.

[Reference Example 1: Correspondence between pH and OD ₆₆₀ ]
(Seed I culture)
In 270 mL of YEL medium (Bacto Yeast Extract 5 g / L, glucose 30 g / L, manufactured by Becton Dickinson) in a 2 L Sakaguchi flask, 5.4 mL of frozen cell stock of a fission yeast S. pombe heterologous protein expression strain (ATCC38399 mutant strain) was added. The mixture was cultured at 30 ° C. for 24 hours while shaking at a speed of 100 to 110 rpm.

(Seed II culture)
Next, the total amount of the medium containing fission yeast after 24 hours of culture was added to 13.23 L of YEL medium in a 30 L culture tank manufactured by Able, and cultured at 30 ° C. with stirring at 250 rpm. PH measurement during culture is performed using a BROADLEY JAMES fermentation pH electrode (Product No .: F-635-B-120-DH) and attached to the culture tank so that the liquid junction at the tip of the pH electrode is immersed in the medium. The time course of pH was measured online in real time. At that time, the pH of the medium, dissolved oxygen concentration, exhaust CO ₂ , OD ₆₆₀ , and glucose concentration were measured over time. The obtained results are shown in FIGS.

  As apparent from FIG. 1, the pH in the medium continued to decrease from the beginning of the culture, but gradually decreased after a lapse of 10 hours from the start of the culture, and began to increase after the pH change reached a plateau. As is clear from FIG. 2, the time when the pH change reached a plateau corresponded to the time when the growth phase of the cells hit the latter half of the exponential growth phase (point A). In addition, the online measurement of dissolved oxygen concentration is difficult to use as an indicator of the growth phase of bacterial cells because of the large fluctuations in the measurement in which the rise and fall of the value are repeated in a short time compared to the measurement value of pH. it is conceivable that.

[Reference Example 2: Correspondence between planting time (OD ₆₆₀ ) and growth rate rise]
(Seed I culture)
2.7 mL of frozen cell stock of fission yeast S. pombe heterologous protein expression strain (ATCC38399 mutant) in 135 mL of YEL medium (Bacto Yeast Extract 5 g / L, glucose 30 g / L, manufactured by Becton Dickinson) in a 1 L Sakaguchi flask And cultured at 30 ° C. for 24 hours while shaking at a speed of 110 to 120 rpm.

(Seed II culture)
Next, 30 mL of the medium containing fission yeast after 24 hours of culture described above was added to 1470 mL of YEL medium in a 2 L culture tank manufactured by Maruhishi Bio-Engine, and cultured at 30 ° C. while stirring at 700 rpm. . At that time, OD ₆₆₀ and glucose concentration were measured over time.

(Planting, main culture)
In the seed II culture, 150 mL of a medium containing fission yeast was collected at each time point (points D, E, and F) shown in FIG. 3 and completely contained in a separate 3 L culture tank manufactured by Maruhishi Bioengine. in addition to the synthetic medium (SMF23 medium + amino acids + compensation vitamins) 1353mL, and incubated with stirring at 30 ° C. in _{250～800Rpm,} we investigated the growth of bacteria by measuring the _{OD 660.} The obtained results are shown in FIG.

As apparent from FIG. 3 and FIG. 4, when the cells are transplanted at the later stage of the exponential growth phase (Point E), when the transplantation is performed at the middle stage of the exponential growth phase (Point D), and , Compared to the case of transplanting 24 hours after the start of seed II culture (point F), the growth rate of the cell growth rate after transplanting was faster, and the time difference at which OD ₆₆₀ reached 10 was more than 5 hours . In addition, when planting was carried out 24 hours after the start of seed II culture (point F), the start of cell growth was slower than point D. This is considered to be because it was in the stationary phase when the cell growth stopped after 24 hours. On the other hand, the rise of point E is faster than point D because the cell concentration of seed II culture is higher at the point E.

[Reference Example 3: Correspondence between pH and OD ₆₆₀ when using fission yeast wild type strain]
(Seed I culture)
Into 5.6 mL of YEL medium (Bacto Yeast Extract 5 g / L, glucose 30 g / L, manufactured by Becton Dickinson) in an L-shaped test tube, 224 μL of a frozen cell stock of fission yeast S. pombe wild strain (JY1 strain) was added. The mixture was cultured at 30 ° C. for 24 hours while shaking at a speed of 95 to 105 rpm.
(Seed I culture)
To 270 mL of YEL medium (Bacto Yeast Extract 5 g / L, glucose 30 g / L, manufactured by Becton Dickinson) in a 2 L Sakaguchi flask, 5.4 mL of the medium containing fission yeast after 24 hours of culture was added, The cells were cultured at 30 ° C. for 24 hours while shaking at a speed of 95 to 105 rpm.

(Seed III culture)
Next, the total amount of the medium containing fission yeast after 24 hours of culture was added to 13.23 mL of YEL medium in a 30 L culture tank manufactured by Able, and cultured at 30 ° C. with stirring at 250 rpm. PH measurement during culture is performed using a BROADLEY JAMES fermentation pH electrode (Product No .: F-635-B-120-DH) and attached to the culture tank so that the liquid junction at the tip of the pH electrode is immersed in the medium. The time course of pH was measured online in real time. At that time, the pH, dissolved oxygen concentration, OD ₆₆₀ , and glucose concentration of the medium were measured over time. The obtained results are shown in FIGS.

  As is clear from FIG. 5, the pH in the medium continued to decrease from the beginning of the culture, but gradually decreased after a lapse of 10 hours from the start of the culture, and began to increase after the pH change reached a plateau. As is clear from FIG. 6, the time when the pH change reached a plateau corresponded to the time when the growth phase of the cells hit the latter half of the exponential growth phase (point indicated by an arrow).

[Reference Example 4: Correspondence between pH and OD ₆₆₀ when using a heterologous protein-expressing yeast strain]
(Seed I culture)
Expression of heterologous protein of fission yeast S. pombe in 5 mL each of YEL + G10 medium (Bacto Deast, Bacto Yeast Extract 5 g / L, glucose 30 g / L, G418 10 mg / L) contained in two L-shaped test tubes 200 μL of each frozen cell stock of a protease-disrupted strain (ATCC38399 mutant strain) was added and cultured at 30 ° C. for 24 hours while shaking at a speed of 95 to 105 rpm.
(Seed II culture)
To 400 mL of YEL + G10 medium in a 2 L Sakaguchi flask, add 8 mL (4 mL × 2) of the medium containing fission yeast after 24 hours of culture, and shake at 30 ° C. while shaking at a speed of 95 to 105 rpm. For 24 hours.

(Seed III culture)
Next, the total amount of the medium containing fission yeast after 24 hours of culture was added to YPD + G100 medium (Bacto Yeast Extract 10 g / L, Bacto Tryptone 20 g / L, Bacto Tryptone 20 g / L, glucose 20 g) in a 30 L culture tank manufactured by Able. / L, G418 100 mg / L) In addition to 19.6 L, the mixture was cultured at 30 ° C. with stirring at 300 rpm. PH measurement during culture is performed using a BROADLEY JAMES fermentation pH electrode (Product No .: F-635-B-120-DH) and attached to the culture tank so that the liquid junction at the tip of the pH electrode is immersed in the medium. The time course of pH was measured online in real time. At that time, the pH, dissolved oxygen concentration, OD ₆₆₀ , and glucose concentration of the medium were measured over time. The obtained results are shown in FIGS.

  As is clear from FIG. 7, the pH in the medium continued to decrease from the beginning of the culture, but after 30 hours had elapsed since the start of the culture, the decrease became gentle and the pH change reached a plateau. Further, as is clear from FIG. 8, the time point at which the pH change reached a plateau corresponded to the time point when the growth phase of the cells hit the latter stage of the exponential growth phase (point indicated by an arrow). In addition, the online measurement of dissolved oxygen concentration is difficult to use as an indicator of the growth phase of bacterial cells because of the large fluctuations in the measurement in which the rise and fall of the value are repeated in a short time compared to the measurement value of pH. it is conceivable that.

[Reference Example 5: Correspondence between pH and OD ₆₆₀ when using an organic acid-producing yeast strain]
(Seed I culture)
YES medium (Bacto Yeast Extract 5 g / L, adenine sulfate 50 mg / L, uracil 50 mg / L, L-leucine 50 mg / L, L-histidine hydrochloride monohydrate each contained in two test tubes 50 mg / L, L-lysine monohydrate 50 mg / L, glucose 30 g / L) In each 5 mL, one colony of an organic acid producing strain (ATCC38399 mutant) of fission yeast S. pombe from an agar plate The cells were inoculated and cultured at 32 ° C. for 24 hours while shaking at a speed of 110 to 120 rpm.

(Seed II culture)
7 mL of the medium containing fission yeast after 24 hours of culture was added to 350 mL of YES medium in a 1 L Sakaguchi flask, and cultured at 32 ° C. for 24 hours while shaking at a speed of 110 to 120 rpm. .

(Seed III culture)
Next, the total amount of the medium containing fission yeast after 24 hours of culturing was added to YAD12 medium (Bacto Yeast Extract 10 g / L, CSL-AST 20 g / L, glucose, Bacton Dickinson) in a 5 L culture tank manufactured by Able. In addition to 3500 mL of 120 g / L), the cells were cultured at 30 ° C. while cascade-controlling the number of revolutions of stirring so that the lower limit value of the dissolved oxygen concentration could be maintained at 1 ppm. PH measurement during culture is performed using a BROADLEY JAMES fermentation pH electrode (Product No .: F-635-B-120-DH) and attached to the culture tank so that the liquid junction at the tip of the pH electrode is immersed in the medium. The time course of pH was measured online in real time. At that time, the pH, dissolved oxygen concentration, OD ₆₆₀ , and glucose concentration of the medium were measured over time. The obtained results are shown in FIGS.

  As apparent from FIG. 9, the pH in the medium continued to decrease from the beginning of the culture, but after 30 hours passed from the start of the culture, the decrease gradually decreased, the pH change reached a plateau, and then began to increase. Further, as is clear from FIG. 10, the time when the pH change reached a plateau corresponded to the time when the growth phase of the cells hit the latter half of the exponential growth phase (point indicated by an arrow).

[Reference Example 6: Correspondence between pH and OD ₆₆₀ when using E. coli strain expressing heterologous protein]
(Seed I culture)
To 100 mL of LB medium (Bacton Yeast Extract 10 g / L, Bacto Tryptone 10 g / L, NaCl 5 g / L, glucose 1 g / L, tryptophan 100 mg / L) in a 0.5 L Sakaguchi flask. 75 μL of a frozen cell stock of a heterologous protein expression strain of Escherichia coli (K12 mutant) was added and cultured at 37 ° C. for 22 hours while shaking at a speed of 120 to 130 rpm.

(Seed II culture)
Next, 30 mL of the medium containing Escherichia coli after culturing for 22 hours was added to 600 mL of LB medium contained in a 2 L culture tank manufactured by Maruhishi Bioengine, and cultured at 37 ° C. while stirring at 420 rpm. For pH measurement during culture, use a METTLER TOLEDO pH electrode (product number: 405-DPAS-SC-K8S) and attach it to the culture tank so that the liquid junction at the tip of the pH electrode is immersed in the medium. The time course was measured in real time online. At that time, the pH, dissolved oxygen concentration, OD ₆₀₀ and glucose concentration of the medium were measured over time. The obtained results are shown in FIGS.

  As is clear from FIG. 11, the pH in the medium continued to decrease from the beginning of the culture, but the pH change began to increase after 3 hours had elapsed since the start of the culture. Further, as is clear from FIG. 12, the time when the pH change started to increase corresponded to the time when the growth phase of the cells hit the latter half of the exponential growth phase (point indicated by an arrow). In addition, the online measurement of dissolved oxygen concentration is difficult to use as an indicator of the growth phase of bacterial cells because of the large fluctuations in the measurement in which the rise and fall of the value are repeated in a short time compared to the measurement value of pH. it is conceivable that.

[Example 1]
(Seed I culture)
2.7 mL of frozen cell stock of fission yeast S. pombe heterologous protein expression strain (ATCC38399 mutant) in 135 mL of YEL medium (Bacto Yeast Extract 5 g / L, glucose 30 g / L, manufactured by Becton Dickinson) in a 1 L Sakaguchi flask And cultured at 30 ° C. for 12 hours while shaking at a speed of 110 to 120 rpm. Two seed I cultures were prepared under the same conditions. They are called lot 1-1 and lot 1-2, respectively. The OD ₆₆₀ of lot 1-1 immediately after the start of seed I culture is 0.381, and the OD ₆₆₀ of lot 1-2 is 0.336, and the OD of lot 1-1 at 12 hours after the start of culture. ₆₆₀ was 7.7, and the OD ₆₆₀ of lot 1-2 was 7.3.

(Seed II culture)
Next, 30 mL of the medium containing fission yeast after culturing for 12 hours is added to 1470 mL of YEL medium in a 2 L culture tank manufactured by Maruhishi Bioengineer, and cultured at 30 ° C. while stirring at 700 rpm. did. At that time, the pH, glucose concentration, and OD ₆₆₀ of the medium were measured over time. The results are shown in FIG. 13 (Lot 1-1) and FIG. 14 (Lot 1-2).

(Planting, main culture)
In the seed II culture, 150 mL of the medium containing fission yeast was collected at each time point (points G and H) shown in FIG. 13 or FIG. 14 and entered into a separate 3 L culture tank manufactured by Maruhishi Bioengine. in addition to the full-synthetic medium (SMF23 medium + amino acids + compensation vitamins) 1353mL, and incubated with stirring at 30 ° C. in _{250～800Rpm,} we investigated the growth of bacteria by measuring the _{OD 660.} The obtained result is shown in FIG.

As is clear from FIG. 15, when transplanting is performed when the pH change reaches a plateau (point H), transplanting is performed 24 hours after the start of seed II culture (point G). The rise of the cell growth was faster than that, and the time difference between the two conditions until the OD ₆₆₀ reached 10 was 10 hours.

[Example 2]
(Seed I culture)
2.7 mL of frozen cell stock of fission yeast S. pombe heterologous protein expression strain (ATCC38399 mutant strain) in 135 mL of YEL medium (Bacto Yeast Extract 5 g / L, glucose 30 g / L, manufactured by Becton Dickinson) in a 1 L Sakaguchi flask And cultured at 30 ° C. for 24 hours while shaking at a speed of 110 to 120 rpm. Two seed I cultures were prepared under the same conditions. These are referred to as lot 2-1 and lot 2-2, respectively. Note that the OD ₆₆₀ of lot 2-1 immediately after the start of seed I culture is 0.348, and the OD ₆₆₀ of lot 2-2 is 0.329, and the OD of lot 2-1 at 24 hours after the start of culture. ₆₆₀ was 20.3, and the OD ₆₆₀ of lot 2-2 was 19.7.

(Seed II culture)
Next, 30 mL of the medium containing fission yeast after culturing for 24 hours is added to 1470 mL of YEL medium in a 2 L culture tank manufactured by Maruhishi Bioengineer, and cultured at 30 ° C. while stirring at 700 rpm. did. At that time, the pH, glucose concentration, and OD ₆₆₀ of the medium were measured over time. The results are shown in FIG. 16 (lot 2-1) and FIG. 17 (lot 2-2).

(Planting, main culture)
In the seed II culture, 150 mL of the medium containing fission yeast was collected at each time point (points I and J) shown in FIG. 16 or FIG. 17 and entered into a separate 3 L culture tank manufactured by Maruhishi Bioengineering Co., Ltd. in addition to the full-synthetic medium (SMF23 medium + amino acids + compensation vitamins) 1353mL, and incubated with stirring at 30 ° C. in _{250～800Rpm,} we investigated the growth of bacteria by measuring the _{OD 660.} The obtained result is shown in FIG.

As is clear from FIG. 18, when transplanting is performed when the pH change reaches a plateau (point J), the transplanting is performed 24 hours after the start of seed II culture (point I). The rise of the cell growth was quicker than that, and the difference in time until the OD ₆₆₀ reached 10 between both conditions was nearly 8 hours.

Claims (9)

  A method of culturing microorganisms in a liquid medium, the step of measuring the pH of the culture solution over time and planting the microorganisms in another liquid medium after the pH value changes to a plateau A method for culturing microorganisms, comprising:   A method for culturing a microorganism in a liquid medium, wherein the pH of the culture solution is measured over time, and the slope of the pH change curve plotted with the pH value on the vertical axis and the culture time on the horizontal axis is a negative value A method for cultivating microorganisms, comprising a step of planting microorganisms in another liquid medium after the time point of zero.   A method for culturing a microorganism in a liquid medium, wherein the pH of the culture solution is measured over time, the change in pH value reaches the inflection point, and the microorganism is removed after the direction of change is reversed. A method for culturing microorganisms comprising a step of planting in another liquid medium.   The method for cultivating a microorganism according to claim 1, wherein the step of planting the microorganism in another liquid medium is performed from the time when the pH value changes to a plateau to the time when the pH changes to 0.05.   The method for culturing microorganisms according to any one of claims 1 to 4, wherein the liquid medium is a rich medium.   The culture method according to any one of claims 1 to 5, wherein the culture temperature is 20 to 40 ° C.   Yeast fungus selected from the group consisting of the genus Saccharomyces, Shizosaccharomyces, Hansenula, Pichia, Ogataea and Candida The method for culturing a microorganism according to any one of claims 1 to 6, which is a filamentous fungus selected from the group consisting of the genus Aspergillus and the genus Trichoderma, or Escherichiacoli.   The method for culturing a microorganism according to any one of claims 1 to 7, wherein the microorganism is a yeast of the genus Shizosaccharomyces or Escherichiacoli.   The method for culturing a microorganism according to any one of claims 1 to 8, wherein the microorganism is fission yeast Shizosaccharomyces pombe.

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