JPS6214270B2 - - Google Patents

Abstract

PURPOSE:To improve the microbial cell production efficiency of yeast, by controlling properly the flow addition rate of a substrate in cultivation. CONSTITUTION:A seed yeast is put into a cultivation tank 1, and a substrate is fed from a substrate tank 7 thereinto by a substrate feed pump 8. Data from an inlet oxygen partial pressure measuring instrument 5, inlet gas flow rate measuring instrument 6, oxygen partial pressure measuring instrument 11 of the outlet gas, gaseous carbon dioxide partial pressure measuring instrument 12 and waste gas flow rate measuring instrument 13 are input to a controlling electronic computer 4 to calculate the amount of yeast cells in the tank 1 from the multiplication model calculation based on the oxygen balance, carbon balance and substrate feed rate. The substrate flow addition rate is then obtained by considering the alpha value (substrate flow addition rate based on the amount of yeast cell), and the substrate feed pump 8 is operated according to the rate. The respiratory quotient (RQ) and ratio of the outlet gas flow rate to the inlet gas flow rate (beta value) are obtained, and the alpha value is changed according to the values to control the substrate feed rate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a novel microorganism aerobic culture control device.

[Background of the invention]

In the production of yeast cells such as food, feed yeast, and baker's yeast, a fed-batch culture method is used in which a substrate is supplied little by little during culture. In order to carry out this culture efficiently, the fed substrate must be completely consumed by the yeast.
In addition, it is essential that by-products such as ethanol be small.

Conventionally, since there is no means to rapidly measure the concentration of bacterial cells or the activity of bacterial cells during culture, a method has been used in which substrates are added to a culture tank according to a predetermined program. In this method, it was not possible to feed the substrate in accordance with the bacterial activity in the tank, and the productivity was not high.

[Purpose of the invention]

An object of the present invention is to provide an apparatus that makes it possible to improve yeast cell production efficiency by appropriately controlling the amount of substrate fed during culture.

[Summary of the invention]

The present invention detects by-products generated during the culture process by using the substrate flow acceleration per bacterial cell amount (specific current acceleration, referred to as α value) as an index for substrate addition to the culture tank,
The method is characterized by having a control device that increases the α value at a predetermined rate when no by-product is generated, and decreases the α value at a rate larger than the predetermined rate when a by-product is generated. It is something.

Next, control of substrate feeding based on the present invention will be explained in detail. First, the α value is expressed by the following equation.

α=FS ₀ /VX ………(1) Here, F = flow rate (/h), S ₀ = substrate concentration (g/), V = culture solution volume (), X = bacterial cell concentration (g/ ) From the equation, the flow acceleration F can be set by determining α, V, and X using the following equation.

F=α·VX/S ₀ (2) The α value, that is, the substrate flow acceleration per amount of bacterial cells, changes depending on the activity of the yeast used. In the case of baker's yeast,
If the activity is high, yeast can grow on the ingested substrate even if the α value is increased and the substrate flow rate is increased, but if the activity is low, the ingested substrate will be converted to ethanol and CO ₂ due to the glucose effect, reducing productivity. do.

The activity of yeast is the ability to completely consume the fed substrate and produce cells without producing byproducts such as ethanol, so there is no way to directly measure it, and in the end it is the ability to produce bacterial cells without producing byproducts such as ethanol. It is necessary to feed the substrate to prevent the formation of On the other hand, there is a method of estimating the activity of yeast using the oxygen consumption rate of yeast as an index, but this alone is insufficient because the enzyme consumption rate is closely related to the substrate flow acceleration and changes depending on the substrate flow acceleration. be.

Therefore, in the present invention, an initial value of α is set at the start of culture, a substrate is fed at a flow rate corresponding to the α value, and control is performed to increase the α value if no by-products are generated. In this case, the amount of increase in the α value is a very big problem, but as a result of the study of the species, the present inventors set the α value at a value of 20% or less, preferably in increments of 10%, as a value that is compatible with changes in the physiological activity of yeast. found increasing control.

The reason why the α value is increased below 20% is because if it is larger than this, the substrate feeding becomes excessive.
On the other hand, if the increase rate is too small, the supply of substrate to the bacterial cells will be insufficient, making it impossible to improve bacterial cell productivity. From now on, the desired increase rate will be set at 10%.

Next, when by-products are produced, unless the substrate flow acceleration is reduced, the by-products will continue to be produced, resulting in a decrease in cell yield and cell productivity. However, even if the α value was lowered at the same rate as the increase, the generation of byproducts could not be stopped. This is because the pathway for substrate decomposition and bacterial growth is different from the pathway for byproduct production, and once the enzyme activity in the byproduct production pathway is high, even if the substrate feeding is slightly reduced, the flow of metabolites will be reduced. This is thought to be due to the direction of by-product production. From now on, the degree of decrease in the substrate feeding rate will become important, and as a result of our study, we found that if the decrease rate in the α value is set to 30% or more, preferably 50%, the production of byproducts will stop and It was discovered that by-products produced in the culture solution can be reused by yeast.

Experimental examples in which various substrate fed-batch methods were investigated using baker's yeast are shown in FIGS. 1, 2, and 3. Here, the respiratory quotient (RQ) is measured from the 1st or 2nd hour of culture.
Started feedback control by α every 15 minutes.
The substrate flow acceleration was controlled by changing the value.

In Figure 1, the α value was increased by 10% and decreased by 10%. When the control range of RQ was set to 0.8 to 1.0 mol/mol, RQ exceeded 1.0 mol/mol when about 1 g/mol of ethanol was produced. From this point on,
Although the α value was lowered by 10%, ethanol production did not stop and 15.4 g/ethanol was produced. In this way, once ethanol is produced, the fermentation physiology of yeast is in a state suitable for ethanol production, and α
If the range of decrease in the value is small, it may be impossible to immediately change the physiological state in favor of bacterial cell proliferation.

In Figure 2, the α value was increased by 20% and decreased by 20%. When the control range of RQ was set to 0.8 to 1.0 mol/mol, when 8.4 g/mol of ethanol was produced,
RQ exceeded 1.0 mol/mol. The reason why the ethanol production is larger than in the case of FIG. 1 is thought to be due to the increase in the α value. From now on, the α value is
Ethanol production continues to decrease by 20% to 14.7
g/g of ethanol was produced.

In Figure 3, the α value was increased by 50% and decreased by 50%. When the control range of RQ was set to 0.9 to 1.0 mol/mol, RQ exceeded 1.0 mol/mol when about 1.5 g/mol of ethanol was produced. Since then, the α value has been raised and lowered repeatedly, but ethanol production did not stop and 10.5g/ethanol was produced. Also, RQ was hunting.

From the above, it was found that if the increase rate of α value is set to 50%, RQ will hunt, so it is better to set it to 20% or less. However, since the amount of ethanol produced is large at 20%, 10% was the optimal increase rate. On the other hand, when the reduction rate of the α value was set to 20% or less, it was not possible to suppress the production of ethanol.

In FIGS. 1 and 2, ethanol was produced even when the α value did not change, and FIG. 4 shows the results of an experiment to confirm this phenomenon. α value in RUN1
0.3g/g・h, 0.39g/g・h in RUN2,
In RUN 3, when feeding was carried out at a nearly constant rate of 0.42 g/g·h, ethanol was produced from the 5th hour, 7th hour, and 11th hour in descending order of α value. This means that even if the α value is kept constant and ethanol is fed, the physiological activity of the yeast changes and ethanol is produced. The reason why ethanol production does not stop simply by returning to the α value before production is thought to be because there is a time delay in the expression of changes in yeast physiological activity, that is, in ethanol production.

Next, the results of examining the rate of decrease in the α value are shown in the fifth section.
As shown in the figure. Culture was started in a state where 12.7 g/ethanol was present at the initial stage of culture. In this case as well, as in Figure 2, when RQ exceeded 1.0 mol/mol, the α value was reduced by 20%, but ethanol could not be reduced. When the α value was lowered by 50% at the 4th hour of culture, ethanol decreased rapidly, and after 20
Even though the α value was increased by 1.5%, the amount of ethanol could be reduced to 2g/%. From the above, if the α value reduction rate is set to 20%, it is possible to prevent an increase in the ethanol concentration in the culture solution, but it is not possible to reduce ethanol. However, it was revealed that when the reduction rate was set to 50%, the physiological activity of the yeast was changed, suppressing ethanol production and causing ethanol consumption. From this, it can be seen that it is better to reduce the α value by 30% or more, preferably by 50%.

Figure 6 shows data when the α value is increased by 10% and decreased by 30%, and the reduction rate is 30%.
Although the effect is recognized even when the amount is reduced by 50%, it is understood that it is slightly inferior to the case where the amount is reduced by 50%.

As described above, in order to feed the substrate to suit the physiological activity of yeast, the present inventors increased the α value by 20% or less, preferably by 10%, and by-products were reduced. We have found that it is effective to reduce the α value by at least 30%, preferably by 50%, in order to shift the physiological state of the yeast from production of byproducts to only bacterial cell growth. .

To carry out the present invention, it is necessary to measure the amount of bacterial cells in the culture tank, but at present there is no effective method for directly measuring the amount of bacterial cells. Therefore, the present inventors (1)
We found that the amount of bacterial cells can be estimated well using oxygen balance, (2) carbon balance, and (3) growth model.

Estimation of bacterial mass based on oxygen balance is expressed by the following formula.

ΔX=(a ₂・Δs−ΔO ₂ −a ₃ ΔP) a ₁
………(3) ΔX = bacterial cell growth amount (g), Δs = substrate feeding amount (g), ΔO ₂ = oxygen consumption amount (mol), ΔP = amount of by-products (g), a ₁ = bacterial cell Amount of oxygen required for complete combustion of by-products (mol/g), a ₂ = Amount of oxygen required for complete combustion of substrate (mol/g), a ₃ = Amount of oxygen required for complete combustion of by-products (mol/g) ).

When estimating the amount of bacterial cells using equation (3), the amount of oxygen required for combustion of the substrate is known, and by-products are excluded from the calculation because they are produced in small quantities, and the amount of oxygen consumed by the bacterial cells is equal to the inlet gas. This is possible because it is determined from the difference in the amount of oxygen in the outlet gas.

Estimation of bacterial mass based on carbon balance is expressed by the following formula.

ΔX=(b ₂・Δs−12・ΔCO ₂ −b ₃・ΔP)/b ₁
...(4) ΔCO ₂ = Carbon dioxide gas production (mol), b ₁ = Carbon content of bacterial cells (g/g), b ₂ = Carbon content of substrate (g/g), b ₃ = By-product Carbon content (g/
g).

Estimating the amount of bacterial cells using equation (4) is possible because by-products are excluded from the calculation because they are produced in small quantities, the amount of carbon in the substrate is known, and the amount of carbon dioxide in the exhaust gas can be measured.

Estimation of bacterial mass using a growth model is expressed by the following equation.

d(VX)/dt=μVX……(5) μ= _μnax・S/K _S +S……(6) d(VS)/dt=FS ₀ −1/Y _G μVX−mVX…… (7
) dV/dt=F (8) S = substrate concentration in liquid (g/), μ = specific growth rate (1/h), μ _nax = maximum specific growth rate (1/h), Y
_G = cell yield constant for substrate (g/g), m = maintenance constant for substrate (g/g), K _S = saturation constant (g/), t = time (h).

After entering the initial liquid volume V ₀ , initial bacterial cell concentration X ₀ and coefficients μ _nax , K _S , Y _G , and m into this equation, the amount of bacterial cells can be calculated by entering the measured value of the substrate flow acceleration F. .

As mentioned above, the amount of bacteria can be estimated using the oxygen balance, carbon balance, and growth model, but in actual use, it is possible to estimate the amount of bacteria more accurately by combining and averaging each of them. .

To perform optimal substrate feeding, it is necessary to detect the formation of by-products, and this detection can be done by using the respiratory quotient (RQ), which is the ratio of Q _CO2 to Q _O2 , or the flow rate ratio of the outlet gas to the inlet gas. It has been found that volatile by-products in exhaust gas, such as ethanol, can be detected well by a method using a gas chromatograph or the like (referred to as β value).

In yeast culture using sugar as a substrate, RQ = 1.0 indicates complete aerobic cell growth, and RQ > 1.0 indicates ethanol production due to the large amount of carbon dioxide gas produced, and RQ < 1.0. In the case of , it indicates that the amount of substrate is insufficient or that the yeast is consuming the produced ethanol. Therefore, if RQ exceeds 1.0, it is assumed that ethanol has been produced, and the α value is decreased, and if RQ is significantly lower than 1.0, it is assumed that there is a shortage of substrate, and the α value is increased.

The reason why ethanol production can be detected by the ratio of the outlet gas amount to the inlet gas amount (β value) is that when ethanol is produced, the outlet gas flow rate increases than the inlet gas flow rate due to the large amount of CO ₂ produced. discovered by the inventors. This method is extremely effective since it is not necessary to measure O ₂ and CO ₂ concentrations as is the case when using RQ.

One way to measure volatile components in the outlet gas is to directly measure by-products such as ethanol. This is because by-products discharged into the culture solution volatilize into the exhaust gas, which is measured using a gas chromatograph, semiconductor sensor, or the like.

Substrates used in the present invention include glucose, fructose, sucrose, and industrial raw materials such as molasses. Further, as auxiliary raw materials, there are ammonium sulfate, urea, aqueous ammonia, potassium phosphate, yeast extract, magnesium sulfate, ferrous sulfate, and various vitamins and minerals used in normal culture.

FIG. 7 is a configuration diagram of the culture apparatus of the present invention. Inoculum is placed in a culture tank 1, and a substrate is supplied from a substrate tank 7 by a substrate supply pump 8. At this time, an inlet oxygen partial pressure measuring device 5, an inlet gas amount measuring device 6, an outlet gas oxygen partial pressure measuring device 11, a carbon dioxide gas partial pressure measuring device 1
2. Enter the data from the exhaust gas amount measuring device 13 into the control electronic computer 4, calculate the amount of bacterial cells in the tank from the growth model calculation based on the oxygen balance, carbon balance, and substrate supply rate, and calculate the α value from this. The substrate flow acceleration is determined by , and the substrate supply pump 8 is operated according to the determined speed. On the other hand, the substrate supply rate is controlled by determining RQ, the ratio of the outlet gas amount to the inlet gas amount (β value), and changing the α value accordingly. During culture, it is necessary to maintain the dissolved oxygen concentration at 2 mg/min or more.
This converts the signal from the dissolved oxygen sensor 9 to the dissolved oxygen meter 10.
and sends the value to the control computer 4,
The dissolved oxygen concentration is maintained at a constant value by controlling the rotation speed with the stirrer 2 and the inlet gas oxygen concentration and inlet gas amount with the oxygen separator 3.

FIG. 8 is a configuration diagram of another culture apparatus of the present invention. This is a method that performs substrate feeding control by installing a volatile component measuring device 18 in the exhaust gas line, and increasing the α value when volatile components that are by-products are not detected, and lowering the α value when they are detected. It is. The configuration other than the exhaust gas line is the same as the device shown in FIG.

[Embodiments of the invention]

Example 1 Yeast was cultured aerobically using the culture apparatus shown in FIG.

Bacterial body: Saccharomyces cerevisiae (baker's yeast) Medium: glucose 500g, urea 53.75g,
_{Na2HPO4 ・ 2H2O25g} , _MgSO4・_7H2O9.5g ,
KCl 5.5g, sodium citrate 62.5g, yeast extract 12.2g, vitamin solution 25ml and mineral solution 25ml
was added to tap water 1, dissolved, and adjusted to pH 5.0.

However, the vitamin liquid contains 0.04g of biotin and vitamin
B ₁ 0.08g, vitamin B ₆ 2.0g, calcium pantothenate 1.0g and inositol 20g in distilled water 1
Mineral liquid is prepared by dissolving _CuSO4 .
_5H2O0.05g , _ZnSO4・_7H2O0.8g and _FeSO4
It was prepared by dissolving 0.3 g of (NH ₄ ) ₂ ·6H ₂ O in 1 part of distilled water.

Culture conditions: Using a 50 jar fermenter, temperature 30℃, pH 5.0, dissolved oxygen concentration, stirrer rotation speed,
4 to 6 mg/depending on the oxygen partial pressure of the ventilation gas and the amount of ventilation
was controlled within the range of . Note that the oxygen partial pressure of the ventilation gas was changed using an air compressor and an oxygen cylinder. The tank internal pressure was controlled to 0.5 Kg/cm ² G, and the exhaust gas carbon dioxide concentration was controlled within 20%. The initial liquid volume is 15, the initial bacterial concentration is 50 g/, and the α value is 0.3 g/
I made it g.h. For the amount of bacterial cells in the culture tank, the average value of the amount of bacterial cells determined from the oxygen balance and carbon balance was used. The coefficients at this time are a ₁ = 0.042, a ₂ = 0.033, b ₁ =
0.47, b ₂ =0.40 was used. The production of ethanol, a by-product, is detected by the RQ value, and RQ<
When 0.8mol/mol, increase α value by 10% and RQ>
At 1.0 mol/mol, the α value was reduced by 50%.

Results: Throughout 12 hours of culture, the ethanol concentration could be reduced to 3.4 g/lower as shown in Figure 9. Moreover, as shown in FIG. 10, the estimated value of the amount of bacterial cells almost matched the actual value. As a result, the bacterial cell concentration reached a high concentration of 104g/, and the bacterial cell yield was
It was 0.43g/g. The final culture solution volume is 25.5
It was hot.

Example 2 Strain: Saccharomyces cerevisiae (baker's yeast) Medium, equipment: Same as Example 1.

Culture conditions: The culture was carried out in the same manner as in Example 1, except that the amount of bacterial cells in the culture tank was estimated using a growth model. In addition, as a coefficient of the proliferation model, μ _nax =
0.371/h, K _S =0.0648g/, Y _G =0.52g/
g, m=0.025 g/g was used.

Results: Ethanol concentration throughout 10 hours of culture
It was possible to suppress the amount within the range of 0.15 to 4.2 g/. As a result, the bacterial cell concentration reached a high concentration of 100 g/g, and the bacterial cell yield was 0.43 g/g. Note that the final culture solution volume was 24.3.

Example 3 Strain: Saccharomyces cerevisiae (baker's yeast) Medium, equipment: Same as Example 1.

Culture conditions: ethanol production is detected by the ratio of the outlet gas amount to the inlet gas amount (β value), and β<
When β was 0.95, the α value was increased by 10%, and when β<1.0, the α value was decreased by 50%. The conditions other than these were the same as in Example 1.

Result: The ethanol concentration was kept at 1 throughout 10 hours of culture.
We were able to keep it below g/g. As a result, the bacterial cell concentration reached a high concentration of 105g/, and the bacterial cell yield decreased.
It was 0.44g/g. The final culture solution volume is 24.1
It was hot.

Example 4 Strain: Saccharomyces cerevisiae (baker's yeast) Medium, equipment: Same as Example 1.

Culture conditions: The outlet gas was introduced into a gas chromatograph to detect ethanol production, and when ethanol was not produced, the α value was increased by 10%, and when the ethanol concentration in the gas exceeded 1 ppm, the α value was decreased by 50%. .
Other conditions were the same as in Example 2.

Results: Ethanol concentration throughout 12 hours of culture
We were able to keep it below 1.4g/. As a result, the bacterial cell concentration reached a high concentration of 106 g/g, and the bacterial cell yield was 0.45 g/g. In addition, the final culture solution volume is
It was 24.4.

The present invention enables substrate fed-batch control with reduced production of by-products, resulting in high bacterial cell yield and 100%
It has the effect of achieving high bacterial cell concentration culture of 1.5 g/g/g/g or more, and can improve the productivity of the culture tank.

〔Effect of the invention〕

According to the present invention, the efficiency of yeast cell production can be improved.

[Brief explanation of the drawing]

Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, and Fig. 6 are diagrams showing changes in α value and ethanol production, and Fig. 7 and Fig. 8 are examples of the culture apparatus of the present invention. FIG. 9 is a diagram representing a culture example of the present invention.
Figure 0 is a diagram showing an example of bacterial cell estimation according to the present invention. 1... Culture tank, 1... Stirrer, 3... Oxygen separator, 4... Control electronic computer, 5... Oxygen partial pressure measuring device, 6... Inlet gas amount measuring device, 7... Substrate tank , 8... Substrate supply pump, 9... Dissolved oxygen sensor, 10... Dissolved oxygen meter, 11... Oxygen partial pressure measuring device, 12... Carbon dioxide gas partial pressure measuring device, 13... Exhaust gas amount measuring device, 14 , 15, 16, 17... conduit,
18...Volatile component measuring device.

Claims (1)

[Claims] 1. A gas flow rate measuring device for measuring the inlet gas flow rate, an oxygen and carbon dioxide gas analyzer for measuring the oxygen and carbon dioxide concentration or partial pressure in the inlet gas and outlet gas, and a substrate flow acceleration based on the input signal. comprising a substrate supply device that can be controlled by a substrate supply device, a control device that has the function of performing calculations using input signals from the gas flow rate measuring device and oxygen and carbon dioxide gas analyzers, and outputting the results as output signals to the substrate supply device. A substrate fed-batch control device characterized by: